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.     

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.     

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.     

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.     

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.     

Rapid virulence typing of Pasteurella multocida by multiplex PCR

The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.     

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.     

Antibiotic Sensitivity Patterns among Indian Strains of Avian Pasteurella multocida

An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains of P. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains of P. multocida among Indian poultry.     

应用双重PCR检测产毒素多杀性巴氏杆菌

参照有关文献设计了2对引物。分别扩增多杀性巴氏杆菌的Kmt及toxA基因。以使同时检测并区分产毒素与非产毒素多杀性巴氏杆菌。该PCR能检出10^3cfu菌量的模板。特异性试验表明。这2对引物都不能从猪的其他6种常见病原菌扩出特异性的带。对扩增的2个PCR产物测序结果表明，2序列都有很高的保守性，从而进一步证明了该PCR方法的特异性及灵敏性。临床应用，从386份病料分离出66株多杀性巴氏杆菌，其中有8株均能同时扩出457bp和864bp的片段，而其他54株只能扩出457bp的片段。     

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.     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

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     

产毒素多杀性巴氏杆菌菌落双重PCR检测方法的建立

为建立快速特异的PCR方法以及同时检测并区分产毒素与非产毒素多杀性巴氏杆菌,本研究根据GenBank登录的多杀性巴氏杆菌KMT1基因和toxA毒素基因序列,设计合成了2对特异引物。特异性试验表明产毒素多杀性巴氏杆菌C51-6扩增出了460bp和1854bp的2条目的片段,而不产毒素多杀性巴氏杆菌、大肠埃希菌、胸膜肺炎放线杆菌、猪链球菌、支气管败血波氏杆菌、副猪嗜血杆菌和鸡白痢沙门菌的扩增均为阴性;敏感性试验表明该PCR方法能从含450CFU的菌液中扩增出相应的目的片段。同时用豚鼠皮肤坏死试验和小鼠致死试验对该PCR方法进行了验证。     

猪源多杀性巴氏杆菌的生物学鉴定与荚膜PCR分型

从表现猪萎缩性鼻炎临床症状的猪群中分离出13株多杀性巴氏杆菌(Pasteurella multocida,Pm),采用Pm种特异性的KMT1-KMT2引物进行PCR扩增,结果与传统的生化反应鉴定完全一致。基于对甘露醇、卫茅醇、山梨醇、海藻糖的发酵能力和产生鸟氨酸脱羧酶的特性,Q_1、Q_3、Q_6、Q_7、Q_(10)、Q_(13)和C_(48-1)鉴定为Pm多杀亚种(Pm subsp.multocida);Q_2、Q_4、Q_5、Q_8、Q_9、Q_(11)、Q_(12)鉴定为Pm败血亚种(Pm subsp.septica)。对13株Pm分离物采用A型、B型和D型引物进行PCR扩增,8株鉴定为A血清型(61.5%);5株鉴定为D血清型(38.5%);没有发现B血清型的菌株。同时对金黄色葡萄球菌抑制试验、中性吖啶黄沉淀试验与荚膜PCR分型的相关性进行了讨论。     

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.     

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     

兔巴氏杆菌和波氏杆菌多重PCR检测方法的建立

参考GenBank中兔巴氏杆菌16SrRNA和波氏杆菌的fim2的基因序列,应用Premier 5.0软件在二者高度保守区设计了2对引物,建立了适合巴氏杆菌和波氏杆菌快速检测的多重PCR检测方法。以该方法对巴氏杆菌和波氏杆菌参考菌株进行PCR扩增,分别能从各自的基因组中扩增出与试验设计相符的644bp和425bp的特异性DNA片段。将扩增所得的DNA片段进行克隆测序,测序结果表明分别为巴氏杆菌16SrRNA和波氏杆菌fim2基因序列。该方法对波氏杆菌的检测下限为4×102 CFU,对巴氏杆菌的检测下限为6×101 CFU。对兔源性沙门菌、葡萄球菌、大肠杆菌、魏氏梭菌扩增结果为阴性。表明所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鉴别诊断兔巴氏杆菌和波氏杆菌以及2者混合感染。