Chapter 1

Introduction to veterinary protozoology

History

Antonie van Leeuwenhoek (1674- 1716), a Dutch microscopist, was the first to observe and
describe the free living protozoa under a microscope fabricated by himself. He was also credited for
the discovery of the first parasitic protozoan, Eimeria steidae whose oocysts were observed in gall
bladder of a rabbit. Subsequently (in 1681) he found in his own stool, the organism which were
suspected to be Giardia lamblia.

1878 - Lewis found the first mammalian trypanosome, Trypanosoma lewisi in the rat in India.

1880 - Evans discovered the first pathogenic mammalian Trypanosoma evansi, causing ‘surra’ in
horses and camels in India.

1888 - Babes discovered Babesia bovis.

1893 - Smith and Kilborne described Babesia bigemina, the causative agent of Texas fever in USA.
They further showed that it was transmitted through the agency of an ixodid tick Boophilus
annulatus as it was first proof of an arthropod transmission of a disease causing agent.
2

1898 - Ross, working in India, discovered that Plasmodium reclictum (P. praecox) a malarial parasite
of birds, transmitted by culicine mosquito, Culex fatigans.

1902 - Dutton discovered Trypanosoma gambiense the causative agent of African sleeping sickness
of man in Gambia, Africa.

1903 - Leishman and Donovan, working independently, discovered Leishmania donovani in India.

1908 - Nicolle and Manceaux described Toxoplasma gondii in a rodent called gundi.

1922 -Tyzzer and Fabian discovered the transmission of Histomonas meleagridis through the caecal
worm (Heterakis gallinarum) of poultry.

1975 - Frankel and Dubey discovered Hammondia whch was structurally similar to Toxoplasma, but
has an obligatory two host life cycle.

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General characteristics of protozoa
A. Nucleus

Protozoa are eukaryotic (nucleus enclosed in a membrane) single celled microscopic

(majority) organisms. The bacteria are prokaryotic with their single chromosome coiled like a skein
of wool in cytoplasm. Usually only one nucleus is present in protozoans with the exception of
ciliates. The nucleus is very similar to that of a metazoan (multicelluar) cell and it is bound by a
perforated double layered unit membrane. This membraneous envelope separates the nucleus from
the surrounding cytoplasam and encloses a variable number of chromosomes (formed by
assemblage of DNA bound to basic proteins called histones).In addition, an intranuclear mass
(nucleolus) consisting of RNA may be present.

Basically the nucleus of protozoans are of two types. The nucleus in the majority of
protozoa (other than ciliates) is described as vesicular type which consists of a nuclear membrane
which bounds the nucleoplasm, chromatin and an intranuclear body. The chromatin is usually
arranged in the inner side or inner surface of nuclear membrane as small beads and also as strands
radiating from the intranuclear body towards the nuclear membrane. The intranuclear body may be
an endosome or karyosome (devoid of DNA) or a nucleolus (posess DNA) eg. coccidia.

The second type of nucleus is called as compact type of nucleus, contains a large amount
of chromatin and a small amount of nucleoplasm. Intranuclear body is absent. This type is found in
the ciliates, the usual number is two. The large sized macronucleus, is a polyploid, asexual nucleus
concerned with the control of the metabolic activities. The small sized micronucleus is diploid and
is concerned with the sexual reproduction.

B. Cytoplasm

Cytoplasm may be differentiated into an outer ectoplasm and an inner endoplasm, the former
often being homogeneous and hyaline in appearance and the latter frequently containing granules,
vacuoles and sometimes pigments.

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 C. Locomotion

In general, the locomotion in the protozoa can be put under four categories which are largely
based on different organelles used for this purpose.

(i) Amoeboid movement is characteristic of the whole phylum Amoebozoa. The organelle
associated with it is called as pseudopodium which literally means a ‘false foot’ implying that is a
temporary structure and can be formed and retracted at will. The pseudopodium also possesses a
phagocytic capacity and can function as a cup which closes enveloping particulate food material in a
vacuole.

(ii) Flagellar movements are the speciality of protozoa namely phylum Euglenozoa, Parabasalia,
Fornicata, Metamonada. Ciliary movements are the characteristic of phylum ciliophora. Flagellum
(whip like organelle) and cilium (eyelash-like organelle ) have basically similar structure.
Flagella are whip-like filamentous structures which arise from a basal granule or
blepharoplast in the cytoplasm of the organism. They are composed of a central axial filament, the
axoneme, which is surrounded by a contractile cytoplasmic sheath. Ultrastructural studies indicate
that the axoneme to be composed of two central filaments surrounded by nine peripheral filaments.
In some forms, the flagellum may be attached to the body of the protozoan by an undulating
membrane. Despite the structural similarity between flagella and cilia, cilia are fine, much smaller
than flagella, present in large numbers and arranged in definite rows. Many free living protozoa also
utilize flagella and cilia as food gathering organelles by directing the flow of water current
containing food particles towards mouth.

iv) Gregarine movement (gliding movement)

It is seen in protozoans like Toxoplasma and Sarcocystis and also in sporozoites and
ookinetes of certain apicomplexan parasites. Due to a small scale contraction of the pellicle, the
organism appear nearly to glide along without the help of any organelle.

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D. Nutrition

Among the protozoa, two principal modes of nutrition are found based on their capacity
whether they can synthesize food from inorganic matter (autotrophic) or can only use preformed
organic food material (heterotrophic). Autotrophic nuitrition is also known as holophytic or
phototrophic nuitrition. This is mainly encountered in plants and free living protozoa.

Heterotrophic nuitrion is seen in two forms (i) osmotrophic or saprozoic (ii) phagotrophic or
holozoic.

In saprozoic type of nutrion, the nutrients in the form of simple molecules, absorbed through
the surface membrane of the organism and no specialized organelles are needed. The digestion have
been done for them by the host.

In the holozoic type of nutrition, preformed food is ingested through a temporary opening
either by pinocytosis and phagocytosis and the complex molecules of protein, carbohydrate, fat etc.
are ingested, broken down in to simple units and they are rebuilt in to protozoan’s own protein,
carbohydrate and fat. However, in ciliates particulate food is engulfed through a permanent mouth
called cytostome and pass in to a food vacuole in the cytoplasam where it is digested. The
undigested food is ejectd out through a permanent opening called cytopyge.

E. Excretion

The metabolic waste products pass out of the body by diffusion through the surface
membrane or by contractile vacuole.

F. Respiration

Depending on the requirement of oxygen, the protozoa are either aerobes or anaerobes or
both. No organelles are needed for this purpose. The majority of the free living protozoa (and some
parasitic forms) behave aerobically. Most of the parasitic protozoa are either obligatory or
facultative anaerobes.

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 G. Reproduction

Reproduction in the protozoan may be either asexual or sexual.

Asexual reproduction cab be

(i) Binary fission

Binary fission is the commonest form of asexual reproduction. In this, two daughter cells result
from a 'parent' cell, division being along the longitudinal axis, although in ciliates it is along the
transverse axis. The nucleus divides first and cytoplasmic division follows.

(ii) Schizogony (merogony)

It is a kind of multiple fission in which the nucleus of an organism divides repeatedly and
rapidly so that its cytoplasm is not able to keep pace with the nuclear division, hence a
multinucleated structure is formed called as schizont or meront or segmenters. Depending on the
species, the schizont daughter nuclei may arrange themselves peripherally, with the membranes of
the daughter cells beneath the cell surface of the mother cell. The nuclei then move to the periphery
of the schizont and cytoplasam gets organized around each daughter nucleus, thus resulting in the
formation of a variable number of uninucleate merozoites.

(ii) Budding

It is an asexual reproductive process in which two or many daughter forms are produced by the
parent cell. There is usually an unequal fragmentation of the nucleus and cytoplasm, but the budded
forms are separated off and then grow to full size. The internal budding may be endodyogeny or
endopolygeny.

The endopolygeny, differs from schizogony only in the location where daughter cells are
formed. In this process daughter cells begin forming within their own cell membranes, distributed
through out the mother cell’s cytoplasm rather than at the periphery. It is seen in forms such as
Toxoplasma and Sarcocystis. During endodyogeny, two daughter individuals are formed inside the
cytoplasm of a parent cell. They are released by rupturing the parent cell.

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Sexual reproduction can be

(i) Syngamy

Two cells called gametes completely fuse with each other resulting in a zygote. The gametes are
produced by a process of gametogony from specialized cells called gamonts or gametocytes. If the
gametes thus produced are similar they are isogametes and if dissimilar, anisogametes. The latter
relevant to parasitic protozoa, are found in two forms. Microgametes (smaller gametes; male
gametes) are producd by a microgamont or microgametocyte in large numbers while a single
macrogamete (a large gamete or female gamete) is the product of macrogamont or
macrogametocyte. Sporogony, the free living maturation phase follows syngamy and a number of
sporozoites are formed with in the walls of a cyst.

(ii) Conjugation

The two cells temporarily fuse and there is nuclear exchange. This type of reproduction is
only seen in ciliates. Two mating ciliates (conjugants) get attached with each other by their oral
surfaces.

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 Classification of protozoa

Classification of protozoa is extremely complex and undergoing constant revision. The most
recent classification of protozoa now recognizes 13 phyla. Under the older system of classification,
as described in the most of the veterinary textbooks, there were four phyla containing parasites of
veterinary interest: Sarcomastigophora (containing Sarcodina and Mastigophora) Apicomplexa,
Microspora and Ciliophora.

Under the new classification system listed in the table, there are now nine phyla containing the
genera of veterinary importance

KINGDOM: PROTISTA
PHYLUM CLASS ORDER FAMILY GENUS
Entamoeba
Amoebozoa Archamoeba Amoebida Entamoebidae Iodamoeba
Endolimax
Percolozoa Heterolobosea Schizopyrenida Vahikampfidae Naegleria
Leishmania
Euglenozoa Kinetoplasta Trypanosomatida Trypanosomatidae
Trypanosoma
Tritrichomonas
Trichomonas
Tetratrichomonas
Trichomonadidae
Trichomitus
Pentatrichomonas
Trichomonadida Cochlosoma
Parabasalia Trichomonadea
Dientamoebidae Histomonas
Monocercomonas
Monocercomonadidae Chilomitus
Dientamoeba
Honigbergiellida Hexamastigidae Hexamastix
Retortamonas
Retortmonadea Retortmonadida Retortamonadorididae
Chilomastix
Fornicata Hexamitidae Spironucleus
Trepamonadea Caviomonas
Diplomonadida
Enteromonadidae Enteromonas

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Metamonada Trepomonadea Giardida Giardiidae Giardia

Preaxostyle Anaeromonadea Oxymondida Polymastigidae Monocercomonoides

Eimeria,
Isospora
Cyclospora
Tyzzeria
Wenyonella
Eimeridae
Caryospora
Hoarella
Octosporella
Pythpnella
Atoxoplasma
Apicomplexa Conoidasida Eucoccidiorida Besnoitia
Hammondia
Sarcosystidae Sarcocystis
Neospora
Frankelia
Toxoplasma
Lankesterellidae Lankesterella
Klossiellidae Klossiella
Hepatozoidae Hepatozoon
Haemogregarinidae Haemogregarina
Haemoproteus
Plasmodiidae Hepatocystis
Haemosporarida
Leucocytozoon
Aconoidasida Plasmodium
Babesiidae Babesia
Piroplasmorida Theileria
Theileridae
Cytauxzoon
Balantididae
Balantidium
Ciliophora Litostomatea Trichostomatorida Pycnotrichidae Buxtonella
Nyctotheridae Nyctotherus

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 Chapter 2

Phylum: Amoebozoa

Subphylum: Sarcodina (No thick pellicle, possess pseudopodia)

Class: Archamoeba (Lobosea)
Order: Amoebida
Family : Entamoebidae
Genus: Entamoeba, Endolimax, Iodamoeba
Genus: Entamoeba

The genus of Entamoeba has adapted to live as parasite or commensal in digestive tract of
vertebrate and invertebrate animals. Nucleus of this genus possess small vesicular nucleus with
small endosome at or near the center. Golgi bodies and mitochondria are absent. Members of Genus
Entamoeba are divided into four groups according to the morphology of trophozite and cyst.

Groups Histolytica Coli Bovis Gingivalis

Mature cyst 4 Nuclei 8 nuclei 1 nuclei Unknown
Chromatoid Rod shaped with Splinter Either Rod unknown
bodies rounded ends shaped
/Splinter
Trophozoite Granular endoplasm The ectoplasm is thin, The cytoplasm Clear ectoplasm
and hyaline cytoplasm possess food is smoothly
ectoplasm, vacuoles containing granular and
pseudopodia is long bacteria, yeast, starch filled with
and finger like, grains and vegetable vacuoles of
active trophozoite debris, movement is various sizes.
contains food sluggish and no finger A large
vacuoles which like pseudopodia. glycogen
contain RBC. vacuole may or
may not be
present.
Endosome Small and central Larger and eccentric Large, central Small and
endosome central
Peripheral Ring of small Ring of coarse peripheral Ring of small Fine, distributed
granules peripheral granules granules coarse irregularly
peripheral
granules

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Entamoeba histolytica

This is the most pathogenic amoeba responsible for amoebic dysentery in man, dog, monkey,
pig, rat, cat etc. and is rarely seen in cattle. First discovered in in 1873 by D. F. Losch, in St.
Petersburg, Russia. In 1903, Schaudinn named Entamoeba histolytica. ‘Histolytica’ literally means
tissue dissolving.

Distribution: worldwide

Location: Caecum and colon but can also invade organs like liver, lung and spleen causing
extraintestinal amoebosis.

Transmission: Infection occurs due to the lack of personal hygiene, faulty water supply,
contamination of food and water with the house flies or blow flies, through cloths of person,
contaminated raw vegetables, etc. (Cysts are transmitted. Trophozoites do not survive for more than
30 min after passing through stool).
Morphology : Several successive stages occur in the life cycle of Entamoeba
1. Trophozoite: The trophozoite (magna) /vegetative form (10- 60 µm) is the growing /feeding stage
of the parasite. It is large and actively motile in freshly passed dysenteric stools, while in
convalescents and carrier hosts it is smaller. The active trophozite exhibits remarkable locomotion
by pseudopodia. There is a thick clear layer of ectoplasm which is hyaline in appearance and well
differentiated from inner granular endoplasm (ground glass appearance). The endoplasm contains
the nucleus, food vacuole and granules. The nucleus is spherical 4 to 6 µm in size and contains a
small central karyosome surrounded by a clear halo. The karyosome is anchored to the inner surface
of the unclear membrane by fine radiating fibrils giving a cartwheel appearance. Typical amoeboid
motility is crawling/gliding movement and not a free swimming one. Division of trophozoite is by
binary fission once in about 8 hours. Trophozoites are delicate organisms and are killed by drying
heat, and chemical disinfectants.
2. Precyst: Some trophozoites undergo encystment in the intestinal lumen. Before encystment,
trophozoite extrudes its food vacoules and becomes round /ovoid to about 10-20µm in size and
form the precystic stage (minuta). It secretes a highly retractile cyst wall around it and becomes the
cyst.

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3. Cyst: Spherical and size varies from 10-20 µm. Early cyst contains a single nucleus, a mass of
glycogen and one to four chromatoid bodies/chromidial bars which are cigar shaped or oblong
retractile rods with rounded ends.
4. Metacyst: (Quadrinucleate cyst): As the cyst matures, the glycogen mass and the chromatoid
bodies disappear and the nucleus undergoes two mitotic divisions to form four nuclei or
quadrinucleate cyst which are mostly found in formed stools.
5. Metacystic trophozoite: After exycystation in the small intestine both cytoplasm and nuclei divide
to form eight small amebulas or metacystic trophozoite basically similar to mature trophozoite
except their size.
Humans are the primary known reservoir for E. histolytica. The main source of transmission is fresh
food or water contaminated with cysts in the the stools of chronically infected humans. Infective
cysts can be spread by arthropods such as cockroaches and flies.
Life cycle:
Infective form of the parasite is the mature cyst passed in the faeces. The cysts can remain
viable under moist conditions about ten days. The cysts ingested thorugh the contaminated food /
water pass through the stomach undamaged, enter the small intestine. Exscystation of cyst occur in
small intestine in alkaline medium by trypsin in the intestine. The cytoplasm gets detached from the
cyst wall and amoeboid movements appear causing a tear in the cyst wall through which the
quadrinucleate amoeba emerges. This stage is called the metacyst. The nuclei of the metacyst
immediately undergo division to form eight nuclei, each of which gets surrounded by its own
cytoplasm to become eight small amoebulae/ metacystic trophozoite. Though excystation occurs in
the small intestine, they colonize in the large intestine. The optimum habitat of the metacystic
trophozoite is the caecal mucosa where they lodge in the glandular crypts and undergo binary
fission. Some develop to precystic forms and cysts, which are passed in faeces to repeat the cycle.
The entire life cycle is thus completed in one host.

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 Pathogenesis:

Host factors such as stress, malnutrition, alcoholism, corticosteroid therapy and immuno
deficiency may influence the outcome of infection. Parasite factors that cause pathogenicity are

 Tiny cytoplasmic extensions from the surface known as filopodia. They are involved in the
pathogenesis for attachment to host cells, release of cytotoxic substances or contact cytolysis of host
cells.

 Gal/GalNAc lectin, (a heterodimer comprised of three subunits, 260 kDa) expressed on the surface
of the trophozoites, cause induction of the enzyme cyclooxygenase-2 (COX-2) in the lining of the
bowel, leading to an increase in the secretion of prostaglandin E2 (PGE2), which contributes to the
stimulation of the inflammatory process.

 Amoebapores the specific polypeptides secreted by trophozoites of Entamoeba histolytica, cause

cytolysis of the lining of the intestine.

 Cysteine proteases on the surface of the trophozoites (EhCP5)

The lumen dwelling amoeba do not cause any illness. Only when they invade the intestinal
tissue they cause disease. The metasystic trophozoite penetrate the columnar epithetical cells in the
crypts of Leiberkuhn in the large intestine. Penetration is facilitated by the tissue lytic substances
released by the amoebae which damage the mucosal epithelium and by the motility of the
trophozoite.

An intestinal lesion occurs by mucosal penetration by the amoeba which produce discreet
ulcers with pinhead centre and raised edges. The amoebae make their way to submucosal layer
when they multiply rapidly and form colonies destroying the tissues around by lytic necrosis and
forms abscess. The abscess breaks down to form ulcer. Amoebic ulcer is the typical lesion seen in
intestinal amoebosis. Ulcers appear initially on the mucosa as raised nodules with pouting edges.
They later break down discharging brownish necrotic material containing large member of
trophozoites. The typical amoebic ulcer is flask shaped. In cross section, mouth and neck being
narrow and the base large and rounded. Multiple ulcers may coalesce to form large necrotic lesions

12
 with ragged or undermined edges and covered with brownish sloughing. Some cases there is deep
penetration of the intestinal wall with secondary invasion of bacteria leading to hyperemia,
inflammation and neutrophil infiltration. Amoeba are mainly found at the periphery of the ulcer
leaving the cavity of the ulcer filled with necrotic tissues. As disease progresses mucosa of the
intestine develop abnormally and rupture, leading to strong, massive hemorrhage.

Amoebic granuloma/amoeboma are granulomatous mass, formed while healing and scaring
of the deep intestinal ulcers and may obstruct the bowel. Superficial lesions generally heal without
scarring.

Amoeba when enter the lymphatics/mesenteric venules and reach other tissues of the body,
secondary lesions are produced. Hepatic amoebiosis results when trophozoites enter mesenteric
venules and travel to liver through hepatoportal system which give rise to amoebic liver abscesses
(ALA) especially in right lobe. Active amoeba may be found at the wall of the abscess. The centre
of the abscess contains choclate brown pus (anchovy sauce pus) which is liquefied necrotic liver
tissue and is bacteriologically sterile and free of amoeba. Jaundice occurs in multiple lesions.
Pulmonary amoebosis develops by metastasis from hepatic lesion. Other ectopic sites include brain,
skin, penis (venereal transmission) and rarely kidney, adrenal, spleen, male and female genitalia
and pericardium.

Clinical signs:

Intestinal amoebosis:

Incubation period is 1-4 months. Amoebic dysentery/ Acute dysentery is characterized by

diarrhoea with lot of mucus and blood in the stool, abdominal pain, nausea, bloating, growling
abdomen and elevated body temperature. Chronic intestinal amoebosis is characterized by alternating
bloodless diarrhoea and constipation of varying severity, symptoms of chronic ulcerative colitis,
hypersensitivity of the intestines (colon irritable), an enlarged liver and soreness, low-grade fever,
wasting and anemia. Chronic condition simulate appendicitis.

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Extraintestinal amoebosis:

a) Amoebic hepatitis: The disease can develop as a consequence of acute intestinal amoebosis as a
result of transfer of trophozoites of Entamoeba histolytica through blood from the intestine to the
liver. This will cause increase the activity of the enzymes ALT and AST.
b) Amoebic liver abscess (ALA): When amoebic abscess of the liver occurs, there will be pain in the
right upper quadrant, hepatomegaly, elevated body temperature, lack of appetite and weight loss.
There is leukocytosis and accelerated ESR.
c) Pleuropulmonary amoebosis: This occurs through extension of hepatic abscess through the
diaphragm affecting lower part of right lung. In Rare cases, lung abscess due to haematogenous
spread and reddish brown pus is produced during cough.
d) Amoebic abscess in brain: Due to hematogenous spread, amoebic abscess may be produced in
the brain. Severe destruction of brain tissue occurs and it is fatal.
e) Cutaneous amoebosis: Occurs around the anus
f) Penile amoebosis : Prepuce and glans penis are affected.

Diagnosis :
1. Clinical symptoms like diarrhea, dysentery and severe abdominal pain.
2. Microscopic examination of stool:
a) Direct smear examination either as wet mount/stained smear (Heavy infections).
b) Concentration of cysts with zinc sulphate floatation solution (In lighter infections).
Cysts in the faeces may be stained using either with iodine (a saturated solution of 1%
iodine in potassium iodide) or trichrome stain or iron alum haematoxylin. Trophozoites, cysts, pus
cells, epithelial cells, red blood cells, Charcot Leyden crystals (products of degenerated
eosinophils)) can be detected in microscopy. Identification of E. gingivalis is made by the finding
of trophozoites in scrapings of the gums and teeth or in rare occasions in sputum. Permanaent slide
is prepared by fixing in the faecal smears in Schaudinns solution and staining by iron haematoxylin
method.
If a fresh stool specimen cannot be examined immediately, it should be preserved with a
fixative such as polyvinyl alcohol or kept cool (4°C). Occasionally motile trophozoites are seen
even after 4 h at this temperature.

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 3. Serological methods including enzyme-linked immunosorbent assay (ELISA)
4. Rapid immunochromatographic cartridge assay: A rapid cartridge is available which detects
antigens of E. histolytica/E. dispar
5. Molecular methods like PCR
6. Culture: Material for culture may be faeces/ content of abscess of the liver. Two types of culture
media are available for the isolation of Entamoeba spp. xenic and axenic media. Xenic cultivation
is cultivation of the parasite with undefined/unknown flora. Eg. Modified Boeck and Drbohlav egg
diphasic medium, Balamuth's medium, Jones's medium and TYSGM-9. Axenic cultivation is
growth of parasites in the absence of any unknown/undefined flora other than the protozoa intended
to be grown. Eg. TP-S-1, TYI-S-33.

Treatment:

Metronidazole is the drug of choice (Human: 750 mg TID for 5-10 days; dogs: 50 mg/kg for 3
days). For humans, secnidazole (2g single dose orally) and tinidazole (2g once daily orally for three
days) can be given. Antibiotics with vitamin B complex therapy may be given to prevent secondary
infections. Ornidazole and Furamide have been shown to use in combination with metronidazole.

Vaccine:

Targets for vaccine against E. histolytica include the Gal/GalNAc lectin, serine-rich protein, 29-
kDa-reductase antigen, heparin sulfate binding protein (HSBP) and the 30-kDa collagen binding
protein (CBP30).

Other species of Entamoeba

Species Host Location Charecters

Non pathogenic, Trophozoite

E. hartmanni Man Intestine
12-15 µm, cysts 5-9 µm.

Non pathogenic, Trophozoite

E. coli Man Intestine
15-50 µm, cysts 10-33 µm.

Non pathogenic, size similar to

E dispar Man Intestine
E. hystolytica

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 River and lake Free living amoeba Free living amoeba,
E. moshkovskii sediments Trophozoite 9-29 µm, cysts 7-
17 µm.
Man Pathogenicity in dispute.
E. gingivalis
Cat, dog, primates Trophozoite 10-20 µm, No
(Syn. E. canibuccalis) Mouth
cysts formed.

Pig, monkey Non pathogenic, cysts 10-18

E. polecki Intestine
µm.

Cattle Trophozoite 5-20 µm, cysts 4-

E. bovis Rumen
15 µm.

E. invadens Reptiles Harmless in turtles, Pathogenic

Intestine in snakes, Trophozoite 9-38
µm, cysts 11-20 µm.

Genus - Iodamoeba

Iodamoeba bütschlii is a nonpathogenic parasitic amoeba, commonly found in the large

intestines of man, pigs and other mammals. The trophozoite (9-14µm) has a single nucleus,
prominent endosome and many cytoplasmic vacuoles. The ectoplasm and the granular endoplasm
are often hard to distinguish. Cysts (6-15 µm)are with a thick wall and a large glycogen vacuole that
stains darkly with iodine. Cysts have an oval shape with single nucleus and a prominent nuclear
endosome.

Genus- Endolimax

Host- mammals, birds, reptiles, and amphibians

Location- colon, appendix
Endolimax nana is a small nonpathogenic intestinal protozoa with vesicular nucleus. The endosome
is comparatively large and irregular and is attached to the nuclear membrane by achromatic threads.
Trophozoites have 8–10 μm size. The ectoplasm is a thin layer surrounding the granular endoplasm.
Pseudopodia are short and if blunt and move very slowly (dwarf internal slug). Cysts of Endolimax
are oval and very small (6–9 μm × 5–7 μm) compared with cysts of other intestinal amoebae and
contains 4 nuclei. Transmission occur through faecal-oral contamination of food or water.

16
 Chapter 3

Phylum: Euglenozoa Genus: Trypanosoma

Phylum: Euglenozoa
Class: Kinetoplasta
Order: Trypanosomatida (Haemoflagellates)
Family: Trypanosomatidae
Genus: Trypanosoma
Genus: Leishmania
Family: Trypanosomatidae
Members of this family are all parasitic of blood stream and tissues of vertebrates (mammals and
birds). All the members except T. equiperdum have arthropod vectors.
Morphology

Trypanosomes have a leaf-like or round body containing a vesicular nucleus and varying
number of subpellicular microtubules lying beneath the outer membrane. A single flagellum arises
from a neuromotor apparatus called basal granule/blepharoplast/kinetosome. Basal body/basal
granule/ blepharoplast is a protein structure found at the base of eukaryotic flagellum or cilia. In
some forms a flagellum may pass along the body, being attached to it by an undulating membrane.
Closely posterior to the blepharoplast there is a deeply staining granule, the disc shaped kinetoplast.
A kinetoplast is a network of circular DNA (called kDNA) inside a large mitochondrion that
contains many copies of the mitochondrial genome. Kinetoplasts are only found in protozoa of the
class Kinetoplasta. Reproduction is by longitudinal binary fission.

Members of the genus Trypanosoma are heteroxenous and pass through amastigote,
promastigote, epimastigote and trypomasitgotes in their life cycle. In some species only
trypomastigotes are found in the vertebrate host; in others, presumably more primitive species, both
amastigotes and trypomastigote forms are present. Different developmental stages of the genera
classified under the family: Trypanosomatidae are

1. Trypomastigote stage: This is a blade like form with kinetoplast posterior to nucleus usually near
the posterior extremity. Undulating membrane is well developed. Free anterior flagellum is present.
Usually found in vertebrate host and is also found in arthropods as infective stage for the vertebrate
host.

17
2. Epimastigote stage (Crithidial stage): Kinetoplast and axoneme lies anterior to nucleus.
Undulating membrane is short. Principally seen in arthropods. In few species as a part of the
vertebrate developmental cycle.
3. Promastigote stage (Leptomonad stage): Kinetoplast and axoneme are seen at the anterior tip of
the body. No undulating membrane. It is found in arthropods or plants.
4. Amastigote stage ( Leishmania stage): Body is rounded. Flagellum absent and is represented by a
short fibril. Kinteoplast is present. Found in vertebrates and arthropods.
Genus: Trypanosoma

Found in vertebrates principally in blood and tissue fluids. Multiplication in the vertebrate
host by multiple fission or binary fission. Division starts at kinetoplast followed by nucleus and
cytoplasm. Transmission is through blood sucking arthropods either cyclically or non-cyclically
except in T. equiperdum which is transmitted through venereal route.

Cyclical transmission: In cyclical transmission, trypanosome multiplication occurs before it is

transmitted.
In Anterior station development, a developmental stage like procyclic trypomastigote
multiply in the mid gut of arthropods. Metacyclic trypomastigote stages accumulate in the mouth
parts or salivary glands and infection is transmitted while taking a blood meal. This is the
inoculative method of transmission. The various species of trypanosome which use this process are
often considered as a group, the salivaria.

In other trypanosomes, multiplication and transformation occurs in the gut and infective
form migrate to the rectum and are passed with the faeces; this is posterior station development.
Hence, the metacyclic trypomastigotes accumulate in the hind gut and are passed in the faeces of
arthropod. Infection of vertebrate occurs by contamination of the skin or skin wounds and the
trypanososme species are grouped together as Stercoraria.

Section: Salivaria

All the trypanosomes transmitted by tsetse (Glossina spp.) flies are under salivaria group.
Kinetoplast is terminal/ subterminal. Posterior extremity is blunt. Multiplication in the vertebrate

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host is continuous in trypomastigote stage. Metacyclic trypomastigote stage accumulate in the
anterior station of the arthropod host. Transmission is by inoculation.

Salivarian trypanosomes are included under 5 subgenus-

1. Sugenus: Duttonella-T. vivax

2. Sugenus: Nanomonas- T. congolense, T. simiae

3. Subgenus: Trypanozoon- T. brucei, T. rhodesiense, T. gambiense, T. equiperdum T. evansi

4. Subgenus: Pycnomonas-T. suis

5. Subgenus: Trypanomorpha- T. gallinarum

When the infected tsetse fly takes a blood meal, the metacyclic trypomastigotes are injected
into the host. Injected metacyclic trypomastigotes transforms into blood stream trypomastigotes
which are carried to other sites. Trypomastigotes multiply binary fission in various body fluids
(blood, lymph, spinal fluid). When a tsetse fly bites an infected host, the bloodstream
trypomastigotes are ingested, which transforms into procyclic trypomastigotes in the fly’s mid gut.
Procyclic trypomastigotes multiply by binary fission and leave the gut and transfoms into
epimastigotes. Epimastigotes multiply in the salivary gland and transform into infective metacyclic
trypomastigotes.

Life cycle of T. congolense is similar except that after it developed in the mid gut it goes
forward to the proboscis directly rather than going to the salivary gland. T. vivax does not have a
mid gut stage at all but develops in proboscis.

Section: Stercoraria

Posterior end of the stercoraraian trypanosomes are pointed. Free flagellum is present.
Undulating Membrane is not well developed. In stercoraria group, organism multiply in
trypomastigote, epimastigote and amastigote stages in a discontinuous manner. Transmission to the
vertebrate host occurs by posterior station development in the arthropods through contaminated
wounds with the faeces of vector. Stercorarian trypanosomes are included under 3 subgenus

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1.Subgenus: Megatrypanum-eg: T. theileri transmitted by Tabanid flies, T. melophagium transmitted
by sheep ked (Melophagus ovinus)

2. Sugenus: Herpetosoma - T. lewisi transmitted by rat flea

3. Subgenus: Schizotrypanum-T. cruzi (cause Chagas disease in south America in man) transmitted
in the faeces of Reduviid bug

Non-cyclical transmission/ mechanical transmission : The trypanosomes are transferred from one
mammalian host to other by interrupted feeding of biting insects notably tabanids and Stomoxys.
Flies immediately after feeding on infective host must feed on another animal for the transmission to
take place. They will be infective for a short period of time in the vector and within this time the
vector has to bite another host. Parasites do not survive in the proboscis of the flies for more than
10- 15 minutes.

• Trypanosoma evansi- transmitted mechanically by tabanid flies and vampire bat.

• Trypanosoma equinum- transmitted mechanically by tabanid flies

• Trypanosma equiperdum- transmitted mechanically by coitus

Mechanically transmitted trypanosomes

Trypanosoma evansi

Trypanosoma evansi was first identified by Griffith Evans (1880). It was the first
trypanosome shown to be pathogenic for mammals. Parasite is found intercellular in blood/ lymph
of variety of mammalian hosts and causes the clinical disease known as trypanosomosis. Depending
on the locality and kind of host affected, the different names are assigned. Disease is known as
SURRA in asia in all domestic animals, has been derived from the Hindi word means rotten. It is
also known by a variety of names elsewhere, such as el debab (northern Africa), mal de
caderas (Brazil), murrina (Central America). In camels, the disease is known as TIBERSA (3 year
disease-disease is tend to be chronic in nature for 3 years).

Hosts: camel, horse, donkey, mule, cattle, buffalo, sheep, goat

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Morphology: Leaf like parasite measuring 18-34 µm in length and 1.5 -2.5 µm in width. T. evansi is
characteristically monomorphic, slender and thin. Kinetoplast is subterminal. Undulating membrane
is well developed with prominent free flagellum.

Transmission: Transmission is mechanically by biting flies like Tabanus spp. (Horse fly). Disease
can also be transmitted by Haematopota spp, Stomoxys spp., Lyperosia spp. and Chrysops spp. The
non-blood sucking flies picking up the infection from infected meat to open lesions or mucus
membrane of susceptible animals. Vampire bats in the central and South America are known to
transmit the infection. Dogs may get the infection by ingestion of tissues from infected carcasses.

In India T. evansi infection is most common in areas where the environment for the breeding
of insect vectors like tabanid flies is most suitable. The worst affected areas in northern India are
West Bengal, Assam, Maharashtra, Gujarat and Rajasthan. Disease commonly occurs from the end
of August and remains prevalent till mid winter season.

Pathogenesis

Stress, malnutrition, vaccination may make the animals more susceptible to trypanosomosis.
Period of infection is variable and depends on the intensity of infection and species and health of the
animals. Symptoms usually appear after 1-2 weeks after infection.

1. Pyrexial stage: This stage correspond to the appearance of large number of trypanosomes in the
peripheral blood. Temperature is raised by 4-5o C above normal. Animal will be dull and depressed.
Pyrexia last for 2 days to 3 weeks. Temperature returns to normal with disappearance of
trypanosomes in the peripheral blood. Then trypanosomes reappear in the peripheral blood with
fever. Intermittent fever is a characteristic feature of T. evansi infection in animals.

2. Anaemic stage: The most common clinico-pathological findings is progressive anaemia. It is due
to haemolysins produced by the trypanosomes resulting in haemolysis of red blood cells and also
due to immunoglobulin specific for trypanosomes which form a complex with the antigen and
complement on red blood cells leading to erythrophagocytosis by macrophage system. Haemo-
dilution due to increased production of plasma and decrease in intravascular colloidal osmotic
pressure. Depression of erythropoiesis and nonspecific factors which increase red cell fragility,

21
formation of microthrombi and subsequent disseminated intravascular coagulation contribute to
aneamia and death.

3. Nervous stage: Not a common feature of T. evansi infection in animals. It may be exhibited in
certain cases characterized by great weakness of lumbar region, in coordination of muscular
movements, staggering gait, paralysis of hind quarters, prostration, inability to feed and drink and
finally death. Although majority of the animals die of exhaustion and severe anaemia in the anaemic
stage. Symptoms of nervous disorders have been observed in horses in the last stage of the disease.

The pathogenesis depends on three main factors anaemia, tissue lesions like myocarditis,
myositis and immunosuppression. The aetiology of tissue lesion in trypanosomosis is unknown.
Muscle fibre degeneration, mononuclear cell infiltration and oedema are common. Chronically
infected animals show emaciation associated with depletion of lymphoid cells.

Hypoglycemia is also a cause of death. The metabolism of carbohydrate is disturbed due to

malfunction of adrenals, pancreas and thyroid rather than due to direct utilization of glucose by the
parasite.

Trypanosomosis in horse:

The disease is severe in horse. Incubation period is 4 to 9 days. The main symptoms include
intermittent fever with temperature rising to 44ºC, anaemia, transient local or general urticarial
eruption specially on the neck and flank, haemorrhages at the junction of the skin and mucous
membrane like nostrils, eyes, anus, oedema of lower legs and lower parts of the body, testicular
oedema, submandibular oedema, petechial haemorrhage on the mucous membrane of eye, and also
in vagina in mare. The animal becomes dull, listless, gait becomes staggering, and respiration is
rapid and labored.

Postmortem lesions include marked anaemia, emaciation, enlargement of lymph nodes,

spleenomegaly and petechiae observed in the serous surface and liver and kidney parenchyma.

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Trypanosomosis in cattle and buffaloes:

Course of the disease may run from asymptomatic to peracute infection. Cattle and water buffalo
acts as a reservoir of the disease for equines. Occasional outbreaks may occur due to introduction of
the parasite or a new strain or additional stress due to vaccination.

Peracute cases show nervous sign within 2-3 hrs of infection. In subacute and chronic cases,
bilateral lacrimation, progressive emaciation rapid pulse, intermittent fever are seen. In buffaloes
abortion was reported. In acute cases, dullness, sleepy with staggering gait, staring and wide open
eyes, circling movements, nervous excitement, stamping of the feet, bellowing, frequent micturition,
twitching of muscle, shivering of the body followed by coma are seen. Death occur within 6- 12 hrs.

Postmortem lesions include splenomegaly, hepatomegaly, enlargement of lymph node and

kidneys.

Trypanosomosis in camel:

The disease is known as tibersa ( 3 yrs disease). Affected animals show intermittent fever, oedema
of pads and abdomen. Their hump disappears and become anorectic. Periodic convulsion is also
seen. Swelling and suppuration of inguinal lymph glands are also shown. The parasite may be seen
in blood even in absence of the symptoms.

Trypanosomosis in dog and cat:

Disease is more severe in exotic breeds than in the native ones. The course of the disease is
more serious in pups than that in adult. Incubation period is 5- 10 days. The disease is marked by
fever, anorexia, oedema of head and throat, corneal opacity, or even blindness. Other symptoms like
muscular spasms, staggering gait, excitement. Untreated dogs may die in 1-2 months. In cats chronic
cases are reported to be fatal.
Trypanosomosis in sheep and goat: intensity of symptom is less
Trypanosomosis in elephant (Thud): signs similar to camels
Trypanosomosis in pigs: the disease is not pathogenic and parasitaemia is low

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Diagnosis
1. History of the prevalence of T. evansi infection and of biting flies especially tabanids
2. Clinical symptoms: There are no pathognomonic clinical sign of T. evansi infection. However, the
characteristics clinical symptoms of emaciation and anemia are still used for provisional
diagnosis.
3. A variety of techniques commonly known as Standard Trypanosome Detection Methods
(STDM) based on direct microscopy (Wet blood film, thick blood film, thin blood films),
concentration (microhaematocrit centrifugation technique/ Buffy coat technique (BCT)) and
animal inoculation methods are generally recommended for identification of the agents.
i) Direct microscopy:
a. Wet Blood Film (WBF): Ten microliters of blood on EDITA collected from the suspected
animals were put on a clean slide, mixed gently with 10 µl of phosphate buffered saline (BPS),
covered with coverslip and the entire preparation was 2 examined at high magnification (400 X)
for 15-20 min for the detection of motile trypanosome.
b. Thick blood film, thin blood films: Preparation of thin or thick fresh blood smears or lymph
node puncture smears in chronic cases. Morphological appearance in Giemsa stained films under
400x and 1000x magnification were used to identify the trypanosome species.
ii) Concentration method:
a) Microhaematocrit centrifugation technique/ Buffy coat technique (BCT):
Microhaematocrite centrifuge tubes (75×1.5 mm) were filled with blood containing anticoagulant
collected from suspected animals and sealed with clay and centrifuged in microhaematocrite
centrifuge at 12,000 rpm for 5 minutes. A smear was prepared by scratching and breaking the
capillary tube 1mm below the buffy-coat and one drop of the buffy coat was expelled onto
microscope slide, smeared and covered with a coverslip (18×18 mm) and examined with 400 X
experiment, all animals were treated with trypanocidal magnification. The slide was examined for
15-20 min with 400 X magnification.

iii) Animal inoculation test:

This test is more reliable than the direct microscopic examination as it can detect subpatent
infection in blood. Albino mice and rats are most suitable hosts. Inoculate heparinised blood

24
intraperitoneally into mice (0.25-0.5 ml) or rats (1-2 ml). Inoculate a minimum of 2 animals. Bleed
the animal from the tails after every 48 hours to detect parasitaemia. Incubation period in mice is
short (5±2 days) but can extend to 2 weeks in rare cases.

4. Polymerase chain reaction (PCR) and Recombinant DNA probes: detect trypanosomes in
infected blood or tissue.

5. Immunodiagnostic tests/ serological tests

a. Indirect immunofluorescent antibody test

b. Antibody detection ELISA: using T evansi RoTat 1.2 variable surface glycoproteins. This test
successfully differentiated T. evansi from T. brucei.,
c. Card agglutination test (CATT): also makes use of T. evansi RoTat 1.2 clone
d. Latex agglutination test
e. Immune trypanolysis test: detects specific ‘trypanolytic’ antibodies directed against a given
parasitic strain able to induce trypanolysis in the presence of complement. It is performed with T.
evansi variable antigen type RoTat 1.2

6. Chemical tests:

Chemical tests are non-specific and less reliable. They are useful in the field for immediate
tentative diagnosis.

1. Mercuric chloride test is based on the quantitative chemical changes in serum of infected camels.
One drop of the undiluted serum is added to 1 ml of freshly prepared 1: 25,000 solution of
mercuric chloride in a test tube. The contents are gently mixed and observed after 15 minutes.
Appearance of distinct opalescence or white precipitate indicative of positive reaction. Utility is
limited to camels and unreliable for recent infections.
2. Thymol turbidity test is similar to mercuric chloride test and detects excess serum fraction.
Alkaline thymol buffer (3ml) is mixed with the 0.05 ml of suspected serum. A turbidity appears
immediately which increases to a maximum in 1 hr in positive cases. Test is more accurate than
mercuric chloride test.
3. Formol gel test was done for the diagnosis of surra in camels. Two drops of formalin is mixed
with 1 ml of test serum in a test tube. After thoroughly mixing the contents, allowed to stand for 2
hrs at room temperature and then kept at 4o C for 24 hrs. Distinct opalescence with or without

25
 gelation indicates positive reaction. The test has limited utility because of variable false positive
reaction and its unsuitability for the detection of surra in cattle, sheep, goat dogs and donkeys.
4. Jones nitric acid test is done to diagnose the African trypanosomosis of chronic or latent cases.
One drops of serum is added to 1 ml of 1.8% w/v nitric acid. Positive reaction is indicated by the
appearance of yellow colour.
5. Stilbamidine test is mainly used in the diagnosis of surra in cattle. One drop of fresh suspected
serum is added to the surface of 0.5 ml of 10% aqueous stilbamidine isothionate solution in a test
tube and the coagulation and sinking pattern of the serum were noted. The drop of serum would
coagulate on touching the solution of chemical. If the coagulam remained at the bottom of the
tube and took 5-10 min or more to dissolve, it indicated an intensely positive reaction. If the
coagulam that floated formed a ring at the surface and dissolved within 2 min., it was read as a
negative reaction.

Treatment

1. Diminazene aceturate (DA)- 7 mg/kg body weight intramuscular injection (I/M) (curative).
Diminazene aceturate is recommended in ruminants. Its use in horses and dogs is limited due to poor
efficacy and tolerance in these species.It is toxic in camels.

2. Quinapyramine (ANTRYCIDE)

Quinapyramine methylsulphate (ANTRYCIDE SULPHATE)- 5 mg/ kg body wt subcutaneous (S/C)

injection is used for therapeutic use in camel and horse

Mixture of quinapyramine methylsulphate and quinapyramine chloride (ANTRYCIDE PROSALT)

(3 parts of Antrycide methylsulphate and 2 parts of antrycide chloride) has prophylactic use in
addition to treatment of Surra. Protection for 3 months. Dose: 8 mg/kg S/C (mainly in horse and
camel).

3. Isometamidium chloride (IMC) (Phenanthridine family)- (Curative and preventive); Curative

(0.5 mg/kg body weight) and preventive (1 mg/kg body weight) treatment of trypanosome infections
in ruminants and horses, via I/M or S/C.

4. Suramin (Sulphonated naphthylamine)-{NAGANOL; ANTRYPOL}-(curative and prophylactic)-

10 mg/kg I/V- mainly used against T. evansi in camel; horse- 4g/ 45 kg body weight

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5. Melarsomine dihydrochloride (CYMELARSAN) is the latest trypanocide developed.

Camels- 0.25 mg/kg body weight deep intramuscular injection (recommended for curative
treatment of camels)

horses -0.25–0.5 mg/kg body weight I/M

cattle- 0.5 mg/kg body weight I/M
buffaloes -0.75 mg/kg body weight I/M
Control

1. Treatment of affected animals.

2. Chemoprophylaxis
3. Regular spray with insecticides, proper disposal of manure and treatment of breeding places
of vectors to reduce the population of flies
4. Identification of enzootic areas by regular examination of blood of animals.

Trypanosoma equiperdum

The parasite is smaller in size (15-30µm) to T. evansi and show lesser motility. It is monomorphic,
slender and with free flagellum.

Host- Equines (Horse, donkey and mules). Imported and thoroughbred horses are more severely
affected.

Distribution- parts of Asia, South America, South Africa and Russia.

The disease caused by T. equiperdum is known as Dourine (an Arabic word signifies
unclean/dirty) or Equine syphilis or breeding paralysis or Covering disease or mal de coït or syphilis
du cheval or morbocoitale malign or Beschälseuche or slapsiekte or sluchnayabolyezni. First case of
T. equiperdum infection in India was recorded from a stallion from Babugarh. After the
implementation of Dourine act V of 1910 of IPC, last positive case in India was during 1920-21.
Dourine was eradicated from Western Europe, North America and India.

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Life cycle of Trypanosoma equiperdum

Multiplication is by longitudinal binary fission. T. equiperdum is not a true blood parasite.

T. equiperdum is mainly seen in the mucosa of genital tract and skin. Normal habitat is lymph
channels of connective tissue. Occasionally gain access to blood stream (only during eruption of
plaques)

Transmission is mainly mechanical by coitus from stallion to mare and vice versa.
Trypanosomes in the secretion of sexual organs penetrate intact mucous membrane. Rarely
mechanical transmission by biting flies is also seen. Foals can gets infected through contamination
of conjunctival or nasal mucous membrane with infective vaginal discharge of mare.

Pathogenesis

Incubation period is between 2-12 wks or even more. Dourine occurs as chronic or acute
form. Disease lasts for 2-6 months and rarely for 1-2 years.

Chronic form: Symptoms of the chronic form of the disease are divided into 3 distinct stages
following the incubation period.

1)1st phase/Primary phase (Stage of oedema):- oedematous swelling of the genital organs

In stallion, after 10-12 days of mating with infected mare, oedema (cold and painless, rarely hot and
painful) of lower parts of sheath were seen, which gradually extends to the scrotum, inguinal region
and to the lower abdomen towards the chest. After 1 month, orchitis sets in, permanently swollen
sheath, protruded penis (due to irritation caused by inflammation), constant drawing and retraction
of penis, enlarged regional lymph nodes are seen.

In mare, primary symptoms of vaginitis, bilateral or unilateral oedmatous swelling of vulva

extending to anus, congested erect and irritable clitoris, rubbing of rump and tails against stable
wall, mucoid or viscid discharge from vulva are seen. Depigmented vulva is seen in old cases.
Vaginal mucosa is hyperaemic and ulcers may be present. In severe cases, frequent micturition and
even abortion is seen. Appetite is not affected. First phase lasts for 4-6 weeks and mild cases go

28
unnoticed. Parasite may enter into circulation and give rise to second stage of the disease
characterized by patchy eruption (plaques) on skin.

2) 2nd phase/ secondary phase (Urticarial stage)- as plaque eruption

Appears after 4-6 wks of the 1st phase when oval or spherical raised plaques of about 3cm or
more in diameter (upto 10cm) appear on the skin on sides of the body especially in sides and hind
quarters, neck, shoulder and thigh regions. They are the pathognomonic signs of the disease and are
classically known as dollar spots as they appear like a silver dollar or rupee coin is inserted under
the skin. The lesions are neither hot nor tender. The course of the disease is few hours or 3-4 days.
As swelling subsides depigmented areas develop. There is anaemia and the animal gets emaciated.
The inguinal lymph nodes are enlarged. Intermittent fever is seen.

3) 3rd phase/ Tertiary phase (Phase of paralysis): marked by paralysis, anaemia and extreme wasting,
loss of coordination, unilateral paralysis affecting hind limbs, lips, nostrils and ears. Later complete
paralysis occurs and recumbency may lead to death. Mortality is 50-70%.

Acute form: Initially there will be oedema followed by paralysis. Paralysis followed by a few days
of eruption of plaques. Animal dies within 8 weeks. Acute form is common in mares.

Post mortem lesions: Oedematous infiltration of the perineal tissue and the abdominal wall

Diagnosis

1. Based on the pathognomonic symptoms and pathagnomonic lesions on clinical cases.

2. Detection of organisms in smears from mucous membrane of genitalia and urticarial
swellings (fluid from the odematous patches, genital organs, plaques)
3. Complememnt fixation test (CFT) for the detection of antibodies to T. equiperdum. It is sure
and specific test.
4. Inoculation of blood into laboratory animals.

Treatment
None of drugs brings complete cure (Antrycide and suramin are useful).

29
Control

 Test and slaughter policy is recommended.

 The affected animal should be castrated to avoid the spread of infection.

 Dourine is dealt with legislative measures aimed at isolation, castration, or destruction of the
infected animals.

Trypanosoma equinum (Present valid name: T. evansi)

This is a large monomorphic form (20-30µm) of trypanosome lacking kinetoplast. Actually a

vestigial kinetoplast can be seen in electron micrograph, but it does not function in the activation of
mitochondrion. This condition is known as dyskinetoplasty. It occurs in central and south America.
Transmission is mechanical by biting flies. It is chiefly an infection of equines (horse, mules and
donkeys), and the clinical condition in horse is referred to as mal de Caderas or disease of Hip.
Mules and donkeys are less susceptible. The parasite is responsible for high mortality of horse in
wet areas, where incidence of blood sucking flies are high.

Pathogenesis and clinical signs:

The disease in horse usually the disease runs a chronic course with death occurring at 2-6
months after infection. The incubation period is 4–10 days after which pyrexia and parasitemia
appear. Emaciation commences early in the disease and a marked weakness of the hindquarter (mal
de Caderas) results in staggering gait. The disease is progressive and the animal finally becomes
recumbent. Transient cutaneous plaques occur over the neck and the flank, these lose their hair and
later scab occurs. Conjunctivitis, keratitis and oedema of the eyelids occur.

Post mortem

Splenomegaly, enlargement of lymph nodes, aneamia, peticheal haemorrhages in kidneys,

ascites and oedmatous infiltration in the spinal canal.

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Diagnosis

1. Demonstration of organism in peripheral bloods smear in acute stage and animal inoculation in
chronic stage

Cyclically transmitted trypanosomes

I. Anterior station forms (Section: Salivaria)

Subgenus: Dutonella: Monomorphic trypanosomes with free flagellum and a terminal large
kinetoplast.

Vivax group:-T. vivax and T. uniforme

1. T. vivax: Monomorphic trypanosome (21 µm to 25 µm) and found in found in ruminants and
horses but not in pigs, dogs, cats.

2. T. uniforme- Similar to T. vivax but smaller (12 to 20 µm), causing disease similar to T. vivax

Host –cattle, sheep &goat, antelopes.

Trypanosoma vivax

Host- cattle, water buffaloes, sheep and goat, camel, horse

Reservoir hosts: Antelope and giraffe
Morphology: Monomorphic trypanosome (21 µm to 25 µm) with incospicous undulating
membrane, large terminal kinetoplast, rounded posterior end and short free flagellum.
Distribution: Throughout Africa where tsetse flies (Glossina morsitans and G. tachinoides) acts as
cyclical vector while in Central and south America, West Indies, it is also transmitted mechanically
by biting flies.
Life cycle of T. vivax: Life cycle of T. vivax does not have a mid gut stage at all but develops in
proboscis of vector. Trypanosomes turn first into epimastigotes and then the metacyclic infective
trypanosomes which pass to hypopharynx and infect new hosts, when tsetse bite and feed.

Pathogenesis

T. vivax is the most important form of trypnosomois of cattle of West Africa and in cattle it
causes asymptomatic infection or chronic, acute or per acute disease. T. vivax causes the disease

31
Souma in cattle in Africa and is found in mixed infection with T. congolense and T. brucei. In acute
cases, extensive hemorrhages on the mucosal and serosal surface of digestive tract, body cavity,
muscles, heart and lymph nodes (acute hemorrhagic disease) are seen.

Diagnosis:-Peripheral blood smear, Lymph node smear.

Trypanosomes in Africa are parasites of wildlife and achieved a modus vivendi and come to
adjust with the host. It is called as trypanotolerance. N’Dama is a trypanotolerant cattle of West
Africa, which rarely develop the disease.

Subgenus: Nannomonas

Trypanosomes under this subgenus are small form without a free flagellum. Kinetoplast is
medium sized and marginal. Development in the tsetse fly occurs in mid gut and proboscis.

Trypanosoma congolense

Trypanosoma congolense is the most important trypanosomes of cattle in tropical Africa. It

is the smallest of all the African trypanosomes. It is small, monomorphic (8-20 µm/9-18 µm)
trypanosome without free flagellum. It shows pointed anterior extremity, medium sized marginal
kinetoplast and blunt posterior end.There is inconspicuous undulating membrane. In fresh blood
film organism moves sluggishly often apparently attached to RBC (non-progressive movement)

Host- cattle, sheep, goat, horse, camel, dog, pig (all domestic animals)
Reservoir host- wild game animals (antelope, giraffe, Zebra, elephant, wart hog, elephant)
Distribution- tropical Africa
Life cycle of Trypanosoma congolense
T. congolense divide in the vertebrate host by longitudinal binary fission. Bloodstream forms
taken up by the tsetse fly differentiate to procyclic trypomastigotes in the fly midgut. Procyclic
trypomastigotes proliferate in the mid gut and elongate to form long trypomastigotes without free
flagellum which move to foregut lumen via. proventriculus before migrating to the proboscis. In
foregut lumen they become epimastigotes and attach to the chitinous lining of the proboscis and
cibarium where they proliferate and subsequently develop into infective metacyclic trypomastigotes

32
(similar in appearance to blood forms). Development in the vector to infective stage takes in 15 to
20 days at 23-34o C.

Disease in cattle: T. congolense is the principal cause of the disease, Nagana (Zulu word meaning to
be in low or depressed spirit) in cattle often seen in mixed infection with T. brucei and T. vivax. The
name 'nagana' is used to describe collectively the tsetse fly (Glossina) transmitted trypanosome
infections of domestic animals in Africa caused by Trypanosoma vivax, T. uniforme, T. congolense,
T. simiae, T. suis and T. brucei.

In cattle the parasite can cause either an acute fatal disease resulting in death within 10
weeks, chronic condition with recovery in 1year or a mild almost asymptomatic condition.
Symptoms of diseases in cattle are similar to those caused by other trypanosomes, but CNS is not
affected.

Treatment: Diminazene acceturate (BERENIL) and homidium salts (Ethidium and Novidium) are
used in cattle.

Subgenous: Trypanozoon (Brucei group)

T. brucei brucei (T. brucei)

T. brucei evansi (T. evansi)- mechanical transmission by mainly by tabanid flies
T. brucei gambiense (T. gambiense)
T. brucei rhodesiense (T. rhodesiense)
T. brucei equiperdum (T. equiperdum)- transmitted mechanically by coitus

Trypanosoam brucei

Commonest and most important parasite of domestic animal of Africa

Hosts: cattle, horse, donkey, sheep, goat, camel, pig, dog, antelope

Predilection site – blood and extravascularly in the myocardium, CNS and reproductive tract. (T.
brucei is a humeral parasite, i.e. it is seen in intercellular tissue fluids such as of connective tissue
and fluids of the body cavity and in the plasma)

33
Vector-Tsetse fly (Glossina spp.)- Cyclical (anterior station development). Development occurs in
the mid gut and salivary gland of vector.

Polymorphic/pleomorphic forms are

(1) Long and slender form (up to 42 µm in length and with pointed posterior end and long free
flagellum)
(2) Intermediate form (23 µm in length; posterior end is more blunt and free flagellum present).
(3) Short and stumpy form (12-26 µm (av: 18µm) in length; posterior end more broad, kinetoplast
more terminal and no free flagellum)
(4) Posterior nuclear form (only in laboratory animals). All forms are seen in blood. Usually one
form predominates.

Life cycle of Trypanosoma brucei

During blood feeding, trypanosomes in blood or lymph are taken by tsetse fly and they lose their
glycoprotein surface coat in the fly and become elongated to become procyclic trypomastigotes and
multiply in the mid gut. Then migrate to salivary gland as epimastigote and after multiplication in
the epimastigote form, transform into small typical metacyclic trypomastigote with surface
glycoprotein coat. After a few days, at the site of inoculation the metacyclic form multiply locally as
the typical blood form producing a raised cutaneous inflammatory swelling known as Chancre.

Procyclins: Procyclins represent the major surface molecules of Trypanosoma brucei insect forms
and consist of two classes of proteins that are characterized by internal tandem dipeptide (EP
procyclin, a form of procyclin rich in Glutamic acid-Proline repeats) or pentapeptide repeats
(GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats) and are attached to the
membrane by a complex glycosylated glycosylphosphatidylinositol (GPI) anchor.

Variant Surface Glycoproteins/Variant-specific surface glycoprotein in the trypanosomes in the

vertebrate host. Activated in the tsetse-fly salivary gland and inactivated upon return to the tsetse-fly
mid-gut, they produce a protective cell coat throughout the mammalian infectious cycle.

34
Trypanosoma gambiense

Host: Man; Domestic animals are not readily infected

Vector: riverine tsetse flies (Glossina. palpalis and G. tachinoides)
They are morphologically indistinguishable from T. brucei. They are polymorphic trypanosome with
slender, intermediate and stumpy forms.
Development: anterior station as in T. brucei and it can also mechanically transmitted by biting flies.
They are responsible for Gambian sleeping sickness in man or trypanosomosis of humans, which is
a chronic disease in man.
Diagnosis

1. Based on symptoms, habitat,

2. Demonstration of organism in blood, lymph nodes and CSF

Trypanosoma rhodesiense

Host: man, wild and domestic animals

Vector: savannah tsetse flies (G. morsitans, G. swynnertoni and G. pallidipes) and development in
vector is anterior station as in T. brucei
They are indistinguishable from T. brucei and T. rhodesiense. They are polymorphic trypanosomes
with three forms (Long and slender, Intermediate and Short and stumpy form). T. rhodesiense
causes rhodesien trypanosomosis or East African sleeping sickness of humans. It cause more acute
disease in man than T. gambiense. Untreated case will end in death. T. rhodesiense infection in man
is a Zoonoses. Parasite is identified in wild and domestic animals and is mildly pathogenic to cattle.
It is also pathogenic for the rats and other laboratory animal.

African sleeping sickness

African sleeping sickness is caused by Trypanosoma gambiense and T. rhodesiense.

Incubation period: T. rhodesiense - days to weeks; T. gambiense - much longer - months to years

Symptoms occur in two stages. The first stage (hemolymphatic phase):

1. Painful chancre (red sore) will develop at the location of the tsetse fly bite.

35
2. Fever is intermittent (attacks lasting from a day to a week, separated by intervals of a few days to
a month or longer), head ache, joint pain, itching.
3. Lymphadenopathy, localized edema, rash
Winterbottom's sign- Swollen lymph nodes along the back of the neck at the base of the skull. It is
the posterior cervical lymphadenopathy associated with early phase of African trypanosomiasis as
trypanosomes travel in the lymphatic fluid and cause inflammation. It may be suggestive of cerebral
infection. Described by Dr. Thomas Masterman Winterbottom in 1803.
4. Anemia, endocrine, cardiac, and kidney dysfunctions
The second stage (neurological phase): Begins when the parasite invades the central nervous
system by passing through the blood–brain barrier and result in CNS involvement and nervous
impairment. This stage is described as meningoencephalitis,characterized by increased apathy,
fatigue, confusion,somnolence; motor changes including tics (uncontrolled, involuntary
movements), slurred speech, incoordination, convulsions, coma.
Alterations in endogenous circadian rhythms correlate with clinical symptoms.
Suprachiasmic nucleus (SCN), the “biological clock” regulates hormonal, sleep, body thermostat
activity. Spontaneous rhythm of Suprachiasmatic nucleus (SCN) is altered with trypanosome
infection. Infected individuals experience a disorganized and fragmented 24-hour rhythm of the
sleep-wake cycle, resulting in day time sleep episodes and night time periods of wakefulness.
('sleeping sickness’).'

Damage caused in the neurological phase is irreversible. T. b. gambiense will cause death after
several years (T. gambiense -more protracted course) while T. b. rhodesiense will cause death
within months (T. rhodesiense- more rapidly fatal). Both forms are always fatal without treatment.

Diagnosis

• Direct examination of aspirate or smear - wet mount for motile trypomastigotes

• Concentration techniques usually required before microscopic examination
• Animal inoculation/isolation - for T. b. rhodesiense
• CATT (Card agglutination trypanosome test)- useful for T. gambiense screening and
surveillance
• Serology: antibodies detection: IFA, ELISA (high levels of IgM is common)

36
II. Posterior station forms (Section: Stercoraria)

Trypanosoma cruzi

In 1910 a Brazilian physician Carlos Justiniano Ribeiro Chagas discovered this parasite and
named it as T. cruzi in homage to Oswaldo Goncalves Cruz, a pioneer in parasitic and infectious
disease in Brazil.

Host- Man (Human infants and children are most affected), dog, cat, pig, ferret, squirrel, wild and
domestic animals. Most mammals are susceptible.

Principal reservoir hosts: Armadillo and the opossum in south America

Vector: Nymph and adult of kissing bug or cone nosed bug or Reduvid bug (Genus: Triatoma) of
the family Reduviidae.

Predilection site: In early infection: blood. Later invade the cells of the reticuloendothelial system,
heart, striated muscle and other tissues, neuroglial cells of CNS.

Morphology: It is monomorphic and crescent shaped with a pointed posterior end. This is the main
pathogenic spp. of Stercoraria and causes American human trypanosomosis or chagas disease in
south America. In blood, no division take place in the trypomastigote stage while division occurs in
amastigote form in cells of infected tissue.

Life cycle of T. cruzi

When reduvid bugs sucks the blood of infected vertebrates, bloodstream trypomastigotes
will be ingested. Once ingested, trypomastigotes transform, in a few days later, either into spherical
stage (known as spheromastigotes) or into epimastigote stage. Epimastigotes migrate to the intestine
where they divide intensely. At the most posterior regions of the intestine and at the rectum, this
noninfective epimastigotes are transformed into highly infective trypomastigotes (known as
metacyclic trypomastigotes) which are then released together with feces and urine 8-10 days after
the initial infection. Kissing bugs usually defecate after feeding and commonly feed on the thin skin
near the eyes and lips. The human or mammalian host is infected when metacyclic trypanosomes are
directly inoculated through the ocular mucosa (conjunctiva) or the lesioned skin (bite wound) or are

37
rubbed into the wound made by the insect. Other important transmission mechanisms are by blood
transfusion, transplacental route and organ transplantation. Once in the vertebrate host, the
metacyclic trypomastigotes invade the cells at the inoculation site (e.g., fibroblasts, macrophages,
and epithelial cells) and transform inside the cell to amastigotes (also known as intracellular
spheromastigotes) and multiply by binary fission in a parasitophorous vacuole. Intracellular
amastigotes transforms to trypomastigotes and burst out of the cell and enter either blood stream or
infect other cells and transforms to intracellular amastigotes in new infection sites.

Pathogenesis

Following the infection of a wound, the metacyclic trypanosomes enter the macrophages and
proliferate in the amastigote form at the local site. There is local inflammatory response and later
encapsulation by fibrous tissue leading to blockage of the lymphatics, producing oedema of the local
area. This primary lesion is called chagoma. Then the amastigotes pass from local site to the local
lymph nodes and then by the lymphatic system to the whole body. The liver, lung, spleen, bone
marrow, cardiac muscle and brain cortex are affected.

Romana’s sign is a unilateral periorbital conjunctival swelling associated with the acute
stage of chagas disease. Named after Cecilio Romaña, an Argentinean researcher who first described
the phenomenon. Romana’s sign will occur if the parasites enter via the conjunctivae (e.g. by the
patient accidentally rubbing triatomine insect faeces into their eye), an eyelid swelling, known as the
Romaña sign, may form. This is a painless swelling of the upper and lower eyelid of the affected
eye, with accompanying conjunctivitis. The Romaña sign may persist for several weeks and be
accompanied by swollen lymph nodes.

The disease in human may be acute or chronic in character. Death occurs in 2-4 wks after
onset of symptoms. The clinical manifestation depends on the location of the organisms but the
cardiac form is common. Megacolon and mega esophagus are also observed.

38
Diagnosis

• Demonstration of the trypomastigote stage in thick blood films

• Puppies, kittens or guinea pigs are used to demonstrate amastigote form by blood inoculation
• Xenodiagnosis (Greek words xenos=foreign and diagnosi=diagnosis) – in xenodiagnosis, the
vector of a pathogenic agent is infected to make the identification of the parasite easier after
a period of multiplication. This is an important method of diagnosis for chronic phase of
Chagas disease where in low number of parasite occurs in the blood. The laboratory reared
parasite free Reduviid bugs (Triatomines) are allowed to feed on the patient suspected to
have Chagas disease. The bugs are then examined 3-4 weeks later for the presence of T.
cruzi in the hid gut/ excreta.

• Serological test includes CF, IFA, IHA, ELISA and molecular test (PCR)

Trypanosoma theileri

Trypanosoma theileri is the largest spp. of the genus Trypanosoma.

Host: cattle, buffaloes, and deer. Prevalent in the cattle in India but it is non pathogenic.
Vector: tabanid flies (Tabanus and Haematopota) and ticks

The final stages of parasite development take place in the lower digestive tract of the vectors
and, therefore, these parasite species are transmitted by contamination with parasite infected insect
vector excreta.

Avian trypanosomes

They are polymorphic, non-pathogenic trypanosomes and transmitted by blood sucking arthropods,
like mosquitoes, simulids, hippoboscids. Eg. Trypanosoma avium, T. calmetti, T. hannai, T.
numidae, T. dafilae, T. gallinarum.

Immunity to Trypanosomes

Trypanosomes produce severe and prolonged infection due to the unique ability to evade (
escape) from the immune response of the host due to their ability to change the nature of the surface

39
coat. It has been found that the trypanosomes contain with their genome 1000 different genes coding
for variant surface glycoproteins (VSG) on their surface. Each VSG provokes for the production of
antibodies which cause the lysis of trypanosomes. By the time the antibodies are produced, a
proportion of trypanosomes might have altered their chemical composition of glycoprotein and now
they will display a different antigenic surface coat and they are unaffected by antibody.

Variant surface glycoproteins or variable surface glycoproteins

The major surface protein of trypanosomes, chemically a glycoprotein dimer of roughly 50kDa
attached to the surface by glycosylphosphatidylinositol linkage.Mature VSGs are 400–450 amino
acids in length with the GPI anchor linked to their C-terminal. Ten million identical VSGs form
homodimers with one another and cover the parasite’s cell membrane like a dense homogenous
forest. They are discovered from Trypanosoma brucei. They are used by the parasites to evade the
host's immune system by mean of antigenic variation.

The genes are located in telomeric and subtelomeric regions and are often activated by the
duplicative transposition of a silent basic copy gene into an unlinked telomerically located
expression site, producing an active expression-linked copy of that gene. The parasite is expressing
a series of antigenically distinct VSGs from more than 2000 complete and partial (pseudogenes)
VSG genes. Only one VSG variant is expressed at a time. There are at least 200 active VSG alleles
and more than 1000 silent alleles and psuedogenes encoding VSG variants. Active genes can
recombine with silent genes to form new unique active genes. Combinatorial processes increase the
diversity of variable surface glycoproteins. VSGs allow trypanosomes to remain undetected by the
adaptive immune system almost indefinitely and escape the complement system. Individual
trypanosomes switch from one VSG to another at a rate of 10−2 to 10−7 switches per generation time
of 5–10 hours in a process called antigenic variation. This antigenetic switching allows
trypomastigotes to continually change their “appearance” to the immune system and remain
undetected through the course of infection. This monotypic surface coat is eventually recognized by
the immune system but by the time the body is ready to react the parasite has already silenced
expression of that particular VSG and begun expressing a new VSG.

40
 Chapter 4

Phylum: Euglenozoa Genus: Leishmania

Phylum: Euglenozoa
Class: Kinetoplasta
Order: Trypanosomatida (Haemoflagellates)
Family: Trypanosomatidae
Genus: Trypanosoma
Genus: Leishmania
Family: Trypanosomatidae

Genus: Leishmania

The genus: Leishmania is divided into 3 subgenera: Leishmania, Viannia and Mundinia. The
flagellates of this genus are primarily found in mammals (especially man, dogs and rodents) and
other hosts like lizards and bats.

The disease caused by this parasite became known as Leishmaniosis after William
Leishman, a Glasgwegian doctor seving with the British Army in India. In Dum Dum, a town near
Calcutta, Leishman discovered ovoid bodies in the spleen of a British soldier who was experiencing
bouts of fever, anemia, muscular atrophy and swelling of the spleen. Leishman described this illness
as “dumdum fever” and published his findings in 1903. Charles Donovan also recognized these
symptoms in other kal-azar patients from Chennai and published his discovery a few weeks after
Leishman. After examining them using Leishman's stain, these amastigotes were named Leishman-
Donovan bodies by Ronald Ross.

Morphology
1. Amastigote stage: Amastigotes occur in vertebrates in the macrophages and other cells of
reticuloendothelial system in the skin, spleen, liver, bone marrow, lymph nodes, mucosa and
other sites. They are ovoid or round (2.5-5µm X 1.5-2µm size).

41
2. Promastigote stage: is found in the invertebrate host (sand fly) and in culture (Novy-MacNeal-
Nicolle medium (NNN media)).They are spindle shaped (14-20µm x 1.5-3.5µm).
Life cycle
Multiplication is by binary fission. Vector is the sand fly of the genera Phlebotomus (old
world) and Lutzomyia (new world). During a blood meal, the Phlebotomus ingests leucocytes and
large mononuclear cells containing amastigotes. These develop in the midgut of the sand fly as
promastigotes by binary fission. They pass to the oesophagus and pharynx of the fly and their
number may be so great as to block the food canal. When a fly attempts to feed, a plug of organisms
may be dislodged and injected into the mammalian host. Infection can also occur when infected
sandflies are crushed on the skin.

Pathogenesis

Two main clinical forms, cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL)
depending on which types of phagocytic cells are invaded. In CL, the parasites infect macrophages
resident in the skin. When the host cell is full of parasites, it bursts and the released amastigotes will
infect neighbouring macrophages. In VL, the released amastigotes are spread by the blood
circulation and they infect cells of the mononuclear phagocyte system (reticuloendothelial system)
of liver, spleen, bone marrow, lymph nodes and the intestine.

Visceral leishmaniosis (kala-azar/ Dum-dum fever/Black Fever/Black Sickness)

Caused by Leishmania donovani (Laveran and Mesnil, 1903; Ross, 1903). The parasite is
responsible for causing kala azar, dun dum fever or visceral leishmaniosis in human.

Three ecological forms of Leismania donovani are recognized:

1. Leishmania donovani donovani of the Indian subcontinent and Burma. In India, epidemics found
in Assam, Bengal and Bihar causes Indian kala azar or dum dum fever. It affects young adults and
children aged 5-15 years. Transmitted by Phlebotomus argentipes. Human is the only known
reservoir of infection and does not affect dogs in nature (but can be experimentally infected).
Indian kala-azar has unique epidemiological feature being anthroponotic.

42
2. L. donovani sensu lato occurs in Kenya, Southern Ethiopia, Somalia, Uganda, causing African
Kala-azar

3. L. donovani sensu lato in Sudan, Western Ethiopia and other parts of Africa and is a zoonoses
where carnivores acts as reservoirs.

Leishmania infantum (Syn. Leishmania chagasi)

Leishmania infantum causes the Chinese kala azar, Mediterranean kala- azar and Russian kala-
azar. Reservoir hosts are fox, wolf, jackal, and porcupine with dog as a domestic reservoir host. L.
infantum responsible for zoonotic cutaneous and systemic diseases within Mediterranean region. It
is transmitted by P. chinensi and Lu. Longipalpis. Leishmania chagasi is responsible for New world
visceral leishmaniosis or American kala-azar.

Pathogenesis of visceral leishmaniosis

Incubation period varies from 10 days to more than a year. Clinical symptoms occur after 3-6
months. The disease starts with an irregular fever followed by malaise, headache, abdominal pain,
dysentery or diarrhoea and bleeding of the mucous membrane of mouth and nostrils. Skin becomes
dry thin, scaly and hair may be lost. Light coloured person show grayish discoloration of the skin of
hands, feet, abdomen and face which gave the name kala-azar in India meaning black fever.
Splenomegaly (spleen enlarges rapidly to massive enlargement, usually soft and non-tender),
hepatomegally (enlargement not to the extent of spleen), anaemia (develops rapidly due to blockage
of RE system) are the characteristic lesions of the disease. Macrophages, myelocytes and neutrophils
of the bone marrow are filled with the amastigote stages of the parasite. These are called Leishman
donovan bodies (LD) bodies. In advanced cases, there is ulceration of the digestive tract and
enlargement of nodules in the skin particularly on the face and neck. The nodules of skin are known
as post kala-azar dermal Leishmanoids (PKDL). These contain numerous leishmanial organisms
which result in premunition state giving a lifelong immunity in Leishmania donovani infection in
man.

43
Post Kala-azar Dermal Leishmanoid (PKADL/PKDL)

PKDL is a skin sequela of visceral leishmaniosis that appears as macular, papular or nodular
rash usually on face, upper arms, trunks and other parts of the body. First appear as whitish spots
which develop into lentil sized nodules in the skin particularly in the face and neck. It usually
appears 6 months to 1 or more years after the recovery or cure from kala-azar. People with PKDL
are considered to be a potential source of kala-azar infection. It is seen in East Africa in 50% of
patients with kala-azar and in Indian subcontinent in 5-10% of patients with kala-azar.

Visceral leishmaniosis in Dogs

Leishmania infantum is the most common and important cause of canine leishmaniosis
worldwide. Other Leishmania spp. reported in dogs include L. mexicana, L. donovani and L.
braziliensis. There is no involvement of dog with Indian kala-azaar. Pathogenesis is similar to that
of leshmaniosis in human beings. Prevalence of disease is less than 10% and only about 1 in 5
infected dogs are considered likely to develop clinical disease. German shepherd, Boxers and
Cocker spaniels are more likely to develop disease. In United States visceral leishmaniosis is
endemic in many foxhound kennels and is most commonly identified in this breed. Disease is most
commonly seen in dogs less than 3 years of age and older than 8. Clinical picture is one of slow and
progressive illness. Syndrome range from mild local self-limiting skin lesion to fatal systemic
disease. Symptoms include anaemia, emaciation, and diarrhea, terminating in death. In chronic
cases, eczema, scurfy desquamation and loss of hair are the characteristic of the disease. Eyelids,
lips and nostrils may show ulceration. Main dermatological lesions of visceral leishmaniosis in dogs
are keratitis, scaling of skin, seborrhea, onychogryphosis, ulceration, alopecia (often in symmetrical
distribution) .

Diagnosis of visceral leshmaniosis

1. Demonstration of organisms in spleen, lymph node, bone marrow (sternal or iliac crest puncture),
liver.
2. Cultivation of biopsy or postmortem material on NNN media to demonstrate the promastigote.

44
 NNN (Novy Mac Neal and Nicolle) medium (NaCl-6g, Agar-15 g, DW-900ml, Defibrinated
rabbit blood up to 1/3 vol. of the above medium base)

3. Immunodiagnostic tests: Complement fixation test, Indirect immunoflurrescence test, Indirect

haemagglutination test

4. Rapid rk39 dipstick test: Immunochromatographic tests (ICTs) based on the recombinant K39
antigen (rK39), comprising the 39 amino acid repeats of a kinesin-like gene of Leishmania. The
recombinant antigen is a 39-amino acid (rK39) cloned in Escherichia coli, from the C terminus of
the kinesin protein of Leishmania major in India. Used for detecting circulating anti-K39, IgG in
blood and serum. Highly sensitive and specific in the diagnosis of both VL and PKDL.

5. Napier’s aldehyde test:-1 drop of formalin is gently added on the side to 1-2 ml of suspected
serum. A gel of milky white opacity is formed in positive cases. A clear gel is not positive. Test
will be positive after 3 months of infection.

6. Chopra’s antimony test: Urea stibamine 4% solution is allowed to trickle down the side of a test
tube containing 1-2 ml of diluted suspected serum (1:10). A white precipitate is formed within 15
minutes.

Treatment of visceral leishmaniosis (VL) in Human

1. Sodium stibogluconate (SSG) (Pentostam® Injection.)- a pentavalent antimonial compound

Dose: pentavalent antimonial compound 20mg / kg iv or im x 30 days

2. Meglumine antimoniate (GLUCANTIME / GLUCANTIM)-a Pentavalent antimonial

compound

3. Sodium antimony gluconate (SAG)-a pentavalent antimonial compound.

4. Antimony gluconate (Solustibosan)- a pentavalent antimonial compound

5. Pentamidine isethionate- a diamidine compound very effective for treatment of Leishmania

donovani infection in the treatment of pentavalent antimonial-resistant cases of kala-azar

45
 6. Amphotericin B-1mg/Kg iv daily or alternate days for 15-20 infusion

7. Liposomal amphotericin B- Dose: a single dose of 10 mg/kg L-AmB intravenously.

8. Miltefosine- the first oral drug registered in India. Dose:100mg PO daily x 4 weeks

Treatment of visceral leishmaniosis (VL) in dogs

All known anti-Leishmania drugs used in dogs can lead to temporary or permanent remission of
clinical signs, but none are sufficient to eliminate the infection.

1. N-methyl-glucamine (meglumine) antimoniate- Dose: 100 mg /kg once a day for 4 weeks.
2. Allopurinol (FDA approved for use in dogs)-Dose: 5-20 mg/kg PO, every 12 hours for 2 to 24
months.
3. Aminosidine (paromomycin) (FDA approved for use in dogs): 5 mg/kg, SC, once a day for 3
weeks, plus 60 mg of meglumine antimoniate / kg, IM, every 12 hours for 4 weeks.
4. Amphotericin B (FDA approved for use in dogs)
5. Miltefosine

Control

 Control of sand flies by using insecticides

 Measures against the breeding places of the fly
 Removal of decaying vegetation and clearing of dense vegetation around the house
 Control of stray dogs when it infects the dog
Vaccine : LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate
against human leishmaniosis

46
Cutaneous leshmaniois

Leishmania tropica (Host: man, dog, gerbils and other wild rodents) causes oriental sore in
Middle eastern and Eastern countries with dry hot climate. In India disease is seen in north-west
India (Rajasthan). Disease transmission is by Phlebotomus sergenti, P. papatasi and P. perfiliewi.
Life cycle is similar to L. donovani except that amastigotes live in the reticuloendothelial
cells and lymphoid tissues of the skin and not of the viscera. The lesion is differentiated into dry or
moist or mildly ecthymatous. Dogs are commonly infected in India and dry form is more common
in dog. Moist form is prevalent in gerbils and other rodents which affect man. Anthroponotic and
zoonotic cutaneous leishmaniosis has been reported from Rajasthan and other adjacent northern
parts of India.
Leishmania tropica is found in more densely populated areas. Its lesion is dry, persists for
months before ulcerating, and has numerous amastigotes within it. By contrast, L. major is found in
sparsely inhabited regions in Africa, Middle East, Soviet Asia. Its papule ulcerates quickly, is of
short duration, and contains few amastigotes. Life cycle and vectors similar to L. tropica.

Cutaneous leishmaniosis occurs in three different forms:

1. Localised cutaneous leishmaniosis (LCL): Parasite is confined to the skin. Incubation period
is of 1 to 12 weeks. A papule or bump develops at the site of the insect bite. The papule grows and
turns into an ulcer. Most people with CL have 1 or 2 lesions varying in size from 0.5 to 3 cm in
diameter, usually on exposed parts of the body such as the face, arms or legs. Most lesions heal
spontaneously over months or years, leaving permanent scarring with skin thinning.

2. Diffuse cutaneous leishmaniosis (DCL) : It is a less common and distinguished from LCL by
the development of multiple, slowly progressing nodules without ulceration involving the entire
body. It affects only the skin but with generalized skin lesions. Seen mainly in Africa and is caused
by L. aethiopica.

3. Mucocutaneous leishmaniosis (MCL) (Espundia, Uta, Chiclero ulcer) manily caused by L.

braziliensis. Other species responsible are L. [V.] panamensis, L. [V.] guyanensis and L.
(Leishmania) amazonensis. MCL is restricted to Latin America. After the initial skin lesion has

47
healed, the disease spreads to the mucous membranes of the nose, mouth and throat. Subsequently,
the mucosal ulcers cause destruction of the nasal septum, lips and palate leading to extensive facial
disfiguring. MCL occurs when cutaneous lesions expand to the mucosal region or through
metastasis. It is a chronic form of leishmaniosis. MCL never heals spontaneously and is very
difficult to treat.

Cutanoeus leishmainiosis can be of two geographical types:

1. Old World (Eastern Hemisphere) cutaneous leishmaniosis (OWCL) (Oriental sore or Delhi boil,
or Aleppo boil or Aleppo Evil, Aleppo ulcer or Aleppo Button) is present in many endemic areas in
North Africa, the Mediterranean, the Middle East, the Indian subcontinent and Central Asia. The
species responsible for OWCL are mainly L. tropica and L. major. L. aethiopica can cause
diffuse cutaneous leishmaniois in Ethiopia and Kenya. All are transmitted by sand flies
(Phlebotomus). L. infantum and L. donovani can also cause cutaneous leishmaniosis in the
Mediterranean areas. Two distinctive clinical and epidemiologic forms of OWCL are recognized:

a. Anthroponotic cutaneous leishmaniosis / dry sore / urban cutaneous leishmaniosis / Dry

anthroponotic CL : An urban, anthroponotic, dry and chronic form of leishmaniosis caused by
Leishmania tropica, without a reservoir host, and now largely controlled. Seen in Middle East,
the Indian Subcontinent, Western Asia (However, both dogs and rodents serves as reseroirs
in Rajastahn)

b. Zoonotic cutaneous leishmaniosis / Wet sore / rural cutaneous leishmaniosis / Wet zoonotic
CL: the more common and wide spread zoonotic rural disease with a moist acute form, caused
by L. major, with reservoir rodent hosts. Seen in Africa, Middle East, Soviet Asia

2. New World (Western Hemisphere) cutaneous leishmaniosis are either due to the L.
mexicana species complex (L. mexicana, L. amazonensis, and L. venezuelensis) or the
subgenus Viannia (L. [V.] braziliensis, L. [V.] guyanensis, L. [V.] panamensis, and L. [V.]
peruviana). American cutaneous leishmaniosis is endemic in widespread areas of Latin
America. The spectrum of disease includes single localized cutaneous ulcers, diffuse cutaneous
leishmaniosis and mucosal disease. The main reservoirs for L. (V.) braziliensis and

48
 other Leishmania (Vianna) spp. are small forest rodents. The vectors are ground-dwelling or
arboreal Lutzomyia sandflies, which are abundant in the forest. A wide variety of skin
manifestations ranging from small, dry, crusted lesions to large, deep, mutilating ulcers may be
seen. The disease process can be divided into-metastatizing form and non-metastatizing form. In
metastatizing form, initial papule formation leads to ulceration with induration. The classical
espundia shows metastasis which takes several years to develop. The nasal septum,
nasopharynx, larynx may be involved with severe disfigurement.

Recidivans: Leishmaniosis recidivans or lupoid or tuberculoid leishmaniosis is a prolonged,

relapsing form of cutaneous leishmaniosis resembling tuberculosis of the skin. This form appears in
around 5% of the patients suffering CL by L. tropica. Leishmaniosis recidivans may last for many
years.

Diagnosis of cutaneous leishmaniosis

• Microscopic examination of materials taken from the edge of an ulcer or a local lymph node
for the demonstration of the organism

• Biopsy examination of the proximal lymph node

• Culture of materials in NNN medium

• Immunodiagnostic tests

• Montenegro skin test (Intradermal Leishmanin Test or Leishmanin skin test (LST): After an
intradermal injection of a suspension of killed promastigotes, delayed hypersensitivity
reaction is measured for detecting asymptomatic Leishmania infections. This test is used for
detection of disease in the New World. The test becomes positive a few weeks after
infection. Skin reactions to MST ≥ 5 mm are considered positive and < 5 mm are considered
negative.

49
Treatment of cutaneous leishmaniosis

1. Cryotherapy and intra-lesional injection of antimony: Apply the liquid nitrogen (-196°C) on the
lesion and up to 2 mm outside the lesion margin.Then, inject the antimony immediately into the
lesion.

2. Systemic treatment of cutaneous leishmaniosis with pentavalent antimonials

• Sodium stibogluconate: Dosage: pentavalent antimony compound 20 mg /kg/per day × 21

days.

• Meglumine antimoniate - (Glucantime® 5 ml ampoules containing 405 mg of pentavalent

antimony compound) Dosage: pentavalent antimony compound 20 mg /kg/day × 20 days.

3. Antibiotics are given to check the secondary bacterial infection

Control of cutaneous leishmaniosis: Use of repellants for protection against the sand fly

Immunity to cutaneous leishmanios: Recovery from cutaneous leishmaniosis is followed by solid

immunity to reinfection. With Espundia, there is less evidence of spontaneous recovery. Diffuse
cutaneous leishmaniosis (DCL) occurs in american cutaneous leishmaniosis associated with either
weak or totally absent cell mediated immunity. Cutaneous and mucocutaneous leishmaniosis give a
delayed skin reaction in montenegro skin test.

50
 Chapter 5

Phylum Parabasalia

(They possess a complex mastigont system i.e., an ultrastructural characteristic of mastigophorans,

comprising all of the organelles associated with the flagella, including basal bodies, axostyle, and
Golgi body.)

Class: Trichomonadea

Order: Trichomonadida (four to six flagella, free or attached to an undulating membrane;

no true cysts )

Family: Trichomonadidae

Genus: Trichomonas, Tritrichomonas, Tetratrichomonas, Pentatrichomonas, Trichomitus,

Cohlosoma

Genus Species Anterior Posterior Host Characters

flagella flagella

Location: In Cows genital tract,

Tritrichomonas T. foetus 3 1 Cattle In bulls, penile and preputial
cavity. No pelta.

Pigeon, Location: Pharynx, oesophagus,

T. gallinae 4 0
turkey crop, proventriculus

Location: Vagina, prostrate

Trichomonas gland, urethra. Largest human

trichomonad. Undulating
T. vaginalis 4 0 Human
membrane extends along one
third to two thirds of body
length

51
 Man, Location: mouth
T. tenax 4 0
Monkey

Pig Location: caecum and colon.

Trichomitus T. rotunda 3 1 Pelta present.

Chicken, Location: Caecum and liver.

turkey Pelta present. Lesions produced
Tetratrichomonas T. gallinarum 4 1
resembles histomonosis.

Human, Location: Large intestine. Pelta

primates, present.
Pentatrichomonas P. hominis 5 1
cats, dogs,
cattle

Genus Tritrichomonas

Species Tritrichomonas foetus ( Syn. Trichomonas foetus)

Morphology

The organism is pear shaped and has a single nucleus and four flagella. Three of the
flagella are free anteriorly and fourth extends backwards to form an undulating membrane along the
length of the organism, then continues posteriorly as a free flagellum. There is a vesicular nucleus
with endosome. Anterior flagella help in the propulsion of body and posterior flagella for rotatory
movement. The axostyle, a hyaline rod with a skeletal function, extends the length of the cell and
usually projects posteriorly. The costa is prominent but there is no pelta.

There are three strains serologically distinct-they are Belfast (Europe, Africa, USA), Brisbane

(Australia) and Manley of which Belfast is most pathogenic.

52
Host : Cattle, buffalo, pig, deer, horses, cats. Experimental infection has been established in
rabbits, golden hamsters, guinea pigs, dogs, pigs and goats.

Location: Cows- Genital tract, Bulls-Penile and preputial cavity

Life cycle

Organism multiplies by binary longitudinal fission. There are no cystic stages and
transmission occurs during coitus. Infected and recovered bulls are regarded as a permanent source
of infection. The parasite may also be transmitted through artificial insemination if bulls are not
properly checked and also thorugh equipments used for artificial insemination.

Pathogenesis and clinical signs

Trichomonosis, commonly referred to as “Trich,” is a highly contagious sexually transmitted

disease in cattle, resulting in abortions and infertility. Most common site of infection in bulls is
preputial cavity. Infected bulls show small red nodules on the preputial membrane and preputial
discharge. If not treated properly in time the infection then extends to testes, epididymis and seminal
vesicle and become permanent infection. Early clinical signs are pain on micturition and
disinclination to serve cows. Though the testes and seminal vesicle are infected, the fertility and
viability of sperm are not affected. There is no permanent recovery in the case of bulls and they act
as carriers.

Cows

Initially the organism multiply in the vagina leading to vaginitis. Later, it will enter to uterus
through cervix, vulva and vagina leading to cervicitis and endrometritis. If the infection is mild, cow
can conceive normally and deliver normally. If the infection causes placentitis, it lead to early
abortion (1 to 6 weeks). If abortion occurs very early (at 1-2weeks) the foetus and membranes are
expelled out unnoticed. Abortion before the fourth month of pregnanacy is the commonest sequale
and this is normally followed by recovery. If infection after 8-16 weeks of infected service,
placentitis with detachment of the placental membranes and death of the foetus with abortion and
uterine discharge occurs. Such a cow will show irregular heat periods.

53
 When foetus and membranes are not eliminated completely, maceration occurs leading to
chronic catarrhal or purulent endometritis. If the cervix is closed, there will be retained corpus
leuteum which lead to closed pyometra and permanent sterility. The uterus is seen filled with several
liters of thin greyish white fluid, swarming with trichominads. In the absence of bacterial infection,
the fluid is odourless. Immunity is short lived, and cows are susceptible to re-infection and abortion
in the following season.

Mechanisms leading to infertility and abortion are related to the parasite’s ability to infect
the mucosal surfaces of the reproductive tract, to bind to spermatozoa and to release a cysteine
protease (CP30) that induces cell death. The cysteine protease is capable of cleaving IgG2 and
evading the host immune response. Adherence of T. foetus to spermatozoa results in loss of motility,
agglutination and release of lysozymes that digest the sperm. Survival of T. foetus in the uterus
occurs for up to 22 months.

Clinical symptoms in cows include irregular oestrus cycle, purulent endometritis, pyometra,
early abortion, anoestrum, low conception rate and sterility.

Diagnosis

1. Field diagnosis of bovine trichomonosis is based on herd history which includes cases of
irregular oestrus cycles, infertility, failure of conception, early abortion and pyometra
2. Microscopic examination by demonstration of organism in placental fluid, stomach content of
aborted foetus, uterine washings, pyometra discharge or vaginal mucus (in bulls-Preputial
washings). Samples should be taken using a long pipette attached to a 20 mL syringe or
another suction device. In bulls, the pipette should be inserted into the sheath and up beside
the penis. The sample is then collected either by gently scraping or flushing with a quantity of
saline while holding the prepuce closed. Suction is then applied to collect the sample.
The identification of Tritrichomonas to the species level can be done with a
commercial Wright-Giemsa staining kit (Diff-Quick). The kit contains fixative (methyl
alcohol), Lugol's iodine, xanthene dye and thiazine. The use of iodine and iodide improves
the staining of the flagella and costa.
3. Serological tests: Vaginal mucus agglutination tests (VMA) and serum agglutination tests
4. Tricin test- An intradermal test for diagnosis of bovine trichomonosis

54
 The injection site is in the skin of the neck, similar to the site used for the tuberculin test. A dose
of 0.1 mLof the ‘Tricin’ antigen is injected intradermally and the reaction is measured 30–60
minutes later. The reaction consists of a shallow plaque observed visually and showing an
increase of >2 mm in skin thickness.
5. Cultivation- Trichomonas medium/ Clausens medium, modified Plastridge's medium, modified
Diamonds medium and InPouch™ TF medium (Field culture test)
6. PCR
7. Animal inoculation using golden hamsters
Treatment
Since the disease is self-limiting in the female, symptomatic treatment and sexual rest for 3
months is normally necessary. In bulls slaughter is the best policy, however dimetridazole (oral or
I/V) can be tried.
Control and Prevention
Control of the infection depends on proper herd management. All bulls should be tested four
times at weekly intervals after a minimum of one week’s sexual rest; before they can be assumed to
be negative. Culling of all positive bulls is adviced. All cows that abort or produce vaginal
discharge, may be investigated for the presence of the organism. Artificial insemination rather than
natural mating ia advisible. Positive bulls may be replaced with virgin bulls, or young bulls less
than three years of age as they are less able to transmit disease.
Vaccination
TRICHGUARD (2 mL administerd subcutaneously) is the first and only vaccine licensed
to reduce shedding of Tritrichomonas foetus. TRICHGUARD V5L against T. foetus, Campylobacter
fetus (vibrio) and five serovars of Leptospira is ( 5 mL administerd subcutaneously) also available.

Genus Trichomonas

Four free anterior flagella and no trailing flagellum

Trichomonas gallinae

Host: Pigeons, turkey, chicken.

Location –Oesophagus, crop, proventriculus

55
Description: The body is pyriform in shape having four anterior flagella, without a free posterior
flagellum and the undulating membrane extend about 2/3rd of the total length of the body.

Pathogenesis

Trichomonas gallinae cause of avian trichomonosis/ canker/frounce/roup. In turkey and chicken

lesions most commonly occur in the crop,oesophagus and phaynx.

Clinical signs
Severely affected birds lose weight and die within 10 days of infection. Yellowish white
circumscribed areas develop in the pharynx, oesophagus and crop and later they occlude the lumen.
The circumscribed disk shaped lesions are described as yellow buttons. The lesions in the liver,
lungs and other organs are solid yellowish caseous nodules up to 1cm in diameter. In turkey poults
and chicken, the infection results in pendulous crop and foul odour in the mouth.
Postmortem lesion
Yellowish necrotic areas in the mouth, crop, oesophagus, proventriculus and liver.
Diagnosis
Diagnosis is basedon post mortem lesions.
Demonstrating the organism in the samples taken from the lesion / fluid.
Treatment
1. Enheptin: 6.3 g of enheptin soluble per gallon of drinking water / 0.125 % enheptin for 7-14 days
2. Furazolidone: 25-30 mg/kg for seven days.
Control

Provision of clean and fresh drinking water on a daily basis, provision of fresh food from accredited
sources, rotation of positions of feeders in the garden to avoid build-up of contamination in single
area and clearance of food remnants that fall onto the ground.

56
 Chapter 6

Phylum Parabasalia Genus: Histomonas

Phylum:Parabasalia
Class: Trichomonadea
Order: Trichomonadida
Family: Dientamoebidae
Genus: Histomonas

Histomonas meleagridis

Host: Turkey, pheasant, chicken, quail. Parasite is ubiquitous in chickens, but not much harmful. It
cause disease in turkey.
Location: Caeca, liver
Intermediate host: Heterakis gallinarum (a caecal nematode)
Morphology:
Highly pleomorphic organisms and shape depends on location of the parasite. When they are
found in the lumen of the cecum (which is rare) or in culture, the stages are ameboid, 5-30 µm in
diameter, and almost always with only one flagellum. Liver stages are mostly amoeboid and do not
show flagella. Reproduction is by binary fission. No cysts or sexual stages occur in the life cycle.
Life cycle:
Birds become infected by ingestion of the embryonated eggs of caecal worm
Heterakis gallinarum. The unhatched larva which carries the organism, when reaches the intestine
of poultry, hatching occurs and histomonads are released from the larva. They enter the caecal
mucosa and cause ulceration and necrosis. They reach the liver through the portal stream and
colonize in the liver parenchyma producing circular necrotic foci which increases in size as the
parasites multiply in the periphery of the lesion. Next phase of the life cycle is not clear ie how the
worm get infection but it is assumed that it is through ingestion of the histomonads. Infection of the
birds may also occur when earthworms are ingested because earth worms are transport hosts for
caecal worm. The naked trophozoites are delicate and do not survive more than few hours when
passed in the faeces.

57
Pathogenesis:

Young turkeys up to 14 weeks old are highly affected and is characterized by necrotic
lesions in the caeca and liver. The earlier lesions are small ulcers in the caeca, but these quickly
enlarge and coalesce so that the entire mucosa becomes necrotic and detaches and forms a ceacal
plug with the caecal contents. The liver lesions are circular and up to 1 cm diameter with yellow
depressed centers.

Pathology:

Pinpoint ulcers in the caeca enlarges and affect the whole mucosa which ulcerate and
perforate the caecal wall causing peritonitis. The mucosa become thickened and necrotic and may be
covered with a characteristic foul smelling yellowish exudate which will form a hard dry caecal core
adhering to the caecal wall. The liver lesions are pathognomonic and consist of circular and up to 1
cm diameter with yellow depressed centers which extend deeply into the liver.

Clinical signs:
Histomonosis (infectious enterohepatitis, black head disease) infection is often mild and
asymptomatic in chicken. Turtkey poults show dullness, ruffled feathers, and droppings become
sulphur yellow coloured. The head and wattle become cyanotic (black head) which is characteristic
sign.
For culture of H. meleagridis, modified Dwyer’s medium (85-95% of Medium 199, 5-10% horse
serum, 0.8% rice powder) can be used.

Treatment:
Nitarsone (Histostat-50 ) is a pentavalent arsenical compound and it is the only drug of choice
available now against histomonosis for preventive use in feed. Nitoimidazole compounds,
dimetridazole (1,2-dimethyl-5-nitromidazole) is useful in the treatment of enterohepatitis in turkeys
(In feed, 150 - 500 ppm and in drinking water 300 - 1230 ppm).

58
Prevention:

• Prevent earthworms from accessing turkey holding area

• Do not run turkeys on range that has previously held chickens for at least 2 years.
• Well drained range
• Control cecal worms
• Preventative in-feed medication if high risk
• Maintain good biosecurity practices

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 Chapter 7
Phylum: Metamonada
Class: Trpomonadea
Order: Giardia
Family: Giardidae
Genus: Giardia
Giardia is a protozoan flagellate that was first observed by Antony Van Leeuwenhoek in
1681 and In 1915, Charles Wardell Stiles and others introduced the name Giardia lamblia to
commemorate the work done by Professor A. Giard in Paris and Dr. Lambl in Prague.
G. duodenalis ( earlier G. lamblia)
Morphology:
Organisms are seen in two forms, trophzoite and cyst. Trophozoites (12 µm -15 µm x 6 µm -
8 µm) are pyriform to elliptical (tear drop shaped) in outline and bilaterally symmetrical. Eight
flagella are arranged in four pairs. The anterior end is broadly rounded and the posterior end is
drawn out and somewhat pointed. The dorsal side is convex and the ventral surface bears a concave
bilobed adhesive disc. There are two anterior nuclei, two slender median axostyle and one pair of
darkly staining bodies placed medially. Adhesive disc along with the ventral flagella placed in the
ventral groove help in the attachment of organism to the cell. Cysts (8µm-12 µm x 7 µm – 10 µm)
are oval or elliptical and possess 2/4 nuclei and number of fibrillar remnants of the trophozoite
organelles.
Trophozoites multiply by longitudinal binary fission. No sexual reproduction occurs.
Transmission is through contaminated food and water mostly by cyst. At 4–10 oC, the cysts may
remain viable for several months. The cysts are relatively resistant to chlorination and to
disinfection by ultraviolet light.
Location: Upper digestive tract of their host.
Pathogenesis:
Animals
Most infections are asymptomatic, particularly in adult animals. Acute, chronic or
intermittent diarrhoea or soft stools may be seen in some dogs and cats. The stools are typically

60
light-colored and mucoid. They are often malodorous, and may contain undigested fat, but blood is
rare. Vomiting occurs occasionally, but fever is not usually present. Infected animals may also have
an unthrifty appearance and occasionally lose weight or fail to gain weight. The diarrhoea is self-
limiting in most immunocompetent dogs and cats. Giardia is a potential cause of diarrhoea and
decreased productivity in livestock, especially calves and other young ruminants, but it is still
uncertain whether this occurs to any significant extent. Clinical giardiosis appears to be uncommon
in horses.
Giardia spp. infections may also cause diarrhoea in some birds. Forms of diarrhoea
described by various sources include voluminous, aerated “popcorn” feces, soft green stools and
mucoid, malodorous diarrhea etc. Dry, scaly skin, which may progress to pruritus, feather pulling,
(especially from the axillae and inner thigh), and alopecia have been attributed to giardiosis in pet
birds. Weight loss, depression, decreased feed and water consumption, dehydration and deaths have
been documented in some case reports.
Man
Incubation period is 1-10 weeks. Children are more susceptible than adults. Acute cases are rare
than chronic cases. In chronic cases there are mechanical interference with absorption particularly
of fats in the intestine because of a parasite layer adhering to its wall. This malabsorption will lead
deficiency of vitamin A and D. There is chronic diarrhea. The faeces condition large amount of
mucus and fat but no blood. Epigastric pain and accumulation of gas in the abdomen are observed.
If gall bladder is involved cholecystitis can result. On postmortem examination, ulcerative lesions on
the digestive tract are seen.
G. duodenalis cysts shed in the feces are infectious. Both symptomatic and
asymptomatically infected animals excrete cysts, for days to months. The duration of shedding was
found to be at least 100 days in some dairy calves, up to 25 weeks in beef calves and more than 10
weeks in lambs and goats.
Diagnosis:
1. Demonstration of cyst and trophozoite in the faecal sample. Direct smears or fecal wet mounts
can be used for detecting trophozoites. Faecal sample is mixed with a drop of lugol’s Iodine and
examined. Samples should be taken from the surface of the feces, where organisms are more
common. Trophozoites are usually detected only in very fresh samples from animals with diarrhoea.

61
Cysts can be concentrated by passive fecal flotation or centrifugal fecal flotation. Zinc sulfate (33
per cent) preserves the morphology better than sugar solutions.
2. Immunodiagnostic tests like IFAT, ELISA.
3. Molecular techniques like PCR.
Treatment for livestock
1. Metronidazole (e.g., Flagyl) can be used in both dogs and cats (not in pregnant animals)
2. Albendazole is quite effective in dogs, and may be more efficacious than metronidazole in
stopping the shedding of cysts. Fenbendazole can also be used in dogs and cats at 50 mg/kg for 3-5
days.
Prevention

Prevention is considered to be impractical in livestock, and difficult in most species, because the
organisms are so prevalent in the environment.
1. Keeping pets indoors can reduce exposure to Giardia sources such as soil, unsafe water and
feces from other animals and wildlife.
2. Water treatments similar to those performed for humans (e.g., boiling or filtering) can
destroy the organism in unsafe water supplies.
3. Indoor housing and avoidance of crowding are expected to reduce transmission in livestock.
4. Rodent control and decreased contact with wild birds might lower the risk of infections in
captive birds and other species.
5. Regular cleaning, prompt removal of feces, and frequent changes of bedding materials
(where applicable) can limit contamination of animal environments.
6. Hard surfaces can be disinfected or steam cleaned after cleansing, and should be left to dry,
as the cysts are susceptible to desiccation.
7. Livestock facilities should be cleaned and dried between introduction of animals.
8. In birds, the use of wire-floors in cages can reduce access to feces and raised food and water
containers in bird cages are less likely to become contaminated by droppings
9. Ensuring that newborn mammals receive adequate colostrum can help build resistance to
disease.

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 Chapter 8
Phylum: Apicomplexa
Class: Conoidasida
Order : Eucoccidiorida
Suborder :Eimeriorina
Family :Eimeriidae
:Cryptosporididae
:Sarcocystidae
:Lankesterellidae
Suborder : Adeleorina
Family :Klossiellidae
:Haemogrigaridae
:Hepatozoidae
Class: Aconoidasida
Order : Haemospororida
Family :Plasmodiidae
Order : Piroplasmorida
Family :Babesidae
:Theileridae
All apicomplexan species are are obligate intracellular parasites and cause disease by
destroying their host cells. Other general characteristics include; micropore(s) generally present at
some stage; cilia absent; sexuality by syngamy; reproduction generally both asexual
(schizogony/merogony) and sexual (gametogony) methods; oocysts generally containing infective
sporozoites resulting from sporogony; locomotion of mature organisms by body flexion, gliding, or
undulation of longitudinal ridges; flagella present only in microgametes of some groups;
pseudopods ordinarily absent, but if present used for feeding, not locomotion.
Following invasion into their respective host cell (erythrocytes, lymphocytes, macrophages or
cells of the alimentary canal), most apicomplexan parasites grow and replicate within the
parasitophorous vacuole (PV), a nonphagosomal membrane bound compartment that is segregated

63
from most cellular trafficking pathways. The parasitophorous vacuolar membrane (PVM) constitutes a
barrier between the parasite and its host cell, conferring protection from host defenses. The PVM also
functions as an interface, which allows host cell–parasite exchanges. However, Babesia and Theileria are not
seen inside PV.
Apicomplexan structure
I. Pellicle :
The parasites are bounded by the pellicle, a composite structure consisting of the plasma
membrane and the closely apposed inner membrane complex (IMC). The IMC lies directly below
the parasite plasma membrane and is closely associated with it, creating a three-layered pellicle
characteristic of the Apicomplexa . In Plasmodium sporozoites, the IMC is constructed from a single
large flattened vesicle joined by a single suture line traversing the long axis of the parasite. In other
apicomplexans, it is made of many flattened vesicles aligned in longitudinal rows and joined in a
patchwork fashion by sutures.
The IMC extends throughout the body of the parasite and provides support for the gliding
machinery, which drives motility. Closely associated to the parasite pellicle is the subpellicular
network, which acts as the parasite’s skeleton and is constituted by the intermediate filaments.
Underneath the subpellicular network, at the apical tip, is the apical complex, after which the
phylum is named and it is present in motile stage of parasite.
II. Apical complex:
All invasive forms of Apicomplexa (referred to as zoites ie merozoites and sporozoites),
possess a unique complex of organelles (the apical complex) located at the anterior end of the
parasite and is not seen in other stages of development. Apical complex organelles have been
implicated directly in the sequence of events leading to host cell invasion, parasitophorous vacuole
membrane (PVM) formation and its maintenance. Apical complex organelles include the rhoptries,
micronemes, polar ring, dense granules and conoid. Its composition can vary depending on the
members of the phylum. The specialized secretory organelles, micronemes, rhoptries and dense
granules, as well as the apical polar ring, are present in all Apicomplexa. The conoid, is only present
in a set of parasites named coccidians.
1. Apical polar ring: Apical thickening of the inner membranes of motile stages of apicomplexa at
which the subpellicular microtubules are anchored. The apical polar ring is a hallmark organelle of

64
all members of the Apicomplexa. It serves as one of the three microtubule-organizing centers
(MTOCs) in these parasites. The spindle pole plaques and centrioles/basal bodies are the other two
MTOCs.
2. Conoid: Truncated cone shaped hollow cylindrical structure formed of spirally arranged
microtubules (composed of a polymer of alpha and beta tubulins assembled into a new type of
protofilament sheets). It can move up and down through the apical polar ring and protrude apically
at the time of cell invasion in a calcium-dependent fashion. Function of it is the mechanical
penetration of the host cells. Conoid is present in only in somome apicomplexan parasites
(Toxoplasma, Eimeria, and Sarcocystis) not found in Plasmodium and Theileria.
3. Micronemes: Unique tiny apical cigar shaped/elliptical secretory organelles that contain products
required for gliding motility, host cell recognition, adhesion to host cells and invasion of host cells.
Micronemes are first discharging proteins thought to participate in host cell recognition and gliding
motility.
4. Rhoptries: Tear drop shaped/club shaped/elongated paired specialized secretary organelles with
an anterior part called rhoptry ‘neck’ extending in the apical end and is characteristic of the motile
stage of the apicomplexan parasite. They discharge/secrete their contents (at least 20 enzymes are
present) through a duct forming parasitiphorous vacuole thereby assisting the penetration of the host
cell.
5. Subpellicular microtubules: Cytoskeletal structures seen beneath the pellicle and help in the
motility of the organism. The subpellicular microtubules radiate from the apical polar ring and run
down the cytosolic face of the pellicle, ending in the region below the nucleus (approximately two-
thirds of the length of the parasite). These spirally arranged microtubules closely follow the
serpentine body shape of apicomplexans. Subpellicular microtubules confer both elongated shape
and apical polarity to apicomplexan parasites
III. Cytoskeleton elements (microtubules and centrocone)
Centrocones: are sub-cellular structures involved in the cell division of apicomplexan parasites.
They exist in close apposition to centrosomes.
IV. Secretory organelles (exonemes, dense granules, micronemes and rhoptries)
Exonemes are secretory organelles involved in host cell egress in Plasmodium. Dense granules
(dense bodies in Sarcocystis) are the secretory vesicles found throughout the organism and is likely

65
involved in the maturation of the parasitophorous vacuole (PV) where the parasite multiplies. An
essential function of some of the dense granule proteins might be the construction of the cyst wall at
the encysted stage.
Micronemes release their contents early during the attachment-invasion process, then the
rhoptries are released as invasion proceeds, and finally the dense granules discharge their contents
when invasion is essentially complete.
V. Non-secretory intracellular organelles (mitochondrion, apicoplast, nucleus, endoplasmic
reticulum (ER) and Golgi)
V. Micropore: is a simple cytostome consisting of an invagination of the plasma membrane. It is
very similar in structure to the flagellar pits that occur in many other protist groups. Flagellar pits
are sites of endo and exocytosis. In apicomplexans material is taken in through micropores to form
endosomes or food vacuoles.
Life cycle of coccidia

Most important members of phylum Apicomplexa are coccidians. Coccidia the class of
Conoidasida, they develop in epithelial cells of alimentary canals and cause a form of enteritis called
coccidiosis. Coccidians are transmitted mainly by faecal contamination and reproduce by rigid
sequences of asexual and sexual phases of multiplication and development. In few cases, require an
alteration of hosts. Haemosporidians, develop in erythrocytes and cause hemolytic anaemia.
Haemosporidians are transmitted by blood sucking arthropods. They include the piroplasms, which
are transmitted by ixodid ticks and Plasmodida which are transmitted by by dipterans. They
complete their sexual phase of life histories in the invertebrate host.

The general form of coccidian life history is represented by the genus Eimeria, species of
which are gastrointestinal parasites of wide range of vertebrate hosts. Life cycle of coccidia involves
endogenous phase (schizogony and gametogony) followed by the infection and exogenous phase
(sporulation). Life history includes both asexual (schizogony) and sexual multiplication
(gametogony). Sexual multiplication culminates in the formation of oocysts, which are discharged in
faeces. During the development within each of these oocysts, eight infective organisms, the
sprozoites develops.

66
Asexual reproduction (schizogony/ merogony)

Infection of suitable host occurs by the ingestion of infective stage called sporulated oocyst,
through contaminated food and water. Inside the host, it undergoes excystation. For this, in animals
it requires two stimuli like CO2, bile and trypsin. In poutry, exystation is assisted by the grinding
action of the gizzard. The sprozoites emerged from the sporulated oocysts on excystation enter the
epithelial cells or lamina propria cells and round up to form trophozoites, grow larger and undergo
repeated division forming schizont (meront). Schizont divides to form large number of merozoites
(1st generation merogony/ schizogony). The number of merozoites formed in the first generation of
schizogony varies according to the species (Eimeria bovis-1,00,000, Isospora bigemina-16). After
completing division in the host cell, the mature meront rupture and merozoites escape to invade
neighbouring cells (initiate 2nd generation schizogony) to become second generation schizont.
Schizogony (merogony) may be repeated (the number of schizogonic generations depends on the
species) but two or three schizogonic generations are the limit for the many important species of
Eimeria. Salient attributes of schizogony are (1) expontial increase in the number of zoites arising
from a single sporozoites, (2) destruction of host cells in proportion to the degree of infection and
(3) automatic suspension of asexual process after fixed number of repetitions.

Sexual reproduction or Gametogony

Merozoites produced by final schizogony enter fresh host cell and develop either as male
gametocyte (microgamont/microgametocytes) or female gametocyte (macrogamont/
macrogametocytes). Macrogamont enlarges, stores food materials, induces hypertrophy of both
cytoplasm and nucleous of its host cells and matures to form the macrogamete or female sex cell.
From a single microgametocyte after repeated nuclear and cytoplasmic division, form many
uninucleate biflagellate microgametes or male sex cells. Microgametes are freed by rupture of host
cell. Microgamete fertilizes a macrogamete to form a zygote (nonmotile). Larger granules present at
the periphery of macrogamete called plastic or wall forming granules form the wall of the oocyst
following fertilization. Unsporulated oocyst which consists of nucleated mass of protoplasm
(sporont) or zygote enclosed by a resistant wall and are passed in faeces. Shape of the oocyst may be
spherical/subspherical /ovoid/ellipsoidal. Wall of the oocyst contains two layers, many species

67
possess a micropyle at one end especially at the pointed end and it may be covered by a micropylar
cap (polar cap). Unsporulated oocysts are expelled along with the faeces. In general, coccidial
infection is self-limiting. This is because, if asexual reproduction does not continue indefinitely and
in the absence of re-infection, only one cycle of development can takes place.

Sporogony

Initially, the zygote almost fills the oocyst cavity but within a few hours outside the host, the
protoplasm contracts from the wall of the oocyst to form the sporont and leaves a clear space
between sporont and wall. Thus, unsporulated oocysts are made. Under suitable conditions of
oxygenation, moisture (humidity) and temperature (27-30°C), sporulation takes place. The sporont
(single cell) in the oocyst divides to into four sporoblasts and remaining protoplasm form the oocyst
residual body. The sporoblasts are initially more or less spherical but later they elongate into ovoid
or ellipsoid bodies which later become sporocyst by laying down of refractive material. The
protoplasm inside each sporocyst further divides to form two banana shaped haploid sporozoites.
The remaining protoplasm within sporocyst form the sporocyst residual body. Sporocyst may have
knob at one end called steida body. Sporulated oocysts are the infective stage of coccidian parasites.

Structure of sporulated oocysts

1. Double layered wall 2. Polar granule 3. Micropylar cap 4. Micropyle 5. Oocyst residual body 6.
Sporocyst 7. Stieda body 8. Sporocyst residual body 9. Sporozoite
Family Eimeriidae
Genus: Eimeria; Cystoisospora; Isospora; Tyzeeria; Wenyonella
Coccidiosis
Infection of the genera Eimeria, Isospora and Cystoisospora are generally referred to as
coccidiosis and is usually an acute invasion and destruction of intestinal mucosa by these
protozoans. Life cycle is homogenous (all stages are seen in single host) ie schizogony and syngamy
occurs inside the same host. The sporogony occurs outside the host in the environment. Coccidiae
are usually host specific. Location of coccidial parasites in the intestinal epithelial cells of host is
either above or below the nucleus.

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 Chapter 9
Coccidiosis of ruminants, pigs, horses and rabbits
Bovine coccidiosis: At least 20 different species of Eimeria are known to infect cattle of which 13
species are more commonly found. ‘Coccidiosis is commonly a disease of young cattle (1–2 months
to 1 year). However outbreaks do occur in cattle up to 2 years of age, most often due to Eimeria
zuernii and E. bovis. Coccidiosis in bovines is sporadic during the wet seasons of the year. The life
cycle is typical coccidian type and it takes between 1-4 weeks depending on the species of Eimeria.
Both E. bovis and E. zuernii have two generation of schizogony.
The most pathogenic species of coccidia are those that infect and destroy the crypt cells of
the large intestinal mucosa. This is because the ruminant small intestine is very long providing a
large number of host cells and the potential for enormous parasite replication and minimal damage.
Pathological changes in most pathogenic species is mainly due to the large number of oocysts
ingested over a short period of time and hence there is low rate of cellular turn over. In calves
denudation of mucosa leads to severe haemorrhage and impaired water restoration leading to
diarrhoea, dehydration and death. Because of loss of villus structure and diminution of brush border
“flat mucosa” is produced which decrease the surface area for absorption of nutrients and leads to
reduced feed efficiency.

Eimeria bovis
It is the most common coccidia infecting cattle
Location - lower small intestine, caecum and colon.
Distribution- worldwide
Host- cattle, buffalo, zebu
Oocyst is ovoidal, blunted across the narrow end and medium sized, wall is smooth
homogenous, transparent, greenish brown in colour. Micropyle is present appearing lighter area of
the wall. Sporulation time is 48-72 hrs. First generation schizonts occur in the endothelial cells of
the lacteals of villi of lower ileum and they are giant (globidial schizont). This megaschizonts
(250µm) of E. bovis are macroscopically visible. Second generation schizonts are microscopic and
located in the epithelial cells of the caecum and colon. Gamonts are also seen in caecum and colon.
Prepatent period is 16-21 days.

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Pathogenesis and clinical signs
Gamonts cause greatest pathogenic effect. Onset of clinical signs coincides with the
beginning of gametogony and results from the mechanical disruption of mucosal cells. In severe
infections many of the crypts of large intestine and small intestine are destroyed. The epithelial layer
becomes denuded and lumen of the intestine is filled with blood. The mucosa becomes necrotic and
sloughed. This cause diarrhoea with or without blood.

E. zuernii
Host- cattle, zebu, water buffalo
Distribution -World wide
Location- Small intestine, caecum, colon, rectum
Most pathogenic coocidia of cattle.Oocysts are sub spherical, colourless, with thin wall,
homogenous, transparent, with no visible micropyle and are very small. Sporulation time is 9-10
days. Prepatent period is 19- 20 days. 1st generation schizonts are giant schizonts and occur in the
lamina propria cells of lower ileum. Mature oocysts are visible as whitish specks in the mucosa.
Oocysts are highly pathogenic for young calves causing red dysentery (very severe diarrhoea
streaked with blood). Later straining and spurting of blood along with diarrhoeic faeces occur. Back
of animal appear as painted red. Acute condition remains for 3-4 days. Survived animals then take a
chronic course with loss of weight, rough hair coat and difficulty in walking.

Winter coccidiosis is caused by E. zuernii in cattle in cold countries during winter. Severe
outbreak is seen in stabled or yarded animals because bedding provides warmth and moisture for
sporulation of oocyst in subzero temperature. Winter coccidiosis in calves is characterized by
bloody diarrhoea and tenesmus. Nervous coccidiosis is caused by E.zuernii during coldest months
of year. In addition to acute diarrhoea, affected animals display muscular tremors, convulsions,
opisthotonus, nistagmus, blindness. Mortality rate is 50%.

Postmortem lesions
In E. zuernii infection, lesions appear as catarrhal enteritis in small and large intestine.
Intestine may be filled with semifluid hemorrhagic material or even frank blood with fibrinous clots.
Epithelium may slough away leaving large denuded areas infiltrated with lymphocytes and

70
leucocytes. Swelling and thickening of mucosa with greyish white areas of necrosis are also seen.
Ininfections due to E. bovis, lesions are similar to E. zuernii but are of less severity. In severe
infection majority of crypts of large intestine and sometimes terminal part of small intestine are
destroyed. Epithelial layer is denuded and lumen of intestine is filled with blood. Mucosa is necrotic
and sloughed and this damage may extend to submucosa.
Bovine coccidial oocyst with heavily granulated (mamillated) oocyst wall : E. auburensis
Largest oocyst of bovine Eimeria: E. bukidonensis
Smallest oocyst of the bovine Eimeria: E. subsperica
Bovine coccidial oocyst with thick brown wall with evenly distributed protuberances: E. pellita

Diagnosis
Diagnosis is made from history, clinical signs like severe diarrhoea in young animals, Post-mortem
findings like inflammation, hyperemia, thickening of caecum with masses of gamonts and oocysts in
scrapings and oocyst count.

Treatment
Drugs that can be used for therapy of clinically affected animals include sulphonamides and
amprolium
1. Sulphonamides: These are more active against 2nd generation schizonts. It inhibits folic acid
synthesis. The sulphonamides can either be coccidiostatic or coccidiocidal depending on the dose,
duration of treatment and combination.
eg: Sulphadimidine, sulphaguanidine, sulphaquinoxaline (6 mg/lb/day for 5 days) and
sulphamethazine (110 mg/kg/ day for 5 days).
2. Amprolium: A thiamine antagonist acting on the 1st generation schizonts to prevent merozoite
production. Continuous use of this drug will lead to polyencephalomyelitis in calves and kids (due
to thiamine deficiency). Amprolium (10 mg/kg/day for 5 days for treatment) and amprolium (5
mg/kg/day for 21 days for prevention/ prophylaxis).
Other drugs can be very useful in helping to prevent bovine coccidiosis are 1. Lasalocid (1
mg/kg per day, max. 360 mg/day); 2. Decoquinate (22.7 mg/100 lb. daily for 28 days),3. Monensin
(100 to 360 mg/head per day); 4. Toltrazuril in water and diclazuril in feed.

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Prevention: Prevention can be achieved by good management practices viz., protecting drinking
water and feed from contamination with manure by raising water and feed troughs above the
ground, not feeding directly on the ground; draining excessive moisture from the pens; providing
dry bedding using well drained pastures; adopting minimum grazing on grasses near the ponds and
streams where overgrazing occurs; and isolating and treating of infected animals.

Coccidiosis of sheep and goats

Fourteen species of coccidia have been identified in goats, of which nine species are
commonly identified based on oocyst morphology and predilection site. Usually, single generation
of schizogony occur in sheep and goat. Oocyst usually contain micropyle and polar cap.
E. arloingi: The most common and pathogeneic coccidia of goats. Gametocyte causes hyperplasia
and pseudo-adenomatous metaplasia of villi in the small intestine causing polyps and focal
hyperplasia of the mucosa.
E. christenseniï: One of the most pathogenic species of domestic goats (kids) affecting small
intestine. Infection causes desquamation of the mucosa and superficial necrosis.
E. ninakohlyakimovae: A moderately pathogenic eimeria of goat (kids) affecting the posterior

small intestine and large intestine. It cause widespread denudation of the mucosa of large

intestine.

E. gilruthi : Occurs in abomasum and rarely in small intestine of sheep and goats. The globidial
schizonts are visible to the naked eye in the as whitish nodules.
E. intricata: is largest species of Eimeria in sheep.
E. bakuensis (E. ovina) is the commonest species in sheep. Papilloma like lesions may occur in the
small intestine.
E. crandallis: Occurs in the small and large intestine of sheep and known to be highly pathogenic.
E. ovinoidalis: Occurs in the small and large intestine of sheep and this species is the most
pathogenic of sheep coccidian. Life cycle and pathogenesis are similar to E.crandallis
Clinical signs: Coccidiosis in sheep and goats is chiefly confined to young animals upto 4-6
months of age. In lambs or kids that become heavily infected, the mucosa becomes completely
denuded resulting in severe haemorrhage and impaired water resorption, leading to diarrhoea,

72
dehydration and death. Brownish to yellowish green diarrhoea which may be streaked with blood
are seen. Abdominal pain, anaemia, inappetance, weakness and loss of weight are the other
symptoms.
Pathological changes: vary with species. Giant schizonts and gametogenous stages produce
enlargement of villi (may be visible to naked eye), polyp like growth on the mucosa of small
intestine and in more acute cases, the wall of intestine is thickened, oedematous and haemorrhagic.

Coccidiosis of pigs

It is a disease of young animals. The older pigs act as carriers. Oocysts produced by the sow during
the periparturient period act as a source of infection. Piglets are infected by coprophagia.
1. Cystisospora suis (Syn. Isospora suis): occurs in the small intestine and is the cause of naturally
occurring severe enteritis in young piglets aged 1-2 weeks. Most of the litter scours at 8-10 days of
age. Diarrhoea ranges from white to cream pasty faeces through watery diarrhoea. Affected piglets
are stunted and hairy. Severely affected piglets show dehydration, continue to suckle but weight
gains are reduced. Mortality is low to moderate.
2. E. debliecki: occurs in the small intestine and is the most common species in pig. It causes clinical
disease and severe pathology in young piglets.
E. polita, E. scabra and E. spinosa cause moderate to mild diarrhoea in piglets
Clinical changes and pathology: Diarrhoea, inappetance, emaciation, depressed growth, occasional
mortality young piglets, catarrhal inflammation of jejunum, villous atrophy and crypts hyperplasia
are seen.
Control
Pens should be kept clean and dry. Ammonia based disinfectants can be used after
thoroughly cleaning farrowing pens by high pressure hosing or steam disinfection. Overcrowding of
piglets and faecal contamination of food and water should be avoided. Prevention is by
administration of amprolium in feed to sows during the periparturient period, from 1 week prior to
farrowing until 3 weeks postfarrowing.

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Coccidiosis of horses

E. leukarti: Occurs in the small intestine of horses and donkeys and has been incriminated as the
cause of intermittent diarrhoea. Oocyst of E. leukarti is the largest oocyst in the genus of Eimeria.
Other species of Eimeria infecting horses are Eimeria solipedum and Eimeria uniungulata.

Klossiella equi- (Sub order: Adeleorina, Family: Klossiellidae) is non‐pathogenic. Meronts are seen
in endothelial cells of the Bowman’s capsule in the kidneys. Second‐generation meronts are found in
epithelial cells of the proximal convoluted tubules. Gametogony and sporogony occur in the
epithelial cells of the thick limb of Henle’s loop and mature sporocysts are surrounded by a thick
wall and pass from the body in the urine.

Coccidiosis in rabbits

E. stiedai occurs in liver and bile duct and so responsible for hepatic coccidiosis. The affected
rabbits loose appetite and become thin. There will be severe diarrhoea, icteric mucous membrane.
Enlarged dark brown liver may contain white circular nodules which later join together resulting in
fibrosis of liver.

E. flavescens: Occurs in the small intestine and large intestine and is highly pathogenic for young
rabbits, causing high morbidity and mortality and is a major problem on commercial farms.

E. intestinalis: Occurs in the small intestine and highly pathogenic causing diarrhoea and
emaciation.

Treatment and Control

Sulphonamides are used for treatment usually given as two 7 day courses in drinking water,
1 week apart to prevent reinfection. Control is by daily cleaning of cages, provision of clean feeding
troughs, rearing of animals on wire floor, incorporation of coccidiostats such as amprolium, clopidol
or robenedine in feed.

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 Chapter 10

Coccidiosis in poultry

Poultry coccidia are generally host-specific, and the different species parasitize specific parts of the
intestine. Coccidia are almost universally present in poultry-raising operations, but clinical disease
occurs only after ingestion of relatively large numbers of sporulated oocysts by susceptible birds.
Micropyle is absent in chicken coccidians.

Sr. Name of the coccidia Predilection site Prepatent period Sporulation

No time
1 Eimeria acervulina duodenum 97 hours 17 hours
2 E. maxima Middle part of small intestine 121 hours 30 hours

3 E. mitis Small intestine, caeca, rectum 93 hours 15 hours

4 E. necatrix Small intestine 138 hours 18 hours
5 E. praecox Small intestine 83 hours 12 hours
6 E. tenella caeca 115 hours 18 hours
7 E. brunetti Lower small intestine, caeca, 120 hours 18 hours
rectum
8 E. hagani Small intestine 99 hours 18 hours
9 E. mivati Small intestine 93 hours 12 hours
Eimeria tenella : It lives in epithelium of intestinal caeca of chickens. It is responsible for acute
haemorrhagic caecal coccidiosis. Mainly affect chicks below 3weeks. Chickens become infected
when they swallow food or water that is contaminated with sporulated oocysts. Following ingestion,
oocyst wall breaks by grinding action of gizzard and activated sporozoites are released from the
sporocyst in the small intestine. Once in caecum, sporozoites enter cells of the surface epithelium
and pass through basement membrane in to the lamina propria. There they are engulfed by
macrophages and that carry them to the glands of Lieberkuhn. They then escape from macrophages

75
and enter in to glandular epithelial cells of the crypt, located between nucleus and basement
membrane. Sporozoites become trophozoites within epithelial cells, feeding on host cells and
enlarging to become meronts. During merogony, meronts separate into about 900 first-generation
merozoites, each about 2 μm to 4 μm. These break out into the lumen of the cecum about two and
half to three days after infection, destroying their host cells. Surviving first-generation merozoites
enter other cecal epithelial cells to initiate a second endogenous generation. Merozoites develop into
meronts that live between the nuclei and free borders of host cells. A great many merozoites will
form meronts in the lamina propria under the basement membrane. About 200 to 350 second-
generation merozoites, each about 16 μm long, are then formed by merogony. Disruption of 2 nd
generation schizonts and overlying epithelium releases merozoites in to the lumen of the caecum.
When large number of 2nd generation schizonts do this, a massive haemorrhage in to the lumen is
evident at about 96th hours of this infection. The 2nd generation merozoites enter the cecal lumen
about five days after infection. Some of these merozoites enter new cells to initiate a third
generation of merogony below the nucleus, producing 4 to 30 third-generation merozoites, each
about 7 μm long. Many merozoites are engulfed and digested by macrophages during these cycles of
merogony. Some of the second-generation merozoites enter new epithelial cells in the cecum to
begin gametogony. Most of them develop into macrogametocytes. Both male and female gamonts
lie between the host cell nucleus and the basement membrane. Microgametocytes bud to form many
slender, biflagellated microgametes that leave their host cell and enter cells containing
macrogametes, where fertilization takes place. Immediately after fertilization plastic granules of
macrogametes pass peripherally toward the zygote’s surface, flatten out, and coalesce to form first
the outer and then the inner layer of the oocyst wall. This coalescence takes place within the
zygote’s cell membrane, and the membrane thus becomes the outer-wall covering. Oocysts are then
released from the host cells, move with caecal contents into the large intestine and pass out of the
body with faeces. Oocysts appear in faeces within six days of infection and are passed for several
days because not all second-generation merozoites reenter host cells at the same time. Freshly
passed oocysts each contain a single cell, the sporont. Sporogony (often called sporulation), or
development of the sporont into sporocysts and sporozoites, is exogenous (occurs outside the host).
The sporont divides into four sporoblasts, each of which forms a sporocyst containing two
sporozoites. Sporulation takes two days in summer, whereupon the oocysts become infective.

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Although unsporulated oocysts can survive anaerobic conditions, as might be found in freshly
passed feces, metabolism of sporulation is an aerobic process and will not proceed in the absence of
oxygen. Oxygen consumption is high at first, but falls steadily as sporulation is completed. The
organisms have large amounts of glycogen, which is rapidly consumed, and measurements of their
respiratory quotient indicate that they depend primarily on carbohydrate oxidation for energy during
sporoblast formation and then change over to lipid for energy as sporulation is completed. A rapid
burst of energy fuels sporulation and then a shift to a low level of maintenance metabolism
conserves resources until a new host is reached.

Theoretically, one oocyst of E. tenella, containing eight sporozoites, can produce 2.52 million
second generation merozoites, most of which will become macrogametes and there by oocysts.
However, many merozoites and sporozoites are discharged with faeces before they can penetrate
host cells, and many are destroyed by host defenses. A complete replacement of caecal epithelium
normally occurs about every two days, so any merozoite or sporozoite that invades a cell that is
about to be sloughed is out of luck. Young chickens are more susceptible to infection and discharge
more oocysts than do older birds

Symptoms and pathology: The chicks are dull and listless with drooping feathers. Poor weight gain
and food conversion rates are observed in subclinical infections.
Lesion: PM lesions are haemorrhage in the caecal lumen followed by thickening of mucosa and
formation of caecal core with clotted blood. In long standing cases, the caecal contents become
caseous and adherent to the mucosa and will get expelled along with the faeces by 7th or 8th day.
E . necatrix: One of the most pathogenic species affecting poultry but shedding of the oocyst is very
poor. It affects the middle part of the small intestine. Three generations of schizogony occurs.
Asexual development occurs in small intestine and gametgonous cycle occurs in caecum. In acute
cases, severe submucosal haemorrhage occurs on 5th and 6th day. The wall of small intestine is
markedly swollen, haemorrhagic and the contents are filled with unclotted blood. The haemorrhage
is associated with large, deeply seated second generation schizonts which appear as white, opaque
foci surrounded by zone of haemorrhage. Necrosis of mucosa leading to scar tissue formation which
affects the feed efficiency especially in broilers.

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Lesion: Ballooning of intestine, haemorrhage in intestine with white spots (meronts) (salt and
pepper apprearance)
E. acervulina: Four generation of schizogony occurs. It is moderately pathogenic. It affects the
anterior part (duodenum) of small intestine. It is responsible for subacute or chronic intestinal
coccidiosis in older birds and chickens at the point of lay. It causes mucosal/catarrhal enteritis with
thickening of intestinal mucosa. The gamonts and immature oocysts form white ladder like lesions
in duodenum.
Clinical signs: weight loss, watery whitish diarrhoea. Haemorrhage is rare.
E. maxima: Three generation of schizogony occurs. It is located in the middle part of small
intestine but has a tendency to spread to both sides. It is moderately pathogenic. The asexual stages
cause little damage but serious damages are due to sexual stages.
Clinical signs: diarrhoea, depression, ruffled feathers, decreased growth rate, weight loss and death
Lesions: It causes catarrhal enteritis and massive ballooning (due to lack of muscle tone).
Thickened mid intestine with petechial haemorrhage and blood tinged exudate.
E. brunetti: It is located in the posterior part of small intestine (lower ileum, upper part of caecum
and cloaca) and results in rectal coccidiosis.
Lesions: It causes haemorrhagic enteritis and haemorrhagic ladder like lesions. Faeces will be blood
stained.

E. mitis: The number of asexual generations are unknown. Older chickens are affected. Lesions
include small petechiae in small intestine and mucoid exudates in the lumen.

E. praecox (lowest prepatent period of 83 hours), E.hagani and E. mivati are less pathogenic

Coccidiosis in turkeys E.adenoides Lower part of small intestine and caeca

E.meleagrimitis Duodenum
E.dispersa Duodenum, upper small intestine

E.meleagridis Caeca
E. gallopavonis Lower part of small intestine, rectum and caeca
Coccidiosis in goose E.truncata Located in the kidney and is responsible for

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 renal coccidiosis
E.anseris Located in the small and large intestine
General factors
Coccidia survive in an inhibited developmental state in the gut and they get activated when the
birds are exposed to stress factors like overcrowding or extreme weather conditions. Main
pathogenic effect usually occurs prior to oocyst formation. Thus the presence of large number of
oocysts is not necessarily correlated with severe pathological changes in the gut. So the mere
presence of oocysts in the faeces does not necessarily mean that the bird is suffering from a clinical
coccidiosis. But we cannot completely rule out the possibility of coccidial infection, by the mere
absence of oocysts in the droppings. Sometimes oocysts get entangled in the caecal contents and
form a plug that would be thrown off along the droppings. Both clinically infected and recovered
birds shed oocysts in their droppings, which contaminate feed, dust, water, litter, and soil.
Pathogenicity is influenced by host genetics, nutritional factors, concurrent diseases, age of the host,
and species of the coccidium.
Eimeria spp. infections are self-limiting; that is, asexual reproduction does not continue
indefinitely. If the chicken survives through oocyst release, it recovers. It may become reinfected,
but a primary infection usually imparts some degree of protective immunity to a host. True age-
immunity does not occur, but older birds are usually more resistant than young birds because of
earlier exposure to infection.
Diagnosis
 It is best achieved at the time of post mortem examination of representative number of birds.
Location of major lesion give a good indication of the species of coccidia concerned and nature
of lesion. Lesion scoring is one of the important method for assessing the severity and intensity
of coccidiosis. It is expressed as 1+, 2+, 3+, 4+ based on the severity of lesion found in the
serosa, mucosa, nature of the intestinal contents and intestinal wall (Lesion scoring chart for
Chicken Coccidiosis (LESCHAR). The birds are randomly selected as 10 out of 1,000 birds (0.1
%). Select bird with average size and fit enough to walking and feeding. It is performed only at
the necropsy of sacrificed birds. Examine within 15 minutes of sacrifice in order to permit
identification of lesions. Advantages of lesion scoring card: The test is rapid, can be done at the
field level, cost effective and no instruments are needed.

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 Examination of morphology of oocysts
 Calculation of sporulation time of oocysts
Prevention and control:
Three aspects of prevention are
 Management and hygiene
 Use of anticoccidials
 Immunisation
Management and hygiene
 Avoid birds from stress and overcrowding (more than 22 birds/sqm)
 Nutrition is very important. The maize diet contains Vitamin A and E and the birds fed
with this are found to be more resistant to coccidial infection. Wheat diet contains niacin and
riboflavin favour the development of schizonts of coccidians.
 Add Ascorbic acid (Vitamin C- 10mg/kg body weight), acetyl salicylic acid/chlorpromazine
hydrochloride to reduce the stress
 To avoid summer stress, it is advisable to give Vitamin B complex and electrolytes
 Isolation of infected birds.
 Place feeders well above the level of ground to avoid contamination. The vessels in cages
should be washed thoroughly with hot caustic soda or 10% ammonia.
 Litter material should be dried. Litter can be sterilized using 0.5% phenol or 10%
formaledehyde. Cleaning of poultry cages with blow lamps is very important and it should be
done before a new stock arises.
Restrict movement of visitors & workers from one shed to another.
• Higher the temperature lower the risk of coccidiosis.
• Higher the CO2 and NH3 increases the risk of coccidiosis
• Intermittent lighting increases the risk of coccidiosis than that of the continuous lighting.
During short light periods the birds become more active and churn the litter which favours
the sporulation and survival of oocysts.
• During dark periods the consumption of feed will be reduced, thereby limiting the
availability of anticoccidials in feed which favours the susceptibility to coccidia.

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Chemotherapy

The agents used for the prevention and control of coccidial infections are termed as
anticoccidial drugs. The agents who destroy the coccidial population are termed as coccidiocidal and
agents who prevent the replication and growth of coccidial population are known as coccidiostatic.
Broadly, anticoccidials can be chemicals or ionophores. Some anticoccidials show strong activity
during the sexual cycle i.e., day 5 and 6 after infection. During these days signs of anorexia and
haemorrhage appear. The drugs effective during these days will provide therapeutic effect. Those
drugs which will affect sporozoites or early schizogonal stages are used for prophylactic purposes.

Anticoccidials are used usually in starter rations for meat type birds raised under floor-pen
management. Selection of an anticoccidial is based on the ability of the drug to improve weight and
feed conversion and to suppress the development of lesions.. Presence of drug residues in eggs and
milk is also a point of concern associated with the anticoccidials, so there is need for specified
withdrawal periods before slaughter. The emergence of drug resistant strains of coccidia presents a
major problem.

When the same anticoccidial drug is added to the starter and grower feeds, this is popularly
referred to as a straight program. These are commonly used in spring and summer. In some straight
programs, the concentration of the anticoccidial may be increased in the grower feed to provide
maximum protection at the time of peak coccidial oocyst shedding (3-4 weeks). This is known as a
step-up program, in other cases, the concentration of the anticoccidial may be decreased in the
grower or finisher feed, this known as a step-down program.

A chemical anticoccidial is added to the starter feed and an ionophore anticoccidial to the
grower feed, this is popularly referred to as a shuttle program. These minimise anticoccidial
resistance because the time of exposure to the same drug is limited. However, other important
factors must be considered: for example, when the starter feed is given for just 14-18 days, the
typically strongest chemical anticoccidial will not be consumed during the time of peak coccidial
oocyst shedding.

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 In rotational programme, the anticoccidial drug is changed every 3, 4 or 6 months resulting
in 4, 3 or 2 rotations per year. Thus the rotation programme eliminate the resistance to a particular
family of drugs by use of drugs with a totally different activity mode of action.

Curative treatment: It should be instituted immediately after the diagnosis of coccidiosis. An

interrupted form of treatment is more satisfactory with sulpha drugs than continuous treatment. This
is to avoid undue concentration of the compounds which inhibit the earlier developmental stages of
the parasite and interfere with the acquisition of immunity. Sodium sulphadimidine is used at 0.2%
in drinking water for two periods of 3 days separated by two days without treatment.
Preventive treatment: Consists of prolonged or continuous use of coccidiostatic compounds in feed
or water
Name of the drug Dose
1 Ionophores
a. Monensin 0.0121%
b. Lasalocid 0.005- 0075%
c. Salinomycin 0.006- 0.01%
2 Chemicals
Amprolium compounds 0.0125%
Clopidol 0.0125%
Nitrofurazone 0.005- 0.01%
Decoquinate 0.003%
Sulphaquinoxaline 0.0125%
Diclazuril 0.0001%
Robenidine 0.0033%.

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1. Ionophores: Ionophores are the fermentation products of Streptomyces and other fungi species,
extensively used as anticoccidials. Monensin, Lasalocid and Salinomycin are the Ionophores which
are used commercially. Ionophores facilitate transport of Na+ ion in cells and elevates the
intracellular concentration of Na+ ion. This increased concentration of Na+ ion inhibits the certain
mitochondrial functions such as substrate oxidation and ATP hydrolysis. Intracellular Na+ ion is
exchanged for extracellular Ca++ and increases intracellular concentration of calcium ions lead to
cytotoxicity.
a. Monensin: It is a fermentation product of Streptomyces cinnamonensis and the 1st antibiotic used
as an anticoccidials. Due to its broad spectrum activity, it acts on trophozoites and first generation
schizonts. It gives protection against all species at 0.01-0.121% concentration in the feed. It is
superior over amprolium, clopidol and zoalene in control of coccidiosis. In the USA, there is a three
day premarketing withdrawal requirement for this compound.

b. Lasalocid: It is an another fermentation product and has a high degree of anticoccidials activity. It
is effective at 0.005-0.0075% concentration.

c. Salinomycin: It was isolated from a culture of Streptomyces albus. It is more closely related to
monensin than lasalocid. It has anticoccidials activity at 0.01% in the feed and it was as effective as
0.0121%.

2. Amprolium: It is quarternized derivative of pyrimidine. It is thiamine antagonist and due to its

close structural similarity it blocks the thiamine receptors. Thiamine pyrophosphate is a cofactor of
several decarboxylase enzymes which play role in cofactor synthesis. It is only agent which can be
used in laying birds both for prevention and treatment of outbreaks. At higher doses, thiamine
deficiency can occur in host but it can be prevented by addition of thiamine. It is effective against
1st generation of trophozoites and schizonts and shows peak activity early in day 3 of cycle. It also
suppresses the sexual stages, gametogony and sporulation of oocyst. Continuous use of amprolium
is resulting into the development of drug resistance which is a major problem and limiting its use.
Amprolium is available as a premix and is given prophylactically to birds in a final concentration of
0.0125 per cent. There is no premarketing withdrawal requirement for this compound.

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3. Sulphonamides: They have longest history of use as anticoccidial drugs. Sulphonamides are
coccidiostaic than coccidiocidal. They have no direct curative effect, but helpful in halting thge
onset of disease in other members of the flock. Sulpha drugs are active against 2nd generation
schizonts of E. tenella and E. necatrix. The common drugs of this group which are used as
anticoccidials are sulphadimidine, sulphaquinoxaline, sulphadimethoxine, sulphanitran and
sulphaguanidine. They are more effective against intestinal than caecal forms of coccidian.
coccidian is synthesizing their own folic acid utilizing PABA (p-amino-benzoic acid) from growing
medium because folic acid is required for growth/replication of DNA. Sulfonamides are structural
analogues (PABA and Sulfonamide is similar in nature) of PABA, inhibit bacterial folate synthetase.
Animal cells also require folic acid but they utilize preformed folic acid supplied in diet and are
unaffected by sulfonamides. Therefore, they prevent proper development of schizonts.

a. Sulphadimidine: This compound is still used as a curative drug in certain parts of the world, but
its use has largely been discontinued in Western Europe and North America where it has been
replaced by other compounds. It is given @ 0.4% in feed or in drinking water as 0.2% solution of
the sodium salt. It is active against E. tenella, E. necatrix and other species of coccidian. It has been
used in the control of clinical outbreaks of coccidiosis. The problem of this drug is that it interferes
with vitamin K synthesis in the intestine and resulting into prolongation of blood coagulation time.
At higher doses it causes loss of egg production in laying hens and hyperplasia of the seminiferous
tubules of testicles of male birds.

B. Sulphaquinoxaline: A level of 0.1 % in ration has no adverse effect. But when this concentration
in feed for longer time ( 30 days), may lead to haemorrhagic syndrome in many organs and necrotic
lesions in spleen. The toxicity is due to interference with vitamin K metabolism. The withdrawal
period is 10 days.

Vaccination against coccidiosis

I. Live vaccines

1. Coccivac-B: A live coccidiosis vaccine for broilers. Contains live sporulated oocysts of anticoccidial
sensitive Eimeria acervulina, Eimeria mivati, Eimeria maxima and Eimeria tenella. This is used for
vaccination of healthy chickens at one day of age or older.

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2. Coccivac-D: A live coccidiosis vaccine for broiler breeders and layer pullets. It contains live oocysts
of E. acervulina, E . mivati, E. maxima, E. tenella, E. necatrix. E. praecox, E. brunetti and
E.hagani.
3. Coccivac-T : Live coccidiosis vaccine for the vaccination of turkeys. It contains live sporulated oocysts
of E. adenoeides, E. meleagrimitis, E. gallopavonis and E. dispersa. Strains of Eimeria in coccivac –T
highly sensitive to all feed anticoocidials.
4. IMMUCOX: Immucox C1- It is an oral coccidiosis vaccine. This vaccine is approved for water and gel
delivery. Targeted species are E. acervulina, E. maxima, E. necatrix and E. tenella. Immucox C2- Oral
coccidiosis vaccine of live oocysts of Eimeria spp. Targeted species are E. acervulina, E. maxima, E.
necatrix. E. tenella and E. brunetti.
5. Nobilis®COX ATM: Live vaccine for the active immunization of broiler chickens as an aid in the
protection against field infections with the most prevalent avian Eimeria species affecting broilers. This is
active even in the presence of ionophores compounds. Nobilis COX ATM contains strains of Eimeria
acervulina, Eimeria tenella and Eimeria maxima.
II. Vaccines based on precocious lines
It involves the production of avirulent strains of Eimeria by sequential selection of parasites
which slows the most rapid development in the host. Strains that have shortest patent period in vivo
and that which produce oocysts in minimum possible time are used (least pathogenic but highly
immunogenic).
1. Livacox – T (E. acervulina, E. maxima and E. tenella) for broilers
Livacox – D (E. acervulina and E. tenella) for breeders and layer pullets
2. Paracox- live attenuated vaccine. This is a stabilised suspension of sporulated oocysts of 7
species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E.
tenella). There is no need of anticoccidials in feed and it can be given at 5-9 days of age. This is
effective in breeders and layers.

III. Subunit maternal vaccines: CoxAbic is the first commercially available subunit vaccine against
coccidiosis. The vaccine consists of affinity purified sexual stage (macrogametocyte) antigens
(APGA) isolated from E. maxima. It is a subunit vaccine (water in oil adjuvant vaccine)
indicated for vaccination in breeders in order to prevent against coccidiosis by different species of

85
Eimeria in broilers. Maternal immunisation provides passive immunity followed by local field
exposure provides life long active immunity.

E. truncata is found in kidney of domestic goose and can cause an acute nephritis. Meronts and
gamonts occur in the epithelial cells of the kidney tubules. Clinical signs include marked weakness,
emaciation, polydipsia, muscular incoordination and death. It is highly pathogenic for young
gooslings and can cause up to 100% mortality within a few days of onset of clinical symptoms.

Klossiella cobayae (Sub order: Adeleorina, Family: Klossiellidae), found kidney of guinea pig.
Sporocysts may be detected in urine sediments or trophozoite stages may be found on postmortem in
the kidney. Infection takes place by the ingestion of the sporulated sporocysts.

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 Chapter 11

Coccidiosis of dogs and cats

Coccidiosis of dogs and cats: is caused by Cystisospora (Isospora) species. Sporulated oocyst of
Cystisospora (Isospora) contains two sporocysts each containing four sporozoites. Cystisospora
(Isospora) species occurring in the small intestine of dogs are C. canis, C. burrowsi and C.
Ohioensis and that in cats are C. felis and C. revolta. Clinical signs include diarrhoea in young
puppies or kittens.

In addition, dogs act as definintve hosts for Sarcocycstis and Neospora. Cat act as
definitve hosts for Toxoplasma.

Phylum Apicomplexa

Class Conoidasida

Order Eucoccidiorida

Suborder Eimeriorina

Family Sarcocystidae

Genus Sarcocystis

Sarcocysts are the most prevalent cyst forming isosporoid coccidia of livestock.
Sarcocystosis was described for the first time by Friedrich Miescher in 1843 in skeletal tissue of
domestic house mice. The cysts were originally termed "Miescher's tubules", after their discovery.
They were also referred to as "milky white threads“. The name Sarcocystis is dervived from Greek:
sarx = flesh and kystis = bladder.

Life cycle of Sarcocystis bovicanis

Definitive host- Dog, wolf, coyote, raccoon

After feeding of infected beef with bradyzoites, gamonts develop directly in the lamina
propria of small intestine without undergoing schizogony. By seventh day after ingestion
unsporulated oocysts are visisble in faeces. Sporulated ooccysts and sporocysts are shed in faeces.

87
When sporulated oocysts/ sporocysts are ingested by the cattle, sporozoites are released in the
intestine. They invade the intestinal wall, enter endothelial cells of capillaries of caecum, large
intestine, kidney, pancreas and mesenteric lymph nodes. First and second generation shizogony
occurs here. Third generation schizogony occur in circulating lymphocytes which result in
merozoites which penetrate in skeletal and cardiac muscles. Asexual reproduction / schizogony /
merogony for 2-3 generations, thus lead to the formation of merozoites (tachyzoites: fast
multiplying merozoites). Merozoites enter muscle cell and the muscle cell change into globular
metrocyte/ mother cell. Internal budding (endodyogeny) occur inside the metrocyte producing two
daughter cells. After several such multiplications, banana shaped bradyzoites (Rainey’s corpusclae)
produced. They are subsequently enclosed by cyst wall, forming muscle cyst / sarcocyst. The cyst
wall is radially striated. Two distinct regions are recognized in these cyst. Peripheral region
containing metrocyte and inner region containing bradyzoite.

Pathogenesis

Principle pathogenic effect is caused by second stage of merogony in vascular endothelium.

There will be breakage of endothelium. Heavy experimental infection of calves with S. bovicanis
have resulted in mortality one month later. At necropsy, petechial haemorrhage in all organs
including heart and generalaised lymphadenopathy are seen. It occurs after one month of infection.

A naturally occurring chronic disease called Dalmney disease has been recogonised in
Canada, USA, Britain, characterised by emaciation, submandibular oedema, and disinclination to
move, exophthalmia, lymph node enlargement and recumbency. In cattle, marked hair loss at the
end of tail giving rat tailed appearance and abortion may occur. At postmortem examination
numerous meronts are found in endothelial cells and deveoloping sarcocysts in areas of
degenerative myositis.

Species of Sarcocystis

Identification of the the Sarcocystis sp. is based on the following characters

1. Based on the size of sarcocyst: generally Sarcocyst of felines are macroscopic and canines are
microscopic.
a. Macroscopic sarcocyst- Large and is able to see with naked eye Eg: S. bovifelis
b. Microscopic sarcocyst- Small and is microscopic Eg. S.ovicanis

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2. The structure of the sarcocyst wall
a. Cauliflower like protrusions: S.ovifelis
b. Sarcocyst without true septa: S. arieticanis
c. Sarcocyst without secondary cyst wall: S. gigantea

Sarcocystosis of different animals

Parasite Intermediate Definitive Sarcocyst Pathogenicity
host host character
S. bovicanis Cattle Dog, fox, Microscopic Dalmney disease in cattle.
Emaciations mandibular
(S. cruzi) wolf,
odema, recumbency,
Cattle coyote exophthalmia, rat tail
appearance
S. bovifelis Cattle Cat Macroscopic Nonpathogenic.
(S. hirsuta)
S. bovihominis Cattle Man, Radially Nonpathogenic
Primates striated wall
S. ovicanis Sheep Dog Microscopic Highly pathogenic for lambs.
Severe myositis and
(S. tenella)
encephalomyelitis.
S. ovifelis Sheep Cat Macroscopic Nonpathogenic
Sheep (S. gigantea)

S. arieticanis Sheep Dog Microscopic Nonpathogenic

S.meduciformis Sheep Cat Macroscopic Nonpathogenic

Highly pathogenic. Fever,

S. capracanis Goat Dogs Microscopic
weakness, anorexia, weight
loss, tremor, abortion and
death

Goat S. hircicanis Goat Dogs Microscopic Nonpathogenic

S. hircifelis Goat Cat Macroscopic Nonpathogenic

(S. moulei)

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 The cyst wall
has
numerous
S.miescheriana
dogs, palisade like Pathogenic. Weight loss,
(S.siucanis ) Pigs
Pigs wolf, fox process with purpura of skin, abortion
(S. porcicanis)
randomly
arranged
filaments.
S. porcihominis
cyanosis of the skin, muscle
(Isospora Pigs Man
spasm, abortion
hominis)
S. porcifelis
Pig Cat Non pathogenic
(S. suifelis)
Microscopic
S.equicanis with no
Equines dogs
(S.bertrami) radial
Horse striations
S. fayeri Horse Dogs Nonpathogenic
equine protozoal
S. neurona Equines Opossums
encephalomyelitis
Water
S. fusiformis cat Macroscopic
buffalo
Water
Water S. buffalonis cat Macroscopic
buffalo
buffalo Water
S. levinei Dog Microscopic
buffalo
S. dubeyi Water Not
Microscopic
(S.sinensis) buffalo known
S. bovihominis Nausea, stomachache,
Cattle Man
(S. hominis) diarrhea in man
Human S. porcihominis Pig Man More pathogenic

S.lindermanni Man unknown

myositis,
S. gallinarum Chicken Dogs
meningoencephalitis
Poultry

S. rileyi Ducks Dogs

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Equine Protozoal encephalomyelitis (EPM)

EPM is caused by Sarcocystis neurona. Opossums are the definitive hosts and horses are the
aberrant hosts; natural intermediate hosts are unknown. S. neurona parasitizes and causes lesions
(dark areas) in the brain and spinal cord of horses. Only schizonts and merozoites are found in the
aberrant hosts. Opossums excrete sporulated oocysts or sporocysts after ingesting muscles infected
with sarcocysts.

EPM is often a progressively debilitating disease affecting the CNS of horses. The clinical
signs may vary from acute to insidious onset of focal or multifocal signs of neurologic disease
involving the brain, brainstem, spinal cord or any combination of the areas of the CNS. Some horses
affected with EPM have abnormal upper airway function, unusual or atypical lameness or even
seizures. In severe cases, the horses may have difficulty in standing, walking, or swallowing. The
disease may progress very rapidly. In some horses, the disease appears to stabilize or remain static
for a time period.

Sarcocystosis in humans

It can be two types based on whether humans are acting as the intermediate host or definitive host.

1. Muscular sarcocystosis: If humans act as the intermediate host, myositis is the primary
syndrome. The spectrum of illness ranges from acute self-limiting infections to chronic,
moderately severe disease. Painful muscle swelling has been reported, accompanied by erythema,
muscle tenderness, generalized muscle weakness, bronchospasm and fever. Other reported
symptoms include cough, arthralgia, transient pruritic rashes, headache, malaise,
lymphadenopathy and muscle wasting. Chronic cases can have persistent or recurrent symptoms
for up to seven years. Many infections may be asymptomatic.
2. Intestinal sarcocystosis: When humans serve as the definitive host (S. suihominis or S.
bovihominis), the clinical signs may include fever, chills, sweating, diffuse abdominal tenderness,
diarrhea, nausea and vomiting. Dehydration may occur as a result of the diarrhoea and vomiting.
Eosinophilic enteritis and rare cases of acute intestinal obstruction have been reported. Intestinal
sarcocystosis is transient and usually selflimiting.

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Diagnosis of sarcocystosis

In Definitive host: Finding oocyst in faeces by clinical sample examination(floatation-ZnSO4/NaCl)

and PCR-based methods for detection of DNA from oocysts/sporocysts in feces

In Intermediate hosts: Samples of the animal’s muscle tissues like thigh, cardiac, jaw, tail,
diaphragm, neck, eye muscles, oesophagus and brain should be collected in polythene bags
containing ice at 4 ˚C. They should be first examined grossly and then subjected to pepsin digestion
method after removing fat and connective tissue.

The diagnosis from muscles can be done by using the following methods

1. Isolation of intact microsarcocysts from muscles

2. Pepsin digestion technique

3. Histopathological examination
4. Serological tests like ELISA, IFAT
5. Electron microscopic studies
Treatment of Sarcocystosis

 Equine protozoal myeloencephalitis can be treated with sulfonamides and pyrimethamine.

 In Calves: amprolium hydrochloride 15 mg/kg Body weight for 3 days
 In pigs: salinomycin 2-4 mg/kg in divided doses for about 30 days
 In human: Pyrimethamine, 37 mg daily for 5 days and then at 25 mg daily for 10 days in
combination with sulfisoxazole at 3 g per day for 14 days
Postmortem lesions: Sarcocysts can be found in skeletal and cardiac muscles, as well as the central
nervous system (CNS) in all species. In most cases, the parasite is an incidental finding at necropsy.
Cattle : Cachexia, hepatitis and myocarditis
Sheep: Haemorrhages of the serosa of the viscera, myocardium and skeletal muscles infected with
S. tenella
Horse: Focal discoloration, haemorrhages or malacia in the CNS
Dogs and cats: Multifocal meningoencephalitis in the CNS

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 Control

 Keep dogs and cats away from cattle/goat shed

 Avoid giving uncooked meat to dogs and cats
 Dead birds and animals should be burried properly.
 Do not eat raw or uncooked meat
 Freeze meat -5 ˚C for 48 hours before ingesting
 Cook meat at 65-70 ˚C for 20 minutes before ingesting
In regions with poor sanitation and hygiene:
 Drink only purified water
 Avoid raw vegetables
 Practice careful hygiene
Vaccine: Killed S. neurona vaccine is under trial in U.S.

Difference between Sarcocystis and Toxoplasma

Sl. No Sarcocystis Toxoplasma

1 Obligatory heteroxenous Facultative/obligatory heteroxenous

2. Cyst only in intermediate host Cyst found both in intermediate and
definitive host
3. Cyst contain noninfectious Cyst contain only bradyzoite
metrocyte and bradyzoite
4. Bradyzoites develops into gametes Bradyzoites undergo a propagative cycle
in gut of final host ie schizogony preceedes gametogony in
gut of final host
5. Cyst seen in muscle Cyst in many cell type
6. Bradyzoites are infective to Sporozoites, baradyzoites and tachyzoites
definitive host and oocysts are are infective to definitive and
infective to intermediate host intermediate host

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 Chapter 12

Genus: Toxoplasma
Phylum Apicomplexa
Class Conoidasida
Order Eucoccidiorida
Suborder Eimeriorina
Family Sarcocystidae
Genus Toxoplasma

Toxoplasma gondii

This intestinal coccidian parasite was first discovered in 1908 by Nicolle and Manceaux from a
rodent (Ctenodactylus gondi). The name Toxoplasma (toxon-arc, plasma-form) is derived from the
crescent shaped tachyzoite stage.

Definitive Host: Domestic cat and other felids

Intermediate host: All warm blooded animals including man

Life cycle

Toxoplasma gondii is an obligate intracellular parasite. There are three infectious stages in the
lifecycle.
1. Tachyzoites
2. Bradyzoites in the tissue cysts
3. Sporozoites in the oocysts

Tachyzoites : The term “tachyzoite” (tachos means speed in Greek) was coined by Frenkel. It is a
stage which divide rapidly and multiply in any cell of the intermediate host and in non-intestinal
epithelial cells of the definitive host. They are also termed as endodyozoites or endozoites.
Aggregates of numerous tachyzoites are called clones, terminal colonies, or groups or pseudocyst
(there is no well-defined membrane). The tachyzoite is often crescent shaped, approximately 2 by 6
µm with a pointed anterior (conoidal) end and a rounded posterior end. The nucleus is usually
situated towards the central area of the cell and contains clumps of chromatin and a centrally-located

94
 nucleolus. They can move by gliding, flexing, undulating, and rotating. Tachyzoites multiply
asexually within the host cell by repeated endodyogeny.

Bradyzoites in the tissue cysts: The term “bradyzoite” (brady means slow in Greek) was coined by
Frenkel to describe the organism multiplying slowly within a tissue cyst. A tissue cyst is the
collection of bradyzoites within a well-defined membrane and is resting stage of the parasite.
Bradyzoites are also called cystozoites. Nucleus is terminal in bradyzoites. Location of the tissue
cysts include liver, lung, kidney, brain, eye, skeletal and cardiac muscle. Tissue cysts in the brain are
often spheroidal and rarely reach a diameter of 70 µm, but intramuscular cysts are elongated and
may be 100 µm long.

Sporozoites in the oocysts: Unsporulated oocysts are subspherical to spherical and are 10 by 12 µm
in diameter. Sporulation occurs outside the cat within 1 to 5 days of excretion depending upon
aeration and temperature. Each oocyst contains two ellipsoidal sporocysts without stieda bodies.
Sporocysts measure 6 by 8 µm. Each sporocyst contains four sporozoites.

There are two multiplicative stages in the life cycle

1. Enteroepithelial cycle/ sexual cycle

2. Extra intestinal/ asexual cycle
Enteroepithelial cycle / sexual cycle in felines
Cats and other felids are infected by the ingestion of all the three infective stages
tachyzoites, bradyzoites in the tissue cysts and sporozoites in the oocysts (sporulated oocysts).
Prepatent periods and frequency of oocyst shedding vary according to the stage of T. gondii ingested.
Prepatent periods are 3 to 10 days after ingesting tissue cysts, ≥18 days after ingesting oocysts and ≥13
days after ingesting tachyzoites. Bradyzoites are more infective to cats. After ingestion of tissue cysts
by the cats, the cyst wall is dissolved by the proteolytic enzymes in the stomach and small intestine.
The released bradyzoites penetrate epithelial cells of small intestine and initiate a series of asexual
generations (endodyogeny) followed by sexual cycle. Then there will be formation of merozoites,
gamonts and then oocysts.

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Extra intestinal/ asexual cycle in the intermediate host and felines

Infection of intermediate host is usually by the ingestion of sporulated oocysts / infected

meat of animals / congenital transmission via placenta. Oocysts are most infectious to intermediate
host. After ingestion of the infective oocysts they rupture in the intestine releasing eight sporozoites.
Sporozoites multiply intracellularly in the intestine and in associated lymph nodes and tachyzoites are
formed. They spread to different parts of the body through blood and lymph. Accumulation of
tachyzoites in different tissue is known as terminal clones / aggregrates / pseudocyst. As the immunity
develops tachyzoites transform to bradyzoites / tissue cysts in brain, heart and skeletal muscle and
persist for years maintaining the infection as chronic.

On ingestion of infected meat having tissue cyst proteolytic enzymes dissolve cyst wall
releasing bradyzoites which then transforms to tachyzoites. Tachyzoites undergo repeated division and
ultimately encyst in tissue. The cycle is completed when tissue cysts are ingested by the cat.

Transplacental transmission can occur when a previously noninfected host becomes infected
during pregnancy. T. gondi multiplies in the placenta and then spread to foetal tissue. Foetus is severely
affected if the dam become infected during the first half of the gestation.

Sources and modes of transmission

1. Domestic cats are the key source of infection. Cats can excrete millions of oocysts at a time and then
undergo sporulation within a day. Sporulated oocysts can remain viable in the soil up to 2 years. Soil is
an important source of infection to ground feeding herbivores and birds by ingestion of oocysts. The
oocysts may be mechanically transmitted by filth flies, earth worms, dung beetles and cockroaches.
2. Carnivorism is another important mode of transmission.
3. Use of uncooked meat are source of infection to human beings (conventional cooking at 70 oC,
salting, curing, pickling, freezing at -20 oC etc will kill the tissue cysts). Meat grinders and butchers
knife also act as source of infection
4. No venereal transmission; Congenital transmission occur in man, sheep, pig and goat
5. Pasteurization of milk prevent transmission through milk
6. Blood and platelet transfusion and organ transplantation

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Pathogenesis of toxoplasmosis
Most infections with T. gondii are asymptomatic. Parasite multiply in the tachyzoite form,
producing areas of necrosis. In acute form organism appear in secretions and excretions such as urine,
faeces, milk, conjunctival fluid and saliva but unable to survive long time outside the host. So little or
no spread of toxoplasmosis from one animal to another. The subacute form of the disease is
characterized by the appearance of antibodies which clear the blood and tissues of the tachyzoites. In
chronic form, persistence of bradyzoites in tissue cysts will occur which can live in the host for long
time.
Clinical signs of toxoplasmosis in animals.
Cats: Infection will be mild causing mild enteritis and ulceration of the intestine. Oocysts appear
in faeces 4-5 days after the infection and may continue for 3-20 days.
Dogs: Fever, paralysis, neurological disorders, respiratory and gastrointestinal disturbance
Goat and sheep: Abortion is the most important symptom. The lambs may be mummified,
macerated, aborted and still born or may be born weak/die within 7 days after birth.
Pig: More severe in congenitally infected piglets than infected after birth. Mild in sow. Most of
the infection in pigs are subclinical. Incoordination of the movement and pulmonary involvement
are noticed.
Equine: Most resistant species to clinical toxoplasmosis
Cattle: Fatal toxoplasmosis has not been reported. Experimental infection revealed fever,
respiratory disturbance and diarrohea.
Human Toxoplasmosis
Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Disease in
humans caused by T. gondii was first recognized in the late 1930s. In 1939, Sabin first proved that
Toxoplasma isolates from humans and animals belonged to the same species. In 1948, the
introduction of the methylene blue dye test by Sabin and Feldman enabled seroepidemiological
studies in humans as well as a broad range of animal species which provided evidence for a wide
distribution and high prevalence of T. gondii in many areas of the world.
Human Toxoplasmosis can be divided into
1. Congenitally acquired toxoplasmosis
2. Postnatally acquired toxoplasmosis

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Congenitally acquired toxoplasmosis

This occur when a non-immune susceptible woman acquire the infection during any stage of
pregnancy. Initially there will be parasitaemia of the mother followed by infection of placental
tissues and then infection of foetus.

Severity of infection depends on

1. The time or stage of pregnancy

2. Virulence of Toxoplasma strain
3. Immune status of the host
4. The amount of infective material consumed by the host
If the mother was infected with Toxoplasma prior to the current pregnancy, babies do not
suffer from congenital transmission. Transplacental transmission of chronic infection do not occur.
If the mother was not previously infected, stillbirths and spontaneous abortions may result. Maternal
infection in the first three months of pregnancy results in more extensive pathogenesis, but
transmission to a fetus is more frequent if maternal infection occurs in the third trimester. (if
infected in first trimester maximum damage is caused to foetus compared to second and third
trimesters). Clinical symptoms of new born foetus that is infected may range from no symptom at all
to death.
Important sign shown by the infected new born are the retinochoroiditis, hydrocephalus,
convulsions and intracerebral calcification. These signs are called as tetrad of signs. Symptoms will
be exhibited as child grown up. Initially there may be learning disabilities. Microcephaly,
organomegaly and mental retardation are the other manifestations. The most common complication
of congenital toxoplasmosis is the focal necrotizing retinochoriditis leading to photophobia and
blurred vision.
Postnatally acquired toxoplasmosis
Postnatal infection occurs
1. By ingestion of contaminated food and water
2. By working in labs
3. Transplantation of organs/transfusion of blood

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 Lymphadenopathy, malaise, fever, sore throat, head ache, maculopapular lesions that does
not affect palm and sole, ophthalmic lesions involving eye, cerebral lesions involving brain and
nervous tissue are the important manifestations. This is a benign and self-limiting disease in
imunocompetant host but serious and life threatening in immunodefecient patients.
Diagnosis
1. Light microscopy: Faecal examination (e.g. flotation) to identify oocysts
2. Bioassay: The isolation of T. gondii using laboratory animals is generally considered as the gold
standard for detection of organism. Secretions, excretions, body fluids, lymph nodes, muscle and
brain tissues are specimens used for the isolation. Mice and cats are commonly used for bioassay of
T. gondii.Tachyzoites can be demonstrated in peritoneal fluid.
3. Serological assays: Sabin-Feldman dye test (DT), ELISA, IFAT etc
4. Molecular methods based on detection of parasite nucleic acid like PCR, LAMP, Microsatellite
analysis
Treatment

There is no approved treatment for toxoplasmosis in cats or dogs. However, the following
medications and regimens have been used successfully.

 Clindamycin hydrochloride (10 to 12 mg/kg orally twice daily for 2 to 4 weeks) can be used to
treat disseminated toxoplasmosis.
 Trimethoprim-sulphonamide combination can also be used at the rate of 15 mg/kg orally every
12 hours for 4 weeks.

Pregnant woman with confirmed infection (> 18th week of gestation )

 Pyrimethamine 100 mg/day in two divided doses, then 50 mg per day until birth
 Sulfadiazine 75mg/day in two devided doses for 2 days, then 100 mg/day in two divided dosed
doses until birth
 Leucovorin 10-20 mg daily during and for 1 weak after pyrimethamine therapy
The most promising drug in the treatment of T. gondii is atovaquone during pregnancy, which has
potent in vitro activity against the tachizoite and the cyst forms. Azithromycin has a partial effect
on T. gondii tissue cysts.

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 Infant
 10 mg pyrimethamine 2 mg/kg per day for 2 days, then 1mg/kg per day for 2-6 month, then every
Monday, Wednesday, Friday for 1 year
 Sulfadiazine 100mg/day in two divided doses for 1 year
 Leucovorin three times weekly during and for 1 weak after pyrimethamine therapy

Prevention

1. Prevent hunting activity by cats (e.g., keep cats indoors). Do not feed raw or undercooked
meat or viscera to cats. Feed them commercially prepared diets.
2. Since oocysts require at least 24 hours to become infective, remove fecal material from litter
boxes daily. Allow only immunocompetent, nonpregnant persons to perform daily litter box
cleaning. Wash hands with soap and water after exposure to soil, sand, raw meat or
unwashed vegetables and wear gloves when gardening.
3. T. gondii in meat can be killed by exposure to extreme heat or cold. Tissue cysts in meat are
killed by heating the meat to 67˚C or by cooling to -13˚ C. Toxoplasma in tissue cysts are
also killed by exposure to 0.5 kilorads of gamma irradiation
4. Do not taste the meat until it is cooked. Wash all cutting boards and knives thoroughly with
hot soapy water after each use.
5. Wash and/or peel all fruits and vegetables before eating them. Avoid drinking untreated
water, particularly when traveling in less developed countries.

Vaccine

The S48 strain Toxovax is a live vaccine originally developed for use in sheep, but when
used in cats inhibits sexual development of T. gondii. This vaccine is used in sheep to reduce tissue
cyst development. The T-263 strain of T. gondii is a live mutant designed to reduce or prevent
oocyst shedding by cats which develop only partially in the intestinal tract. Parasite surface
antigen, SAG1, involved in invasion of tachyzoites to host cell is considered an important antigen
for the development of effective diagnostic tests or subunit vaccines.

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 Chapter 13
Genus: Neospora

Phylum: Apicomplexa
Class: Conoidasida
Order: Eucoccidiorida
Family: Sarcocystiidae
Genus: Neospora
Species: Neospora caninum
Final host: Dog (can also act as intermediate host) and other canids
Intermediate host: cattle, sheep and goat
Predilection site: brain, heart, liver, placenta
Transmission: Horizontal transmission and transplacental infection (cattle, sheep, goat, dog)

Neospora caninum is obligate intracellular tissue cyst-forming coccidian parasite infecting

dogs and other animals. Neosporosis like diseases was first reported from Norway in 1984 in Boxer
dogs which developed neurological disorders 2-6 months after birth. Parasites were found in lesions
in the brain and muscles. In 1988, the canine parasite was named Neospora caninum (Dubey et al.,
1988). It causes severe neuromuscular disease in dogs, and abortion and neonatal mortality in cattle.

Structure and Biology

1. Tachyzoites: Tachyzoites are rapidly dividing (endodyogeny) stages that cause tissue damage
and spread the infection in the tissues of the host. Tachyzoites are ovoid, lunate or globular (6 x
2µm). In infected dogs, tachyzoites are found in the cytoplasm of neural cells, macrophages,
fibroblasts, vascular endothelial cells, myocytes, renal tubular epithelial cells, hepatocytes and other
cells of body. Centrally placed nucleus is characteristic of tachyzoites.
2. Tissue cysts: Tissue cysts are round or oval and up to 107 µm long with thick wall (4µm) and
found only in neural tissues (brain, spinal cord and retina). It contains infective bradyzoites which
are orally infectious for intermediate hosts and dogs. In bradyzoites, nucleus is located subterminally
at the narrow nonconoidal end. Tissue cysts can survive up to 14 days at 4°C but are rendered non-
infective at -20°C for 1 day.

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Tachyzoites and tissue cysts are present in the intermediate hosts and the definitive host.
3. Oocysts: Dogs pass small numbers of unsporulated isosporan type of oocysts in faeces after 8-23
days of infection. It sporulate within 24 hours at 37ºC or at room temperature. Sporulated oocysts
are spherical to subspherical with no micropile or oocysts residuum. Enteroepitheilal cycle in dogs is
unknown.

Dogs are serologically negative for IgG antibodies when they are excreting oocysts of N.
caninum. They seroconvert 2 to 3 weeks after oocysts are appeared from faeces. N. caninum isolates
obtained from dogs or cattle behave similarly in experimental animals and cultures.
Transmission
Ingestion of bradyzoite laden tissues of cattle, particularly placenta and fetal membranes, and
transplacental transmission are thought to be the predominant routes of infection to dogs in nature.
Adult cattle become infected by ingesting sporulated oocysts, which may be present in feed and
water sources or soil contaminated with dog feces. Congenital infections in dogs and cattle result
from the active dissemination of tachyzoites across the placenta, which may result from exogenous
infection or recrudescence of a persistent infection during pregnancy.
Life cycle of Neospora Caninum:
Oocysts are generated by sexual replication in the intestinal epithelial cells of the definitive
host ( dogs) and expelled in the feces in an unsporulated (noninfectious) form. Outside the host,
oocysts undergo sporulation in 24–72 hours ( isosporan type). On ingestion of sporulated oocysts
sporozoites are released by excystation and parasitize the intestine, where they transform into
tachyzoites. Tachyzoites divide and quickly spread to other host cells, which they invade and often destroy.
Tachyzoites are found in neural cells, macrophages, fibroblasts, vascular endothelial cells,
hepatocytes, and muscular cells including those of myocardium and the placenta in pregnant cows.
Infection of mononuclear cells that likely aid in parasite dissemination via leukocyte trafficking.
Within host cells, tachyzoites reside and replicate within a parasitophorous vacuole. During the
acute phase of infection, due to intracellular replication, infected host cell lysis occur, tachyzoites
are released and infection of surrounding cells ensue. In an immunocompetent host, tachyzoites
replicate for an estimated 20 divisions before they differentiate into bradyzoites in tissue cyst.

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 Natural N. caninum infections occur via horizontal or vertical transmission. Vertical
transmission / transplacental / congenital transmission in bovines can occur through two ways.
Exogenous transplacental transmission following ingestion of sporulated oocysts by naïve cattle and
is associated with epidemic abortion storms in a herd. Endogenous transplacental transmission
follows recrudescence of infection in a persistently infected cow during pregnancy and is the
principal mechanism responsible for maintaining the parasite within cattle populations, resulting in
fetal transmission rates as high as 95%. Once infected, a cow remains infected for life and may pass
infection to consecutive generations of offspring. Seropositive cattle have an increased risk of
abortion.
In the definitive canid host and other carnivores, horizontal transmission may result from
ingestion of tissue from infected intermediate hosts (bovines) containing tissue cysts, or from
sporulated oocyst contaminated food or water. Dogs have been shown to shed oocysts following
consumption of infected offal or expelled placental membranes from infected cows. Vertical
transmission also occurs in dogs. Subclinically infected bitches can transmit N. caninum to their
offspring in successive litters; however, transplacental infection on its own is considered less
important in sustaining infection in canine populations.
Pathogenesis:
Clinical neosporosis is a result of the proliferation of tachyzoites within tissues
leading to inflammation, granuloma formation, and necrosis. Serve neuromuscular disease is seen
in dogs. A variety of neural cells including those of cranial and spinal nerves are destroyed. Disease
is most severe in congenitally/ transplacentally infected puppies, characterised by progressive
ascending paralysis particularly of hind limbs, polymyositis and hepatitis. More than one littermate
will develop paralysis of rear limbs, often with hyperextension, beginning 3 to 9 weeks after birth.
Muscle atrophy and contracture become evident. Cervical weakness and dysphagia lead to death.

Neosporosis is often observed in dogs less than six months of age, but dogs of any age can
be affected. Neosporosis in adult dogs may result in multifocal central nervous system signs,
polymyositis, myocarditis, dermatitis, and pneumonia. Sudden death due to myocarditis is also seen.

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Clinical signs in dogs: Progressive (ascending) paralysis is seen in young dogs. The hind limbs of
some dogs will be in rigid hyperextension. Other dysfunctions include difficulty in swallowing,
paralysis of jaw, muscle flaccidity, muscle atrophy and even heart failure.

Diagnosis
• History of neurological signs like muscle weakness and progressive ascending paralysis
• Detection of antibodies in serum
• IFA of CSF or serum IFA titer1:50 is suggestive of exposure and a titer1:200 is seen in
dogs with clinical signs.
• Direct agglutination tests
• Microsopic detection of tachyzoites in tissue aspirates
• Muscle biopsy (bradyzoites in biopsy samples of affected muscles) and isolation of parasites
in cell culture from biopsies
• Increased levels of serum enzymes associated with necrosis of myocytes and occasionally
liver.
Postmortem diagnosis
Demonstration of the parasites in the lesions of infected dogs. Most parasites are in the
cerebrum so this tissue should be collected at necropsy for histological examination.
Immunohistochemical tests will confirm the diagnosis. PCR tests using tissues collected at necropsy
are also used for diagnosis.
Treatment: Treatment has been successful in some dogs with early neosporosis-induced limb
weakness. The following treatment regimens are used to control clinical neosporosis
• Trimethoprim sulfadiazine (15-20 mg/kg PO every 12 hours for 4 weeks) in combination
with pyrimethamine (1 mg/kg PO every 24 hours for 4 weeks) reversed N. caninum
associated paralysis in some dogs.
• Clindamycin (12.5-25 mg/kg PO or IM every 12 hours for 4 weeks)

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 Nerosporosis in Cattle
Presence and number of dogs are the contributing factor for abortions on a bovine farm.
Horizontal transmission can occur via ingestion of food and water contaminated with dog faeces
containing N. caninum oocysts. Vertical transmission of N. caninum from cows to their foetus in
utero (transplacental) and lactogenic route can also occur. Tachyzoites and tissue cysts are seen
intracellularly in CNS and retina of affected cattle. Infection is seen in many organs with
commonest site being brain.
Neosporosis is one of the most common causes of bovine abortion in dairy and beef cattle.
Abortion may be sporadic or epidemic occurring year round from 3 months of gestation to term (5-6
months mostly). Fetuses may die in utero, resorbed, mummified, autolyzed, stillborn, born alive but
diseased, born clinically normal but chronically infected. Neospora infected calves may be born
underweight, unable to rise, with neurological signs like ataxia, decreased patellar reflexes, hind
limbs and / or forelimbs flexed or hyper extended, exophthalmia and asymmetrical eyes. The most
characteristic lesion of neosporosis is the nonsuppurative focal encephalitis characterized by
multifocal cerebral necrosis and nonsuppurative inflammation. Myocarditis is also seen in aborted
foetus. Hepatitis is more common in epizootic than sporadic abortions.
Diagnosis
1. Histological examination of aborted foetus -Lesion of heart and CNS are characteristic of the
disease
2. Immunocytochemistry for further confirmation
3. ELISA
Control: No effective treatment in cattle. Protection of food and water from contamination with
faeces of dogs or any animals, disposal of aborted foetus and placenta by incineration and prevention
of dog eating aborted foetus and placenta are the control measures.
Bovilis® NeoGuard is the first and only vaccine for the control of abortions caused by Neospora
caninum in cattle. This vaccine contain killed tachyzoites of Neospora caninum with SPUR adjuvant.
DOSAGE: During the first trimester inject 5 mL subcutaneously, followed by a second dose of 5 mL
after 3 to 4 weeks. Revaccination with two doses are recommended for subsequent pregnancies.

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 Chapter 14

Genus: Cryptosporidium

Phylum: Apicomplexa
Class : Conoidasida
Order : Eucoccidiorida
Family: Cryptosporidiidae
Genus : Cryptosporidium
Species: C. parvum, C. muris, C. meleagridis, C. felis

Host Site of infection Zoonoses

Parasites of small intestine

1 C. hominis humans, Small intestine Yes
monkey
2 C. parvum humans and Small intestine Yes
other
mammals
3 C. bovis cattle Small intestine
4 C. canis dogs Small intestine Yes
5 C. felis cats Small intestine Yes
6 C. suis pigs Small intestine Yes
7 C. meleagridis turkeys and Small intestine Yes
humans
8 C. baileyi chickens Small intestine, Large intestine,
nasopharynx, sinus, trachea, bursa of
Fabricius, cloaca
Parasites of stomach
1 C. andersoni cattle Stomach

2 C. muris mice /rodents Stomach Yes

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 Cryptosporidium was first described by E. E. Tyzzer in 1907. He identified the parasite in
the gastric glands of a mouse. Cryptosporidium spp. have a monoxenous life cycle completed within
the gastrointestinal tract of a single host. The parasite develops just under the surface membrane of the
host cell or within its brush border of epithelial cells rather than inside the cell (intracellular, but
extracytoplasmic). Among all coccidian parasites, Cryptosporidium spp. is having smallest oocyst
(4-6µ) and lack a micropyle and this oocysts sporulate in situ. Sporulated oocyst contains four
sporozoites without sporocyst (naked sporozoites).
Life cycle
Infection occurs by ingestion or inhalation of sporulated oocyst. Four naked sporozoites
inside each oocysts excysts and these sporozoites parasitize the epithelial cells of GI or respiratory
tract. Sporozoites and subsequent development stages are intracellular beneath the host cell
membrane and extracytoplasmic. Sporozoite differentiates into spherical trophozoite with a
prominent nucleous. Schizogony results in two morphological types of schizonts viz., Type I and
Type II. Type I schizont contain 6-8 nuclei which mature to become 6-8 merozoites. Each type I
merozoite can penetrate another host cell where it can develop into type I or Type II schizont.
Type II schizont produce four merozoites. Merozoites from type II schizont initiate
gametogony in new host cell. Merozoite differentiates into macrogamonts or microgamonts.
Microgamont becomes multinucleate and upon maturation each nucleus becomes a microgamete.
Microgamete is small bullet shaped and lack flagella and mitochondria. Microgamete fertilize
macrogamont to form two types of oocysts i.e. 1. Thick walled oocysts 2. Thin walled oocysts.
Oocysts sporulate in situ (sporulated oocyst with 4 sporozoites). Thick walled oocysts leave the
body in faeces or in respiratory or nasal secretion to infect other hosts while thin walled oocyst
cause autoinfection.

Pathogenesis
The sites of infection are gastrointestinal and respiratory tract. There can be subclinical to acute
infection depending upon species and isolate, age, immunologic status of the host etc. Young
animals, especially less than 30 days old (including poultry) are more susceptible. In humans, more
reports are in children less than 2 years of age. Immunodeficient individuals can chronically and
terminally ill. Entire length of gastrointestinal tract (oesophagus to rectum including pancreas, liver

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and gall bladder) and respiratory tract can be affected. Watery cholera like diarrohoea along with
anorexia, weight loss, dehydration, abdominal discomfort, interference in the absorption of nutrients
(due to decreased level of microvillar disaccharidase), large amount of gas in the colon and fluid in
the small and large bowel, oedema of descending and sigmoid colon and rectum, acute colitis,
sometimes swollen mesenteric lymphnodes and hyperemic intestine can result. Lesions are most
severe in lower jejunum and ileum. In the respiratory tract, there will be excess mucous, swollen
mucosa and deciliation / hypertrophy / hyperplasia of epithelium.
Cryptosporidiosis in cattle

C. parvum:

Predilection site: Small intestine of cattle, sheep, goat, horse, deer, man

common in young calves. It can cause swelling and eventually fusion of the villi of ileum affecting
the membrane bound enzymes. Finally result anorexia, diarrhea (often intermittent), poor growth.
Predisposing factors are overcrowding, stress due to early weaning, transporting and marketing
together with low level of hygiene. No treatment is known. Spiramycin is of some value.
Symptomatic treatment includes antidiarrhoeal and fluid replacement therapy. Halofuginone at the
rate of 1 mg/10 kg can be used for prevention. Infection is difficult to control as oocyst is resistant
to most disinfectant except formol saline and ammonia.

C. andersoni

Predilection site: abomasum of cattle

Route of infection is fecal oral route. Oocysts develop in 72 hours. C. andersoni is almost
nonpathogenic. Depressed weight gain and decreased milk yield may result.

Cryptosporidiosis in birds (Chicken, turkey, duck, quail, ostrich, cockatiel)

Cryptosporidiosis due to C. baileyi is a disease of epithelial lining of bursa of fabricius and cloaca of
chicken. Infection of bursa of fabricius and cloaca will produce no clinical signs. Trachea and
conjunctiva are lesser sites of infection. Conjunctivitis is also seen in birds. Prepatent period-3 days.
Patent period-10-20 days.

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Pathology

Enteric cryptosporidiosis: Villous atrophy, shortening of microvilli and enterocyte detachment are
the major pathological changes associated with intestinal cryptosporidiosis.

Respiratory cryptosporidiosis: There will be excess mucous into trachea, nasal mucosal congestion
and atrophic bursa of fabricius. Cryptosporidia can be seen in nasopharynx, trachea, bronchi, and
bursa but not in intestine. There will be epithelial cell deciliation, mucosal thickening, discharge of
mucocelluar exudate into airways and bronchopneumonia in severe cases. In respiratory form, 50%
of broiler flock will show symptoms and there will be 10% mortality. Syptoms include sneezing,
coughing and head extension to facilitate breathing.

Diagnosis:

Ooocysts can be detected by concentration and staining method. To maximize recovery of

oocysts, stool specimens in formalin, or other fixatives, should be concentrated prior to microscopic
examination.

Formol ether concentration technique: Sample ( faeces 1 g) is placed in a centrifuge tube containing
7 mL of 10% formalin. If the stool is liquid, dispense about 750 µl into the centrifuge tube. Break
up the sample thoroughly and emulsify using the applicator stick. Filter the resulting suspension
through a sieve into a beaker, then pour the filtrate back into the same centrifuge tube. Add 3 mL of
ethyl acetate to the formalinised solution, Seal the neck of the tube with a rubber bung and shake the
mixture vigorously for 30 seconds. Invert the tube a few times during this procedure and release the
pressure developed gently by removing the rubber bung slowly. Centrifuge the tube at 1100 g for 2
minutes. Loosen the fatty plug with a wooden stick by passing the stick between the inner wall of
the tube and the plug. Discard the plug and the fluid both above and below it by inverting the tube,
allowing only the last one or two drops to fall back into the tube. Discard this fluid, containing ethyl
acetate and formalin, into a marked re-sealable liquid waste container. Re-suspend the pellet by
agitation. Transfer the re-suspended contents on to a microscope slide with a disposable pipette, and
air dry.

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 For concentration by floatation, Sheathers sugar solution (sp. gr. 1.18)/ Zinc sulphate
(sp.gr.1.12), or Sodium chloride (sp.gr 1.12) can be used.

Staining techniques:

1. Modified acid-fast stain (Ziehl Nielson) (Kinyoun acid fast staining method):

i) Fix the air-dried faecal smear in methanol for 3 minutes.

ii) Immerse the slide in cold strong carbol-fuchsin and stain for 15 minutes.

iii) Rinse the slide thoroughly in tap water.

iv) Decolourise in 1% acid methanol for 10–15 seconds

v) Rinse the slide in tap water

vi) Counterstain with 0.4% malachite green for 30 seconds

vii) Rinse the slide in tap water

viii) Air-dry the slide

ix) Examine for the presence of oocysts by using the ×40 objective of a microscope.
Cryptosporidium spp. oocysts stain red on a pale green background. Oocysts (4 to 6 μm)
often have distinct oocyst walls and stain from light pink to bright red.

2. Auramine phenol (AP) method

i) Fix air-dried faecal smears in absolute methanol for 3 minutes

ii) Immerse the slides in Auramine phenol stain for 10 minutes

iii) Rinse in tap water to remove excess stain

iv) Decolourise with 3% acid alcohol for 5 minutes

v) Counter stain in 0.1% potassium permanganate for 30 seconds

vi) Air dry slide at room temperature

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 vii) Examine for the presence of oocysts, using an epifluorescence microscope equipped with
FITC filters, by scanning the slide under the ×20 objective lens and confirm under the ×40
objective lens. Cryptosporidium spp. oocysts appear ring or ovoid shaped and exhibit a
characteristically bright apple-green fluorescence against a dark background.

II. Serological tests like IFAT, ELISA

III. Molecular tests like PCR
Treatment and control

1. No specific treatment for cryptosporidiosis. Supportive therapy for dehydration and

replacement of feed are suggested.

2. Minimize contact with the feces of all animals, particularly young animals. Wear
disposable gloves while handling animals, animal feces, their living areas or during
gardening and wash hands after the procedure.

3.Practice good hygiene and management (feed and water containers should be kept high
enough to prevent fecal contamination, feeding of young animal with colostrum within 24
hrs of birth, avoidance of overstocking and overcrowding.)

4. As a control measure, dairy calves should be isolated in individual pen or kept in similar
age groups. It also includes prophylactic use of halofuginone for 7 consecutive days
commencing at 24-48 hrs after birth.

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 Chapter 15
Genus: Hepatozoon

Phylum Apicomplexa
Class Conoidasida
Order Eucoccidiorida
Family Hepatozoidae
Genus Hepatozoon
Hepaotzoon canis (Syn. Leucocytozoon canis)
Host: Dog, jackal, palm civet, cat, amphibians, reptiles
Location: Asexual stages in the endothelial cells of spleen, bone marrow and liver
Geographic Distribution: South Europe, Africa, Аsia and South Аmerica
Vector: The primary vector of H. canis is the brown dog tick, Rhipicephalus sanguineus (three-host
tick). In Japan, potential and additional tick vectors viz., Haemaphysalis longicornis and
Haemaphysalis flava are reported.

Life cycle: The life cycle involves two hosts. Sexual reproduction occur in ticks and the asexual
reproduction occurs in dogs. Nymhal ticks engorge with gamont infected leucocytes in an infected
dog. Later, gamonts are freed from the leucocytes in the intestine of ticks. They associate in pairs
(syzygy) and transform in to male (microgamont which later give rise to 2-4 microgametes) and
female gametes (macrogamont). Their fusion results in zygote which transforms to ookinite which
penetrate the intestinal wall and lie in the haemocoel. Moulting of tick occurs and ookinite grows to
become oocyst in the adult tick. Sporogony takes place resulting in numerous sporocysts each with
10-20 banana shaped sporozoites.When the adult ticks are ingested by the dog, the sporozoites are
released from the sporocysts, penetrate the intestinal wall and are transported to target tissues and
organs through blood and lymph. They primarily infect the spleen, liver, lymph nodes and bone
marrow where merogony occurs in the macrophages and endothelial cells. Two forms of meronts
are formed. One type containing 2-4 macromerozoites and second type containing 20 elongated
micromerozoites. When the meronts mature, rupture occur with release of merozoites which
penetrate the circulating neutrophils where they develop to gamonts and circulate in peripheral

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blood. The period of development in dog from an infection to appearance of gamonts is about 28
days.

Pathogenesis: The principal gross pathological finding in dogs, infected with H. canis is cachexia.
Muscle atrophy is most visible in the temporal region. Also, non-regenerative anaemia, mildly
icteric mucous coats and slightly enlarged spleen and liver are observed. Congestive changes in
the lungs and the gastric mucous coat, lymphadenopathy and pale kidneys are also reported.
Histologically, schizonts are observed in the skeletal and cardiac muscles, lymphnodes, spleen,
liver, and kidneys.

Clinical signs: In high parasitaemia, lethargy, fever, severe weight loss and thrombocytopaenia are
seen.
Pathology: Splenomegaly and hepatomegaly, with a diffuse pattern of small white necrotic foci 1 to
2 mm in diameter. Necrotic foci may be larger and nodular in appearance and are also found in
other tissues, including the pancreas and on the pleura. Pneumonia may be evident, and lymph
nodes are typically enlarged.
Diagnosis: Microscopic detection of intracellular H. canis in stained blood smears can be done.
Gamonts of H. canis are found in the cytoplasm of neutrophils and monocytes (rarely). They have
an ellipsoidal, brick-like (gelatin capsule) shape and are about 11 x 4 µm in size.
Histopathology of internal organs of dogs reveals a varying number of developing or mature
meronts with their “wheel spoke” pattern in the affected tissues.

Treatment
 Imidocarb dipropionate at 5-6 mg/kg every 14 days until no parasites are seen in blood smears.
 Oral doxycyline at 10 mg/kg/day for 21 days in combination with imidocarb dipropionate.
Elimination of H. canis from peripheral blood may require treatment for eight weeks.

Control: Tick control

Hepatozoon americanum
Definitive host and tick vector- Amblyomma maculatum (Gulf Coast tick)
Intermediate host- coyotes and domestic dogs
Distribution- United States, Central and South America
Clinical signs: Include fever, myalgia, myasthenia, wasting and reluctance to move

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Gamont: tail like appendage, lacks fine fibril-like structure as that of H. canis
The trophozoite found in macrophage-like cells (mainly in striated muscle) apparently transforms
the host cell into a mucopolysaccharide producing structure called “onion skin cysts”. The meronts
of H. americanum are usually found within these “onion skin” cysts .
Treatment- trimethoprim/sulfadiazine, pyrimethamine, clindamycin, decoquinate

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 Chapter 16
Genus: Haemoproteus
Phylum: Apicomplexa
Class: Aconoidasida
Order: Haemospororida
SubOrder: Haemosporina
Family: Plasmodiidae
Genus: Haemoproteus
Haemoproteus columbae
Host: Domestic and wild pigeon, dove, wild birds, reptiles (snakes or lizards)
Vector: Flies belonging to Hippoboscidae (Hippoboscid or louse flies) like Pseudolnychia
canariensis, Pseudolynchia brunnea ( Brazil) and Ceratopogonidae (Culicoides sp.) (wild birds)
Location: Erythrocytes and endothelial cells of blood vessels and inner organs.
Description- Gametocytes are present in the erythrocytes in the form of tiny ring forms to elongate
crescent shapes (sausage shaped) that curve in around the host cell in the form of halter. Contain
pigment derived from the erythrocytes. Host cell may get enlarged and the host cell nucleus may get
laterally displaced.
Life cycle: The infective stage is the sporozoite which is present in the salivary glands of the vector.
Once the vector bites a new host, the sporozoites enter the blood stream and invade endothelial cells
of blood vessels. Within the endothelial cells, the sporozoites undergo asexual reproduction
becoming early schizont. The early stages are minute cytoplasmic bodies with a single nucleus, but
by growth and nuclear division, 15 or more small unpigmented masses or cytomers each with a
single nucleus are produced. Each cytomers continues to grow and its nucleus undergoes repeated
division until the greatly enlarged endothelial cell is filled with a large number of multinucleate
bodies or cytomers, surrounded by a fine cyst wall. Each cytomere produces an enormous number of
minute merozoites. Subsequently, the endothelial cells breaks down and the cytomers are liberated,
which accumulate in the capillaries and block them. But soon after liberation, the cytomers rupture
and the merozoites escape into the blood stream. The merozoites enter the red blood cells and
become gamonts. Some merozoites enter endothelial cells and continue the asexual cycle. The

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haemozoin granules of male gametocyte are collected into spherical masses while those of female
gametocytes are scattered in the cytoplasm. Further development takes place in the insect host after
blood feeding. Exflagellation of male gamonts occur in the mid gut of the fly and after fertilization
zygote forms in the insect mid gut. The zygote becomes elongate, called ookinete which later
migrates to outer surface of the mid gut. Sporogony takes place and the sporozoites are released into
the body cavity of the insect and pass to the salivary glands. When the vector bites a new host, the
sporozoites enter the blood stream of the host.

Pathogenesis and clinical signs: Majority of the infections does not show any symptoms. In severe
cases, the younger birds show restlessness, loss of appetite, anaemia and even death. Sometimes
birds may die without showing any symptoms. Post-mortem changes include spleenomegaly,
hepatomegaly and pneumonia, necrosis of the muscle fibres which cause muscle damage and
mortality.

Diagnosis: Clinical signs and demonstration of gametocytes in the blood smears, schizonts in the
endothelial cells.

Chemotherapy and control: Antimalarial drugs like chloroquine, primaquine, quinacrine reduce
the parasitemia but do not eliminate the parasite. Insecticidal spray will eliminate the hippoboscid
fly.

Species Features

Host: Turkey. Non-pathogenic/slightly pathogenic.

Haemoproteus meleagridis Cause white streaks and bloody spots (cyst like bodies)
in the muscle tissue like Sarcocyst

Haemoproteus sacharvoi Host: Pigeon, dove

Haemoproteus nettionis (H. Host: Duck, goose, wild duck and swans
anatis/H.anseris/H.hermani) Vector: Culicoides

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 Chapter 17
Genus: Leucocytozoon
Phylum: Apicomplexa
Class: Aconoidasida
Order: Haemospororida
SubOrder: Haemosporina
Family: Plasmodidae
Genus: Leucocytozoon
Location: Erythrocytes or leucocytes
Description: The gametogonous stages occur in the circulating blood, and the infected host cells
become grossly distorted and assume a spindle shape. No pigment is produced.

Life cycle
The sporozoites injected by the black fly ( Simulium spp.) enter the blood stream and invade
various cells of the body. The first asexual generation takes place in Kupffer cells of the liver. The
schizonts are small, and they produce merozoites, some of which may enter blood cells to form
gamonts while others initiate hepatic schizonts and megaloschizonts. Hepatic schizonts occur in the
parenchyma liver cells (Kupffer cells) forming a number of cytomeres which in turn form small
merozoites by multiple fission. Megaloschizonts are found in the brain, liver, heart, kidney, gizzard,
intestine and lymphoid tissues. They are more common than the hepatic schizonts. With the rupture
of megaloschizonts and hepatic schizonts, merozoites are released into blood and enter the blood
cells to form gamonts. But some merozoites initiate further asexual reproduction.
In the mid gut of the black fly, four to eight microgametes are formed by exflagellation
from the microgamonts. These fertilise the macrogametes to form a motile zygote or ookinete.
Ookinete are present in the midgut of black fly 2-6 hours after ingestion of the infected blood. They
develop to produce slender sporozoites with one end round and one end pointed and pass to the
salivary glands, where they accumulate.

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 Leucocytozoon caullery
Leucocytozoon simondi Leucocytozoon smithi
(Akiba caulleryi)
Ducks, geese, and
Host Turkey Chicken and guinea fowl
swans
Simulium spp. Simulium spp.
Vector Culicoides spp.
(blackfly)
Round hepatic meronts Round hepatic meronts Megalaomeronts only
Meronts
and megalaomeronts only
Gamonts are elongate. Gamonts are elongate. Gamonts are round.
Infected host cell is Host cell is greatly Host cell is not much distorted.
grossly distended and distorted and elongated. Host cell nucleus is compressed to
elongated. Host cell The host cell nucleus is one side of the cell forming a band
nucleus is elongate and elongated forming a extending about one third of the way
Gamonts
forms a long thin dark dark band along one around the parasite.
crescent along one side side of the parasite
of the cell. often splitting to form a
band on each side of the
parasite.
Markedly pathogenic Markedly pathogenic Some strains are highly pathogenic,
for ducks and geese. for turkeys. some non-pathogenic. Produce
Ducklings show Recovered birds may Bangkok’s haemorrhagic disease.
listlessness, anorexia, continue to carry
rapid breathing and infection in their blood.
Pathogenesis anaemia Persistent infection lead
and clinical to lethargy, lack of
signs libido in male birds and
persistent coughing.
Anorexia, difficulty in
moving, incoordination,
birds may collapse, and
die
Spleenomegaly, Emaciation, spleen and Haemorrhages, in the lung, liver and
liver hypertrophy and liver enlarged, enteritis kidney. There may be gross
degeneration haemorrhage from the kidney lesions
Postmortem in to the peritoneal cavity due to the
examination rupture of megalameronts.
Splenomegaly and hepatomegaly.
White dots will be visible due to the
presence of meronts in many organs.

Treatment- Not usually effective. Pyrimethamine 1ppm and sulphadimethoxine 10ppm or Clopidol
125 ppm in feed can prevent L. caulleryi in chicken.

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 Chapter 18
Genus: Plasmodium
Phylum: Apicomplexa
Class: Aconoidasida
Order: Haemospororida
SubOrder: Haemosporina
Famiy:Plasmodidae
Genus:Plasmodium
Plasmodium gallinaceum in chicken was described by Brumpt in 1935.
Malaria parasites are micro-organisms that belong to the genus Plasmodium. There are more
than 100 species of Plasmodium, which can infect many animal species such as reptiles, birds, and
various mammals. The transmission of Plasmodium requires two hosts, an invertebrate host (vector),
and a vertebrate host (mammals, birds and lizards).
Human Malaria
Major species causing malaria in humans include
1. Plasmodium malariae (quartan malaria)
2. Plasmodium vivax (benign tertian malaria)
3. Plasmodium falciparum (malignant tertian malaria, subtertian malaria)
4. Plasmodium ovale (ovale tertian malaria)
5. Plasmodium knowlesi (which usually infects macaque monkeys and is zoonotic)
Transmission: By the bite of an infected female Anopheles mosquito, exposure to infected blood
products (transfusion malaria) and by congenital transmission
Life cycle
All Plasmodium species undergo the general haemosporina developmental cycle.
a. Iinitial or continual schizogony (reproduction by multiple asexual fission) in the
vertebrate host with initiation of gametogony (the formation or production of gamonts)
b. Formation of gametes in the arthropod host and subsequent fertilization and formation of
a zygote

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 c. Formation of sporozoites from the zygote by repeated nuclear division followed by
cytoplasmic divisions
The life cycle of all species of human malaria parasites is essentially the same. It comprises
an exogenous sexual phase (sporogony) with multiplication in certain Anopheles mosquitoes and an
endogenous asexual phase (schizogony) with multiplication in the vertebrate host. The latter phase
includes the development cycle in the red cells (erythrocytic schizogony) and the phase taking place
in the parenchyma cells in the liver (exoerythrocytic schizogony). Many species produce haemozoin
pigment granules as a byproduct of haemoglobin metabolism.
Vertebrate Phases: Schizogony comprises erythrocytic schizogony and exoerythrocytic
schizogony. When the infected Anophelus mosquito takes a blood meal, sporozoites are inoculated
into the bloodstream of vertebrate host. Within an hour, sporozoites enter hepatocytes and begin to
divide into exoerythrocytic merozoites (tissue schizogony). The dormant forms (hypnozoites)
typically remain quiescent in the liver in the case of P. vivax and P. ovale after recovery form the
disease. Plasmodium falciparum does not produce hypnozoites. Once merozoites leave the liver,
they invade erythrocytes and develop into early trophozoites, which are ring shaped, vacuolated and
uninucleated. Soon, the vacuole disappears and parasite changes to amoeboid form. This stage is
called ameboid stage (late trophozoite). In this stage, parasite feeds on the haemoglobin and other
contents of RBC. Haemozion pigments are produced. The trophozoites develop to schizonts
(cryptozoite) consisting of many daughter merozoites (blood schizogony). The stage in the
erythrocytic schizogony at which the cytoplasm is coalescing around the individual nuclei, before
cytokinesis, is called a segmenter. The infected erythrocytes are lysed by the merozoites, which
subsequently invade other erythrocytes, starting a new cycle of schizogony. Rupture of mature
schizont release pyrogens and cause febrile paroxysms. After an indeterminate number of asexual
generations, some merozoites enter erythrocytes and become macrogamonts (macrogametocytes)
and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each
species and they also contain haemozoin. Unless they are ingested by a mosquito, gametocytes soon
die and are phagocytized by the reticuloendothelial system. The length of the developmental stage in
the mosquito not only depend on the Plasmodium species but also the mosquito host and the
ambient temperature. This may range from eight days in Plasmodium vivax to as long as 30 days in
Plasmodium malariae.

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Invertebrate Stages: When erythrocytes containing gametocytes are imbibed, gametocytes develop
into gametes. After release from its enclosing erythrocyte, a macrogametocyte matures to a
macrogamete. The microgametocyte undergo transformation like exflagellation to become
microgamete. A microgamete swims about until it finds a macrogamete, which it penetrates and
fertilizes. The resultant diploid zygote quickly elongates to become a motile ookinete. The ookinete
is reminiscent of a sporozoite or merozoite in morphology. It is 10 μm to 12 μm in length. The
ookinete penetrates the peritrophic membrane in the mosquito’s gut and migrates intracellularly and
intercellularly to the hemocoel side of the gut. There it begins its transformation into an oocyst. An
oocyst is covered by an electron-dense capsule and soon extends out into the insect’s hemocoel. The
initial division of its nucleus is reductional, meiosis takes place immediately after zygote formation.
The oocyst reorganizes internally into a number of haploid nucleated masses called sporoblasts and
the cytoplasm contains many ribosomes, an endoplasmic reticulum, mitochondria, and other
inclusions. Sporoblasts, in turn, divide repeatedly to form thousands of sporozoites. These break out
of the oocyst into the haemocoel and migrate throughout the mosquito’s body. On contacting the
salivary gland, sporozoites enter its channels and can be injected into a new host at the next feeding.
Plasmodium falciparum (Malignant tertian/falciparum malaria/pernicious malaria, Falx-sickle,
parere- to bring forth)
Most virulent species of Plasmodium spp. in humans. Falciparum malaria reigns supreme as
the greatest killer of humanity in the tropical zones of the world today, accounting for about 50% of
all malaria cases. Disease is concentrated in tropics and sub tropics. True relapses do not occur
however, recrudescence of the disease may follow remissions of up to a year, occasionally up to two
or three years, after initial infection, because small populations of the parasites remain in red blood
cells. Merozoites of P. falciparum can invade erythrocytes by different pathways and so falciparum
malaria usually has much higher levels of parasitaemia than the other types. Soon after invasion of
an erythrocyte, a trophozoite produces proteins that are deposited beneath and within the erythrocyte
surface membrane in deformations called knobs. One or more of these proteins bind to certain
glycoproteins on the postcapillary venular endothelium. This binding causes sequestration of
infected erythrocytes along the venular endothelium. Cerebral malaria occurs as a result of
sequestration of infected RBC in brain.

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 Gametocyte-infected erythrocytes have no knobs and do not stick to endothelium. Infected
red cells are antigenically distinct from uninfected erythrocytes, and sequestration may decrease
clearance of infected cells by phagocytes in the spleen. Infected erythrocytes can also bind to
uninfected red cells, forming rosettes, which may also play a role in clogging venules. Because of
sequestration, parasites may be difficult to be demonstrated in the circulating blood, and
examination of skin biopsies may be helpful in diagnosis. The early ring-stage trophozoite of P.
falciparum is the smallest of any Plasmodium spp. of humans (about 1.2 μm). An infected
erythrocyte develops irregular blotches known as Maurer’s clefts. They are apparently associated
with the tubovesicular membrane network, which is continuous with the parasitophorous vacuole
and extends toward the erythrocyte membrane. This network has a significant role in transport of
nutrient molecules to the parasite and export of P. falciparum molecules to the surface of the red
cell.

Plasmodium vivax (Benign tertian malaria, vivax malaria or tertian ague)

Widest in geographical distribution. It is called tertian because fever paroxysms typically recur
every 48 hours. P. vivax merozoites invade only young erythrocytes, the reticulocytes, and
apparently are unable to penetrate mature red cells because receptor sites change as the cells mature.
Merozoites can only penetrate erythrocytes with mediated receptor sites, such sites being genetically
determined and known as Duffy blood groups, two co dominant alleles, Fya and Fyb . Relapse occurs
because of presence of hypnozoites. Soon after invasion of erythrocytes and formation of ring
stages, the parasites become actively ameboid, throwing out pseudopodia in all directions. As the
trophozoite grows, the red cell enlarges, loses its pink colour, and develops a peculiar stippling
known as Schüffner’s dots.

Plasmodium malariae (Quartan malaria)

It is a cosmopolitan parasite and febrile paroxysms occurs every third day with 72 hours
interval. Their nucleus divides into 6 to 12 merozoites at 72 hours. The segmenter is strikingly
symmetrical and is called a rosette or daisy-head. Merozoites can invade only aged erythrocytes.
Gametocytes develop in internal organs, since immature forms are rare in peripheral blood. They are
slow to develop in sporozoite-induced infections. Recrudescences of quartan malaria can occur up to

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53 years after initial infection. Because P. malariae can live in blood so long, it is the most
important cause of transfusion malaria. Older trophozoites later stretch across the erythrocytes as a
band and fine dots are seen in erythrocytes, known as Ziemann’s dots.

Plasmodium ovalae (Tertian fever, ovale, or mild tertian malaria)

Rarest of the four malaria parasites of humans and is confined mainly to the tropics.
Schüffner’s dots appear early in infected blood cells. Gametocytes of P. ovale take longer to appear
in blood than with other species.

Plasmodium knowlesi (monkey malaria): Fifth Human Malaria Parasite

Host: long-tailed and pig-tailed macaques (Macaca fasicularis and Macaca nemestrina)

Vectors: Anopheles leucosphyrus group, Anopheles latens and Anopheles cracens.They are forest
feeders, bite both humans and macaques at evening or during the night. They have daily (quotidian)
cycle. Distributed in Malaysia and Southeast Asian countries.

Clinical signs include daily fever and chills. Other frequent symptoms like headache, rigors,
malaise, myalgia, abdominal pain, breathlessness, and productive cough. Tachypnea, pyrexia, and
tachycardia also occur. Thrombocytopenia is common.

Pathogenesis

Most major clinical manifestations of malaria may be attributed to two general factors:

(1) The host inflammatory response, which produces the characteristic chills and fever as well as
other related phenomena

(2) Aanemia, arising from the enormous destruction of red blood cells.

Malaria is characterized by overproduction of proinflammatory cytokines of the innate

immune system. The characteristic paroxysms of fever in malaria closely follow maturation of each
generation of merozoites and rupture of red blood cells that contain them (schizont burst). Glycosyl
phosphatidyl inositols (GPIs) specific to the parasite are released along with cellular debris, host and
parasite membranes, and hemozoin. GPIs are the dominant parasite associated molecular pattern

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recognized by host monocytes and macrophages. From the activated macrophages a burst of pro-
inflammatory mediators like TNF, IL-6, IL-12, IL-1, IFN-γ, and nitric oxide synthase will occur and
responsible for majority of clinical signs in malaria. Excessive TNF and quinine (therapy) stimulate
pancreatic islet cells for increased insulin secretion and thus lowering blood glucose
(hypoglycaemia). The main causes of the anemia are destruction of both parasitized and
nonparasitized erythrocytes, inability of the body to recycle the iron bound in haemozoin, and an
inadequate erythropoietic response of the bone marrow.

Three basic types of malaria

a) Benign tertian (P. vivax and P. ovale) with a fever every 2nd day (48hours)
b) Benign quartan (P. malariae) with a fever every 3rd ( 72 hours)
c) Malignant tertian (P falciparum), in which the cold stage is less pronounced and the fever
stage is more prolonged and intensified (if the fever is recurring it occurs every 2nd day or 48
hours). The fever is usually continuous or only briefly remittent. There is no wet stage. This type
of malaria is more dangerous because of the complications caused by capillary blockage (i.e.,
convulsion, coma, acute pulmonary insufficiency, and cardiac failure). Large numbers of
erythrocytes are parasitized and destroyed, which may result in dark coloured urine (blackwater
fever; intravascular hemolysis, hemoglobinuria, and kidney failure.).
Clinical syndromes associated with P. falciparum
1. Prodromal period (An early symptom indicating the onset of an attack or a disease which
include malaise, myalgia, headache, fatigue, chest pain, abdominal pain, arthralgia)
2. Malarial paroxysm fever (interleukines1, TNF, cytokines) chill, rigor
3. Anaemia
4. Hepatospleenomegaly
Complications of severe malaria
Neurologic complications- Cerebral malaria is the most common clinical presentation and cause
of death in adults with severe malaria. It shows evidence of mild cerebral swelling; marked
oedema or focal lesions are unusual. Delirium, agitation, and even transient paranoid psychosis
may develop as the patient recover consciousness. Apart from cerebral malaria, other neurologic
sequelae can occur, such as cranial nerve abnormalities, extrapyramidal tremor, and ataxia.

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 Pulmonary oedema: This is very much common and lead to death in 80% cases. The first
indications of impending pulmonary edema include tachypnea and dyspnoea, followed by
hypoxemia and respiratory failure.
Renal failure: Acute renal failure is usually oliguric (<400 ml/day) or anuric (<50 ml/day), rarely
nonoliguric, and may require temporary dialysis. Urine sediment is usually unremarkable. In severe
cases, acute tubular necrosis may develop secondary to renal ischemia. ‘Blackwater fever’ refers to
passage of dark red, brown, or black urine secondary to massive intravascular hemolysis and
resulting hemoglobinuria. Usually, this condition is transient and not accompanied by renal failure.
Hypoglycaemia: especially in Quinine treatment with lactic acidosis and clinical signs like anxiety,
dyspnea, tachycardia, sweating, and coma, abnormal posturing, generalized convulsions.
Hematologic abnormalities: Severe anaemia is more common in children in highly endemic areas
due to repeated or chronic Plasmodium infections. Thrombocytopenia is common, but usually not
associated with bleeding. Disseminated intravascular coagulation is reported in less than 10% of
patients with severe malaria
Tropical splenomegaly syndrome (Hyperreactive malarial splenomegaly)
Diagnosis
a) Microscopy
1.Light microscopy - demonstration of parasites in the peripheral blood – at least twice
daily- during fever is the best ( Ring forms and gametocytes)
2. Fluorescence microscopy-Kawamoto technique- Acridine orange staining
3. QBC: fluorescence microscopy-based malaria diagnostic test –Acridine orange based–
malaria brilliant green
b) Serological tests
1. IFAT, IHA, ELISA, ELISA, Inhibition test
2. Dipstick test- Enzyme immunoasaay- detect histidine rich protein 2 antigen- a metabolic
product produced by P. falciparum
c) Molecular diagnosis- PCR

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Treatment
A. Uncomplicated (less severe) infection- Chloroquine-10 mg/kg (maximum 600 mg, oral)
Uncomplicated infection with Chloroquine resistance- Combinations of quinine, Pyrimethamine,
sulfadiazine- oral)
B. Complecated (severe) malaria
1. During severe malaria- Chloroquine- i/v
2. In adults with severe cerebral malaria : artesunate i/v
3. Severe malaria with Chloroquine resistance- Quinacrine- i/v
Chemoprophylaxis
a) Chloroquine-5mg/kg /week max 300 mg(oral)
b) Primaquine- 45mg once a week for 8 weeks
c) Mefloquine 250 mg once a week
d) Doxycycline 10 mg in a single dose
Vaccine candidates
a) Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate
molecule for inclusion in a human malaria vaccine.
b) GMZ2 - fusion protein of parts of P. falciparum glutamate-rich protein (GLURP) and merozoite
surface protein 3 (MSP3)
c) Merozoite surface protein 3 (MSP-3), glutamate-rich protein (GLURP), serine repeat antigen
(SERA), known as P126 antigen

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Malaria in birds

Plasmodium in avian hosts Haemoproteus and Leucocytozoon in

Characteristics avian hosts

From infected to uninfected hosts by Arthropod vector is mandatory

Transmission simple blood inoculation

Vector Blood-sucking mosquitoes Biting midges and hippoboscid flies

Both erythrocytic schizonts and Schizogony only in fixed non-circulating
Schizogony gametocytes cells in the host

Smaller tissue schizonts that produce Megaloszhizonts in host tissues that yield
Schizont tens to hundreds of merozoites millions of merozoites

Species with round or irregular gamonts which displace the nucleus of the host cell:

Plasmodium cathemerium, Plasmodium gallinaceum, Plasmodium juxtanucleare, Plasmodium

relictum, Plasmodium griffithsi

Species with elongate gamonts which do not usually displace the host cell nucleus:

Plasmodium circumflexum, Plasmodium durae, Plasmodium elongatum, Plasmodium fallax,

Plasmodium hexamerium, Plasmodium lophurae, Plasmodium polare, Plasmodium rouxi,
Plasmodium vaughani

P. gallinaceum
Infections are seen in chickens in Asia and probably Africa. Highly pathogenic to domestic chicken.

Morphology: The trophozoite is small round form containing a large vacule.The nucleus of host
cells is rarely expelled during infection, but may be displaced by the parasite. The nucleus is situated
at one of the poles giving the young form a signet ring appearance. Both gametocytes and schizonts
of P. gallinaceum can be round, oval or irregular in shape. Usually 16 - 20 merozoites are seen in
erythrocytic schizonts of P. gallinaceum.

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Life cycle: Following the introduction of sporozoites from infected culicine mosquitoes, numerous
preerythrocytic schizonts are formed in the macrophages and fibroblasts of the skin near the point
of entry. They are cryptozoites. Merozoites from this first generation preerythrocytic schizonts, form
a second generation of preerythrocytic schizonts, the metacryptozoites. Merozoites form them, enter
erythrocytes and other cells of the body and in the latter form exoerythrocytic schizonts (endothelial
cells for P. gallinaceum, P. relictum and P. cathemerium; cells of haematopoetic system for P.
elongatum). Avian malarial parasites does not use liver parenchyma for EE schizogony. In some
species like P. elongatum and P. gallinaceum, the merozoites of schizonts of erythrocytic cycle
develop to exoerythrocytic developmental stages. They are phanerozoites.

On entering red cell, merozoites round upto form a trophozoite. The small vacuolated forms which
displaces the cytoplasm of the parasite to periphery of the cell. The nucleus is situated at one pole –
signet ring appearance, when stained by Rhomanosky stains. The early trophozoites undergo
schizogony to produce merozoites, the number produced depend on species of parasites. During
schizogony, the parasites takes in host cell cytoplasm, by invagination, haemoglobin is digested and
residual haematin pigment is deposited in granusles within the food vacuoles. The release of
merozoites from schizonts in erythrocytes occurs. After a number of asexual genrations has
occurred, some merozoites undrgo sexual development with the formation of micro and macro
gamonts. Further development occur in mosquitoes.

Neurological complications are caused by obstruction of capillaries in the brain by extraerythrocytic

meronts.

P. juxtanucleare
Host- chickens and turkeys in South America, Africa and Asia
The schizonts of P. juxtanucleare are round, ovoid or irregular and small compared to P.
gallinaceum' s schizonts and usually in contact to the hosts cell nucleus. Three to five merozoites are
seen in erythrocytic schizonts. The gametocytes are usually round or ovoid, but may be irregular or
slightly elongated. Often the host cell is distorted.

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P. durae
Host: turkeys and gallinaceous birds other than chickens
Highly pathogenic to turkeys. Trophozoites are amoeboid in appearance. Mature meronts rarely
displace the host cell. Gamonts are elongate at the end side of the host cell and often displace the
host cell nucleus. The host cell is not usually enlarged.
P. relictum
Highly pathogenic to pigeons and doves.
Host: Pigeon, dove, duck
Vector: Culex and Ades sp.
Treatment of avian malaria: Captive bird populations may be treated with chloroquine,
primaquine, sulphonamide drugs or halofuginone.

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 Chapter 19
Genus: Babesia

Phylum: Apicomplexa
Class: Aconoidasida
Order: Piroplasmorida
Families: Babesiidae
Genus: Babesia; Theileria

Characters of order: Piroplasmorida

Members of order Piroplasmorida are blood parasites of vertebrates (cells of hemopoietic
system) and often referred to as “piroplasms” (Piroplasm: a collective term for phenotypically
similar protozoan parasites that utilize mammalian erythrocytes in their life cycle. Piroplasms are
pleomorphic i.e. pyriform, round, amoeboid, or rod shaped. Piroplasms of domestic animals
encompass two main genera, Babesia and Theileria). They are present in RBC (not enclosed by
parasitophorous vacuole) and sometimes in other cells. These organisms do not form oocyst or
pseudocyst and lacks flagella. Ticks acts as vectors. They are heteroxenous parasites with asexual
reproduction (binary fission or Budding or merogony) in vertebrate host and sexual reproduction
(gamogony, syngamy and sporogony) in invertebrate host. Pigments are not formed in the host cells
of these parasites. Apical complex is reduced without conoid but with polar ring and rhoptries.
Locomotion in them is by gliding.
Family: Babesiidae
Organisms are relatively large, round to pyriform or amoeboid forms occurring in the
erythrocytes and locomotion is by gliding. They multiply by binary fission, endodyogeny,
endopolygeny (budding) or merogony to form merozoites in RBC. Apical complex reduced to polar
ring, rhoptries, and subpellicular microtubules (micronemes present in some stages). The vectors are
ixodid ticks (hard tick).

History
In 19th century (1888) Victor Babes discovered microorganisms in erythrocytes of cattle in
Rumania and associated them with bovine haemoglobinuria or red water fever. He incorrectly

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believed it to be due to a gram negative bacterium and named Haematococcus bovis. Later, he found
similar organisms in red blood cells of sheep. In 1893, the agent of Texas fever of cattle in the USA
was identified as a parasite, Pyrosoma bigeminum by Theobald Smith and Fred Lucius Kilborne,
who also showed that it was transmitted by a tick. This appears to have been the first report of the
transmission of a protozoan parasite by an arthropod. In the same year, Starcovici gave these
parasites, the names of Babesia bovis, Babesia ovis and Babesia bigemina respectively.
Genus: Babesia
Organisms multiply in the erythrocytes by asexual division, producing two, four or more
nonpigmented amoeboid parasites. They are pear shaped forms lying at an angle with the narrow
ends in apposition. They can be stained with Romanowsky stains resulting in blue cytoplasm and a
red chromatin mass usually at one pole. A string of chromatin granules may extend from the larger
mass. B. bigemina paired forms often have two discrete red-staining dots in each parasite (B. bovis
and B. divergens always have only one). Babesia spp. are classified into large forms (having
average length of more than 3µm) and small forms (having average length of less than 2.5 µm).

Large forms Small forms

Size ≥3µm length ≤2.5µm

Morphology in RBC Pairs usually subtended at an acute Obtuse angle
angle
Pathogenesis Generally more pathogenic pathogenic

Amenable to treatment Generally amenable Not amenable

with trypan blue
Bovine B. bigemina B. bovis, B. divergens, B.
major
Canines B. canis B. gibsoni
Equine B. caballi
Ovine B. motasi B. ovis
Goat B. foliata B. taylori
Pig B. trautmanni B. peroncitoi

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General life cycle of Babesia spp.
The life cycles of the Babesia spp. are very similar. All species of Babesia are naturally
transmitted by the bite of infected ticks (ixodids) and the main difference in the life cycle amounts to
the presence of transovarial transmission in some species [Babesia spp. sensu stricto i.e.,
Babesia spp. from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis, and other Babesia spp. from
cattle)) and tansstadial in others (B. microti-like).
Babesia life cycles consist of merogony, gamogony and sporogony. Infection is acquired
when sporozoites are transferred during tick feeding. Sporozoites then invade erythrocytes and
develop into trophozoites. Preferential invasion of young erythrocytes by merozoites occurs in acute
Babesia bigemina infections. Trophozoites divide by binary fission and produce merozoites which
continue infection by invading other RBCs and reinitiate the replicative cycle in the host until a
large percentage of red blood cells are parasitized. Occasionally, a cell shows multiple infection with
a large number of trophozoites, but it is considered that this represents a series of binary fissions
rather than a multiple invasion of the cell. Some trophozoites develop into gametocytes which can
initiate infection in the tick vector. In the tick gut, gametocytes develop into “Strahlenkörper” bodies
(ray bodies), which fuse to form a zygote. The zygote develops into an infecting stage and penetrates
the tick intestinal cells to form fission bodies and from them motile kinetes (large
merozoites/vermicules /sporokinetes) develop. Kinetes destroy the intestinal cells, escape into the
haemolymph and distribute into the different cell types and tissues, including the ovaries. In the
ovary, embryo cells are infected by kinetes. When the female tick lays her eggs, the embryos are
already infected. When the infected larvae attach to a bovine host, the kinetes migrate to the salivary
glands of the tick, where sporogony is initiated to form multinucleated sporoblasts. Thousands of
sporozoites develop from each sporoblast. When tick larvae feed on bovines, the sporozoites are
liberated with saliva into the animal’s circulatory system.
The blood forms are readily transmissible by mechanical means to another animal, and these
then initiate a further cycle of asexual reproduction.

Transovarial / transovarian transmission: occurs in one host tick. All the stages of tick (larvae,
nymph, and adult) remain in one animal and do all their feeding on the same animal. Transmission
occurs through the eggs of tick. B. bigemina transmitted by nymphal/adult stages of one-host

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Rhipicephalus (Boophlus) spp. ticks. B. bovis transmitted by larval stages of one-host
Rhipicephalus(Boophlus) spp. ticks.

Transstadial transmission: Occurs in two or three host tick. Adult stages transmitting infection
which they acquire as nymphs or nymphs transmitting the infection which they acquired as larvae.
Infrequently, calves can become infected in utero.
Babesia of cattle: Seven species of Babesia have been reported from cattle. The principal species of
Babesia that cause bovine babesisosis are Babesia bigemina, B. bovis, B.divergens. Other Babesia
that can infect cattle include B. major, B. jakimovi, B. ovata and B. occultans.
Babesia bigemina: B. bigemina occurs throughout the tropics and subtropical areas such as
Central and South America, North and South Africa, Australia and southern Europe and is the cause
of cattle tick fever, red water fever, piroplasmosis and Texas fever.
Host: bovine, zebu, water buffalo, deer.
Vectors: The following ticks have been incriminated as vectors: One-host ticks: Rhipicephalus
(Boophilus) microplus (tropical cattle tick/southern cattle tick) (in Australia, Panama, South
America, Asia); Rhipicephalus (Boophilus) annulatus (North America). Other competent one host
tick vectors are R. (Boophilus) calcaratus (North Africa) and R. (Boophilus) decoloratus (South
Africa). B. bigemina transmitted by feeding of nymphal and adult stages of one-host Rhipicephalus
(Boophlus) spp. ticks. Two-host ticks: Rhipicephalus evertsi (South Africa); Rhipicephalus bursa
(South Africa) and Three-host ticks: Haemaphysalis punctata (Europe and Eurasia) and
Rhipicephalus appendiculatus (South Africa) are acting as vectors.
Morphology: Large piroplasm, 4-5 µm in length by about 2 µm wide, round forms 2-3µm in
diameter. The organisms are characteristically pear-shaped and lie in pairs forming an acute angle in
the red blood corpuscle. Round, oval or irregularly shaped forms may occur, depending on the stage
of the development of the parasite in the red cell.
Pathogenesis: The main sequelae of the disease are:
1. Anaemia due to haemolysis; haemoglobinaemia and haemoglobinuria, icterus.
Haemolytic anaemia: due to emergence of parasites from RBC (attributable to mechanical rupture of
RBC by parasites), direct removal of non-infected erythrocytes by phagocytosis, increased osmotic

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fragility of non-infected RBC and adsorption of circulating antigen antibody complexes to the
surface of RBC leading to RBC removal by phagocytic activity of reticuloendothelial system.
2. Pharmacologically active substances such as kinins and catecholamines released in babesiosis
lead to increased vascular permeability and dilatation of blood vessels resulting in oedema and
hypovolaemic shock. Babesia bovis produces an in vitro activator of kallikrien. Kinin produces
increased vascular permeability and vasodilatation (changes muscle tone) leading to circulatory
stasis and shock.
3. Centrilobular liver degeneration and degeneration of kidney tubule epithelium are caused by
hypoxia and possibly by immunopathologic reactions and glomerulonephritis.
4. Selective concentration of parasitized cells occurs in brain capillaries leading to the obstruction of
blood flow. Central nervous system damage is a feature of Babesia bovis and Babesia canis
infections. Cerebral form of Babesia bigemina is also reported.
The disease caused by Babesia bigemina resembles a haemolytic anaemia and while with Babesia
bovis infection kinin production is more important.
Symptoms/ Clinical signs: Incubation period is 1-2 weeks. First evidence of the disease is rise in
body temperature to 41-42°C. The high fever lasts from two to seven days or more. At the height of
fever, up to 75% of red blood cells may be destroyed and during this period of pyrexia a profound
anaemia frequently develops. Haemoglobinuria (coffe / dark brown coloured urine) is ordinarily
present but may be absent also. Initially there will be profuse diarrhoea and later followed by
marked constipation. Affected animal will be dull and listless, fail to eat and stop rumination, faces
are yellowish brown and cardiac palpitation seen. Affected animal may be emaciated and icteric.
Mortality may be very high (50-90%) in acute cases and death occurs after 4-8 days of onset of
clinical signs. There is haemoglobinuria and cardiac palpitation. Survival of acute phase goes into a
chronic disease. Chronic disease may extend over several weeks with an irregular course and
intermittent temperature. Temperature is not very high, rises up to 40-40.6°C and animals become
thin and emaciated. In chronic disease, usually there will not be any marked haemoglobinuria but
diarrhoea or constipation with hard yellow faeces and finally animal will recovers.

Post-mortem lesions: Lesions seen are subcutaneous and intramuscular oedema with icterus,
yellow and gelatinous fat, thin and watery blood, spleenomegaly (enlarged spleen) with a soft, dark

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red splenic pulp and prominent splenic corpuscle, moderately swollen and dark reddish-brown
coloured kidney in (haemoglobinuric nephrosis), enlarged pale and yellowish liver and distended
gall bladder with thick and dark bile. Blood plasma will be tinged red on sedimentation and contains
haemoglobin. The urine in the bladder is frequently red (haemoglobinuria) or dark brown. Mucosa
of abomasum and intestine is edematous and icteretic with patches of haemorrhage. In cerebral
form, there will be perivascular, perineuronal and interstitial oedema throughout the brain and spinal
cord.
Microscopically, centrilobular necrosis in the liver, deposits of haemosiderin in Kupffer cells
and congestion in a variety of organs, such as lungs, heart, spleen and kidney. Degeneration of the
tubular epithelium and cast formation are seen in the kidneys, along with deposits of haemosiderin
in various cells of this organ. Depletion of germinal centers of spleen, lymph node, hyperplasia of
reticular tissue and large number of marcophages with haemosiderrin are also seen.

Cerebral form of B. bigemina infection: Onset of sudden onset of the disease is sudden with body
temperature raising to 41-42oC within few hours. Death will occur within 12-36 hours after onset of
signs. Parasite accumulates in cerebral capillaries and organisms are rarely seen in blood smear.
Incoordinations and other CNS signs are the main symptoms.
Diagnosis: Diagnosis is maninly based on clinical signs and blood smear examinations. Both thick
and thin blood smears may be employed, being stained by one of the Romanowsky stains (Giemsa
stain). In the cerebral form, examination of cerebral capillaries is necessary. In endemic areas, high
fever with haemoglobinuria and anaemia is suggestive of Babesia infection. Immunodiagnostic tests
are used specially in the subclinical situation when organisms are not demonstrable in the blood.
Indirect fluorescent antibody test (IFAT) is considered as the gold standard.

Treatment

1. Diminazene aceturate (4,4-diamidinodiazoaminobenzene aceturate) (BERENIL), Dose: 2-3.5

mg/kg body wt by deep IM injection is used to treat all species of Babesia.

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2. Amicarbilide. (3, 3' -diamidinocarbakilid'e diisethionate) (DIAMPRON), Dose: 10 mg/kg IM or
SC. This durg is introduced for the treatment of B. divergens infection, but also effective against B.
bigemina and will eliminate infection with this species.
3. Imidocarb [3, 3’-bis (2-imidazolin-2-yl)-carbanalidae] (IMIZOLE), Dose: 0.5-1 mg/kg given SC.
It is used as a therapeutic and prophylactic drug against B. bigemina and B. bovis.
3. Quinuronium sulphate (PIREVAN 5% Sol):1-2ml/100kg b.w, 1-2 injection at 24h interval S/C
Prevention and control
Adequate tick control measures including, anti-tick vaccines can be used as part of an integrated
program for the control of ticks. The inverse age resistance of cattle to B. bigemina has been
exploited for control by immunization. Young animals are inoculated, with a mild strain of B.
bigemina, and the subsequent infection, if necessary, is controlled by drug treatment.

Babesia bovis (Syn. B. argentina; B. berbera)

Hosts: cattle, roe deer and stag.

Distribution: Southern Europe, Africa, Asia and Central and South America and Australia. In
Australia and Mexico it is more important than B. bigemina and not seen in US / Canada
Morphology: A small piroplasm, 2.4 µm by 1.5 µm and slightly larger than B. divergens. Sometimes
round or irregular, vacuolated signet rings are common.
Vectors: Ixodes ricinus, I. persulcatus, Rhipicephalus (Boophilus) calcaratus, R.(Bo). microplus and
R.bursa. B. bovis transmitted by larval stages of one-host tick Rhipicephalus Boophilus) spp.

Pathogenesis: B. bovis is responsible for major outbreak in Australia. More pathogenic in Austraila
and produce twice mortality of those infected than with B. bigemina. It is relatively uncommon in
animals under one year of age. Animal over two years of age are only affected. The disease caused
by B. bovis infection reveal kinin production and massive mobilization and activation of kallikrein.
Kallikrein causes increased vascular permeability and vasodilatation leading to circulatory stasis and
shock. It also triggers intravascular coagulation. Initial fall in packed cell volume (PCV) is due to
these disturbance rather than erythrocyte destruction. High fever is evident about a week to ten days
after infection and shortly afterwards haemoglobinuria occurs. Anaemia also occurs.

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Clinical signs: Incubation period is 4-10days. High fever for 7-10 days (104-106oF for 2-3 days),
haemoglobinuria, anemia, diarrhoea, icterus, rapid heartbeat are the symptoms.
Cerebral form in B. bovis infection: the most common form characterized by convulsion,
incoordination, comma, death. Brain capillaries are the most common predilection site of B. bovis in
healthy animals. Selective concentration of parasitized cells occurs in brain capillaries leading to
obstruction of the blood flow. Infected cells stick to one another and to the vessel endothelium, and
the increased stickiness has been ascribed to a parasite enzyme or antigen which alters the surface
charge.
Post-mortem lesions: Congestion of the grey and white matter of the brain, generalized dilatation
of capillaries by RBC with B. bovis, perivascular, perineuronal and interstitial oedema occurred
throughout the brain and spinal cord.
Diagnosis: Diagnosis is similar to that done for B. bigemina.
Brain smears: a small sample of grey matter of the cerebral cortex is placed on a slide and the tissue
is smeared using another slide. The brain tissue is fixed and stained for the detection of the
organism.
Treatment: Treatment is similar to that against B bigemina. Imidocarb (3, 3’-bis (2-imidazolin-2-
yl)-carbanalidae): 1-3 mg/kg S/C single dose and is the only compound which eliminates B.bovis
from carrier cattle.
Control: Control is same as B. bigemina. A culture derived vaccine gives promise of effectiveness
against B.bovis infection.
Inverse age resistance: An inverse age resistance occurs in Babesia infections, young animals
(calves below 9 months) being naturally resistant while older animals are fully susceptible. The
natural resistance of the young calf to infection usually disappears at 9-12 months of age. Earlier it
was thought that it was due to colostrual antibodies. Actually the innate resistance in bovine
babesiosis is antibody-independent.

During primary B. bovis infection, is chiefly controlled by a T-helper cell 1 (Th1) immunue
response involving IFN-γ and TNF-α. The cytokines together with parasite antigen activate
mononuclear phagocytes/ marcophages to release reactive nitrogen (and oxygen) intermediates. The
oxidative and nitric radicals cause oxidation of haemoglobin in the infected and noninfected

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erythrocytes to nitrosothiolhaemoglobin or methhaemoglobin and H2O2. This enhance the clearance
of these cells in spleen. Infected calves respond very rapidly with an early appearance of IL-12 and
IFN- γ transcripts in the spleen followed by a brief period of iNOS (inducible nitric oxide synthase)
expression leading to clearance of infected cells in the spleen. In addition, calf leucocytes also have
much higher baseline or constitutional levels of cNOS than adult animals. Thus invading parasites
encounter an elevated NO levels from the point of infection as they are carried to spleen and almost
immediately NO levels begins to rise further. This early response is rapidly counter-regulated by the
release of IL-10, TGF-β and negative feedback by NO itself.

In contrast adult animal suffer transient immunosuprression and fail to mount type I immune
response at the appropriate time and appropriate location. In addition, expression levels of IL-10 in
adult animals seem to be grater and remain prominent for longer period than in calves.

Thus the innate resistance in calves to B. bovis infection appears to depend on early type I
response that is largely confined to lymphoid organs, particularly spleen. In calves, oxidative burst
activity in peripheral circulation is likely to remain low while in adult cattle oxidative activity in
peripheral circulation by production of oxidative radicals by peripheral monocytes is substantial. In
adult cattle, this account for untimely and systemic over production of inflammatory mediators that
could account for some of the severe organ damage observed in B. bovis infections.

Thus the difference in the outcome of infection is explained by the localization and timing of
inflammatory response: in calves Nitric oxide production occurs early and appears to be
concentrated in the spleen. On the other hand, there is delayed and systemic inflammatory response
occurs in adult animals and that is ineffectual and probably contributes to pathogenesis.

Premunity: One infection with B. bigemina (and B. bovis) in cattle confers species-specific
protection for life. Recovered cattle are premunized. Premunition is due to latent infection. There is
strain difference, however sterile immunity rather than premunition may also exists. Bos indicus has
been suggested to be more resistant than Bos taurus. Antibodies are detectable 7-21 days after tick
transmitted infection.

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Acquired immunity: In latent infections, the Babesia spp. may persist for several years in animal
body and be demonstrable by the injection of blood into splenectomized animals. The latent
infection, is responsible for premunition.

Endemic stability: It is possible to have Babesia spp. and their vectors present in a cattle population
without measurable economic losses or clinical disease. This is known as endemic stability, which is
defined as the state where the relationship between host, agent, vector and environment is such that
clinical disease occurs rarely. An important factor in the establishment of endemic stability is the
age of first exposure: Calves have a natural resistance during the first six to nine months of life and
rarely show clinical signs, yet develop solid, long-lasting immunity.

Culture of Babesia: Cattle parasite Babesia bovis was the first one to be established in continuous
cultures using an agitated suspension culture technique. Later the microaerophilous stationary phase
(MASP) culture system was developed by Levy and Ristic using a stationary layer of erythrocyte.
The culture medium was HEPES-buffered medium 199 (60%) and bovine serum (40%), and an
inoculum of infected and normal erythrocytes was used, adjusted to pH 7 and incubated at 37- 38°C
in an atmosphere of 5% CO2-95% air. Medium was replaced at daily intervals, and subculturing was
performed at 48 to 72 h. MASP culture technique was utilized to produce exo-antigens released by
the parasite for immunization in cattle. Recently, a novel method for the culture system
for Babesia canis involving use of Ham's medium F10 or F12 incubated first under higher
temperature of 34°C-38°C followed by lower temperature of 0°C-10°C, has also been developed.
Babesia divergens
It is a European form found in cattle of western and central Europe. B. divergens is smaller than B.
bovis, 1.5 µm by 0.4 µm, and commonly appears as paired, divergent forms lying superficially on
the red blood cell.
Hosts: Cattle, red deer and roe deer. The wild game may serve as natural reservoirs.
Developmental cycle: Transmitted by Ixodes ricinus (3 host tick). Transovarian transmission and
transstadial transmission are present. Ixodes ricinus is a three-host tick with only adult stages
feeding on vertebrates (eg. cattle).
Babesia major

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Babesia major is smaller than B. bigemina and trophozoites lies in the centre of the erythrocyte. The
pyriform bodies are 2.6 µm by 1.5 µm, and form acute angle. Round forms about 1.8 µm in diameter
may occur.
Hosts: cattle of South America, north and west Africa, southern Europe, Great Britain, Soviet
Union.
Vectors: Rhipicephalus (Boophilus) calcaratus, (one host tick) and Haemaphysalis punctata (3 host
tick) are the tick vectors.
Developmental cycle and pathogenesis: Comparable to B. bovis.

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Babesia of sheep and goat
Four species (1 large form and 3 small forms) of Babesia have been reported: Babesia
motasi (large form), Babesia ovis (small form), Babesia foliata (small form), Babesia taylori (small
form).

Babesia motasi
Hosts: sheep and goats
Distribution: Southern Europe, Middle East, Soviet Union, south-east Asia, Africa and other parts of
the tropics.
Morphology: large form (2.5-4 µm x 2 µm), pyriform stages resemble B. bigemina. They may occur
at singly or in pairs forming at acute angle.
Developmental cycle: Similar to that of B. bigemina.
Tick vectors: Dermacentor silvarum, Haemaphysalis punctata and Rhipicephalus bursa.
Both transovarian and stage to stage transmission have been demonstrated in R. bursa.

Babesia ovis
Hosts: sheep and goats.
Vector: Rhipicephalus bursa, a two-host tick
Distribution: Tropical and subtropical areas, southern Europe and the Soviet Union.
Developmental cycle: Transovarian and stage to stage transmission. Transplacental infection may
occur.
Much smaller than B. motasi, (1-2.5 µm). The majority of organisms are round, occurring at the
margin of the red cell. Pyriform organisms are comparatively rare, and when they occur in pairs the
angle between them is obtuse, the organisms usually lying at the margin of the erythrocyte.
Babesia foliata: This species has been recorded from sheep in India. It resembles B. ovis, but is leaf
shaped. Trophozoite lies more centrally in the erythrocyte. Life cycle and pathogenesis is not
known. The vector has not yet been identified. It may be a synonym of B. ovis.
Babesia taylori: Host: goats in India. This is a small form (1.5-2 µm in length), but usually it is
ovoid to round, about 1 µm in diameter, and appears to undergo several fissions to produce 8 or
even 16 parasites per erythrocyte. The host red cell is often enlarged, and dividing forms of the
organism may be seen in the plasma. The vector of this species has yet to be identified.

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Babesia of horses (Equine piroplasmosis)
Babesia caballi is a large form of Babesia. Babesia equi is now regarded as Theileria equi.
Both are found in India.
Babesia caballi
Hosts: horse, also donkey and mule.
Distribution: Southern Europe, Asia, Soviet Union, Africa, Panama and USA.
Morphology: Large pyriform ( 2.5-4µm) pairs at acute angle. Rarely, round or oval (1.5-3 µm)
Tick vectors: Dermarentor marginatus (southern and eastern Soviet Union, Germany), D.
reticulatus and D. silvarum (European Soviet Union), D. nitens (Florida, Panama), Hyalomma
excavatum and H. dromedarii (North Africa), H. scupense (Ukraine), Rhipicephalus bursa
(Bulgaria), R. Sanguineus (Greece), etc.
Pathogenesis: Incubation period is 6-10 days. Mixed infection with B. equi occur. Changes in the
coagulation parameters due to activation of kallikrein is observed. Common symptoms are persistent
fever, anaemia, icterus and gastroenteritis. Haemoglobinuria is rare. Acute, subacute or chronic
forms are present. In acute cases, death may occur from 1-4 weeks after the onset of clinical signs.
Involvement of the central nervous system is common and result in paralysis of hind quarters.
Restlessness, nervousness and walking in circles with incoordination are also seen. Disease is more
marked in older horses may be due to the inverse age resistance (comparable to B. bigemina).
Recovered animal is premune upto 1-2 years after recovery in the absence of reinfection.
Post-mortem lesions: Emaciation, anemia, jaundice, oedema, gelatinous fat, bile stained internal
tissues, spleenomegally with soft pulp, hepatomegally with brownish yellow colour, endocarditis,
enlargement of lymph nodes and haemorrhagic gastro-intestinal mucosa.
Diagnosis: Based on clinical signs, the presence of tick vectors, history of the disease in the area
and the demonstration of the organism in the peripheral blood (blood smear).
The most satisfactory site to obtain the blood sample is the skin of the ear, and frequently the first
drop of blood contains the greatest number of organisms. A reduction in haemoglobin (Hb) amount
and erythrocytes (TEC) and an increase in erythrocyte sedimentation (ESR) rate supply supportive
evidence. Serological tests are IFAT, CFT, and ELISA (acute & latent infection).
Treatment: Diminazine (5 mg/kg, twice at 24 hour interval) i/m
Imidocarb (1-2mg/kg twice at 24 hour interval) i/m or s/c

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Babesia of dogs (canine babesiosis, canie piroplasmosis)

I. Large Babesia spp. (large piroplsam 4-5µm, amoeboid or ring):

1. B. vogeli (Syn: B. canis vogeli): seen worldwide
2. B. canis (Syn: B. canis canis): seen in Europe
3. B. rossi (Syn: B. canis rossi): seen in South Africa
4. B. coco: seen in United States (in splenectomized or immunosuppressed dogs).
Canine babesiois due to B. canis, and its subspecies are called as biliary fever, malignant jaundice
and Nambiuvu.

II. Small Babesia spp:

1. B. gibsoni: worldwide (anular, oval or signet ring common)
2. B. conradae: southern California only
3. B. annae (Theileria annae ?): B. microti-like organisms seen in Spain; also prevalent in North
American foxes; a single case reported in a dog (Mississippi)

Mode of transmission:
1. Tick bite:
Parasite Tick vector Reamrks
B. vogeli R. sanguineus Transtadial and transovarian
transmission
B. canis Dermacentor reticulatus, D. variabilis, R.
sanguineus
B. rossi H. leachi
B. gibsoni R. sanguineus, H. bispinosa, H. longicornis

2. Mechanical transmission through bite wound due to fighting between dogs in B. gibsoni-
(common in American Staffordshire and American Pit Bull Terrier breeds of dogs)
3. Iatrogenic transmission due to blood transfusion and blood contaminated fomites.

Pathogenesis

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 Lagre form: Most pathogenic species is B. rossi, and least pathogenic is B. vogeli while
others are moderate to severe in pathogenicity. Incubation period is 10-20 days. Haemolytic anaemia
is the principal pathogenic mechanism. Immune mediated destruction of RBC also occurs. Direct
parasite induced red cell damage, increased osmotic fragility of infected RBC, oxidative and
secondary immune mediated injury of RBC membrane result in a combination of intravascular and
extravascular haemolysis. Clinical signs can vary substantially because of differences among
Babesia spp. and individual patient response to infection.
First sign is fever (38.9-40.6oC) followed by anaemia, icterus, inappetance, marked thirst,
weakness, prostration and death. Heaemoglobinuria (red or orange urine) is seen in peracute cases
when loss of RBC is marked, but not frequently seen. Faeces will be yellow in later stage.
Splenomegaly, lethargy, pale mucous membranes, waxing and waning pyrexia, bounding pulses,
lymphadenopathy, generalized weakness, jaundice (yellow tinge to skin, gums, whites of eyes,
etc),vomiting (more commonly reported with B. conradae infection) are also seen. In acute cases,
death occurs in 4-5 days due to respiratory failure. During acute cerebral babesiosis, locomotor
disturbances, paresis, epileptiform fits are seen. Dog scream with pain if their head is touched or
mouth is opened.

Small form : Babesia gibsoni infection in more chronic in nature than that caused by large forms.
Periodic exacerbation of fever, progressive anaemia and haemoglobinuria are the symptoms. Death
occurs after several weeks. Post mortem lesion in B. gibsoni include enlargement of spleen and
liver. Jaundice is not frequently seen.

Diagnosis
(1) Diagnosis by light microscopy of blood smear
(2) Indirect fluorescent antibody test (IFA): Cannot differentiate various Babesia spp.
(3) Polymerase chain reaction (PCR)
Treatment of large Babesia
Intravenous fluid can be given for correction of dehydration and hypovolemia. Imidocarb
dipropionate (IMIZOL) (6.6 mg/kg IM once, repeat in 7–14 days) reduces morbidity and mortality
in most cases of Babesia spp. infection. (Side effects of imidocarb include pain at the injection site,

144
salivation, lacrimation, gastrointestinal signs, and tremors). Pre-treatment with atropine (0.04 mg/kg
SC) may prevent these cholinergic side effects. Diminazene aceturate (3.5–7 mg/kg SC or IM q1–
2wk) is effective against B. canis.
Treatment of small babesia
Imidocarb is less effective against B. gibsoni. Atovaquone (13.3 mg/kg PO q8h) and
azithromycin (10 mg/kg PO q24h) combination therapy for 10 days, has effectively cleared B.
gibsoni and B. conradae infections. Atovaquone should be given as liquid suspension with a fatty
meal to ensure adequate absorption.
Clindamycin (25 mg/kg PO q12h), metronidazole (15 mg/kg PO q12h), and doxycycline (5
mg/kg PO q12h) have been associated with clearance of B. gibsoni after administration for ~3
months, but true treatment efficacy is unknown.
Vaccine
Pirodog (Manufacturer: Merial) is a first generation vaccine prepared from culture
supernatants of B. canis with a saponin adjuvant, used for active immunisation of dogs against
Babesia canis. Primary vaccination (first vaccination) is given to dogs at the age of 5 months or
older. A booster dose is given after 3-4 weeks and then revaccinated every 6 months. Vaccine gives
protection only for 6 months.
Nobivac© Piro (Manufacturer: Intervet): This vaccine contains soluble parasite antigen
(SPA) of supernatant of in vitro culture of B. canis and B. rossi. This vaccine is now withdrawn
from use in the European Union.

Babesia of swine

Two species: Babesia trautmanni (large form) and B. perroncitoi (small form). Babesia trautmanni
causes severe disease in pigs. It is reported from Europe and Africa. B. perroncitoi is similar in
pathogenicity but has a limited distribution in Europe and Africa. Vectors are unknown, although
Rhipicephalus spp. have been shown to transmit B. trautmanni. Fever with anaemia,
haemoglobinuria, jaundice, oedema of the dependent parts and incordination are also seen. Pregnant
sows may abort.

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 Chapter 20
Genus: Theileria
Phylum: Apicomplexa
Class: Acoindasida
Order: Piroplasmorida
Family: Theileriidae
Genus: Theileria

Species Host Disease Vector Distribution

Theileria parva/ East coast fever(ECF), Rhipicephalus
cattle Africa
T. lawrencei corridor fever appendiculatus
Tropical piroplasmosis,
Asia, Africa,
Tropical bovine
Hyalomma anatolicum, Europe
T. annulata cattle theileriosis (TBT),
H. marginatum
Mediterranean coast
fever
T. mutans Benign African Amblyomma variegatum,
Africa
cattle theileriosis, Benign R. appendiculatus
Asia
bovine theileriosis Hyalomma spp.

T. orientalis Asia, Europe,

cattle Oriental theileriosis Haemaphysalis
Australia
Sheep/
goat Malignant ovine/caprine R. bursa Asia, Africa,
T. hirci (T. lestoquardi)
theileriosis Hyalomma spp. Europe

T. velifera/
Haematoxenous veliferus Cattle Amblyomma variegatum Africa

Sheep/ Benign ovine/caprine R. bursa, Asia, Africa,

T. ovis
goat theileriosis Haemaphysalis Europe
Ixodes sp., Rhipicephalus
T. annae
Dogs, Severe, regenerative sp.,
(Related to Babesia foxes anaemia Dermacentor sp.
microti )

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 Theileria are either small/round/ovoid/rod/comma shaped or pleomorphic protozoan
parasites seen in RBC, lymphocytes and histiocytes of cattle, sheep, goats and other ruminants. It
multiply by schizogony in lymphocytes and finally invade RBC as piroplasms. These are
transmitted by ixodid ticks of the genera Amblyomma, Haemaphysalis, Hyalomma and
Rhipicephalus, where gametogony and sporogony takes place. Theileria can be grouped into
“transforming” and non-transforming” species. Transforming species are responsible for the
malignant form due to the uncontrolled proliferation of schizonts and infected lymphocytes. eg,
Theileria parva, T. annulata, T. lestoquardi. Non-transforming species do not cause any schizont
associated pathology in lymphocytes and are regarded as being benign, but still able to cause disease
as a result of anaemia induced by the piroplasm stage. eg. T. Taurotragi.

Life cycle

A generalised lifecycle for the Theileria genus include liberation of infective sporozoites
during tick feeding into the feeding site. Sporozoites invade and infect host lymphocytes and
macrophages. T. parva infects both B and T lymphocytes. T. annulata infects macrophages, dentritic
cells and B cells that express major histocompatibility (MHC) class II antigens. Cell entry occurs
through receptor-mediated parasite-directed phagocytosis and reproduce by schizogony in
lymphocytes (leukocytic or tissue phase). Infected lymphocytes are transformed into lymphoblastic
cells. Macroschizonts develop in the cytoplasm of the transformed cells. Schizont divides
synchronously with the host lymphocytes and infect their daughter cells. Thus, in the parasitized
lymphocyte clonal expansion takes place, i.e. as the lymphocytes divide into two, parasite also
divide and infect both lymphocytes. After a few days, macroschizonts transform into
the microschizont stage. Macroschizonts/macromeronts contain large chromatin granules.
Microschizonts contain smaller chromatin granules and these produce merozoites.

Macro and microschizonts with their merozoites constitute Koch’s blue bodies (KBB) found
in the cytoplasm of lymphocytes and are circular or irregular shaped bodies. At microschizont stage,
host cells are destroyed and merozoites are released to invade erythrocytes, establishing the
piroplasm stage (erythrocytic phase). Binary fission in red cells in the case of T. annulata produces
various forms of piroplasms. In the case of T. parava the erythrocyte form do not divide. Some

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forms of piroplasms dominate in certain species: round and oval forms in T. annulata; rods in T.
parva; rods and elongate forms in T. orientalis.

Larval or nymphal ticks ingest piroplasms during feeding on an infected animal and the
released parasites undergo gametogony followed by syngamy in the tick gut, forming a zygote, the
only diploid stage. The zygote undergoes meotic division into motile kinetes that infect the tick gut
epithelial cells. During or just after the tick moults to the next instar the kinete escapes from the gut
cell and migrate to the haemocoel and later migrate to the salivary glands. In these cells, the parsite
develop into a multinucleate sporont. Sprozoite development (sporogony) occurs during the tick
feeding. Thousands of sporozoites are released into mammalian host.

Transmission is transtadial (from one life stage to the next). In transtadial transmission,
parasite is acquired during larval or nymphal feeding and transmitted in the next stage by nymphs (if
acquired by larvae) or adults (if acquired by nymphs). There is no transovarial transmission as seen
in Babesia. The incubation period following tick transmission is 8-24 days.

Cattle

Theileria parva, T. annulata, T. taurotragi, T. mutans, T. velifera and T. orientalis are the
six Theileria species known to infect cattle.

Theileria annulata

Host: cattle and water buffalo

Vector: Hyalomma anatolicum

Disease: Tropical bovine theileriosis (TBT)

Occurrence of TBT is seasonal and coincides with incidence of ticks on host, very high in
summer and immediately after rain. Indigenous cattle mostly act as carriers. Buffaloes are resistant
and act as carriers. Congenital transmission in calves is also reported. Trophozoite forms in the RBC
are round, oval, rod shaped or comma shaped with round and oval forms dominating. Division by
binary fission occurs in RBC. Koch’s blue bodies (KBB) are in the lymphocytes of spleen or lymph
nodes or even free in these organs. Macro and micromeronts are present. Micromeronts produce the
merozoites.

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Theleria orientalis complex (Theileria mutans, and Theileria orientalis)

Theileria orientalis (Synonym: T. Sergenti, T. buffeli) is responsible for benign or non-

transforming theileriosis, and exerts its major effect through erythrocyte destruction. Rods and
elongate forms dominated in the piroplsam stage. It is transmitted by the tick, Haemaphysalis
longicornis in Japan. The life cycle of T. orientalis is similar to that of other Theileria species,
except that the schizonts do not induce transformation and fatal lymphoproliferation. Theileria
orientalis Ikeda strain (most pathogenic) was reported from Kerala too.

Theileria parva

A highly virulent Theileria with three recognized subspecies, viz. T. parva parva causing
classical East Coast Fever (ECF), T. parva lawrencei responsible for Corridor disease transmitted
from buffalo to cattle and T. parva bovis, the causing a more benign form called “January disease“
(Zimbabwe theileriosis). The main vector for T. Parva is the tick Rhipicephalus appendiculatus.
Trophozoite forms in the erythrocyte are predominantly rod shaped (1.5–2.0 × 0.1–1.0 μm), but may
also be round, oval and comma shaped with rod forms dominating. Koch’s blue bodies are found in
the lymphocytes and endothelial cells of the spleen or lymph nodes. Parasite can cause disorder
called turning sickness due to nervous signs resulting from the blockage of the cerebral capillaries
by the meronts.

T. velifera (Synonym: Haematoxenus veliferus)

T. velifera is a minor and mildly pathogenic species infecting cattle in Africa and mainly a
parasite of African buffalo (Syncerus caffer). A great majority of piroplsams have a characteristic
rectangular ‘veil’(a parasites associated structure consisting of haemoglobin derived substance) 1–
3.5 μm extending out from the side.

Sheep and Goats

Theileria lestoquardi (T. hirci) is responsible for malignant ovine theileriosis (MOT) or malignant
small ruminant theileriosis in sheep and goats. T. uilenbergi and T. luwenshuni are the newly
identified Theileria species (nontransforming), highly pathogenic for sheep and goats in China. T.
ovis, T. separata and T. recondita are considered as low or non-pathogenic species in small ruminants.

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Pathogenesis of theileriosis

Virulence of the different Theileria species is strongly influenced by the degree to which
they multiply during the schizont stage of development. Some features of pathogenesis of theieriosis
is attributed to the expression of matrix metalloproteinases (MMPs) by the macroschizont-infected
cells. Pathogenesis include lymphoproliferative (lymphotrophic) and haemotrophic changes.
Lymphoproliferative changes:
The sequence of events in a typical acute and fatal infection progress through three phases,
each spanning about 1 week.
Phase I (incubation period): In the first week of infection, neither parasite nor lesions can be
detected.
Phase II: During the second week, there will be marked hyperplasia and expansion of the
infected lymphoblast population initially in the regional lymph node draining the site of the tick bite
(parotid), and ultimately throughout the body. Theileria-transformed host cells home to the draining
lymph node where they proliferate and disseminate into various organs, causing lymph node
swelling, fever, anorexia, and frothy nasal discharge. In fact, pulmonary oedema is often the cause
of death for cattle infected with Theileria.
T. annulata-infected macrophages invade tissues via an amoeboid invasion mechanism for
which matrix metalloproteinase 9 (MMP-9), transforming growth factor β (TGF- β) and TNF-α are
essential. Theileria parasites have been shown to have a close association with host microtubules,
which play a crucial role in metastasis.
PhaseIII: (Phase of lymphoid depletion) There is lymphoid depletion, massive
lymphocytolysis and depressed leucopoiesis.
Haemotrophic changes are characterised by haemolytic anaemia and icterus.
Haemoglobinuria is seen in some cases (T. annulata).

Clinical signs

Disease lasts for 4-20 days. The first clinical sign is fever (body temp.104-107°C) which
may be continuous or intermittent with swelling of superficial lymph nodes. Inappetence, cessation
of rumination, rapid heartbeat, difficult breathing, nasal discharge, lacrimation, paleness of mucus
membrane due to marked anaemia, and poor productivity and diarrhoea. Faeces may contain blood

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and mucus. Conjunctiva may be icteric with petecheal haemorrhages. In few cases, urticarial skin
lesions are seen. Affected animals are greatly emaciated (rapid muscle wasting or cachexia). These
symptoms (as well as the pyrexia, anorexia and panleukopenia) can be explained by the action of
macroschizont-infected cells induced secretion of tumour necrosis factor α (TNF-α) by
macrophages. Matrix metalloproteinases (MMPs) secreted by the macroschizont-infected
leucocytes are essential components in the processing and activation of type II TNF- α.

Death is due to anoxic anaemia. Mortality varies from 10-90%. Occasionally nervous signs
are seen because of blockage of cerebral capillaries by the meronts. This may results in a condition
known as turning sickness as seen in T. parva infection. In sub-acute cases there will be intermittent
fever. Sometimes pregnant animals may abort. In chronic cases there will be intermittent fever,
inappetence, marked emaciation, anaemia and icterus.

Post-mortem lesions

Carcass will be emaciated. Splenomegaly, hepatomegaly, infarcts in kidney, lung oedema,

swollen superficial lymph nodes with extensive haemorrhage, icteretic mucous membrane with
petechial haemorrhages, haemorrhages on the pericardium, epicardium and myocardium are the
common lesions.
The pathegnomonic lesion in theileriosis is “punched out (cigarette burn) abomasal ulcers
with necrotic centre and haemorrhagic border” on the mucosa. Necrotic punched out ulcer in
abomasum can result in haemorrhage and catarrhal diarrhoea. Destruction of connective tissue due
to digestion of the extracellular matrix by matrix metalloproteinases (MMP) secreted by
macroschizont-infected cells present in these lesions are responsible for the abomasal ulcer. Lesions
in cerebral theileriosis are acute congestion and haemorrhages in the meninges, cerebral
hemispheres and cerebellum.

Immunity: Animals recovered from Theileria infection may develop premunity. Such animals may
act as reservoirs of infection to ticks. No cross immunity is seen between T. parva and T.mutans and
T.annulata.

Diagnosis: Diagnosis can be done by observing the clinical signs, demonstration of piroplasms in
RBC by microscopy of the Leishman/Giemsa’s stained peripheral blood smear, and demonstration

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of KBB in lymphocyte cytoplasm in the stained lymph node aspirate smear. In addition serological
assays (IFAT and ELISA) and molecular assays (PCR) can also be done.

Treatment

Chemotherapeutic agents such as parvaquone, buparvaquone and halofuginone are available

for treatment of T. annulata and T. parva infections in cattle. Parvaquone is mainly active drug
against schizonts; it should be injected intramuscularly at dose of 20 mg/kg.

Buparvaquone is active against both schizontes and piroplasms. Single dose of

Buparvaquone at the rate of 2.5mg/kg, deep i/m, is the drug of choice. In severe cases, repeated dose
is needed after 48-72 hrs interval or at initially 5mg/kg i/m. This drug act on the mitochondria of the
parasites blocking the oxidative phosphorylation in them. Do not use milk for human consumption
48 hrs following treatment.

Halofuginone lactate at the dose of 1.2 mg/kg b.w. PO and repeated after 48 h. the drug has
potent effect on schizonts but only moderate effect on erythrocytic forms.

Oxytetracycline (OTC) at the dose of 10-15 mg/kg for 4-7 days, will arrest macro and micro
schizont formation. Hence, it must be given at the time of initial phases of infection, but has only
minor effect on clinical cases.

Supportive treatments like vitamin B complex with liver extract injection, blood transfusion
and Iron-dextran injection etc for correcting anaemia can be given.

Control
1. Chemoprophylaxis: Oxytetracycline with diminazine aceturate at a ratio of 10:20 mg/kg to
develop premunity.

2. Vector control: Dipping cattle, spraying walls, sheds and buildings with acaricides

3. Managemental methods-rotational pasture, skip infected pasture for 18 months, regular tick
control programmes

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Schizont vaccine

Rakshavac-T is a commercially available attenuated schizont infected lymphocyte culture vaccine

(SIL) against Theileria annulata infection, developed by NDDB, Anand and marketed by Indian
immunologicals, Hyderabad. Attenuation of T. annulata to produce live vaccine is achieved by the
prolonged passage of the macroschozont infected leucocytes. Attenuation is due to reduced matrix
mellaoproteinase (MMP) activity consequent to the lost ability of macroschizont to induce MMP.
This freeze dried vaccine consisting of 106cells/dose are stored in liquid nitrogen at -196°C. Single
dose @ 3ml/animal s/c for adults and calves above 4 months. Vaccine gives immunity for one year.

Theileria equi
It is smaller, about 2 µm in length, and merozoite in RBC are round, amoeboid, or most often
pyriform. Pyriforms characteristically divides into four daughter organism (Maltese cross).
Hosts: Horse, mule, donkey, zebra.
Distribution: Asia, Africa, Europe, South America, USSR.
Developmental cycle: The tick vectors are: Deracentor reticulatus (European Soviet Union), D.
marginatus (Eastern Europe), Hyalomma excavatum, H. plumbaeum (Greece, Central Asia) and H.
dromadarii (North Africa), Rhipicephalus bursa (USSR), R. Sanguineus (Central Asia, North
Africa), R. evertsi, R turanicus etc.
Exoerythrocytic schizogony has been demonstrated in vivo and in vitro in lymphocytes with the
development of macro and microschizonts. Merozoites invade the RBCs.

Pathogenesis

T. equi is more pathogenic than B. caballi, but mixed infection may occur. Incubation period
is 8-10 days. First clinical signs include marked increase in body temperature up to 41.70C and it
may be remittent and intermittent. Following rise in body temperature, organisms appear in large
number.
In per acute cases death occurs in 1-2 days after the onset of clinical signs. In acute cases
fever lasts 8 -10 days, recovery may occur and animals become carriers. Marked anaemia and
icterus. Haemoglobinuria may be present and there is listlessness, depression and inappetence.
Oedema of the dependent parts of the body and head may occur. There may be gastrointestinal

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disturbances. Constipation is well marked and common and hard faeces covered with yellowish
mucus. Abortion of pregnant mare is seen. Subacute cases develop slowly and is more prolonged.
Posterior paralysis, common in B. caballi is not seen in T. equi infection.

Pathological changes: General jaundice, petechial haemorrhage, enlargement of spleen and liver,
flabby kidneys, pneumonia of lung. Recovered animals are immune to infection.

Diagnosis
On the basis of clinical signs, blood smear examination and immunological tests (IFAT).
Treatment
1. Diminazene aceturate @ 3.5 mg/kg IM every 48 hours for 2 treatments. No chemosterilization has
been reported.
2. Imidocarb @ 4 mg/kg body weight 4 times at 72 hours interval. Imidocarb is contraindicated in
donkeys.

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 Chapter 21

Phylum Ciliophora

Ciliophora possess simple cilia or compound ciliary organelles in at least one stage of their
life cycle. Most species have one or more macronuclei and micronuclei. Reproduction is asexual by
transverse binary fission and some species exhibit sexual reproduction involving conjugation (two
organisms join together undergo nuclear exchange), autogamy (fusion of two gametes that come
from one individual) and cytogamy (two organisms join together but do not undergo nuclear
exchange). Most ciliates are free living, but many are commensals of vertebrates and invertebrates,
and a few are parasitic.
Class Litostomatea
Order Trichostomatorida
Family Balantidiidae
Genus Balantidium
The family has a single genus Balantidium, species of which are found in the intestine of
crustaceans, insects, fish, amphibians, and mammals. Balantidium coli appears to be primarily a
parasite of pigs, with strains adapted to various other hosts including several species of primates. In
pigs it is generally regarded as a commensal, since under natural conditions it is found in the lumen
of large intestine and is associated with no change in the mucosa. This is the largest protozoan
parasite of humans, live in the caecum and colon. Life cycle involves the trophozoite and dormant
cyst stages.
Morphology
Balantidium coli trophozoites are oblong, spheroid, or more slender, 30 μm to 150 μm long
by 25 μm to120 μm wide. Body is covered with cilia (the swimming organism exhibits a rotary-type
motion by means of its somatic cilia that may facilitate movement through the contents of the
colon). A Funnel shaped depression is located at the tapering anterior end leads into cytostome/
mouth and a weakly developed cytopharynx. Macronucleus is kidney/sausage shaped for
cytoplasmic activities of the organism and micronucleus, is much smaller lies in the notch of the
macronucleus, which is a vesicular type of nucleus for reproductive process. Two contractile
vacuoles and food vacuoles which contain erythrocytes, cell fragments, starch granules, and fecal

155
and other debris. Cytopyge is a pore near the posterior end and contents of food vacuoles are
evacuated through this structure. Encysted stages of the organism most commonly found in stools
are spheroid or ovoid in shape, measuring 40 μm to 60 μm in diameter. Cyst contains the macro and
micronucleus, contractile vacuoles and covered with a double layered wall. Living trophozoites and
cysts are yellowish or greenish in colour.

Life cycle

Life cycle completes in a single host. Reproduction is asexual by transverse binary fission
or in sexual phase by conjugation. Infection occurs when cysts are ingested, usually in contaminated
food or water. Pigs are probably the usual source of infection for humans, but the relationship is not
clear. The cyst is destroyed by a pH lower than 5 and the infection is most likely to occur in
malnourished persons with low stomach acidity. Cyst excyst to form trophozoite in large intestine
and trophozoite multiply by binary fission each producing two daughter trophozoites. Large number
of trophozoites are produced by successive divisions. Sometimes trophozoites replicate sexually by
conjugation also. After a period of growth and multiplication, trophozoites encyst to thick walled
cyst in the lower part of intestine and are passed in faeces. Encystment is instigated by dehydration
of faeces as they pass posteriorly in the rectum. These protozoa can also encyst after being passed in
stools, this is an important factor in their epidemiology. The ciliates’ ability to encyst after being
passed increases the number of potential infections from a single reservoir host, and cysts can
remain alive for weeks in pig faeces, if the feces do not dry out.

Pathogenesis, clinical signs and Treatment

Man
Balantidium coli can produce proteolytic enzymes like hyaluronidase that digest away a
host’s intestinal epithelium. This enzyme could help in enlarging an ulcer. Ulcers usually are flask
shaped, like amoebic ulcers, with a narrow neck leading into an undermining sac like cavity in the
submucosa. Colonic ulceration produces lymphocytic infiltration with few polymorpho nuclear
leukocytes, and haemorrhage and secondary bacterial invasion may follow. Fulminating cases may
produce necrosis and sloughing of the overlying mucosa and occasionally perforation of the large
intestine or appendix, as in amoebic dysentery. Secondary foci, such as liver or lung may become
infected. In mild cases in man, intermittent diarrhoea is alternating with constipation is observed.

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Severe cases, blood and mucous is observed in the stool with the severe abdominal pain, tenesmus,
nausea, vomiting, headache and fever. Diagnosis is done by clinical symptoms and by the
demonstration of trophozoites and cyst stages in the stool. Several drugs are used to combat
infections of B. coli, including carbarsone (mainly in captive animals), Metronidazole (800 mg 3
times daily for 5 days)`and tetracycline (500 mg 4 times daily for 10 days).

Family Pycnotrichidae

Genus Buxtonella

The genus Buxtonella, which has an ovoid, uniformly ciliated body with a prominent curved groov .
It has world wide distribution. Buxtonella sulcata is found in the cattle, buffalo, goat, sheep, deer,
camel, rarely human in the large intestine. Recent studies showed that B. sulcata may be a potential
causative agent of periodical recurrent diarrhoea of unknown etiology in cattle.

Balantidium coli Buxtonella sulcata

Cyst A. Cysts are ovoid to spherical A. Cysts are regular round or slightly oval; it
and measure 40- 60 μm in reaches 52–131 μm in diameter. After three days
diameter and covered with a its size changes to 2/3 of its initial size and is
double layered wall. covered by a two-layer capsule,
Trophozoite A. These are oblong, spheroid, or A. These are asymmetric and oval, 60 – 138 μm
more slender, 30 μm to 150 μm long by 46 – 100 µm wide. Body is covered with
long by 25 μm to120 μm wide. short, thick cilia with a prominent curved groove
Body is covered with cilia. bordered by two ridges running from end to end.
B. Cytostome is near the anterior B. The position of the cytostome near the
end and Cytopyge at the posterior permanently open cytopyge and within the
end. peristome which continues from the cytostome
C. Macronucleus is large around the oral end of the body to the extreme
kidney/sausage shaped and aboral end which during locomotion is anterior
micronucleus, is much smaller C. The macronucleus being relatively shorter and
lies in the notch of the plumper and the micronucleus relatively larger
macronucleus. than in Balantidium coli.

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 Chapter 22
Order: Rickettsiales
The organisms in the order Rickettsiales are classified based on biological characteristics and
genetic analyses of 16S rRNA genes, groESL (Heat shock operons) and surface protein genes.
Family 1. Anaplasmataceae
They are obligate intracellular bacteria found exclusively within the membrane bound
vacuoles in the host cell cytoplasm. They have wider variety of in vivo targets ranging from
haemopoetic cells including phagocytes like monocytes and macrophages.
Family 2. Rickettsiaceae
They are obligate intracellular bacteria that grows freely within the cytoplasm of eukaryotic
cells. They infect predominantly in endothelial cells in mammals including humans.

Family: Anaplasmataceae
Genus1. Anaplasma
A. marginale, A. centrale, A. ovis, A. phagocytophilum ( Syn: E. equi), A. platys (Syn: E.
platys), A. bovis (Syn: E. bovis). Aegyptionella pullorum is presently included in this genus.
Genus 2. Ehrlichia

Ehrlichia chaffeensis, E. ruminantium (Syn: Cowdria ruminantium), E. ewingii, E. ovis, E.

canis, E. muris,
Genus 3. Wolbachia

Wolbachia pipientis

Genus 4. Neorickettsia

Neorickettsia helminthoeca, N. risticii (formerly Ehrlichia risticii), N. sennetsu (formerly

Ehrlichia sennetsu)
5. Canditatus (Candidatus Neoehrlichia mikurensis)

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Family Anaplasmataceae

Genus Anaplasma

A. marginale

The intracellular pathogen Anaplasma marginale described by Sir Arnold Theiler in 1910, is
endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes
bovine anaplasmosis (gall sickness), a mild to severe haemolytic disease that results in considerable
economic loss to both dairy and beef industries. In Giemsa stained blood smear, the organism appear
as small, round, dark red inclusion body within the red blood cell. Usually there is only one
organism in an RBC.

Host: Cattle, sheep, goat, wild ruminants

Transmission

1. Biologically by ticks – Rhipicephalus (Boophilus) annulatus, B. decoloratus, B. microplus,

Dermacentor albipictus, D. andersoni, D. variabilis, Hyalomma excavatum, H. rufipes, Ixodes
ricinus, I. scapularis, Rhipicephalus sanguineus
Tick transmission can occur from stage to stage (inter- stadial or tran- stadial or within a stage
(intra- stadial). Transovarial transmission from one tick generation to the next does not occur.
2. Mechanically by biting flies tabanids, hornflies, deerflies, stable flies and mosquitoes and by
blood-contaminated fomites, needles, surgical instruments
3. Transplacental transmission
Life cycle of A. marginalae

The organism enters in the red cell by invaginating the cell membrane so that a vacuole is
formed, thereafter it divides to form an inclusion body containing up to eight initial bodies packed
together. The inclusion bodies are most numerous during the acute phase of infection, but some
persist for years afterwards.

Geographical distribution: - Asia, Africa, Southern Europe, Australia, South America

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Pathology

Anaemia is mainly due to due to erythrophagocytosis especially in the spleen. The carcasses of
cattle that die from anaplasmosis are generally markedly anaemic and jaundiced. Blood is thin and
watery. The spleen is characteristically enlarged and soft, with prominent follicles. The liver may be
mottled and yellow-orange. The gallbladder is often distended and contains thick brown or green
bile. Hepatic and mediastinal lymph nodes appear brown. There are serous effusions in body
cavities, pulmonary oedema, petechial haemorrhages in the epi- and endocardium, and often
evidence of severe GI stasis. The spleen shows a decrease of lymphoblasts and increased
vacuolation and degeneration of reticular cells and there is reduction in white pulp and accumulation
of pigment resembling haemosiderin.

Pathogenesis

After an incubation period of four weeks there will be accute febrile reaction accompanied by a
severe haemolytic anaemia. Anaplasmosis can be divided into four stages viz., incubation stage,
developmental stage, convalescent stage, and carrier stage.

Incubation stage: The incubation stage begins with the original infection with A. marginale and lasts
until 1 per cent of the animal’s red blood cells (RBCs) are infected. The average incubation stage
ranges from 3 to 8 weeks. During this period the animal remains healthy and shows no signs of
being infected. Finally, after the parasite has reproduced many times and established itself in the
RBCs of the animal, the body attempts to destroy the parasite.

Developmental stage: During the developmental stage, which normally lasts from 4 to 9 days, most
of the characteristic signs of anaplasmosis appear. As the infected animal’s body destroys the
parasite, RBCs are destroyed as well. When a substantial loss of RBCs has occurred, the animal will
show signs of clinical anaemia. The body temperature will commonly rise to 104oF to 107oF (400 C
to 41o C) and a rapid decrease in milk production will occur in lactating cows. It refuses to eat or
drink water. The skin becomes pale around the eyes and on the muzzle, lips, and teats. Later, the
animal may show constipation, excitement, rapid weight loss, and yellow tinged skin. The animal

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may fall or lie down and be unable to rise. Affected cattle either die or begin a recovery 1 to 4 days
after the first signs of the disease.
Convalescent stage: Cattle that survive the clinical disease, lose weight, abort calves, and recover
slowly over a 2 or 3 month period. This is known as the convalescent stage, which lasts until normal
blood values return. This stage is differentiated from the developmental stage by an increase in the
production of RBCs (erythropoiesis) in the peripheral blood, shown as increase in haemoglobin
levels and total white blood cell counts. Death normally occur during the late developmental stage
or early convalescent stage.
Carrier stage: Unless adequately medicated, cattle that recover from anaplasmosis remain reservoirs
(carriers) of the disease for the rest of their lives. During the carrier stage, an animal will not exhibit
any clinical signs. Unidentified carriers in a herd are the most likely source of infection for future
outbreaks of the disease.
Clinical signs
Cattle on higher plane of nutrition develop more severe anaplasmosis than animals kept on lower
energy plane. Clinical anaplasmosis is more commonly encountered in cattle older than 1 year of
age. Clinical signs include severe anemia, depression, weakness, fever, laboured breathing,
inappetance, dehydration, constipation, jaundice, reduction in milk yield, abortion in advanced
pregnancy.
Diagnosis

Microscopic examination of Giemsa-stained thin and thick blood films, reveal Anaplasma spp.
as dense, homogeneously staining blue-purple inclusions 0.3–1.0 μm in diameter. A. marginale
inclusions are usually located toward the margin of the infected erythrocyte, whereas A. centrale
inclusion bodies are located more centrally.

The major surface proteins (MSPs) of A. marginale include MSP1a, MSP1b, MSP2, MSP3, MSP4,
and MSP5. MSP1a, MSP4, and MSP5 are encoded by single gene, while MSP1b, MSP2, and MSP3
are encoded by multigene families. MSP5 is a highly conserved surface protein and that has been
proven effective as a diagnostic antigen and used in a competitive enzyme linked immunosorbent
assay (ELISA). In addition, DNA-based detection methods (PCR) are mostly used for species and
strain differentiation.

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Treatment

Oxytetracycline, at the rate of 22 mg/kg of body weight given 3-4 days IM or IV.
Vaccines
Live and killed vaccines rely on the use of A. marginale from infected bovine erythrocytes.
Both types induce protective immunity that reduce or prevent clinical disease, but these vaccines do
not prevent cattle from becoming persistently infected with A. marginale. Live vaccines involve the
infection of cattle via inoculation with erythrocytes infected with less pathogenic isolates of A.
marginale or A. centrale. Vaccination strategies using live organisms include (i) infection and
treatment (with tetracyclines at the onset of elevated body temperature or at the detection of
parasitaemia) (ii) live vaccines containing attenuated strains of A. marginale and (iii) live vaccines
containing the less pathogenic A. centrale.
Killed vaccines developed in the United States in the 1960s were marketed until 1999 and later they
were withdrawn.
Cultivation: A cell culture system was developed for A. marginale in which the Rickettsia was
propagated in a continuous culture in a cell line, IDE8, derived from embryos of the tick Ixodes
scapularis.
A. centrale
The organisms are found in the centre of the erythrocyte.
Host: Cattle, wild ruminants, sheep
Less pathogenic than A. marginale.
A. phagocytophilum (Syn: Ehrlichia phagocytophilum) causes human granulocytic anaplasmosis,
“tick-borne fever” or “pasture fever”. Blood smears stained with Giemsa or Wright’s stain reveal
one or more loose aggregates (morulae or inclusion bodies, 1.5–5 mm in diameter) of blue–grey
to dark blue coccoid, coccobacillary or pleomorphic organisms within the cytoplasm of
neutrophils. The organism was identified as a human pathogen in 1994.
Host: Granulocytes of various mammals, including humans, sheep, goats, horses, dogs, cattle,
llamas, and rodents.

Vector: Ixodes scapularis (I. scapularis) and I. pacificus, I. ricinus

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 Fever, anorexia, reduced growth, decline in milk production are seen in cattle. Abortion and still
birth can also be seen. Leucopenia and transient thrombocytopenia are characteristic feaures.

A. platys (Syn: Ehrlichia platys) causes infectious canine cyclic thrombocytopenia (ICCT) in dogs.
IThe organism infect platelets. Clinincal signs include pale mucus membrane, lethargy, epistaxis etc.
The organisms appear as round, oval or bean shaped blue cell inclusions in platelets and range from
0.35 to 1.25 µm in diameter

Vector- Rhipicephalus sanguineus, Dermacentor spp.

A. bovis (Syn: E. bovis)

Organisms are seen as small, medium and large sized compact or diffuse particles(2–10 μm in
diameter), in the cytoplasm of mononuclear cells particularly monocytes. They cause a chronic
condition charecterized by rise in temperature, swelling of parotid glands, drastic drop in production
and severe leucopenia. Transmitted by Haemaphysalis bispinosa.

Host: Cattle

Vector: Hyalomma anatolicum, Rhipicephalus appendiculatus, Amblyomma variegatum

Genus 2. Ehrlichia

The genus is named after German microbiologist Paul Ehrlich. They are small pleomorphic
coccoid to ellipsoidal intracytoplasmic forms in circulatory leucocytes of various mammals.
Organisms may occur singly or in compact colonies as a morulae which is the characteristic form of
the organism. Organisms are transmitted by Ixodid ticks. Transstadial transmission occurs but
transovarian transmission does not occur.

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Summary of ehrlichial diseases and their related Ehrlichia pathogens.

Common name
Species Natural host Cells infected Primary Vector Distribution
of disease
Dogs and Primarily
Canine Rhipicephalus
other mononuclear
monocytic sanguineus,
E. canis members of cells (monocytes World wide
ehrlichiosis Dermacentor
the family and
(CME) variablis
Canidae lymphocytes)

USA,Europe,
Human Dermacentor
Human, deer, Africa, South
E. monocytic Monocytes, variablis,
horse, and Central
chaffeensis ehrlichiosis macrophages Amblyomma
rodents America,
(HME) americanum
Korea

Canine
granulocytic
Amblyomma
ehrlichiosis
Neutrophils and americanum, USA, Africa,
E. ewingii (CGE), Human Dog, human
eosinophills Otobius Korea
granulocytic
megnini
ehrlichiosis
(HGE)

Ehrlichia canis

Canine monocytic ehrlichiosis, Tracker dog disease, Tropical canine pan cytopaenia, Canine
haemorrhagic fever, Canine typhus

Location- Monocytes of peripheral blood

Description: Ehrlichia canis is a small, pleomorphic, Gram‐negative, coccoid, obligatory

intracellular bacterium that parasitises circulating monocytes, intracytoplasmically in clusters
(morulae). The earlier stages are elementary bodies (0.2–0.4 μm in diameter) followed by initial
bodies (0.5–4 μm in diameter) and later larger inclusion bodies (4–6 μm in diameter).

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Life cycle: Infection is transmitted to the dog through the bite of an infected Rhipicephalus
sanguineus tick. Transmission in the tick occurs transstadially, but not transovarially. Larvae and
nymphs become infected while feeding on rickettsaemic dogs and transmit the infection to the host
after moulting to nymphs and adults, respectively.

Pathogenesis
The incubation period is 8- 20 days. The organism multiply in macrophage of mononuclear
phagocytic system by binary fission and spread throughout the body. Thrombocytopenia is the most
common and consistent hematological abnormality of dogs naturally or experimentally infected
with E. canis. Thrombocytopenia in the acute phase of the disease include increased platelet
consumption due to inflammatory changes in blood vessel endothelium, increased splenic
sequestration of platelets, and immunologic destruction or injury resulting in a significantly
decreased platelet life span. Platelet bound and circulating antiplatelet antibodies are detected in
whole blood and serum. Platelet migration inhibition factor also exist in infected dogs and will lead
to reduction in platelets.
Canine monocytic ehrlichiosis has three phases
1. Accute ( febrile ) phase : Usually develops 1-3 weeks after the tick bite and lasts for 2-4 weeks.
Signs include fever, anaemia, depression, loss of appetite, stiffness and joint pain. As a result of
infection, lymph nodes, liver and spleen are enlarged. Due to the destruction of platelets, blood
clotting mechanisms are destroyed.
2. Subclinical phase: The animal may appear normal or show only slight anaemia. Dogs remain as
persistent carriers in this phase. Platelets are subnormal
3. Chronic phase: Tropical panctopaenia occurs (deficiency of all three cellular components of the
blood (red cells, white cells, and platelets) due to bone marrow hypoplasia. Lower leucocyte and
platelet counts are a risk factor for mortality. The affected animal develop extensive serosal and
mucosal haemorrhage. Epistaxis, bleeding from the inner aspect of thigh and forearm also occurs.
Animals are highly prone to pneumonia and renal infection in this phase.
Clinical signs
Fever, lethargy, loss of appetite, weight loss, abnormal bleeding (e.g., nosebleeds, bleeding
under skin looks like little spots or patches of bruising), enlarged lymph nodes, enlarged spleen, pain

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and stiffness (due to arthritis and muscle pain). Ocular signs like anterior uveitis, chorioretinitis,
papilledema and retinal haemorrhages. Subretinal haemorrhages may lead to retinal detachment and
blindness.

Diagnosis of ehrlichiosis

1. History and clinical signs

2. Buffy coat smear examination to detect the morula stage
3. Serological tests like IFAT, ELISA
4. Western immunoblot is a more specific test, which can distinguish between infections with
the different organisms causing ehrlichiosis, anaplasmosis, or neorickettsiosis as well as between
Ehrlichia spp.
5. PCR : PCR can be performed on whole blood, serum, splenic aspirates, lymph nodes, or
bone marrow. The spleen is the organ most likely to harbor E. canis parasites during the
subclinical phase.
6. Detection of serum antiplatelet IgG antibodies (Coombs test positive)
Treatment
1. Oxytetracycline (7.5- 10 mg/kg given every eight hours) for 21-28 day or doxycycline (5
mg/kg every twelve hours) for 21-28 days.
2. Supportive treatment should include multi-vitamin supplements.
3. In severe cases blood transfusions can be done.

Prophylaxis: No effective anti-E. canis vaccine has been developed and tick control remains
the most effective preventive measure.

Cell Culture for E. canis: It is possible to culture Ehrlichia species in specific macrophage cell
lines (canine macrophage cell line or mouse macrophage cell line.

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