Prevalent Serotypes of Pasteurella multocida Isolated from Different Animal and Avian Species in India

Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused by P. multocida, a total of 206 bacterial cultures were identified as P. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping of P. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.     

Molecular Variability among Strains of Pasteurella multocida Isolated from an Outbreak of Haemorrhagic Septicaemia in India

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.     

8株禽多杀性巴氏杆菌外膜蛋白H基因序列分析

为了解国内禽多杀性巴氏杆菌（Pasteurella multocida,Pm）流行株外膜蛋白 H（OmpH）基因的变异情况,参考GenBank中已发表的多杀性巴氏杆菌序列设计1对特异性引物,采用PCR方法对不同来源的8株禽Pm菌株和3个荚膜型参考株（A、B、D）的OmpH基因进行扩增、测序。结果显示,11个菌株的OmpH基因开放阅读框在1002~1071 bp 之间;SignalIP 4.0预测结果表明,信号肽均为N端20个氨基酸残基,成熟蛋白氨基酸残基数量在314~337 aa之间,推测的分子质量在33.76~37.04 ku之间。与GenBank中15个菌株OmpH基因序列比对结果发现,核苷酸同源性在84.9%~100.0%之间;氨基酸同源性在81.5%~100.0%之间;其中C48-1、1010、9003、890920、921012、XJ-e 6个国内禽Pm分离株OmpH序列同源性为100.0%。试验结果表明,国内禽Pm菌株OmpH基因非常保守。     

Identification of Pasteurella multocida isolates of ruminant origin using polymerase chain reaction and their antibiogram study

A total of 100 isolates of Pasteurella multocida from various ruminant species (cattle, buffalo and sheep) belonging to different parts of country were identified using Pasteurella multocida-PCR (PM-PCR) and capsular PCR assays. PM-PCR revealed an amplicon of approximately 460 bp in all the isolates tested. As regards capsular PCR, 36 of 38 cattle isolates and 30 of 34 buffalo isolates were found to belong to capsular serogroup B whereas rest of the cattle and buffalo isolates belonged to serogroup A of P. multocida. In case of sheep, a total of 26 out of 28 isolates were positive for serogroup A specific PCR while remaining 2 amplified a PCR product specific for serogroup F of P. multocida. All the isolates were subjected to antibiotic sensitivity testing using 17 different antibiotics. Enrofloxacin was found to be most potent antibiotic as it was effective against 94% of the isolates followed by ofloxacin (93%), chloramphenicol (93%), doxycycline (89%), tetracycline (86%) and ciprofloxacin (84%). Vancomycin, bacitracin and sulfadiazine were ineffective against P. multocida isolates showing 84%, 75% and 82% resistance, respectively. Further, the antibiogram also revealed the development of resistance against multiple drugs among various isolates of the organism.     

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

Evaluation of different API systems for identification of porcine Pasteurella multocida isolates

An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.     

Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay

A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A. A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies. This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P. multocida serogroup-A. A nested PCR method yielded a single 374 bp product. All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII.     

REP-PCR Analysis of Pasteurella multocida Isolates from Wild and Domestic Animals in India

Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation. Saxena, M.K., Singh, V.P., Kumar, A.A., Chaudhuri, P., Singh, V.P., Shivachandra, S.B., Biswas, A. and Sharma, B., 2006. REP–PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India. Veterinary Research Communications, 30(8), 851–861     

Protective effect following intranasal exposure of goats to live Pasteurella multocida B:2

This study aimed to determine the effect of intranasal exposure to low doses of Pasteurella multocida B:2 on survival of goats challenged with high doses of the same organism. Eighteen goats were selected and divided into three groups. Goats of group 1 were exposed intranasally twice, with a two-week interval, to 7× 10⁶ cfu/ml of live P. multocida B:2. Goats of group 2 were not exposed to P. multocida B:2 but were kept together with the exposed group 1. Goats of group 3 remained as unexposed controls and were kept separated from the other two groups. Serum samples were collected at weekly intervals to determine the antibody levels. At week 5 post exposure, all goats were challenged subcutaneously with 3.7× 10¹⁰ cfu/ml of live P. multocida B:2. Following challenge exposure, 8 (67%) goats (4 goats from each of groups 1 and 2) were killed owing to haemorrhagic septicaemia. Four goats were killed peracutely within 48 h post challenge, while the other four goats were killed acutely between 2 and 4 days post challenge. None of the goats of group 3 were killed for haemorrhagic septicaemia. Goats of groups 1 and 2 showed significantly (p<0.05) higher antibody levels following the first intranasal exposure to P. multocida B:2. However, only group 1 retained the significantly (p<0.05) high antibody levels following a second intranasal exposure, and remained significantly (p<0.05) higher than groups 2 and 3 at the time of challenge. P. multocida B:2 was successfully isolated from various organs of goats that were killed between 1 and 4 days post challenge.     

In vitro study of Pasteurella multocida adhesion to trachea, lung and aorta of rabbits

In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.     

Biochemical characterization of lipopolysaccharides extracted from a hydrophobic strain of Pasteurella multocida

Lipopolysaccharides were extracted from freeze-dried cells of Pasteurella multocida strain P-1581 (serotype 8) by the phenol-chloroform-petroleum ether method and biochemically analysed using standard procedures. The primary neutral sugars were glucose, galactose and heptose. No deoxy sugars were detected. Amino sugars included galactosamine, glucosamine and glucosamine-6-phosphate. 3-Deoxy-d-manno-2-octulosonic acid was present at a relatively low concentration. The analyses included identification and quantification of phosphate and alanine. The primary fatty acids and their approximate relative ratios were 3-hydroxytetradecanoate and tetradecanoate 2:1. Tetradecanoic acid was bound almost exclusively by ester linkages. 3-Hydroxytetradecanoic acid was bound primarily by amide linkages, although significant numbers of ester-bound residues were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses indicated that the lipopolysaccharides were of low molecular weight.Abbreviations KDO 3-deoxy-d-manno-2-octulosonic acid - LPS lipopolysaccharide(s) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

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.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

鸭源巴氏杆菌的分离鉴定及药敏试验

本试验为确定江西新余地区鸭发病的病原,对3个鸭场的病死鸭进行细菌分离,并通过形态特征、培养特性和生化试验对分离菌进行鉴定。从病死鸭的肝脏和心脏中分离到5株鸭多杀性巴氏杆菌,且分离菌对试验鸭和小白鼠有较强的致病性。药敏试验结果表明,分离的5株菌对左氧氟沙星、卡那霉素、头孢西丁、先锋必和大观霉素较为敏感。     

Ultrastructural Observation of Nasal and Pulmonary Intracellular Pasteurella multocida A:3 in Rabbits

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multocida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the cilated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.     

Specific Antibodies of Pasteurella multocida in Newborn Calves of Vaccinated Dams

Twenty-five newborn Holstein Friesian calves, from dams vaccinated against haemorrhagic septicaemia (HS), were tested repeatedly over the first 6 months of life to monitor the transferred antibody levels against HS. Enzyme-linked immunosorbent assays were used to measure the specific HS antibodies with antigens from Pasteurella multocida strains B:2 and E:2. There was a significant curvilinear relationship between the monitored IgG response and the age of the calves. Peak serum IgG levels were obtained during the period from 8 to 16 weeks of age. Beyond this age, the concentration of IgG in the serum fell away.     

Adaptive Acquisition of Novobiocin Resistance in Pasteurella multocida Strains of Avian Origin

Naturally occurring strains of Pasteurella multocida are atypically susceptible to hydrophobic antibiotics such as novobiocin, despite their Gram-negative cell envelope ultrastructure. Four strains adaptively resistant to 1000 g/ml of novobiocin were obtained by sequentially subculturing cell surface hydrophobic variants of avian origin in the presence of increasing antibiotic concentrations. Adaptive novobiocin resistance was accompanied in all cases by the concomitant acquisition of resistance to coumermycin, a hydrophobic antibiotic possessing the same mechanism of action, but not to the functionally disparate hydrophobic antibiotic rifamycin. The acquisition of resistance was not accompanied by alterations in the lipid composition of the cell envelope. Subsequent growth of adaptively resistant strains in the absence of novobiocin did not result in the restoration of susceptibility to either novobiocin or coumermycin. Acquisition of adaptive resistance in encapsulated parental strains resulted in an inability to synthesize capsular material and enhanced cell surface hydrophobicity; however, parental encapsulation and decreased cell surface hydrophobicity were restored upon removal of novobiocin. These data suggest that acquisition of adaptive resistance to novobiocin conferred in this manner is the result of a stable genetic event affecting the mechanistic target of both novobiocin and coumermycin rather than a physiological adaptation involving outer membrane impermeability.