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.     

The molecular biology of pasteurella multocida

Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis. Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent application of molecular methods such as the polymerase chain reaction, restriction endonuclease analysis, ribotyping, pulsed-field gel electrophoresis, gene cloning, characterisation and recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic mapping, have greatly increased our understanding of P. multocida and has provided researchers with a number of molecular tools to study pathogenesis and epidemiology at a molecular level.     

Pharmacokinetics of penicillin G in the turkey

Parmacokinetics of penicillin G was determined for the turkey. The study was prompted by the isolation of a sulfonamide-resistant strain of Pasteurella multocida from tissues of turkeys involved in an outbreak of fowl cholera and the subsequent discovery that little pharmacologic information was available concerning other antimicrobial agents in that species. Penicillin G was chosen for study because P multocida is susceptible to this antibiotic. The elimination of the antibiotic followed first-order kinetics, and the half-life was found to be 0.5 hours. Parenteral administration of benzathine-procaine penicillin G resulted in higher concentrations, which persisted for longer periods than did procaine or potassium salts of the antibiotic.     

Pasteurella multocida as the cause of disease outbreaks in commercial poultry flocks

Six cases of fowl cholera in growing turkeys and 3 in adult breeder chickens of the broiler type as well as one case each of a Pasteurella (P.) multocida-associated disease in ducklings and goslings were described in consideration of own laboratory findings and available informations of the case history. Furthermore a report is given on a treatment strategy successfully used in turkeys with highly acute fowl cholera. All the P. multocida strains isolated culturally could be assigned to the subspecies multocida. In one case Bordetella avium, Salmonella (S.) arizonae and S. hadar were additionally cultured form part of turkeys submitted. P. multocida and Moraxella (Pasteurella) anatipestifer could be determined as the causative agents of the disease of ducklings and goslings. P. multocida strains from turkeys were identified serologically as serovars A:3.4 (3x), F:3.4 (2x) and A:3 (1x); those from the breeder chickens as A:3 (3x); and one each from ducklings and goslings as F:3.4 and -:3. (uncapsulated). No death occurred in turkeys with clinical signs of a highly acute fowl cholera if the treatment of the affected birds was started with an intravenous injection of sulfadimethoxine and continued with a combination of sulfachlorpyridazine (SCP) and trimethoprim (TMP) given in the drinking water for 5 days. However relapse occurred 2-3 days after withdrawal of the drug, although the therapy was clinically highly effective. The recurrence of the disease could be prevented reliably if the turkeys were vaccinated with an effective oil-based bacterin and subsequently treated with the SCP-TMP combination given in drinking water over a 12 day period.     

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.     

Fowl cholera: influence of Bordetella avium on vaccinal immunity of turkeys to Pasteurella multocida

Turkeys exposed to Bordetella avium were vaccinated against fowl cholera with live Pasteurella multocida vaccine. Previous exposure to B. avium resulted in impairment of systemic immunity conferred by the vaccine: 86% of the vaccinated turkeys exposed to B. avium at 1 day old developed lesions or died of fowl cholera after challenge at 15 weeks old with virulent P. multocida. Of vaccinated turkeys not previously exposed to B. avium, only 26% had lesions or died of fowl cholera.     

Comparison of various antigens and their ability to detect protective antibodies against Pasteurella multocida using enzyme-linked immunosorbent assay

Various antigenic extracts of the CU strain of Pasteurella multocida were prepared to determine their suitability as plate antigens for use in the enzyme-linked immunosorbent assay (ELISA) for the detection of fowl cholera antibodies. Antisera from two separate broiler breeder flocks with known fowl-cholera-vaccination histories were collected just before the birds were challenged with virulent strain X-73 P. multocida. A potassium thiocyanate (KSCN)-extracted antigen, a capsular (CAP) antigen, a lipopolysaccharide-protein antigen, and heat-stable, salt-soluble antigen were all suitable as ELISA plate-coating antigens. Filtered and unfiltered sonicates of the CU strain of P. multocida were also suitable ELISA plate antigens. The results suggested that different plate antigens were detecting different populations of antibodies formed in response to fowl cholera vaccinations. When antibody titers were correlated with survival after challenge, the KSCN and the CAP plate antigens placed more nonsurvivors into low-antibody-titer ranges and more survivors (protected birds) into the high-antibody-titer ranges than the other plate antigens.     

The response of broiler breeder chickens to parenteral administration of avirulent Pasteurella multocida

Broiler breeder chickens were exposed to avirulent Pasteurella multocida at 14, 22, and 34 weeks of age either by stick wing 1 to 3 times or subcutaneously 3 times. Fowl pox vaccine was mixed with the first P. multocida exposure in some groups. Exposure did not impair egg production or hatch of fertile eggs. Challenge with pathogenic P. multocida serotype 1 at 68 weeks indicated that exposure to avirulent P. multocida 2 or 3 times provided better protection than 1 exposure. Mixing fowl pox vaccine with the avirulent P. multocida did not reduce immunity to fowl cholera or fowl pox.     

Diversity of avian Pasteurella multocida strains based on capsular PCR typing and variation of the OmpA and OmpH outer membrane proteins

One hundred avian Pasteurella multocida isolates recovered from cases of fowl cholera and related infections in England and Wales over a 13-year period were characterised by capsular PCR typing and analysis of outer membrane protein (OMP) profiles. Sixty-eight percent of the strains were of capsular type A, 14% were type F, 5% were type D, 4% were type B and 9% were untypable. Nineteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Fifty-six percent of the isolates were represented by 15 OMP-types, whereas 44% of the isolates were associated with four OMP-types. The extensive molecular mass heterogeneity of the OmpA and OmpH proteins supports previous findings that avian P. multocida strains are very diverse. Furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. The high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of P. multocida are opportunistic pathogens of relatively low virulence. Strains of capsular types B, D and F, as well as the untypable isolates, were associated exclusively with specific OMP-types and represent distinct and widely disseminated clonal groups. These observations support the view that avian strains of P. multocida have a clonal population structure. Based on previous studies, the molecular mass heterogeneity of the OmpA and OmpH proteins might provide a selective advantage to P. multocida by generating antigenic variation.     

Diagnostic and typing options for investigating diseases associated with Pasteurella multocida

Pasteurella multocida is responsible for major animal diseases of economic significance in both developed and developing countries whereas human infections related to this bacterium are infrequent. Significantly, development of a carrier status or latent infections plays a critical role in the epidemiology of these diseases. Aiming at increased knowledge of these infections, we examine potential diagnostic and selected typing systems for investigating diseases caused by P. multocida. Detection of P. multocida from clinical specimen by; (i) isolation and identification, (ii) polymerase chain reaction (PCR), iii) specific hybridisation probes, (iv) serological tests and (v) other alternative methods is critically evaluated. These detection systems provide a wide spectrum of options for rapid diagnosis and for detecting and understanding of latent infections in herd/flock health control programmes, though PCR methods for detecting P. multocida in clinical specimen appear increasingly preferred. For establishing the clonality of outbreak strains, we select to discuss macromolecular profiling, serotyping, biotyping, restriction enzyme analysis, ribotyping and multiplex PCR typing. Although P. multocida infections can be rapidly diagnosed with molecular and serological tests, isolation and accurate species identification are central to epidemiological tracing of outbreak strains. Our review brings together comprehensive and essential information that may be adapted for confirming diagnosis and determining the molecular epidemiology of diseases associated with P. multocida.     

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.     

Characterization of an avian cholera epizootic in wild birds in western Nebraska

Avian cholera killed an estimated 2500 birds in western Nebraska and eastern Wyoming from 28 November 1985 to late January 1986. Wild mallards (Anas platyrhynchos) suffered the most losses. Other wild waterfowl, wild turkeys (Meleagris gallopavo), a few domestic fowl, and a bald eagle (Haliaeetus leucocephalus) also died. Pasteurella multocida serotype 1 was the predominant isolate from these carcasses. Cold, wet weather persisted throughout the outbreak, but daily losses in the flock of 50,000 mallards using the area were low. Pasteurella multocida was isolated from nasal swabs of 35 of 37 cattle from a feedlot in which many of these mallards were feeding. Eighty percent of the cattle isolates had antigenic characteristics of serotype 3 or serotype 3 with cross-reactivity. Isolates from wild mallards, wild turkeys, and the bald eagle were virulent to game-farm mallards when inoculated subcutaneously, but P. multocida isolates from cattle were not.     

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.     

Characterisation of Pasteurella multocida isolated from fowl cholera outbreaks on turkey farms

SUMMARY: Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate Pasteurella multocida isolates from outbreaks of fowl cholera on 7 turkey farms in New South Wales. While only a single isolate was available from 5 of the farms, multiple isolates, 4 and 12 respectively, were available from the other 2 farms. The available field evidence suggested that 8 outbreaks had occurred with one farm suffering 2 outbreaks. The isolates obtained were all confirmed as Pasteurella multocida . Biochemical profiles allocated the isolates to 4 groups, 3 being variants of P multocida subsp multocida and the fourth being P multocida subsp septica . REA performed with Hpall established 7 groups. Ribotyping using the Hpall digests probed with the 16S rRNA operon of Haemophilus paragallinarum recognised the same 7 groups as REA. Unlike the biochemical profiles, both REA and ribotyping provided a fine subdivision that identified outbreaks as either related or unrelated. The REA and ribotyping patterns as well as biochemical profiles were stable for all isolates from the outbreaks in which multiple isolates were obtained from either the same bird or from different birds. REA and ribotyping were found to be superior to biotyping methods for the investigation of fowl cholera outbreaks.     

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.     

鸡禽霍乱疾病流行病学特点及防控措施

鸡养殖中禽霍乱疾病的出现主要是由于多杀性巴氏杆菌感染的一种传染性疾病,主要会导致鸡只出现急性败血症和机体炎症反应等,慢性型感染会导致鸡只出现皮下及机体内脏器官、关节等的化脓性炎症反应。禽霍乱疾病在鸡养殖全年均会出现,尤其是在气温变化较大的时期疾病爆发的可能性更高。禽霍乱疾病在急性爆发的情况下病死率相对较高,同时短期内即会出现死亡的情况,难以进行有效的治疗,易导致鸡养殖户出现严重的经济损失。该文主要对鸡禽霍乱疾病的流行病学特点、临床症状及相关的防治措施进行论述。     

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production.Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group 1 was orally infected with 1000+/-50 embryonated A. galli eggs. Group 2 received 10(4) cfu P. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for 11 weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded.The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion.In conclusion a negative interaction between A. galli and P. multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli.     

Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania

Investigation was done to determine the presence of Pasteurella multocida and related species in free ranging chickens and ducks, dogs, cats and pigs in three climatic zones (cool, warm and hot) of rural Morogoro, Tanzania. A total of 153 isolates of P. multocida ssp. multocida and related species were obtained by direct culture on blood agar, selective medium and mouse inoculation. P. multocida ssp. multocida was isolated from 0.7% of chickens and 7% of ducks. In dogs and cats, P. multocida ssp. multocida was isolated from 1 and 68%, respectively. One isolate of Pasteurella gallinarum was isolated from a duck. Other species obtained were; P. multocida ssp. septica, Pasteurella stomatis and taxon 16 from dogs and cats, while Pasteurella dagmatis and Pasteurella canis were found in dogs only. Prevalence of P. multocida ssp. multocida was significantly higher (P<0.01) in ducks of the warm zone (22%) than in ducks of other zones (0%). No significant difference was observed between the prevalence of P. multocida ssp. multocida in chickens of the warm zone (2%) and chickens of the cool and hot zones (0%). Extended phenotypic characterization revealed phenotypic similarities between two isolates from chickens and the duck strains. Mouse inoculation appeared to be more sensitive in detecting P. multocida ssp. multocida than blood agar and selective medium. Direct culture on blood agar recovered most of the isolates from dogs. This study has demonstrated for the first time the presence of P. multocida and related species in the village free ranging chickens, ducks, dogs and cats in Tanzania. Other non-classified Pasteurella spp. were also observed in the study, but further characterization is required before the final classification can be made. This paper reports for the first time the isolation of unclassified Pasteurella from dogs and cats in Africa. The results implies that fowl cholera might be occurring in free ranging poultry, and dogs and cats kept in contact might serve as sources of P. multocida to chickens and ducks. Subsequent applications of molecular techniques to analyse the epidemiological relatedness of clones isolated from different host species is indicated.     

A review of hemorrhagic septicemia in cattle and buffalo

Hemorrhagic septicemia (HS), an acute, fatal and septicemic disease of cattle and buffaloes caused by Pasteurella multocida, is important in tropical regions of the world, especially in African and Asian countries. The prevalence of disease has been well documented with predominant isolation of P. multocida serotypes B:2 and E:2. Conventional methods of identification such as serotyping, biotyping, antibiogram determination and pathogenicity as well as molecular methods (P. multocida-specific polymerase chain reaction (PCR), a serogroup B-specific PCR assay, multiplex capsular typing system and loop-mediated isothermal amplification techniques) and characterization (restriction endonuclease analysis, randomly amplified polymorphic DNA analysis, repetitive extragenic palidromic PCR and enterobacterial repetitive intergenic consensus PCR analysis) are applied in parallel for rapid epidemiological investigations of HS outbreaks. Although several vaccine formulations including alum precipitated, oil adjuvant and multiple emulsion vaccines are commercially available, the quest for suitable broadly protective HS vaccines with long-lasting immunity is on the upsurge. Concurrently, attempts are being made to unravel the mysteries of the pathogen and its virulence factors, pathogenesis and determinants of protective immunity as well as diversity among strains of P. multocida. This review highlights the advances in these various aspects of HS.     

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.