Sampling

Sampling Methods For
Different Diseases

Dr. Tariq Mustafa Mohamed Ali
Al Ain Veterinary lab
Animal Health section
Agriculture Sector
Department of Municipalities and Agriculture

‫طرق جمع العينات للتشخيص‬
‫المرضى‬

‫د. طارق مصطفى محمد على‬
‫المختبر البيطرى- قسم الثروة الحيوانية‬
‫قطاع الزراعة – دائرة البلديات والزراعة‬

APPROACH TO
DIAGNOSIS

Success of diagnostic veterinary
laboratory depends on submission
samples of good quality which will
provide optimal opportunity for the
diagnosis of disease.

Sampling standards :
 Provide epidemiological and clinical details with the
samples.
 Always sample several animals in an outbreak.
 Collect samples from live animals in acute stage of
the disease.
 Keep samples cool during transfer to the laboratory
(preferably on melting ice) and reduce the time in
transit to the minimum.
 Mark sample bottles carefully with an indelible pen
and record details of each sample's origin for
submission to the laboratory.

Accession or submission
forms
1. Provide the requested information on the Lab
form.
2. Brief, concise, complete histories are required and
aid in providing diagnoses and pertinent advice.
3. Please use black ink and write or print legibly.
4. List the tissues submitted, also the number of
tumors. This will help insure that all submitted
specimens are identified

Submission of Serum and
Blood samples

 Blood samples should be collected in sterile tubes
containing no anticoagulants.
 These should be submitted to the laboratory in
specially designed Styrofoam holders to avoid
breakage.
 Blood samples should not be frozen or allowed to
overheat.
 If samples cannot be delivered to the laboratory
within a reasonable time, serum should be removed
and refrigerated or frozen.

Blood samples.
 Blood submitted for culture should be
submitted in blood culture bottles or sterile
vacutainer.
 Serum must be fresh, clear, unhemolyzed,
and uncontaminated.
 Submit at least 1.0 ml of serum for each test
requested. Refrigerate the serum until
shipment.
 Identify specimen in a way that will avoid
confusion when results are reported.

Blood samples
 Be careful that writing will be legible
 Avoid using animal names to avoid
duplication and confusion.
 Label each tube with tube number and
vet code.

Submission of swabs
 Swab material from the more advanced
lesions.
 Collect swabs from acutely ill animals .
 Collect swabs from several animals in
different stages of the illness .
 Two swabs should be collected each
time

Submission of swabs
 Swabs for virus isolation should placed in
viral transport medium .
 Most of these virus transport media are
balanced salt solutions containing high
protein content and antibiotics to prevent
bacterial overgrowth.
 Swab for electron microscopy should be
placed in a screw-capped tube containing one
or two drops of distilled water.

Feces
 Feces should be collected from acutely ill
animals and placed in leak proof containers.
 Well-saturated swabs are adequate for many
individual examinations.
 Several milliliters or grams of feces permit a
more complete diagnostic work-up including
bacteriologic and parasitological
examinations. Samples should submitted to
the laboratory using cold packs as coolant.

Tears
Cotton buds or swabs of absorbent
cotton wool are inserted into the
conjunctival sac and swirled around to
collect tears. The bud/swab is broken
off into a container and about 150
microlitres of sterile phosphate-buffered
saline (PBS pH 7.2 to 7.6) are added (if
available).

Pus
 Collect samples aseptically and submit
in culturette swab.
 When abscess material is available
submit the exudates in a sterile
container.
 Exudates should be collected from non-
draining lesion

Tissues
 It is recommended that the following tissues be
collected during post mortem examination: lymph
nodes found around the lungs (mediastinal) and
alimentary tract (mesenteric); portions of the spleen
and the lungs.

Two sets of each tissue are required; one set is
chilled but not frozen, and the other is put in 10
percent formalin solution to preserve the samples.

Gum debris

 This material can be collected by a
spatula or finger rubbed across the gum
and inside the upper and lower lips. The
material collected is then scraped into a
container and 150 microlitres of PBS
are added (if available).

NECROPSY SUBMISSION
STANDARDS
 Dead animals should be cooled as soon
as possible after death.
 Large animals should be thoroughly
hosed down with cold water.
 Birds, rabbits, and other fur bearing
animals should be soaked in cold,
soapy water, placed in a plastic bag,
and refrigerated.

SPECIMENS FROM NECROPSIED
ANIMALS
1. Collect all specimens as aseptically as
possible. Liberal portions of each
organ should be collected. If the
outside of the specimen is accidentally
contaminated, wash the specimen
with clean tap water.
2. Refrigerate (wet ice packs) all
specimens to prevent saprophytic
growth.

ANIMALS ( cont.)

3. Collect observable lesions or
suspected target organs

4. For neonatal diarrhea, submit a tied off
4-5 cm segment of jejunum, ileum, and
colon with the accompanying lymph
nodes for culture of pathogenic
bacteria.

ANIMALS ( cont.)

5. Tissue specimens should be placed in
individual leak-proof plastic bags and
identified (use water-proof ink on bags)

MASTITIS MILK SPECIMENS
1. Wash udder to remove dirt and allow to dry.
2. Scrub teat end with alcohol soaked cotton
and let dry.
3. Samples should be collected in a sterile
container immediately prior to regular milking
without discarding any streams of milk (since
the foremilk usually contains the greatest
number of the infecting micro-organisms.

Diarrhea/Enteritis
 Feces
 Blood samples
 Serum samples
 Food material
 Affected intestine , Liver, Intestinal LN

ABORTIONS
 Diagnosis cause of abortion is difficult
and complex.
 Fetus, placenta, fetal stomach contents,
uterine contents and serum are the
favorite specimens.
 Submit multiple specimens to increase
the probability of diagnosis.

ABORTIONS (cont.)
 Rinse the fetus and placenta with clean tap
water and place them in a plastic bag. Force
the air out of the bag before sealing it.
 All specimens should be refrigerated .
 If a toxic condition is suspected, submit
samples of the aborting animal’s feed and
water.
 If you suspect nitrate toxicity send Eye or
aqueous humor.

ABORTIONS (cont.)
 Collect and submit the first of paired
serum samples from the suspected
aborting animal. The second serum
sample should be collected and
submitted in 2-3 weeks.

SPECIMENS for ANAEROBIC AND
MICROAEROPHILIC culture.
 The success of culture for anaerobic and
microaerophilic organisms is heavily
dependent on sample selection and
shipment.
 Sample should be taken from a living animal
or a fresh carcass.
 Specimens for Campylobacter isolation
should be submitted in a transport media that
limits or excludes air from the sample such as
Amies media, containing Cary-Blair or
thioglycolate broth.

MYCOLOGY
1. Submit skin scrapings from the outer edges
of a lesion and submit plucked (not cut)
hairs.
2. Skin, hair, and nails should by shipped to
the laboratory without refrigeration.
3. Submit internal organs or internal lesions
suspected of fungal infection.
4. Internal specimens should be sent
refrigerated (wet ice packs) and not frozen.

Surveillance
 Continuous investigation of a given
population to detect the occurrence of
disease for control purpose

Active surveillance

 Advantage of Active surveillance
 Better information quality

 Reflect the true situation

 Faster

 Cheaper

Passive surveillance
 Compulsory notification
 Laboratory submission data
 Disadvantages of Passive surveillance
 Under reporting system
 Expense
 Non representative report

Monitoring
 Constitutes on going programmes directed at
the detection of changes in the prevalence of
a disease in a given population
 What you are looking for??????
 Estimate disease prevalence
 Estimate disease incidence
 Detect disease or demonstrate freedom from
disease

Prevalence
 The proportion of number of sick
animals at a single point in time to the
total population at risk at the same point
of time
 In the previous example, Prevalence is
50%

Incidence rate
 It is a measure of average speed at
which the disease is spreading
 Incidence rate
= Total new cases during a period of time
av. No. of animals at risk X time period

Example :
A small animal farm consists of 2000 goat
suffer from an outbreak of PPR. The first
animal start to get sick on the 3rd of March. By
5th of march many animal are dying. The
owner contact the veterinarian on the 6th of
march . The veterinarian count 56 sick
animals and the owner said that 143 animal
have already died and 28 animals had been
sick but recovered. A number of 1801
apparently healthy .

Calculation of Prevalence percent.
 Prevalence at 6th March
= 56 / (2000-143 ”DEAD”)
= 56 / 1857
= 3%

Calculation of Incidence rate
 New cases =(143 + 28 +56) =227
 Av.Population at risk in the 4 Days
=(2000 – 227) + 2000/2=1886.5
 Incidence rate :
= 227/1886.5 X 4 “Period of time”
= 0.03 animal /day
= 21 animal /100 animals/ week

Sample size
 The sample size is independent of the
total number of animals in the
population
 It depends on 3 factors :
1. Expected prevalence.
2. Level of confidence wanted (90 0r 95 or
99%).
3. Desired absolute precision.

Sample size in infinite population
 Suppose the true prevalence is thought
to be about 40% and the desired
estimate at precision of 5% at 95%
level of confidence.
 From the table, the sample size will be
369 animals.

Sample size in infinite population
 Suppose we have 900 animals at the
same prevalence ,precision of 5% and
confidence level .
 The sample size (1/n)=1/n∞ + 1/N
=1/369 +1/900
=1/262

Sampling frame ( Stratified
Random sampling)
Prepare a list of camel owner for each
clinic , avoid repletion of names .
e.g.
Clinic # 1 : 37
Clinic # 2 : 101
Clinic # 3 : 76 .
Calculate the total No of owners e.g. 214

Sampling frame (Cont.)
Determine the proportion allocation of owners belonging
to each clinic ( Considering that the number of
owners reflect the animal population density in each
clinic )
i.e. Clinic # 1 37 / 214 = 17%
Clinic # 2 101/214 = 47%
Clinic # 3 76/214 = 36%

As far as we determine the sample size as 15000 animal
from total true animal population 95000
i.e. we select 15000/95000 = 1/6
i.e. one owner from every 6 owner randomly .

Calculate the total no of owners that will be
sampled , in our example it will be as
follows:
214 / 6 = 36 owners
Calculate how many owners will be sampled
from each clinic by multiplying the
obtained proportion allocation of each
clinic by the total No of owners that
should be sampling

 Clinic # 1:
17% X 36 = 6 owners will be selected on random basis
 Clinic # 2 :
47 % X 36= 47 owners will be selected on random basis
 Clinic # 3 :
36% X 76 = 13 owners will be selected on random basis

 If we get less number of animals than that
required we should go back to re-select
randomly another ? This will depend
mainly on the animal density exists for
each clinic .

Avian Influenza
 SPECIMEN
Serum , cloacal, tracheal, oropharyngeal
Swabs
 TYPE OF TEST
AGID , Imunochromatography, PCR ,HI

Avian Chlamydia infection
 SPECIMEN
 Spleen, liver, lung,
 Air sac, conjunctival swab
 TYPE OF TEST
 FA
 ELISA

Avian Mycoplasma Spp.
 SPECIMEN
 Serum
 TYPE OF TEST
 HI
 Plate agglutination test

Salmonella pullorum
 SPECIMEN
 Serum

 TYPE OF TEST
 Micro agglutination

Canine Corona virus
 SPECIMEN
 Serum
 Feces / small intestine
 TYPE OF TEST
 Imunochromatography
 ELISA
 IF

Canine Distemper Virus
 SPECIMEN
Lung, kidney, spleen, urinary bladder, brain,
stomach, liver, blood smear
Serum
 TYPE OF TEST
IF, IIF ,chromatography

Canine Parvovirus (CPV)
 SPECIMEN
 Intestine (jejunum, ileum), spleen,
mesenteric lymph node
 Serum
 TYPE OF TEST
 IF , IIF , chromatography

Bluetongue disease in a sheep
 Note the bluish
discoloration of the
coronary bands of
the hoof. The lips
will usually be found
to be swollen and
discolored blue at
the same time

Blue tongue
 SPECIMENS
 Serum
 TYPE OF TEST
 AGID , ELISA

Bovine Leucosis (BLV)
 SPECIMEN
 Serum
 TYPE OF TEST
 AGID

Bovine Respiratory Syncytial Virus
(BRSV)
 SPECIMEN
 Lung, bronchial
 lymph node
 Serum
 TYPE OF TEST
 IF ,IIF , ELISA

 Serum Samples are tested at 1:50 dilution

Bovine Viral Diarrhea (BVD)
 SPECIMEN
 Lung, intestine, turbinate, trachea, swabs
from lesions, fetal organs
 Ear notches are the samples of choice in
case of persistent infection
 Serum
 TYPE OF TEST
 IF , SNT , ELISA

Infectious Bovine Rhinotracheitis
(IBR) .
 SPECIMENS
 Lung, trachea, turbinate, aborted fetal
tissues
 Serum
 TYPE OF TEST
 IF , SNT , ELISA

Caprine Arthritis-Encephalitis
(CAE) / Ovine Progressive Pneumonia
(OPP)
 SPECIMENS
 Serum
 TYPE OF TEST
 AGID

Chlamydia
 SPECIMENS
 Lymph node,
 tissues of aborted fetus, joint fluid ,
 TYPE OF TEST
 IF , ELISA

Clostridium
 SPECIMENS
 Intestinal content
 Affected lesions (Liver , Int., Muscles)
 Serum
 TYPE OF TEST
 IF , ELISA

Bovine Corona virus , Rotavirus,
Cryptosporidium Infection
 SPECIMENS
 Intestinal content , Feces
 TYPE OF TEST
 IF , ELISA , Immuno chromatography

Salmonellosis
 SPECIMENS
 Feces,
 Feed,
 Water,
 Environmental samples
 TYPE OF TEST
 Culturing
 Agglutination test

E.coli Pilus (K 99 ,F 5
Serotype)
 SPECIMENS
 Intestinal content
 Feces
 TYPE OF TEST
 ELISA

Johne’s Disease (Mycobacterium
Para tuberculosis).
 SPECIMENS
 Fecal swabs, mucosal scrapings, intestinal lymph
nodes
 Serum
 TYPE OF TEST
ELISA , CFT, PCR

 Titers:
 1:8 – Negative,

1:16 – Suspicious,

1:32 – Positive

Typical lesions of contagious
caprine pleuropneumonia (CCPP)
in a goat
 Note the yellowish,
fibrinous deposit on
the surface of the
lungs and adhesions
to the inside of the
rib cage.

Contagious caprine pleuropneumonia
(CCPP)
 SPECIMENS
 Lung
 Serum
 TYPE OF TEST
 IF , L Agglutination

Leptospirosis
 SPECIMENS
 Kidney
 Serum
 TYPE OF TEST
 IF , Micro agglutination

 Test for 6 serovars - canicola, grippotyphosa,
hardjo, icterohemorrhagiae, pomona, and
bratislava. Samples are tested at an initial dilution
1:100.

Listeria
 SPECIMENS
 Cerebellum, pons, medulla, fetus, uterine
secretions
 Serum
 TYPE OF TEST
 Card Agglutination

 Test for Type 1 and Type 4 serotypes.
 Serum is screened at an initial dilution 1:20.

Ruminant Anaplasmosis
 SPECIMENS
 Serum
 TYPE OF TEST
 Card Agglutination , CFT , ELISA

Toxoplasmosis
 SPECIMENS
 Serum
 TYPE OF TEST
 Latex agglutination , IHA

 A titer > 1:64 is considered positive.

Specimens for FMD
 The preferred sample for virus isolation is the
epithelium (at least 1-2 cm square) from
unruptured or freshly ruptured vesicles.
 Vesicular fluid should be added if available.
 Samples should be collected into a transport
medium consisting of equal amounts of
phosphate buffer and glycerol at pH 7.2-7.6
(with added antibiotics).

PPR in a goat
purulent eye and nose
discharges
Discharges from the
nose and eyes in
advanced PPR
infection; the hair below
the eyes is wet and
there is matting together
of the eyelids as well as
partial blockage of the
nostrils by dried-up
purulent discharges

PPR in a goat
Inflamed (reddened)
eye membranes
Reddening of the
mucous membranes of
the eye (the
conjunctiva) in the early
stages of infection. Note
the purulent eye
discharges

PPR in a goat
Early mouth
lesions showing
areas of dead
cells
Early pale, grey
areas of dead cells
on the gums

PPR in a goat
later mouth
lesions
The membrane
lining the mouth is
completely obscured
by a thick cheesy
material; shallow
erosions are found
underneath the dead
surface cells.

Specimens for RFV
 Blood in anticoagulant from any animals
with a fever of 40.5-42°C
 Liver and spleen from any freshly dead
animals, on ice, in glycerol buffered
saline and/or in buffered formalin
 Liver, spleen and brain from fresh
fetuses

RFV
 Sheep, fetus. Both
the pleural and
peritoneal cavities
contain excessive
clear, straw-colored
fluid.

RFV
 Sheep, fetus,
kidney. There is
severe perirenal
edema.

RFV
 Sheep, liver. The cut
surface of the
swollen liver is pale
and contains many
petechiae.

RVF
 Sheep, colon.
Severe hemorrhagic
colitis.

RVF
 Sheep, colon. There
is severe locally
extensive mucosal
hemorrhage.

RVF
 Sheep, liver. Section
reveals that the liver
is pale, swollen and
contains multiple
foci of hemorrhage.

RFV
 Sheep, liver. Liver is
pale and swollen
and contains many
areas of severe
congestion.

Sampling

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