Outbreaks Of Fowl Cholera Research Articles

Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing Pasteurella multocida OmpH Protein for Fowl Cholera Prevention in Ducks.

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.

Immunogenicity and protection of a Pasteurella multocida strain with a truncated lipopolysaccharide outer core in ducks

Pasteurella multocida infection frequently causes fowl cholera outbreaks, leading to huge economic losses to the poultry industry worldwide. This study developed a novel live attenuated P. multocida vaccine strain for ducks named PMZ2 with deletion of the gatA gene and first four bases of the hptE gene, both of which are required for the synthesis of the lipopolysaccharide (LPS) outer core. PMZ2 produced a truncated LPS phenotype and was highly attenuated in ducks with a > 105-fold higher LD50 than the wild-type strain. PMZ2 colonized the blood and organs, including the spleen, liver and lung, at remarkably reduced levels, and its high dose of oral infection did not cause adverse effects on body temperatures and body weights in ducks. To evaluate the vaccine efficacy of the mutant, ducklings were inoculated orally or intranasally with PMZ2 or PBS twice and subsequently subjected to a lethal challenge. Compared with the PBS control, PMZ2 immunization stimulated significantly elevated serum IgG, bile IgA and tracheal IgA responses, especially after the boost immunization in both the oral and intranasal groups, and the induced serum had significant bactericidal effects against the wild-type strain. Furthermore, the two PMZ2 immunization groups exhibited alleviated tissue lesions and significantly decreased bacterial loads in the blood and organs compared with the PBS group post-challenge. All the ducks in the PMZ2 oral and intranasal groups survived the challenge, while 70% of ducks in the PBS group succumbed to the challenge. Thus, the P. multocida mutant with mutation of the gatA gene and part of the hptE gene proved to be an effective live attenuated vaccine candidate for prevention of fowl cholera in ducks.

Phase variation in the glycosyltransferase genes of Pasteurella multocida associated with outbreaks of fowl cholera on free-range layer farms.

Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene (htpE) (ST7 and ST274) or galactosyltransferase gene (gatG) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG. Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.

First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera.

Background and Aim:Fowl cholera (FC) caused by Pasteurella multocida is a highly contagious bacterial disease of global importance for poultry production. The severity and incidence of FC caused by P. multocida may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment, and the bacterial strain. This study aimed to investigate the genetic diversity of multidrug-resistant P. multocida strains isolated from FC outbreaks in laying hens from commercial farms of Bangladesh.Materials and Methods:We collected 57 samples of suspected FC, including 36 live and 21 dead laying hens. P. multocida isolates were characterized by biochemical and molecular-biological methods.Results:Twenty-two strains of P. multocida were isolated from these samples through phenotypic and genotypic characterization. The strains were grouped into two distinct random amplification of polymorphic DNA (RAPD) biotypes harboring a range of pathogenic genes; exbB, ompH, ptfA, nanB, sodC, and hgbA. In this study, 90.90% and 81.82% P. multocida strains were multidrug-resistant and biofilm formers, respectively. Whole-genome sequencing of the two representative RAPD phylotypes confirmed as P. multocida type B: L2:ST122, harboring a number of virulence factors-associated genes (VFGs), and antimicrobial resistance (AMR) genes (ARGs). In addition, pan-genome analysis revealed 90 unique genes in the genomes of P. multocida predicted to be associated with versatile metabolic functions, pathogenicity, virulence, and AMR.Conclusion:This is first-ever report on the association of P. multocida genotype B: L2:ST122 and related VFGs and ARGs in the pathogenesis of FC in laying hens. This study also provides a genetic context for future researches on the evolutionary diversity of P. multocida strains and their host adaptation.

Genomic diversity and molecular epidemiology of Pasteurella multocida.

Pasteurella multocida is a bacterial pathogen with the ability to infect a multitude of hosts including humans, companion animals, livestock, and wildlife. This study used bioinformatic approaches to explore the genomic diversity of 656 P. multocida isolates and epidemiological associations between host factors and specific genotypes. Isolates included in this study originated from a variety of hosts, including poultry, cattle, swine, rabbits, rodents, and humans, from five different continents. Multi-locus sequence typing identified 69 different sequence types. In-silico methodology for determining capsular serogroup was developed, validated, and applied to all genome sequences, whereby capsular serogroups A, B, D, and F were found. Whole genome phylogeny was constructed from 237,670 core single nucleotide variants (SNVs) and demonstrated an overall lack of host or capsular serogroup specificity, with the exception of isolates from bovine sources. Specific SNVs within the srlB gene were identified in P. multocida subsp. septica genomes, representing specific mutations that may be useful for differentiating one of the three known subspecies. Significant associations were identified between capsular serogroup and virulence factors, including capsular serogroup A and OmpH1, OmpH3, PlpE, and PfhB1; capsular serogroup B and HgbA and PtfA; and capsular serogroup F and PtfA and PlpP. Various mobile genetic elements were identified including those similar to ICEPmu1, ICEhin1056, and IncQ1 plasmids, all of which harbored multiple antimicrobial resistance-encoding genes. Additional analyses were performed on a subset of 99 isolates obtained from turkeys during fowl cholera outbreaks from a single company which revealed that multiple strains of P. multocida were circulating during the outbreak, instead of a single, highly virulent clone. This study further demonstrates the extensive genomic diversity of P. multocida, provides epidemiological context to the various genotyping schemes that have traditionally been used for differentiating isolates, and introduces additional tools for P. multocida molecular typing.

Molecular characterisation and antibiotic sensitivity profile of Pasteurella multocida isolated from poultry farms in Malaysia

Fowl cholera has caused significant economic losses in many poultry producing countries worldwide. In Malaysia, outbreaks of fowl cholera are frequently reported and encountered in different types of poultry productions. The objective of this study was to characterise 13 avian Pasteurella multocida, isolated from fowl cholera outbreaks in Central Peninsular Malaysia in the period between 2000 and 2018. The isolates were subjected to multiplex polymerase chain reaction (PCR) for capsular serotyping, disc diffusion method for antimicrobial susceptibility profiles, and molecular genotyping using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The capsular serotyping showed all 13 Pasteurella multocida isolates belonging to capsular serotype A. The antimicrobial susceptibility showed several multidrug resistance strains among the P. multocida isolates. All the isolates were resistant to erythromycin (100%), streptomycin (68%), tetracycline (37%), enrofloxacin (37%), florfenicol (23%), penicillin G (14%), gentamicin (14%), and amoxicillin (14%). The PFGE analysis clustered the isolates into three clones. Group A included isolates with a similarity of 87% from the year 2000, 2013, and 2018. Three sequence types were identified using MLST typing namely, ST129, ST231, and ST355. The ST355 was assigned for the first time in the Rural Industries Research and Development Corporation (RIRDC) database. Besides, ST129 has been reported in India, China, and Sri Lanka, which highlights the possibility of transmission between Asian countries. This study provides an insight into epidemiological information of Pasteurella multocida that causes fowl cholera outbreaks in the central region of Peninsular Malaysia.

Multilocus Sequence Typing of Pasteurella multocida Isolates from Acute Fowl Cholera Outbreak in Layer

Fowl cholera is an infectious disease caused by <i>Pasteurella multocida</i> that contributes to high economic loss in the commercial chicken industry. Three <i>Pasteurella multocida</i> strains were isolated from outbreaks of acute fowl cholera in the Korean layer farms from 2018 to 2019. One strain was identified and serotyped using capsular PCR typing. This strain was also genotyped by lipopolysaccharide (LPS) PCR typing as A: L3, whereas other strains were non-typable. The multilocus sequence typing (MLST) result showed that the A: L3 strain is sequence type (ST) 134; the non-typable strains were recorded as the following new STs: ST 366 and ST 374. Using phylogenetic tree analysis based on MLST sequences, we determined that ST 366 and ST 374 are closely related to the reference strains that were previously isolated from duck and chicken in Korea, and they were highly prevalent within the Korean cluster. In conclusion, <i>Pasteurella multocida</i> strains were identified and isolated in this study. Furthermore, this is the first report of using MLST to determine the prevalence of fowl cholera in Korea.

Using genomics to understand inter- and intra- outbreak diversity of Pasteurella multocida isolates associated with fowl cholera in meat chickens.

Fowl cholera, caused by Pasteurella multocida, continues to be a challenge in meat-chicken-breeder operations and has emerged as a problem for free-range meat chickens. Here, using whole-genome sequencing (WGS) and phylogenomic analysis, we investigate isolate relatedness during outbreaks of fowl cholera on a free-range meat chicken farm over a 5-year period. Our genomic analysis revealed that while all outbreak isolates were sequence type (ST) 20, they could be separated into two distinct clades (clade 1 and clade 2) consistent with difference in their lipopolysaccharide (LPS) type. The isolates from the earlier outbreaks (clade 1) were carrying LPS type L3 while those from the more recent outbreaks (clade 2) were LPS type L1. Additionally, WGS data indicated high inter- and intra-chicken genetic diversity during a single outbreak. Furthermore, we demonstrate that while a killed autogenous vaccine carrying LPS type L3 had been successful in protecting against challenge from L3 isolates it might have driven the emergence of the closely related clade 2, against which the vaccine was ineffective. The genomic results also revealed a 14 bp deletion in the galactosyltransferase gene gatG in LPS type L3 isolates, which would result in producing a semi-truncated LPS in those isolates. In conclusion, our study clearly demonstrates the advantages of genomic analysis over the conventional PCR-based approaches in providing clear insights in terms of linkage of isolate within and between outbreaks. More importantly, it provides more detailed information than the multiplex PCR on the possible structure of outer LPS, which is very important in the case of strain selection for killed autogenous vaccines.

Genetic diversity of different avian strains of Pasteurella multocida recovered during four outbreaks in India

Pasteurella multocida causes fowl cholera in avian species and type A is predominantly reported from outbreaks of fowl cholera. Biochemical, serological, and molecular methods are employed for its diagnosis and typing. Utilizing rapid molecular tools like repetitive extragenic palindromic (REP) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR, P. multocida isolates from four outbreaks (three from Chennai and one from Ahmadabad, India) were characterized and typed to determine their relationships. A total of 36 isolates were recovered from the outbreaks, including one isolate from a parakeet, which was also subjected to characterization by conventional and molecular analysis. All of the isolates were found to be capsular type A based on PCR assay capsular typing. ERIC- and REP-PCR showed differences in the banding patterns among different outbreak isolates, and also among the geographical regions. Differences were also noticed among different host species, as the banding pattern in the ERIC- and REP-PCR differed; the analysis of results also revealed the same. All of the isolates were found to be sensitive to enrofloxacin and cefotaxime among the antibiotics used in the study. It was found that different strains might have been involved in the different outbreaks reported in the study. The results show that molecular typing methods like ERIC- and REP-PCR are useful epidemiological tools for classifying the strains.

Genome sequencing of a virulent avian Pasteurella multocida strain GX-Pm reveals the candidate genes involved in the pathogenesis

Pasteurella multocida (P. multocida) was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. First genomic study was performed on an avirulent avian strain Pm70, and until 2013, two genomes of virulent avian strains X73 and P1059 were sequenced. Comparative genome study supplied important information for further study on the pathogenesis of fowl cholera. In the previous study, a capsular serotype A strain GX-Pm was isolated from the liver of a chicken, which died during an outbreak of fowl cholera in 2011. The strain showed multiple drug resistance and was highly virulent to chickens. Therefore, the present study performed the genome sequencing and a comparative genomic analysis to reveal the candidate genes involved in virulence of P. multocida. Sequenced draft genome sequence of GX-Pm was 2,292,886bp, contained 2941 protein-coding genes, 5 genomic islands, 4 IS elements and 2 prophage regions. Notability, all the predicted drug-resistance genes were included in predicted genomic islands. A comparative genome study on virulent avian strains P1059, X73 and GX-Pm with the avirulent avian strain Pm 70 indicated that 475 unique genes were only identified in either of virulent strains but absent in the avirulent strain. Among these genes, 20 genes were contained within genomes of all three virulent strains, including a few of putative virulence genes. Further characterization of the pathogenic functions of these genes would benefit the understanding of pathogenesis of fowl cholera.

Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks

Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.

Evidence of circulation of an epidemic strain of Pasteurella multocida in Jiangsu, China by multi-locus sequence typing (MLST)

Pasteurella multocida, the causative agent of fowl cholera, is a serious threat to poultry farming. In this study, we isolated and identified 40 P. multocida strains in fowl cholera outbreaks in Jiangsu province, China. The identified P. multocida was further characterized using multi-locus sequence typing (MLST). All of the 40 P. multocida strains studied are genetically identical and belong to the ST129 sequence type based on seven MLST loci. Our study provides evidence of a circulating epidemic strain of P. multocida in Jiangsu, China.

Molecular Epidemiologic Investigation of Polish Avian Pasteurella multocida Strains Isolated from Fowl Cholera Outbreaks Showing Restricted Geographical and Host-Specific Distribution

Molecular Epidemiologic Investigation of Polish Avian Pasteurella multocida Strains Isolated from Fowl Cholera Outbreaks Showing Restricted Geographical and Host-Specific Distribution

Molecular Epidemiologic Investigation of Polish Avian Pasteurella multocida Strains Isolated from Fowl Cholera Outbreaks Showing Restricted Geographical and Host-Specific Distribution

Molecular epidemiologic analyses of the 42 clinical isolates of Pasteurella multocida from various avian hosts (geese, ducks, turkeys, and laying hens) in Poland from 2001 to 2011, including a single reference strain, were performed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, single primer PCR, and repetitive extragenic palindromic (REP)-PCR. Forty-two isolates were identified as P. multocida (serotype A). The majority of P. multocida strains were obtained from waterfowl clustered within one genotype, and they were not consistent with the genotypes obtained from the turkey strains. Pasteurella multocida showed genetic homogeneity between the host species, especially when isolated on the same farm. Some of the clones also were characteristic to the particular farm. The strains obtained in different regions represent distinct molecular patterns. The present findings demonstrate that some clones of P. multocida are restricted in geographical and host distribution. In addition, this study suggests that ERIC-PCR, single primer PCR, and REP-PCR are suitable techniques for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

An outbreak of chronic fowl cholera in broiler breeder chickens in Korea

Fowl cholera is a contagious acute and chronic disease caused by Pasteurella multocida in both domesticated and wild birds. Acute fowl cholera in both chickens and wild birds has recently been documented in Korea, but the chronic form has not been reported in Korea until now. This study describes the first outbreak of chronic fowl cholera in 13-week-old Arbor Acre broiler breeder chickens submitted to the College of Veterinary Medicine, Kyungpook National University in April 2006. The clinical signs of the affected flock of 9,621 chickens were lameness caused by swollen hock joints, diarrhea, ruffled feathers, and an average weekly mortality of 1.0%. At necropsy, purulent or caseous exudates were found in the hock and wing joints, humerus, and eyes, and severe pneumonia and pericarditis were discovered. Eleven bacterial isolates obtained from the liver, joint, infraorbital sinus and sternal bursa of the submitted chickens were all identified as Pasteurella multocida based on their physiological and biochemical characteristics. Five isolates were examined for antimicrobial susceptibility against 21 different antimicrobial agents including ampicillin. All were resistant to kanamycin, neomycin, and streptomycin, and some were resistant to gentamicin. The tested isolates were all susceptible to the other 17 antimicrobial agents. All 11 isolates were capsular serogroup A based on multiplex polymerase chain reaction. In addition, two of five isolates used in the antimicrobial susceptibility test were identified as somatic serotype 1 by an agar gel diffusion precipitin test, while the others were non-typable.

Causes of Mortality in Commercial Organic Layers in Denmark

A longitudinal study investigated the courses of mortality in commercial free-range organic layer flocks in Denmark. In total, 15 organic egg-producing flocks from 11 farms were randomly selected among 80 farms registered in Denmark. Four farms with confined egg production on deep litter were included for comparison. Flock sizes ranged from 2260 to 5940 layers. The flocks were monitored from introduction to the layer farm until slaughter. Flock mortalities ranged from approximately 2% to 91%, with a mean of 20.8% for organic flocks compared with 7% for confined flocks on deep litter. In total, 4608 layers were submitted for postmortem examination, representing > 40% of all the dead layers in the investigated flocks. Outbreaks of erysipelas (Erysipelothrix rhusiopathiae) and fowl cholera (Pasteurella multocida) were observed in two and three organic flocks, respectively. The mortality rate reached 91% in one organic flock dually affected by erysipelas and fowl cholera. In six organic flocks, outbreaks of blackhead were diagnosed. Concurrent infections of erysipelas and blackhead were diagnosed in one organic flock. Escherichia coli infections in the form of septicemia were identified in all organic flocks. In addition, cannibalism and constipation contributed significantly to the mortality in some organic flocks. In the confined deep litter flocks, E. coli infection, constipation, and cannibalism represented the most common causes of mortality.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes – adk, est, gdh, mdh, pgi, pmi and zwf – and internal fragments of 570–784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

Investigations On The Carrier Rate Of &lt;i&gt;Pasteurella Multocida&lt;/i&gt; In Black Rats (&lt;i&gt;Rattus Rattus&lt;/i&gt;) In A Commercial Quail Farm

The aim was to investigate the level of Pasteurella Multocida infection from two anatomic sites of black rats ( Rattus Rattus ), popularly referred to as house or roof rats in a commercial quail farmhouse with recurrent fowl cholera outbreaks and also to evaluate the association between the Pasteurella Multocida found in rats co-habiting quail poultry houses and isolates from outbreaks of fowl cholera. Thus 100 pharyngeal and 100 rectum swabs samples taken from rats co-habiting farmhouse were obtained and evaluated bacteriologically for isolation of P. multocida; 54% of pharyngeal swabs and 62% of rectum swabs were positive for P. multocida. Extended phenotypic characterization of the isolates confirmed the presence of subspecies P. multocida multocida. Subspecies Pasteurella Multocida septica and gallicida were not encountered. Ramdom serotyping of 5 isolates each from the two sites confirmed serotypes A:4. Fowl cholera outbreaks were confirmed on the quail houses and carrier rats had the same Pasteurella Multocida subspecies and serotype as the infected quail. The public health significance of the finding is also discussed. African Journal of Clinical and Experimental Microbiology Vol. 10 (1) 2009: pp. 2-9

Molecular characterization of Avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR

Repetitive extragenic palindromic (REP)-PCR (polymerase chain reaction), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and single primer PCR assays were employed to characterize 66 strains of Pasteurella multocida serogroup A:1 isolated from avian species belonging to different regions of India. REP-PCR resulted in amplification of REP sequences from the genome which were in the range of approximately 200 to approximately 3000 bp and accounted for a total of 54 distinguishing profiles (D=0.99). ERIC-PCR analysis also generated amplified products in the range of approximately 200 to approximately 3200 bp categorizing strains into a total of 50 different profiles (D=0.98). Amplification of repetitive regions using a microsatellite primer (GTG)(5), resulted in clear distinctive bands ranging from approximately 200 to approximately 2400 bp. Strains were assigned to 43 profiles (D=0.96). No correlation could be drawn between genotypic profiles and avian hosts with their geographical area of origin. Avian strains of P. multocida serogroup A:1 were found to be highly heterogeneous with diverse profiles. REP-PCR was found to be highly discriminatory and simple method for differentiation of phenotypically similar strains. The present study also indicated that PCR based amplification of repetitive regions of P. multocida is a rapid technique with good discrimination and could be employed directly for routine typing of field isolates from fowl cholera outbreaks.

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.