Detection and Characterization of Pathotypes, Serotypes, Biotypes, Phenotypes and Genotypes.pptx

Detection and Characterization of Pathotypes, Serotypes, Biotypes, Phenotypes and Genotypes.pptx

• 1.
  Detection and Characterizationof Pathotypes, Serotypes, Biotypes, Phenotypes, and genotypes of Pathogens: An Integral Element in Disease Investigation and Control DOI: 10.13140/RG.2.2.25741.10727 Available at: https://www.researchgate.net/publication/372787130_Detection_and_Characterization_of_Pathotypes_Serotypes_Bioty pes_Phenotypes_and_genotypes_of_Pathogens_An_Integral_Element_in_Disease_Investigation_and_Control Bhoj R Singh Division of Epidemiology, ICAR-IVRI, Izatnagar-243122, India [email protected] 1
• 2.
  Definitions • Pathotype: Apopulation of a parasite/ pathogen species in which all individuals have a stated pathosystem (host) character (pathogenicity or parasitic ability) in common. Though possible but rare to have a single pathotype of pathogen species. The pathotypes are related to the pathogenicity potential of a pathogen based on the presence of virulence factors. • Virotype: A subset of individuals of a pathogen species sharing common virulence factors/ deterninats. • Serotype/ Serovar: Group of individuals of a pecies having a distinct variation in antigenic/ immunogenic epitopes within a species of bacteria or virus or of different individuals. • Biotype/ Biovar: Biotypes is a populations within a species that differ in their ability to utilize a particular trait as ability to use or produce specific biomolecule(s) in a particular genotype. Two biotypes may differ jus through one codone change leading to observable change (phenotype) as one-codon change in chloroplast GS, making it less sensitive to glufosinate. • Toxinotype: A population of a species of pathogen producing specific type of toxin or a defined set of toxins. • Phenotype: Phenotype is the external or physical characteristics that appear in an organism. A subpopulation in a species having a set of observable characteristics resulting from the interaction of its genotype with the environment is defdined as a single phenotype. • Genotype: A sub-population of a pathogen or a species having (a set of genes that it carries) common DNA/ RNA markers represent a specific genotype. Some developed DNA marker methods are RFLP, RAPD, AP-PCR, PFGE, AFLP and now WGS/ NGS. It is often called as Quasispecies theory or Darwinian Evolution model, is primarily a theory of mutation-selection balance at extremely high mutation rates to separate it from Classical population. 2
• 3.
  Primary requirement fora pathogen to be Characterization of Pathotypes, Serotypes, Biotypes, Phenotypes, and genotypes • Pure isolate/ culture • Efficient system: – Host for pathotyping, though now in-vitro methods are used but they are just predict the pathotype not really determine the pathotypes. – Antibodies and antigenicity of the pathogen for serotyping. Now, some genotypic methods are also in practice to predict serotype. – Ability of the pathogen to grow in laboratory environment under defined conditions for Biotyping. – Integrity and Purity of the pathogen for Phenotyping – Background knowledge of genotypes and suitable platform for genotyping of an established/ known pathogen. For Novel pathogens, a suitable genotyping platform as NGS. • Expertise 3
• 4.
  Pathotyping Host: Experimental/ Modeland real host/ Quasispecies models Epidemiologically Hosts may be : Primary host, Secondary host (Intermediate), Paratenic host, Accidental host, and Reservoir host etc. • Primary host (a.k.a. definitive/final host): a host in which an organism partially/completely sexually develops, and disease or effect of parasitism is apparent. • Secondary host (intermediate host): a host in which an organism partially/completely asexually and may be sexually developing without having a disease. Both definitive and intermediate hosts are obligate to the life cycle of a parasite/ pathogen. • Accidental host: a host that shelters an organism which does not usually parasitize that host • Incidental host (a.k.a. dead-end host): a host that shelters an organism but is unable to transmit the organism to a different host. They may permit development of a pathogen to some extent but not permit development of its all statges. • Partenic/ transport host: Paratenic hosts may span a wide range of fauna and are not needed in the nematode's life cycle, but act as reservoirs in which different asexual/ larval stages of the parasite/ Pathogen can persist but not develop further. • Reservoir host: a host that shelters an organism without harm to itself. A pathogen hosts span a wide range of fauna and are not needed in the pathogen's life cycle, but act as reservoirs in which different stages of the pathogen can persist but not develop further. • Mechanical host (fomites): No development/ growth of the pathogen but pathogen is just mechanically carried from one host to another host like flies and insects transfer LSD. • Symbionts: Members of relationship called symbiosis where both partners have benefits of living together. • Commensals: Commensalism is a type of symbiotic relationship where one partner benefits whereas the second partner (the host) are neither helped nor harmed. The organism that receives the refuge and nourishment is called the ‘commensal’. Most of the normal floras of the human body can be considered as commensals. 4
• 5.
  Pathotyping • Experimental pathotyping:Understanding pathophysiology in Different types of hosts and experimental model hosts. The Genetic make up of host and pathogen boith decides the outcome and may not be true for the population in a defined spatiotemporal situation. • Observational studies : Case Controls/ Cohorts, always specific in spatotemorality. Common pathovars of E. coli are Enterotoxigenic E. coli (ETEC), Enterohaemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), Enteropathogenic E. coli (EPEC), Enteroadhesive E. coli (EAEC), and Necrotoxigenic E. coli (NTEC). • In Vitro studies: For determination of virulence (virotyping) and predicting pathogenic potential. May be misleading and hypothetical because host-pathogen interaction element is missing. • Genotypic methods: detection of genetic markers of pathogenicity to predict a pathotype. The egg-contaminating phenotype of Salmonella enterica serotype Enteritidis was linked to single-nucleotide polymorphisms (SNPs) occurring in cyaA, which encodes adenylate cyclase that produces cAMP and pyrophosphate from ATP. • Virotyping can be done using PCR methods, and also using in-vivo or in-vitro determination of virulence factors of pathogens. • HA/ Hly Typing: haemagglutination and Haemolysin typing requires detections of haemagglutination and haemolysisis of different types of RBCs (from different host species, tanned / fresh, in presence of Mannose etc.) 5
• 6.
  Serotyping Methods for determiningserotype of a pathogen Conventional and Genotypic • Conventional: Using Polyclonal antibodies and Using Monoclonal antibodies for detection of specific ‘O’, ‘H’, ‘K’ and ‘F’ antigens. • Serological Tests used: Agglutination/ Coagglutination/ Precipitation/ ELISA/ IHA/ CFA etc. • Genotypic: Detection of genes through PCR for specific ‘O’, ‘H’, ‘K’ and ‘F’ antigens. 6
• 7.
  Biotyping Biochemical and physiologicalmethods are used for Biotyping of pathogens There may be several biotypes within a serotype and vice-versa. Can be done for culturable microbes. • Common Biotyping methods: – Production/ synthesis of specific biomoleculaes/ detectable (visually or with aids) physiological traits (growth/ colour/ size) – Utilization of specific biomolecules/ organic and inorganic chemicals. – Degradation of specific biomolecules/ organic and inorganic chemicals. – Resistance to different biomolecules/ organic and inorganic chemicals, antibiograms are of common use – Expression of specific detectable (visually or with aids) physiological traits (growth/ colour/ size) in presence or absence of biomolecules/ organic and inorganic chemicals. – Requirement of specific biomolecules/ organic and inorganic chemicals for physiological functions and/ or pathogenesis. – Specific environmental conditions required for physiological functions and/ or pathogenesis e.g. aerobic, microaerobic, anaerobic, capanophilic, acidophilic, halophilic, photophilic, photophobic etc. 7
• 8.
  Genotypig • Can bedone even for non-culturable microbes. • The most fastest growing epidemiological tool having the highest discretionary power, consistency (repeatability), an vitro tool. • Genotyping is becoming a potent in vitro tool for biotyping, virotyping, serotyping and pathotyping. • Requires biotechnologically efficient laboratories and expertise to use and understand the outcome (results). • Bioinformatics and sequencing (targeted or whole genome) methods are the backbone of genotyping. 8
• 9.
  Methods in Genotyping https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.727 Partialgenotyping methods (only a small fraction of an individual's genotype is determined) • Restriction fragment length polymorphism (RFLP) of genomic DNA: IS6110-based restriction fragment length polymorphism typing for MTB • Random amplified polymorphism detection (RAPD) • Amplicon polymorphism-PCR (AP-PCR) • Pulse field gel electrophoresis (PFGE) Targeted Genotyping • Amplified fragment length polymorphism (AFLP), specific primers are used • MLVA (Multiple‐Locus Variable number tandem repeat Analysis) • ERIC PCR (Enterobacterial repetitive intergenic consensus (ERIC) sequences are 127-bp imperfect palindromes that occur in multiple copies in the genomes of enteric bacteria and vibrios). • Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR): polymerase chain reaction (PCR) amplification of a highly polymorphic direct repeat locus in the M. tuberculosis genome. • Allel specific oligonucleotide probe (ASO) hybridization • Single nucleotide polymorphism (SNPs) using qPCR • Copy Number Variation Analysis • Pyrosequencing (allelic typing) • (epi)GBS or RAD seq. Complete Genotyping: Whole genome sequencing (WGS) possible through new generation sequencers (NGS), Genome-Wide, Microarray of whole genome 9
• 10.
  Other typing methodsused for pathogens in epidemiology • Phage-typing • Bacteriocin/ Colicin typing • Antibiograms • Fimbiae typing • Morphotyping • Multilevel Genome Typing • Plasmid profiling and Genotyping of plasmids • Whole Genome Multilocus Sequence Typing (wgMLST) 10
• 11.
  Quiz • Name biotypesof Salmonella and Gallibacterium having belonging to the same serotypes. • Enlist the methods used in epidemiological genotyping of SARS CoV-2 and FMD viruses. • Name the bacteria where toxinotyping is used for discerning epidemiologically different strains. • Name the different pathotypes of – Escherichia coli – Clostridium perfingence – Bacillus anthrecis – Avian influenza virus – SARS CoV-2 • Name the methods used for biotyping, and biotypes of – Brucella melitensis and B. abortus – Yersinia enterocolitica – Salmonella Typhimurium and S. Gallinarum – Bacillus cereus and B. subtilis – Gallibacterium anatis – Vibrio cholerae – BVDV • Name the methods of genotyping for – Enterobacteriaceae members – Vibrionaceae members – Mycobacterium tuberculosis – Brucella abortus • Name the antigens and methods commonly used for serotyping of – Pasteurella – Salmonella – Escherichia – Klebsiella – Streptococci – Influenza viruses – FMDV 11