The molecular epidemiology of four outbreaks of porcine pasteurellosis

Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate a total of 38 Pasteurella multocida isolates from four separate outbreaks of pasteurellosis in Australian piggeries. Six isolates were obtained from Outbreak 1, 16 from Outbreak 2 and eight each from outbreaks 3 and 4. Outbreaks 1 and 2 were cases of pneumonic pasteurellosis while outbreaks 3 and 4 involved systemic pasteurellosis. Biochemical characterisation established that a number of different types of P. multocida were present in outbreaks 1 and 3 while outbreaks 2 and 4 were associated with a single type of P. multocida. Outbreaks 1 and 3 yielded isolates of P. multocida that belonged to the subspecies multocida and gallicida, with the subspecies multocida isolates being identified as biovar 3 (6 in total) or 12 (1 in total) and the subspecies gallicida isolates (7 in total) being identified as biovar 8. All 24 isolates from outbreaks 2 and 4 belonged to the subspecies multocida and were all biovar 3. REA and ribotyping showed that, in outbreaks 1 and 3, there were three different types of P. multocida in each outbreak with no common strains between the outbreaks. The molecular methods showed that only a single strain of P. multocida was associated with outbreaks 2 and 4, although the outbreaks were associated with strains that differed in REA profiles but shared a ribotype profile. This study has shown that both, systemic and pneumonic pasteurellosis can be associated with either a single strain or multiple strains of P. multocida. The results also indicate that the molecular typing methods of REA and ribotyping are superior to biochemical characterisation for epidemiological investigation of porcine pasteurellosis.     

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.     

Ribotype diversity of porcine Pasteurella multocida from Australia

OBJECTIVE: To use the technique of ribotyping to investigate the genetic diversity of Australian isolates of Pasteurella multocida associated with outbreaks of clinical disease in Australian pigs. DESIGN: One hundred and seven porcine P multocida isolates were analysed by ribotyping using the restriction enzymes HpaII and HindIII. The genetic population structure of the Australian porcine P multocida isolates was determined through statistical analysis of the joint ribotype patterns, and this was then compared with biochemical and epidemiological data available for the population. RESULTS: A total of 25 combined ribotypes were recognised, which were grouped into five ribotype clusters. Despite the deliberate selection of diverse isolates, the study revealed only a limited degree of genetic diversity. Fourteen of the ribotypes contained multiple isolates, and 12 of these ribotypes were present on more than one farm. Three of the seven biovars analysed in the study showed very limited diversity. All fifteen biovar 2 isolates (subsp multocida) were found in a single cluster (III), while all four biovar 8 isolates, which correspond to P multocida subsp gallicida, were allocated by themselves to a single cluster (IV). All nine of the biovar 12 isolates (lactose-positive subsp multocida) were assigned to a single cluster (I), together with the single biovar 14 isolate, which was the only other lactose-positive isolate in the population (ODC-negative). CONCLUSION: A limited number of ribotypes of P multocida are associated with Australian pigs. The majority of these ribotypes are widely distributed across multiple farms, and across multiple states. Individual farms can possess multiple ribotypes of P multocida. Some of the unusual biochemical variants of P multocida present in Australian pigs have a very limited genetic diversity. The nature of pig production in Australia, primarily involving continuous flow systems with few closed herds, has possibly contributed to the widespread distribution of a limited number ribotypes.     

Pasteurella multocida in wild mammals and birds in California: prevalence and virulence for turkeys

Samples collected from the oropharynx of wild mammals and birds trapped on 36 turkey farms in California were evaluated for the presence of Pasteurella multocida. A total of 966 animals were collected from 18 premises that had experienced an outbreak of fowl cholera within the past 2-8 months; samples were collected from 16 of these 18 premises within 2-8 weeks of outbreak notification and while the infected flock was still present. A total of 939 animals were trapped from an additional 18 premises that had not reported any outbreaks of fowl cholera within at least 4 months, if ever. Forty-eight isolates of P. multocida, of a variety of somatic serotypes, were recovered from 6 species of mammals and 3 species of birds. On only 2 of 7 premises was the somatic serotype of the isolates obtained from wildlife the same as the isolate obtained from tissues of turkeys that had died of fowl cholera on the same premises. Tests for virulence to turkeys were conducted with 31 of the isolates. Seventeen of these isolates caused mortality in turkeys. Wide ranges in mortality rates and median times to death were observed.     

Restriction endonuclease analysis of Pasteurella multocida isolates from three California turkey premises.

Three California turkey premises that had repeated outbreaks of fowl cholera were studied for periods of 2 to 4 years. Using biochemical, serologic, plasmid DNA, and restriction endonuclease analyses of isolates of Pasteurella multocida from turkeys and wildlife on the premises, strains of the organism were found to be enzootic on two of the premises. On the third, a variety of strains of P. multocida were isolated from fowl cholera outbreak flocks.     

DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys

The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related.     

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.     

Transmission of Pasteurella multocida on California turkey premises in 1988-89.

Restriction endonuclease analysis (REA) of whole-cell DNA was used to determine possible sources of Pasteurella multocida for each outbreak of fowl cholera occurring in turkey flocks in eight commercial poultry companies in California from October 1988 to September 1989. Over this period, 179 isolates of P. multocida were obtained from dead turkeys in 80 meat and breeder flocks on 43 premises. P. multocida was isolated from wildlife on five premises. Isolates were characterized by subspecies, serotype, presence of plasmid DNA, and REA type. In 52 (65%) flocks, all isolates of P. multocida had the same REA pattern as the M9 live vaccine strain following digestion of DNA with the restriction enzyme SmaI. Field strains of P. multocida were obtained from 27 (34%) flocks, and one flock (1%) yielded both M9 and a field strain of the organism. REA of field strains of P. multocida revealed 17 different SmaI REA types. Based on matching SmaI REA types, potential sources of P. multocida were identified for 15 of the 28 flocks infected with field strains of the organism, and transmission between turkey premises was a possibility in only seven flocks.     

Plasmid and restriction endonuclease patterns in Pasteurella multocida isolated from a swine pyramid.

Restriction endonuclease analysis (REA) and plasmid profile were used to study the epidemiology of Pasteurella multocida in a swine pyramid structure. The studied pyramid was comprised of a group of 12 swine farrow-to-finish farms related by unidirectional animal movement. P. multocida isolates were obtained from the lungs of 275 slaughtered pigs. Serotyping was performed by hyaluronidase sensitivity test and toxicity was investigated by the ELISA test. HpaII was used to cleave the P. multocida extracted DNA. REA patterns relationships were studied using the Sokal-Michener coefficients, and the dendrogram was built using the UPGMA system. The 218 P. multocida isolates obtained were distributed in 17 REA patterns. In 9 of the 12 farms studied only 2-3 REA patterns were detected, with one clearly predominant pattern. The 81 strains with plasmids were assigned to six plasmid profiles. REA and plasmid profiles proved to be good epidemiological tools for identifying different strains of P. multocida with the same phenotype.     

Homogeneity of characteristics of Pasteurella multocida isolated from turkeys and wildlife in California, 1985-88

Five hundred twenty isolates of Pasteurella multocida, collected in California from September 1985 to November 1988, were characterized in the laboratory. Characteristics examined included serotype, capsular type, biotype (subspecies), and possession of plasmid DNA. Three hundred thirty-three isolates recovered from turkeys dying from fowl cholera, 88 isolates from liver turkeys in flocks with fowl cholera outbreaks in the recent past, and 99 isolates from wildlife captured on fowl cholera-outbreak and non-outbreak turkey premises were studied in this manner. Characteristics were fairly homogeneous among isolates, especially those obtained from turkeys. The majority of isolates were serotype 3,4, capsular type A, subspecies multocida, and lacked plasmid DNA. Common serotypes of isolates from turkeys and wildlife sampled on the same premises were noted in eight of 13 cases examined.     

Differentiation of field isolates of Pasteurella multocida serotype 3,4 from live vaccine strain by genotypic characterization

Fifty-five serotype 3,4 isolates of Pasteurella multocida, isolated from turkeys dead from fowl cholera, were characterized (fingerprinted) genotypically for comparison with the serotype 3,4 live fowl cholera vaccine principally used in turkeys in California. Twenty-three isolates were obtained from turkeys vaccinated with the M9 live vaccine, and 32 additional isolates were from turkeys not vaccinated for fowl cholera. Methods of characterization included restriction endonuclease analysis of chromosomal DNA and ribotyping, a technique for highlighting restriction site heterogeneity of highly conserved ribosomal RNA genes and associated sequences using a radiolabeled rRNA probe. Eight different genotypes or ribotypes were detected in these isolates by the above methods. Of 23 isolates from M9-vaccinated turkeys flocks, 19 were the same ribotype as M9. Thirty of 32 isolates recovered from unvaccinated turkeys were different ribotypes from M9. The remaining two isolates resembled M9 and were recovered from two different flocks placed in succession on a turkey farm where a flock placed previously had been vaccinated with M9, suggesting interflock transmission. Ribotyping and restriction endonuclease analysis appear to be useful tools to aid in the determination of the role that the live vaccine plays in fowl cholera epidemiology.     

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.     

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.     

Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida

Eighty-one isolates presumptively identified as Pasteurella multocida from a variety of diseases in animals in Zimbabwe were subjected to biochemical characterization, capsular typing and RAPD analysis. The majority of isolates (over 80%) were assigned into named taxa and were predominantly P. multocida subsp. multocida and P. multocida subsp. septica, whilst the remainder were unassigned. Serogroup A was predominant among the three capsular types (A, B and D) of P. multocida detected. Three main RAPD clusters and three subclusters were observed among the majority of isolates (93.8%), whilst the remainder was found to be weakly related. Nine different groups of strains with similar RAPD profiles (100% similarity) were also observed. The reference strain of capsular serogroup F clustered with the reference strain of P. multocida subsp. septica, whilst all other serogroups clustered with reference strains of subsp. multocida and gallicida. Notably, serogroups A and D were observed to be closely related to the reference strain of subsp. multocida. The relationship between biotype, capsular type, host origin and disease manifestation was not clear-cut. However, most pig isolates of subsp. multocida clustered together as did most cattle isolates of subsp. multocida. RAPD tended to separate subsp. multocida from septica.     

Case control study of fowl cholera in meat turkeys in California from August 1985 through July 1986 [corrected

From Aug 1985 through July 1986, 720 meat turkey flocks on 160 California premises were monitored and outbreaks of fowl cholera (Pasteurella multocida) were investigated. Data from 43 outbreak (case) flocks were compared with data from 43 nonoutbreak (control) flocks. Outbreak flocks, compared with control flocks, were more likely to be located on premises with higher maximal bird capacity and history of fowl cholera outbreaks. The overall impression was that flocks in larger, newer, more intensively managed premises were at greater risk of fowl cholera outbreaks than were other flocks.     

Epidemiologic studies on infectious coryza outbreaks in northern New South Wales, Australia, using serotyping, biotyping, and chromosomal DNA restriction endonuclease analysis

The epidemiology of 16 cases of infectious coryza, an upper respiratory tract disease of chickens caused by Haemophilus paragallinarum, was investigated in a retrospective study. The cases occurred over a 14-month period on 10 farms in northern New South Wales. The available field data indicated that the cases formed six unrelated outbreaks. The 16 isolates of H. paragallinarum were subjected to serotyping by the Page and Kume schemes and biotyping based on carbohydrate fermentation and antimicrobial drug-resistance patterns. As well, newer fingerprinting techniques--plasmid profiles, whole-cell protein profiles, immunoblots of whole-cell protein profiles and total DNA restriction endonuclease analysis (REA)--were evaluated. Antimicrobial biotyping and REA profile typing proved most useful, allowing the recognition of three groups among the isolates. The other techniques gave either limited or no subdivision among the isolates. The combined results of the laboratory study indicated that, rather than six unrelated outbreaks, the 16 isolates represented three pairs of related outbreaks. This study represents the first application of sensitive biotyping and fingerprinting techniques to outbreaks of infectious coryza. The results have established that farms can be repeatedly infected with a single strain of H. paragallinarum that re-emerges at intervals. This study also obtained the first detailed evidence that replacement stock are a major source of infectious coryza.     

Molecular epidemiology of Pasteurella multocida in dairy and beef calves

The molecular epidemiology of Pasteurella multocida has rarely been studied at the farm level in cattle. The aim of this study was to determine whether single or multiple strains of P. multocida tend to exist within farms. Molecular characterisation was carried out on isolates obtained from nasal swabs from 105 calves from 32 randomly selected beef and dairy farms located throughout Scotland, and from 131 calves from 20 farms in the Mayenne region of France, where sampling occurred in response to respiratory disease outbreaks. P. multocida isolates were characterised by random-amplified polymorphic DNA (RAPD) typing and pulsed-field gel electrophoresis (PFGE) using restriction enzyme ApaI. In addition, isolates representative of each farm/RAPD profile combination were typed by multilocus sequence typing (MLST). Among 105 Scottish isolates, 15 RAPD profiles were distinguished. The majority of farms (27/32) had indistinguishable profiles in all positive animals. Five farms had two profiles. Among 140 French isolates, 23 RAPD profiles were distinguished. More within-farm heterogeneity was observed although 10/20 farms had just one profile (E4) in sampled calves. Profile E4 accounted for 60% (84/140) of French isolates. PFGE was more discriminatory than RAPD but confirmed results with respect to within farm homogeneity or heterogeneity of strains, whereas MLST was not discriminatory enough for farm level epidemiology. As in other host species, either several strains or one dominant strain of P. multocida may exist within farms, with evidence for a role of management factors such as movements onto the farm in the number of strains detected.     

The first outbreak of fowl cholera in Muscovy ducks (Cairina moschata) in Japan.

The first outbreak of fowl cholera occurred in a flock of Muscovy ducks (Cairina moschata) in Okinawa Prefecture of Japan in November 1990. Fifty (25%) of 200 birds in a farm died of an acute disease. Remaining birds recovered after treatment with oxytetracycline. Pasteurella multocida subsp. multocida was isolated in pure culture from all tissues tested from two dead birds. Serovars of the isolates were identified as Carter's capsular type A. Heddleston's type 3.4.12, and Namioka's type 5:A which have not been demonstrated in Japan. Pathologically, multiple necrosis and bacterial aggregates were prominent in several organs, particularly in the liver. The isolate killed chickens when inoculated intravenously at a concentration of 10(8) colony forming units.     

A survey of potential virulence markers from avian strains of Pasteurella multocida

Twenty-four isolates of Pasteurella multocida from clinical cases of fowl cholera and the Clemson University vaccine strain were surveyed for the presence of potential virulence markers. Membrane proteins, enzymatic activity of the membrane proteins, and carbohydrate fermentation patterns were also determined to demonstrate phenotypic relationships within the groups. Few differences were found in these phenotypic characteristics among the isolates. Almost all the organisms produced siderophore and were hemolytic on turkey red blood cells. No extracellular enzyme or bacteriocin activity was detected and little antibiotic resistance was found. However, many organisms contained plasmids and demonstrated some degree of resistance to complement. Both characteristics were correlative markers in Pasteurella multocida isolated from birds with fowl cholera.     

禽巴氏杆菌Pm-HB株的分离与鉴定

湖北省某鸡场疑似巴氏杆菌(Pasteurella)感染,从送检病鸡的肺脏和肝脏中分离到1株细菌,通过菌落形态、培养特性、生化试验、16S rRNA序列比对鉴定为禽多杀性巴氏杆菌,并命名为Pm-HB株。动物回归试验表明,10 CFU细菌即可100%致死30日龄SPF鸡,可确诊为巴氏杆菌病。