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.     

Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes.

Three panels of monoclonal antibodies (MAbs) were prepared against the spike (S) proteins of infectious bronchitis virus (IBV) strains Arkansas 99, Connecticut 46, and Massachusetts 41. Based on enzyme-linked immunosorbent assay (ELISA), the MAbs were grouped into three categories: 1) group-specific, which reacted with a broad spectrum of homologous and heterologous IBV serotypes; 2) serotype-specific, which reacted only with strains of the homologous serotype; and 3) strain-specific, which reacted "selectively" with only certain strains of homologous and heterologous serotypes. MAbs that displayed serotype specificity were all specific to S1 fractions of the homologous serotype, confirming that epitopes that determine virus serotype are associated with the S1 protein. An excellent correlation was found when the results of IBV serotyping by MAb-based indirect ELISA were compared with those from the conventional virus-neutralization test. This confirms that the MAbs described here will serve as valuable tools in epizootiological studies and serotype-specific diagnosis of IBV infection.     

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.     

Avian cholera in a gyrfalcon (Falco rusticolus)

A 2-year-old female gyrfalcon (Falco rusticolus) was being used to hunt ducks in southeastern Wyoming during an outbreak of avian cholera in waterfowl and wild turkeys. While out overnight, the falcon consumed a bird. Within 24 hours the falcon was anorectic, and it was found dead approximately 48 hours following ingestion of wild prey. Gross and microscopic lesions were typical of avian cholera, and Pasteurella multocida serotype 1 was isolated from tissues of the falcon.     

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.     

Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.     

禽源多杀性巴氏杆菌多位点序列分型研究

为了解国内禽源多杀性巴氏杆菌流行情况,对分离自18省份的84株多杀性巴氏杆菌采用荚膜多重PCR分型和多位点序列分型对其血清型和基因型进行鉴定。结果表明:禽源多杀性巴氏杆菌主要以血清A型为主,占96.4%(81/84);多位点序列分型可将禽源多杀性巴氏杆菌分为5种ST型,其中ST129为主要流行型,占94.0%(79/84)。本研究为我国禽源多杀性巴氏杆菌的流行病学监测和基因多样性提供了数据支持。     

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.     

Virulence of raptor-origin Pasteurella multocida in domestic chickens.

Pasteurella multocida belonging to somatic serotype 1 and capsular type A has been known to cause avian cholera in domestic poultry. Pasteurella multocida serotype 1 has also been isolated from raptorial birds. However, the capsular type for these raptorial isolates remains unknown. Moreover, the virulence of these raptorial isolates for domestic poultry has not been determined. The objectives of this study were to determine the capsular type of raptorial P. multocida serotype 1 isolates and to determine if these isolates were virulent for domestic chickens. Study chickens were inoculated with one of three P. multocida isolates. Isolate WESO-1 was obtained from a western screech owl (Otus kennicottii) and isolates RTHA-2 and RTHA-4 were isolated from two red-tailed hawks (Buteo jamaicensis). These isolates were given by either the oral, intravenous, or intraocular route. Control birds were given brain-heart infusion broth. The capsular serotypes of three isolates were also determined. The RTHA-2 and RTHA-4 isolates belonged to P. multocida capsular type A. The WESO-1 isolate belonged to capsular type F. Results also demonstrated that, for the isolates examined, the intraocular route did not cause mortality in chickens. There was mortality in all groups for the intravenous route. However, various mortality patterns were observed when P. multocida was given orally for the three different isolates. The RTHA-4 isolate (serotype 1:A) was the most virulent for domestic chickens. The WESO-1 isolate (serotype 1:F) was the least virulent for chickens among the raptorial isolates examined.     

Genomic distribution of a serotype 1-specific antigen-coding DNA fragment of Pasteurella haemolytica.

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.     

Molecular epidemiology of Pasteurella multocida in turkeys.

Pasteurella multocida isolated from turkeys during an outbreak of fowl cholera was characterized by serotype and heterogeneity of genes encoding rRNA (ribotype) to investigate the epidemiology of the organism. Isolates were collected between October 1985 and July 1986. The M9 or Clemson University fowl cholera vaccine-like strain was detected in 17% of the flocks with fowl cholera. One particular strain, isolated only from breeder flocks, was recovered from 7 of the 10 breeder flocks examined in this study. Intracompany transmission appeared to be common, implying a failure in biosecurity. Circumstantial evidence indicated that in the field; the incubation period of P multocida in a turkey flock may be between 2 to 7 weeks. Wildlife did not appear to be an important reservoir of P multocida for turkeys during this study period. Ribotyping results tended to discount several of the possible interflock transmissions, as suggested by examination of serotyping results alone; however, serotyping in combination with ribotyping proved helpful in understanding the epidemiology of P multocida in turkeys.     

Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks

Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry. An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R. anatipestifer infection in ducks. The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R. anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein. The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R. anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain. Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms. Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01). The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E. coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica. In addition, the DNA sequence encoding P45 was detected in R. anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R. anatipestifer serotypes. Thus, the ELISA described is applicable to the detection of R. anatipestifer infection in ducks.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Somatic serotypes of Pasteurella multocida strains isolated from avian hosts (1976-1988)

During the period of 1976-88, 733 strains of Pasteurella multocida isolated from avian hosts were submitted to the National Animal Disease Center for somatic serotyping. The serotypes were determined and are presented in a summarized form in this report. The most common serotypes were 3 (29%); 1 (18%); 3,4 (12%); and 3,4,12 (9%). The 733 strains had been isolated from 25 species of avian hosts; 400 (55%) were from turkeys. The most common serotypes of strains from turkeys were 3 (38%); 3,4 (18%); 3,4,12 (11%); and 4 (4%).     

Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.     

Infection of duck plague carriers with Pasteurella multocida and P. anatipestifer

Mallards (Anas platyrhynchos platyrhynchos) and white pekin ducks (Anas platyrhynchos domesticus) were infected with duck plague virus and challenged with LD20's of Pasteurella multocida and P. anatipestifer. There was no difference between mortality rates of duck plague-infected ducks and controls, suggesting that these organisms do not act synergistically under the conditions of our experiments. There was a difference of about 500-fold between the LD20 of P. multocida for mallards and that for white pekin ducks, indicating that mallards are much more susceptible to avian cholera than white pekin ducks.     

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam. The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P. multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry.     

Molecular epidemiology of two fowl cholera outbreaks on a free-range chicken layer farm.

Two outbreaks of fowl cholera on a multiage free-range egg farm were investigated. The outbreaks occurred in 1994 and 2002. A total of 22 strains of Pasteurella multocida were available for study, 11 from the 1994 outbreak and 11 from the 2002 outbreak. Lesions typical of acute fowl cholera were seen in the 1994 outbreak, whereas both acute and chronic fowl cholera occurred in the 2002 outbreak. The isolates were examined in an extended phenotypic typing methodology, by a P. multocida-specific polymerase chain reaction (PCR), by the Heddleston somatic serotyping scheme, and by restriction endonuclease analysis (REA) typing using the enzyme HpaII. All 22 strains had the same phenotypic properties, all were confirmed as P. multocida by PCR, all were Heddleston serovar 4, and all had the same REA pattern. The results indicate that these 2 outbreaks were caused by the same clone of P. multocida--despite the 8-year time period between the outbreaks.     

鸭疫里默氏菌基因分型

在鸭疫里默氏菌 (RA)血清分型的基础上 ,以提取的 RA基因组 DNA为模板 ,进行 Rep- PCR扩增。根据扩增产物的琼脂糖凝胶电泳图谱 ,将分属于 8个血清型的 4 0株 RA区分为 12个不同的基因型 ,不同血清型 RA的图谱有明显差异 ,相同血清型 RA的图谱也存在差异。这些结果显示 ,RA基因组中重复的基因外的回文结构可用于区别相同血清型的不同分离株 ;Rep- PCR作为一种实用的分子生物学方法 ,在分子流行病学研究中可对 RA分离株进行基因定型     

A new serotype of Pasteurella multocida associated with fowl cholera

Gel-diffusion precipitin tests demonstrated an additional Pasteurella multocida serotype, designated serotype 16. Isolate P-2723, antigenically distinct from the other (previously reported) 15 serotypes, was from a turkey affected with fowl cholera. This serotype is not widely distributed. Isolate P-2723 was of mild virulence in turkeys, resulting in local infections in the hock joint and sternal bursa of only 1 of 9 turkeys exposed.