Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera.

A 5' Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5' Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5' Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

An outbreak of infectious hepatitis in commercially reared ostriches associated with Campylobacter coli and Campylobacter jejuni.

A disease causing high morbidity and mortality was observed in young ostriches from six properties in southeast Queensland, Australia. The disease affected birds from 2-8 weeks of age and was characterised clinically by bright-green urates and pathologically by severe necrotic hepatitis. The liver lesions resembled those of vibrionic hepatitis in other avian species. Campylobacter coli was isolated from the livers of affected ostriches from five of the six properties. Campylobacter jejuni subsp. jejuni was isolated from birds from the remaining property. Pulsed-field gel electrophoresis-based (PFGE) typing of representative isolates indicated that trade of infected birds between farms was an important factor in the spread of C. coli. Phenotypic and genotypic data suggest a clonal variant of the principal outbreak type may account for the remaining cases from which C. coli was found. Conventional biochemical test results and PFGE clearly distinguished the C. jejuni strain isolated from the geographically remote farm from the outbreak of C. coli type. We believe this to be the first definitive report of avian hepatitis associated with C. coli.

Intestinal spirochete infections of chickens: a review of disease associations, epidemiology and control.

This paper presents an overview of intestinal spirochete infections of chickens. It focuses particularly on studies in Australia, where recent surveys of 136 layer and broiler breeder flocks have revealed a high rate of infection (>40%) with intestinal spirochetes. Infection was not detected in broiler flocks. Approximately 50% of isolates from infected flocks were Brachyspira (Serpulina) intermedia or B. pilosicoli, with the other isolates being B. innocens, B. murdochii or the proposed species 'B. pulli'. No isolates of B. alvinipulli were found. Intestinal spirochetes were significantly associated with wet litter problems and/or reduced egg production. Experimental infection of point-of-lay birds with either B. intermedia or B. pilosicoli caused reduced egg production, and, with B. intermedia, a significant increase in fecal moisture content. Infection with B. innocens caused no significant changes. In-water treatment of a flock with a mixed spirochete infection using lincospectin resulted in a slimy diarrhea lasting for 2-3 weeks, followed by absence of spirochetes for 3 months. Birds treated with tiamulin remained healthy, and had a reduced level of infection with intestinal spirochetes (30%) for 3 months. Trials are under way to test the efficacy of antimicrobials in point-of-lay chickens experimentally infected with either B. intermedia or B. pilosicoli.

Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia varigena.

OBJECTIVE: To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella-Actinobacillus and obtained from cattle and sheep. DESIGN: The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia--M. haemolytica, M. glucosida, M. granulomatis, M. ruminalis and M. varigena. RESULTS: Thirty-four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty-nine were M. haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M. haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M. haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M. haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M. granulomatis--one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M. varigena--one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia. CONCLUSION: The study represents the first time that M. haemolytica, M. granulomatis and M. varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella-Actinobacillus-like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.

Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis.

OBJECTIVE: To validate a polymerase chain reaction (PCR) based method. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), for the fingerprinting of Haemophilus parasuis strains and to use that method to differentiate isolates from apparently related outbreaks of Glässer's disease on three pig farms. DESIGN: ERIC-PCR was evaluated by comparing 15 different strains that represented all 15 recognised serovars in the Kielstein-Rapp-Gabrielson (KRG) scheme for serotyping H parasuis. Next, ERIC-PCR was used to examine 14 Australian field isolates of H parasuis; 12 collected from three farms suffering apparently related outbreaks of Glässer's disease and two from two other farms with no known connection. RESULTS: The 15 serovar reference strains all gave unique, reproducible ERIC-PCR fingerprints. The 12 isolates from the three apparently related outbreaks all gave a single fingerprint, which was distinct from any seen in the 15 serovar reference strains and the two other Australian field isolates in the studied farms. The confirmation that all 12 isolates were the same strain allowed the development of a prevention and control program that has prevented the emergence of any further outbreaks of Glässer's disease on the three farms. CONCLUSION: ERIC-PCR is a suitable technique for the differentiation of unrelated strains of H parasuis. The finding that the 12 field isolates of H parasuis all shared the same fingerprint is strong evidence that there was a common source of infection on all three farms. This study has shown, for the first time, that ERIC-PCR is a suitable technique for the sub-typing of H parasuis and useful for studying the epidemiology of outbreaks of Glässer's disease.

Polyarthritis in wild mice (Mus musculus) caused by Streptobacillus moniliformis.

Mice trapped on farms in south-eastern Queensland had chronic abscessating osteoarthritides mainly involving the carpi and tarsi. Pleomorphic bacteria were shown by silver staining to be plentiful within lesions. Streptobacillus moniliformis was isolated from the joints, and streptobacillary polyarthritis was diagnosed. Other lesions observed included subcutaneous and hepatic abscesses.

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens.

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n = 25) and Brachyspira pilosicoli (n = 17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC > 4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC > 32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Evaluation of tiamulin and lincomycin for the treatment of broiler breeders experimentally infected with the intestinal spirochaete Brachyspira pilosicoli.

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 30 individually caged 22-week-old Cobb 500 broiler breeder hens. Another 10 birds were sham-inoculated with sterile broth. All birds failed to become colonized. At 29 weeks of age, all birds were transferred to a diet containing 50 parts/10(6) zinc bacitracin (ZnB) and were re-challenged with the same B. pilosicoli strain at 32 weeks of age, weekly for 5 weeks. The majority of the inoculated birds then became colonized, confirming previous findings that ZnB can increase susceptibility to colonization with B. pilosicoli. The control group remained uninfected. Infected groups tended to have an increased faecal water content and faecal staining of eggshells. Ten birds were then treated by crop tube with 25 mg/kg body weight tiamulin for 5 days, and 10 birds with 20 mg/kg body weight lincomycin for 5 days. Both treatments removed the infection, while untreated birds remained infected. The results support previous observations that ZnB at 50 parts/10(6) in the diet increases the susceptibility of birds to B. pilosicoli infection, and demonstrated the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.

Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia.

Faecal samples (n = 1786) from chickens in broiler breeder (n = 28), layer (n = 22) or broiler (n = 19) flocks in the eastern states of Australia were cultured for intestinal spirochaetes. Overall, birds in 42.9% of broiler breeder and 68.2% of layer flocks were colonized with spirochaetes, but no birds in broiler flocks were infected. Colonization rates in infected flocks ranged from 10 to 100% of birds sampled. Faeces from colonized flocks were on average 14% wetter than those from non-colonized flocks. There was a highly significant association between colonization with spirochaetes and the occurrence of wet litter and/or reduced production. A subset of 57 spirochaete isolates from birds in 16 flocks were identified to the species level using a panel of polymerase chain reaction tests. Isolates from nine (56%) of these flocks were spirochaetes that are known to be pathogens of poultry: Serpulina pilosicoli was isolated from birds from five flocks, birds from two flocks were infected with Serpulina intermedia, and in two other flocks both species were identified. Isolates from the other seven flocks belonged to other Serpulina species, which are currently of unknown pathogenicity. This study indicates that infections with intestinal spirochaetes are a common but currently under-diagnosed cause of wet litter and/or reduced egg production in broiler breeder and layer flocks in Australia.

Influence of in-feed zinc bacitracin and tiamulin treatment on experimental avian intestinal spirochaetosis caused by Brachyspira intermedia.

Thirty individually caged layer hens were inoculated with Brachyspira intermedia, and 20 control birds remained unchallenged. Birds received a diet containing 100 parts/10(6) zinc bacitracin (ZnB), and were monitored for 10 weeks. B. intermedia was recovered sporadically from five of the inoculated birds, and there were no significant effects on body weight, faecal water or egg production. ZnB was presumed to be indirectly inhibiting spirochaete growth, and when removed from the diet, 18 of the 30 inoculated birds rapidly became culture positive. After 4 weeks, 10 of the 30 infected birds were treated with tiamulin at 25 mg/kg for 5 days, and 10 were returned to the diet containing ZnB. Birds receiving tiamulin became spirochaete negative and maintained their egg production, but re-infection occurred. The other 20 infected birds had a significant drop in egg production, but those receiving ZnB showed a reduced colonization by B. intermedia after 3 weeks.