Pathogens, patterns of pneumonia, and epidemiologic risk factors associated with respiratory disease in recently weaned cattle in Ireland.

We examined the pathogens, morphologic patterns, and risk factors associated with bovine respiratory disease (BRD) in 136 recently weaned cattle ("weanlings"), 6-12 mo of age, that were submitted for postmortem examination to regional veterinary laboratories in Ireland. A standardized sampling protocol included routine microbiologic investigations as well as polymerase chain reaction and immunohistochemistry. Lungs with histologic lesions were categorized into 1 of 5 morphologic patterns of pneumonia. Fibrinosuppurative bronchopneumonia (49%) and interstitial pneumonia (48%) were the morphologic patterns recorded most frequently. The various morphologic patterns of pulmonary lesions suggest the involvement of variable combinations of initiating and compounding infectious agents that hindered any simple classification of the etiopathogenesis of the pneumonias. Dual infections were detected in 58% of lungs, with Mannheimia haemolytica and Histophilus somni most frequently recorded in concert. M. haemolytica (43%) was the most frequently detected respiratory pathogen; H. somni was also shown to be frequently implicated in pneumonia in this age group of cattle. Bovine parainfluenza virus 3 (BPIV-3) and Bovine respiratory syncytial virus (16% each) were the viral agents detected most frequently. Potential respiratory pathogens (particularly Pasteurella multocida, BPIV-3, and H. somni) were frequently detected (64%) in lungs that had neither gross nor histologic pulmonary lesions, raising questions regarding their role in the pathogenesis of BRD. The breadth of respiratory pathogens detected in bovine lungs by various detection methods highlights the diagnostic value of parallel analyses in respiratory disease postmortem investigation.

Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease.

Bovine respiratory disease (BRD) causes severe economic losses to the cattle farming industry worldwide. The major bacterial organisms contributing to the BRD complex are Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, and Trueperella pyogenes. The postmortem detection of these organisms in pneumonic lung tissue is generally conducted using standard culture-based techniques where the presence of therapeutic antibiotics in the tissue can inhibit bacterial isolation. In the current study, conventional and real-time polymerase chain reaction (PCR) assays were used to assess the prevalence of these 5 organisms in grossly pneumonic lung samples from 150 animals submitted for postmortem examination, and the results were compared with those obtained using culture techniques. Mannheimia haemolytica was detected in 51 cases (34%) by PCR and in 33 cases (22%) by culture, H. somni was detected in 35 cases (23.3%) by PCR and in 6 cases (4%) by culture, Myc. bovis was detected in 53 cases (35.3%) by PCR and in 29 cases (19.3%) by culture, P. multocida was detected in 50 cases (33.3%) by PCR and in 31 cases (20.7%) by culture, and T. pyogenes was detected in 42 cases (28%) by PCR and in 31 cases (20.7%) by culture, with all differences being statistically significant. The PCR assays indicated positive results for 111 cases (74%) whereas 82 cases (54.6%) were culture positive. The PCR assays have demonstrated a significantly higher rate of detection of all 5 organisms in cases of pneumonia in cattle in Northern Ireland than was detected by current standard procedures.

An aetiopathological study of chronic bronchopneumonia in lambs in Ireland.

Chronic bronchopneumonia in lambs, also known as 'atypical' or 'chronic, non-progressive' pneumonia is a common, frequently sub-clinical disease affecting animals under 12-months-old in intensive production systems. Infection with both Mycoplasma ovipneumoniae and Mannheimia haemolytica have been implicated in the aetiology of this condition and a variety of pulmonary lesions can result. In this study, detailed laboratory examination of 30 abattoir-derived lungs with the characteristic gross features of atypical pneumonia (AP) was carried out with a view to refining and correlating the histopathological and microbiological criteria required for the diagnosis of this disease. For the first time a broad range of laboratory detection techniques including bacterial and virus isolation, fluorescent antibody tests and immunohistochemistry were used in parallel to identify potential causative pathogens such as M. ovipneumoniae, M. haemolytica, parainfluenza type-3 (PI3) virus and respiratory syncytial virus (RSV) in AP lesions. The most consistent finding was the association of gross AP lesions with M. ovipneumoniae, identified by either culture or immunohistochemistry in 27 (90%) of the 30 cases. However the presence M. ovipneumoniae organisms or antigen did not consistently correlate with particular histopathological changes. Furthermore, peri-airway lymphoid hyperplasia, intra-alveolar exudation and nodular 'hyaline scars', which are all previously reported microscopic lesions of AP, were not identified in 12 (40%) of the cases and isolation of M. haemolytica was over-represented in lungs exhibiting suppurative lesions. These findings illustrate the complex aetiopathogenesis of this disease and highlight the requirement to use a combination of diagnostic criteria in its laboratory diagnosis.

The anti-nuclease humoral immune response of broiler chickens exposed to Staphylococcus aureus, infectious bursal disease virus and chicken anaemia virus in an experimental model for bacterial chondronecrosis and osteomyelitis.

This study aimed to develop an enzyme-linked immunosorbent assay to detect antibody associated with Staphylococcus aureus that is produced during the chicken immune response to this organism. The protein nuclease was tested for suitability as an antigen to detect antibody in sera from broiler chickens that had been exposed to aerosolized S. aureus on day 1 post hatch during experiments to reproduce bacterial chondronecrosis and osteomyelitis. An enzyme-linked immunosorbent assay was developed to measure the levels of nuclease antibody in 509 chicken sera from various experiments, which also enabled the examination of the influence of factors such as the S. aureus infection status and co-infection with chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) on nuclease antibody levels. Positive levels of nuclease antibody were detected in 71% of serum samples from chickens inoculated with S. aureus, CAV and IBDV, while positive levels were detected in 35% of chickens inoculated with S. aureus only. The influence of CAV and IBDV on the number of chickens with positive levels was most prominent in chickens aged up to 42 days. The study showed that nuclease-specific antibodies form part of the humoral immune response in broiler chickens that have been exposed to S. aureus. Co-infection with CAV and IBDV appeared to promote development of antibody in birds younger than 42 days; however, the presence of antibody did not necessarily prevent systemic infection.

Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland.

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.

Identification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis.

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells.

Detection of Mycoplasma mycoides subspecies mycoides SC in bovine lung and lymph node tissues by culture, sandwich ELISA and polymerase chain reaction systems.

Cattle from Northern Portugal, many with pulmonary lesions typical of contagious bovine pleuropneumonia, were investigated for the presence of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), which is the causative agent of CBPP, with several detection tests. Sandwich ELISA that included a culture enrichment stage, and 2 different PCR diagnostic systems were used to detect MmmSC in lung and mediastinal lymph node tissues from these animals. The comparison of typical CBPP pathology with the results of detection revealed that no single one of these methods provided a perfect match to the pathological data. Best performing tests were the PCR with laser induced fluorescence and PCR with pleuroTRAP kit (Chemicon, Australia), which are diagnostic systems based on amplification of genomic MmmSC DNA followed by sensitive detection of the amplified products. These were followed by the broth-enriched sandwich ELISA, which uses a monoclonal antibody specific to the M. mycoides cluster, to capture the antigen.

Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows' milk from approved dairy processing establishments in the United Kingdom.

Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semi-skim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence of Mycobacterium paratuberculosis by immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall, M. paratuberculosis DNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. Confirmed M. paratuberculosis isolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74 degrees C for 15 s; the remainder had been treated at 72 to 75 degrees C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and post-process contamination was considered unlikely to have occurred. It was concluded that viable M. paratuberculosis is occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.

Presence in bovine enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli of genes encoding for putative adhesins of human EHEC strains.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells. The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium. A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E. coli (NTEC). In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups. Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC. Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.