Using amplified fragment length polymorphism analysis to differentiate isolates of Pasteurella multocida serotype 1

Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida, kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission.     

Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

Characteristics of Pasteurella multocida isolated from waterfowl and associated avian species in California

Characteristics of Pasteurella multocida isolated from tissues of dead waterfowl and associated avian species found at 23 sites located in northern and central California, from January 1986 through January 1988 are reported. Two hundred ninety five isolates of P. multocida were obtained from 23 avian species. Most of the isolates belonged to the subspecies P. multocida multocida (63%), followed by P. multocida gallicida (37%), and by P. multocida septica (less than 1%). There appeared to be a higher prevalence of P. multocida multocida in Ross' geese (Chen rossi) and Snow geese (Chen coeruleus). All of the isolates belonged to somatic serotype 1, possessed the A capsule type and were susceptible to the 8 antimicrobial agents tested. None contained plasmid DNA.     

Cloned duck hepatitis B virus DNA is infectious in Pekin ducks

Approximately 10% of German-bred Pekin ducks were found to be chronically infected with duck hepatitis B virus (DHBV). The genomes of three German DHBV isolates analyzed were closely related but showed substantial restriction site polymorphism compared with U.S. isolates. We tested the infectivity of three sequence variants of cloned DHBV DNA by injecting them into the liver of virus-free ducklings. Most of these animals injected with double-stranded closed-circular or plasmid-integrated dimer DHBV DNA developed viremia, demonstrating the infectivity of all three cloned DHBV DNA variants. The cloned viruses produced were indistinguishable from those from naturally infected animals, implying that our experimental approach can be used to perform a functional analysis of the DHBV genome.     

Associations between water quality, Pasteurella multocida, and avian cholera at Sacramento National Wildlife Refuge

We studied patterns in avian cholera mortality, the presence of Pasteurella multocida in the water or sediment, and water chemistry characteristics in 10 wetlands at the Sacramento National Wildlife Refuge Complex (California, USA), an area of recurrent avian cholera epizootics, during the winters of 1997 and 1998. Avian cholera outbreaks (>or=50 dead birds) occurred on two wetlands during the winter of 1997, but no P. multocida were recovered from 390 water and 390 sediment samples from any of the 10 wetlands. No mortality events were observed on study wetlands during the winter of 1998; however, P. multocida was recovered from water and sediment samples in six of the 10 study wetlands. The pH levels were higher for wetlands experiencing outbreaks during the winter of 1997 than for nonoutbreak wetlands, and aluminum concentrations were higher in wetlands from which P. multocida were recovered during the winter of 1998. Water chemistry parameters (calcium, magnesium, sodium, and dissolved protein) previously linked with P. multocida and avian cholera mortality were not associated with the occurrence of avian cholera outbreaks or the presence of P. multocida in our study wetlands. Overall, we found no evidence to support the hypothesis that wetland characteristics facilitate the presence of P. multocida and, thereby, allow some wetlands to serve as long-term sources (reservoirs) for P. multocida.     

Molecular characterization of Pasteurella multocida isolates from rabbits

Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.     

Duck Enteritis Virus Glycoprotein D and B DNA Vaccines Induce Immune Responses and Immunoprotection in Pekin Ducks

DNA vaccine is a promising strategy for protection against virus infection. However, little is known on the efficacy of vaccination with two plasmids for expressing the glycoprotein D (gD) and glycoprotein B (gB) of duck enteritis virus (DEV) in inducing immune response and immunoprotection against virulent virus infection in Pekin ducks. In this study, two eukaryotic expressing plasmids of pcDNA3.1-gB and pcDNA3.1-gD were constructed. Following transfection, the gB and gD expressions in DF1 cells were detected. Groups of ducks were vaccinated with pcDNA3.1-gB and/or pcDNA3.1-gD, and boosted with the same vaccine on day 14 post primary vaccination. We found that intramuscular vaccinations with pcDNA3.1-gB and/or pcDNA3.1-gD, but not control plasmid, stimulated a high frequency of CD4⁺ and CD8⁺ T cells in Pekin ducks, particularly with both plasmids. Similarly, vaccination with these plasmids, particularly with both plasmids, promoted higher levels of neutralization antibodies against DEV in Pekin ducks. More importantly, vaccination with both plasmids significantly reduced the virulent DEV-induced mortality in Pekin ducks. Our data indicated that vaccination with plasmids for expressing both gB and gD induced potent cellular and humoral immunity against DEV in Pekin ducks. Therefore, this vaccination strategy may be used for the prevention of DEV infection in Pekin ducks.     

Outbreaks of avian cholera in Hope Bay, Antarctica

During austral summers 1999-2000 and 2000-01, two outbreaks of avian cholera occurred in the Hope Bay area (63 degrees 24'S, 56 degrees 59'W), located on the tip of the Antarctic Peninsula. Eighty-six dead birds were found: five kelp gulls (Larus dominicanus), 36 skuas (Stercorarius sp.), and 45 Adelie penguins (Pygoscelis adeliae). The carcasses were studied using clinical, pathological, and microbiological criteria. Water samples from ponds where birds were settled and samples from 90 healthy birds also were analyzed during the second outbreak. Pasteurella multocida isolates were identified by biochemical tests, capsular type, somatic serotype, and susceptibility to nine antibiotics. Molecular subtyping was performed by ApaI and SmaI pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC-PCR). In February 2000, mortality in skuas was 16% and 2% in kelp gulls. In the 2000-01 breeding season, mortality in south polar skuas was 47%, 24% in brown skuas, 1.4% in kelp gulls, and 0.01% in Adelie penguins. All birds had lesions of avian cholera. In kelp gulls the presentation was chronic, whereas skuas and penguins suffered subacute and acute disease, respectively. Fifty-five isolates recovered from dead birds and one from water were identified as P. multocida gallicida, type A:1. The strains presented a unique molecular pattern by PFGE and ERIC-PCR. A possible hypothesis to explain the origin of the outbreaks was that nonbreeder kelp gulls carried P. multocida gallicida to Hope Bay, and avian cholera was transmitted through water to skuas and penguins. This study reports avian cholera in new bird species, their potential role in the transmission of the disease, and the different responses of these species to the disease.     

Effect of saline intake on water flux and osmotic homeostasis in Pekin ducks (<Emphasis Type="Italic">Anas platyrhynchos</Emphasis>)

The physiological regulation of body water volume and concentration was evaluated in Pekin ducks, Anas platyrhynchos, slowly acclimated to increasingly saline drinking water (six equal 75 mM NaCl increments). Body mass, total body water (TBW), water flux, plasma osmolality (Osm(pl)), and ionic and osmoregulatory hormone concentrations were measured at the end of each increment. The salinity at which each variable deviates from its homeostatic set point was calculated by continuous two-phase linear regression. We hypothesized that, as drinking water salinity increases: (1) body water increases in concentration before it decreases in volume and (2) that regulating variables that help determine homeostatically set values (plasma hormone concentrations and water flux) deviate from values of freshwater ducks at lower drinking water salinities than the variables they regulate (Osm(pl), hematocrit, TBW). Osm(pl) was the first variable for which we could calculate a deviation from its homeostatically controlled value. It increases at much lower drinking water salinity than that at which TBW decreases, supporting our first hypothesis, but not our second hypothesis. We further hypothesized that, because the concentration of Pekin duck salt gland secretion is only slightly higher than that of their drinking water, they increase water flux (drinking) as salinity of drinking water increases, until the latter exceeds the secretion concentration and then they drink less. There was no change in water flux until it decreases when TBW decreases, 329 mM NaCl and 335 mM NaCl, respectively. The results do not support our hypothesis that Pekin ducks increase drinking as the salinity of their drinking water increases, but do indicate that, at tolerable salinities, Pekin ducks maintain body water volume while allowing body water osmolality to increase. At higher salinities, ducks decrease drinking and use body water to get rid of the excess salt.     

Prevalence of serologic types of Pasteurella multocida from 57 species of birds and mammals in the United States

The serologic types of 265 isolates of Pasteurella multocida collected from 50 species of wild birds and seven species of wild mammals over a 22-yr period were determined with the gel diffusion precipitin test. Antigens prepared from these isolates reacted with reference P. multocida antisera representing serotypes 1, 2, 3, 4, 5, 6, 7, 8, 12, and 15. Antigens from some isolates reacted slightly with antisera from more than one serotype. Overall, gel precipitin reactions involving serotype 1 (65%) and 3 (20%) were the most prevalent.     

Serologic methods for detection of Pasteurella multocida infections in nasal culture negative rabbits

An agar gel-diffusion test (AGDT) and an enzyme-linked immunosorbent assay (ELISA) were utilized to detect serum antibodies against Pasteurella multocida in naturally infected rabbits derived originally from a Pasteurella-free colony. The antigen used in both assays was purified from a serotype 3 (P-1059) strain of P. multocida. Among 47 serum samples tested 15 (32%) were seropositive; 12 (26%) of which were both AGDT and ELISA-positive, while 3 (6%) were ELISA-positive only. All rabbits examined were normal clinically and negative to repeated nasal cultures, but subsequent cultures at necropsy demonstrated the presence of P. multocida in 11 of the AGDT-positive rabbits and in 14 of the ELISA-positive rabbits. The organism was isolated most frequently from the naso-oropharynx and the tympanic bullae. Serotyping of isolates recovered from the nasopharynx were determined to be serotype 3 or 3,12. Ten seronegative rabbits also were necropsied and none were found harboring P. multocida. These preliminary data indicate that the application of an enzyme-linked immunosorbent assay may prove efficacious in identifying apparently healthy, consistently nasal culture-negative rabbits as subclinical carriers of P. multocida.     

Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

Migratory aquatic birds play an important role in the maintenance and spread of avian influenza viruses (AIV). Many species of aquatic migratory birds tend to use similar migration routes, also known as flyways, which serve as important circuits for the dissemination of AIV. In recent years there has been extensive surveillance of the virus in aquatic birds in the Northern Hemisphere; however in contrast only a few studies have been attempted to detect AIV in wild birds in South America. There are major flyways connecting South America to Central and North America, whereas avian migration routes between South America and the remaining continents are uncommon. As a result, it has been hypothesized that South American AIV strains would be most closely related to the strains from North America than to those from other regions in the world. We characterized the full genome of three AIV subtype H11N9 isolates obtained from ruddy turnstones (Arenaria interpres) on the Amazon coast of Brazil. For all gene segments, all three strains consistently clustered together within evolutionary lineages of AIV that had been previously described from aquatic birds in North America. In particular, the H11N9 isolates were remarkably closely related to AIV strains from shorebirds sampled at the Delaware Bay region, on the Northeastern coast of the USA, more than 5000 km away from where the isolates were retrieved. Additionally, there was also evidence of genetic similarity to AIV strains from ducks and teals from interior USA and Canada. These findings corroborate that migratory flyways of aquatic birds play an important role in determining the genetic structure of AIV in the Western hemisphere, with a strong epidemiological connectivity between North and South America.     

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.     

Avian cholera exposure and carriers in greater white-fronted geese breeding in Alaska, USA

We conducted a 3-yr study (2001-03) on greater white-fronted geese (Anser albifrons frontalis) breeding in Alaska, USA, to determine the exposure of this population to Pasteurella multocida and the potential role of these birds as disease carriers. We tested sera from nearly 600 adult geese for antibodies to P. multocida serotype 1. We found a low prevalence (<5%) of positive antibodies in adult geese, and based on the short duration of detectable antibodies, these findings indicate recent infection with P. multocida. Prevalence was similar to serologic results from both breeding and wintering lesser snow geese. We also collected oral (n=1,035), nasal (n=102), and cloacal (n=90) swab samples to determine the presence of avian cholera carriers in this population. We were unable to isolate P. multocida serotype 1 from any of the birds sampled. Based on comparison with other waterfowl species, we concluded that these geese may be exposed to avian cholera during the winter or spring migration but are unlikely to play a significant role as carriers of the bacterium causing avian cholera.     

Safety and efficacy of a streptomycin dependent live Pasteurella multocida vaccine in rabbits

The safety of and protection provided by a streptomycin dependent live Pasteurella multocida (serotype 12:A) vaccine was evaluated in New Zealand white rabbits. The vaccine strain was isolated from two of twelve rabbits 24 hours after intranasal administration. Streptomycin independent P. multocida isolates were not recovered for 4 weeks after vaccination, indicating a lack of reversion to the wild type. Thirty days after a single intranasal administration of vaccine, eight rabbits were challenged with either P. multocida serotype 3:A or serotype 12:A. Eight non-vaccinated rabbits were challenged in the same manner. Vaccinated rabbits challenged with serotype 12:A had nasal infections for only 2 weeks following challenge. Vaccinated rabbits challenged with serotype 3:A developed chronic nasal infections but were protected from severe disease. Immunoglobulin A or G antibodies against P. multocida were not detected after vaccination in nasal lavages or sera using an enzyme-linked immunosorbent assay. However, both antibodies increased following challenge with either serotype 3:A or serotype 12:A. These studies indicated that the streptomycin dependent pasteurella strain colonized rabbits briefly and was genetically stable in vivo. The results in challenged rabbits suggest that the vaccine provided protection against chronic infection by a homologous pasteurella serotype and protection against severe disease by a heterologous pasteurella serotype.     

Genetic Structure of Avian Influenza Viruses from Ducks of the Atlantic Flyway of North America

Wild birds, including waterfowl such as ducks, are reservoir hosts of influenza A viruses. Despite the increased number of avian influenza virus (AIV) genome sequences available, our understanding of AIV genetic structure and transmission through space and time in waterfowl in North America is still limited. In particular, AIVs in ducks of the Atlantic flyway of North America have not been thoroughly investigated. To begin to address this gap, we analyzed 109 AIV genome sequences from ducks in the Atlantic flyway to determine their genetic structure and to document the extent of gene flow in the context of sequences from other locations and other avian and mammalian host groups. The analyses included 25 AIVs from ducks from Newfoundland, Canada, from 2008–2011 and 84 available reference duck AIVs from the Atlantic flyway from 2006–2011. A vast diversity of viral genes and genomes was identified in the 109 viruses. The genetic structure differed amongst the 8 viral segments with predominant single lineages found for the PB2, PB1 and M segments, increased diversity found for the PA, NP and NS segments (2, 3 and 3 lineages, respectively), and the highest diversity found for the HA and NA segments (12 and 9 lineages, respectively). Identification of inter-hemispheric transmissions was rare with only 2% of the genes of Eurasian origin. Virus transmission between ducks and other bird groups was investigated, with 57.3% of the genes having highly similar (≥99% nucleotide identity) genes detected in birds other than ducks. Transmission between North American flyways has been frequent and 75.8% of the genes were highly similar to genes found in other North American flyways. However, the duck AIV genes did display spatial distribution bias, which was demonstrated by the different population sizes of specific viral genes in one or two neighbouring flyways compared to more distant flyways.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Partial characterization of plasmids from rabbit isolates of Pasteurella multocida.

Plasmids have not been reported for isolates of Pasteurella multocida from rabbits. We assayed 28 isolates of rabbit P. multocida for plasmids and sought to determine whether or not plasmid presence correlated with clinical or pathologic findings, serotype, toxin production, possession of pili, or biochemical characteristics. Fourteen isolates bore a single 1.6 Md (covalently closed circular form in 0.7% agarose gels) plasmid. An additional isolate had two plasmids which migrated as a closely-spaced doublet, centered around 1.6 Md. Eleven isolates appeared to have identical plasmids, according to Hae III and Hinf I digests. The apparent linear size of this common plasmid in 2% agarose gels was 2.1 Md, as calculated from the sums of the sizes of Hae III or Hinf I digestion fragments. Linearization of the common plasmid with Msp I produced an apparent size of 2.5 Md in 0.7% agarose gels. No correlations between presence of the common plasmid and somatic serotype, toxigenicity, presence of pili, antimicrobial resistance, selected biochemical characteristics, anatomic site from which the bacteria were cultured, or disease status of the host were found.     

Effects of heat stress on antioxidant defense system,inflammatory injury,and heat shock proteins of Muscovy and Pekin ducks: evidence for differential thermal sensitivities

Rising temperatures are severely affecting the mortality, laying performance, and meat quality of duck. Our aim was to investigate the effect of acute heat stress on the expression of heat shock proteins (HSPs: HSP90, 70, 60, 40, and 10) and inflammatory factors (nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)) and antioxidant enzyme activity (superoxide dismutase (SOD), malondialdehybe (MDA), catalase (CAT), total antioxidant capacity (T-AOC)) in livers of ducks and to compare the thermal tolerance of Pekin and Muscovy ducks exposed to acute heat stress. Ducks were exposed to heat at 39 ± 0.5 °C for 1 h and then returned to 20 °C for 1 h followed by a 3-h recovery period. The liver and other tissues were collected from each individual for analysis. The mRNA levels of HSPs (70, 60, and 40) increased in both species, except for HSP10, which was upregulated in Muscovy ducks and had no difference in Pekin ducks after heat stress. Simultaneously, the mRNA level of HSP90 decreased in the stress group in both species. Morphological analysis indicated that heat stress induced tissue injury in both species, and the liver of Pekin ducks was severely damaged. The activities of several antioxidant enzymes increased in Muscovy duck liver, but decreased in Pekin duck. The mRNA levels of inflammatory factors were increased after heat stress in both duck species. These results suggested that heat stress could influence HSPs, inflammatory factors expression, and the activities of antioxidant enzymes. Moreover, the differential response to heat stress indicated that the Muscovy duck has a better thermal tolerance than does the Pekin duck.     

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.