Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood.

Theileria orientalis is an emerging bovine pathogen in Australasia. PCR-based detection methods for this parasite are sensitive but relatively expensive, partly due to costs associated with DNA extraction. An inexpensive and efficient technique was developed for the extraction of T. orientalis DNA from blood based on hypotonic erythrocyte lysis and detergent-proteinase K treatment (DPK method). The DPK method compares favourably to a commercial extraction kit when paired with a T. orientalis multiplex qPCR.

Development and validation of a quantitative PCR assay using multiplexed hydrolysis probes for detection and quantification of Theileria orientalis isolates and differentiation of clinically relevant subtypes.

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.

Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep.

An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p < 0.05). The sensitivities were 5.9% (95% CI: 1-26.9), 27.9% (95% CI: 14.7-45.7) and 35% (95% CI: 18.1-56.7), for low grade, paucibacillary and multibacillary lesion grades, respectively. The cost of the commercial assay kit was 2.7 to 5.2 times greater than that of the 316v ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

Distribution and temporal prevalence of Theileria orientalis major piroplasm surface protein types in eastern Australian cattle herds.

OBJECTIVE: To determine the distribution of theilerial types in eastern Australian cattle herds and their changing prevalence in regions of New South Wales (NSW). DESIGN: Survey testing of herds in 2010-11 in Queensland (QLD), NSW and Victoria (VIC) where clinical theileriosis was not evident and ongoing surveillance in NSW through laboratory submissions. METHODS: Blood samples were tested by PCR targeting the Theileria orientalis p32 gene and positive tests were examined for the Ikeda, Chitose and Buffeli types. Survey samples from 516 cattle in 50 herds and diagnostic submissions from 434 suspect field cases in 116 herds were analysed. RESULTS: In clinically normal survey cattle, T. orientalis prevalence was high (NSW 23.7%, QLD 56.8%, VIC 34.0%), with variability among regions of each state. Chitose was the most common and widespread type (19.1-43.7% per state), with Buffeli present in all states at a lower prevalence (10.8-24.8% per state). Ikeda was detected in three of five regions in QLD (North, South and South East; prevalence 3.4-15.4%), only one of the surveyed regions in NSW (North Coast; prevalence 74.2%) and in only one animal in VIC. Evidence of clinical disease and laboratory confirmation of Ikeda infection in diagnostic submissions were predominant in several NSW regions, with increasing numbers of affected herds particularly in the coastal Mid-Coast and Cumberland areas. CONCLUSIONS: In those regions where prior evidence of theileriosis was uncommon, Ikeda infection was evident in a limited number of NSW regions and multiple regions in QLD. However, clinical disease has continued to become widespread in NSW and VIC, involving Ikeda strains in many regions.

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs following vaccination or experimental challenge with respiratory pathogens and natural A. pleuropneumoniae serovar 1 challenge.

OBJECTIVE: To compare the sensitivity and cross-reactivity of six serological enzyme-linked immunosorbent assays (ELISAs) based on serovar-independent antigens of Actinobacillus pleuropneumoniae (App). DESIGN: An experimental pig trial using direct or natural challenge with App and direct challenge or vaccination using other common respiratory pathogens. PROCEDURE: A 39-kDa outer membrane protein antigen and five recombinant antigens from the ApxIVA gene of App were evaluated. The latter were derived from the ApxIVA N terminus (ApxIVA-N, ApxIVA-NP, ApxIVA-NPS) or C terminus (ApxIVA-C, ApxIVA-CP). Pigs were sampled after direct challenge with App, Pasteurella multocida or Haemophilus parasuis, after vaccination with these organisms and after natural App infection. Clinical and necropsy findings were evaluated. RESULTS: The 39-kDa ELISA had high sensitivity, but cross-reactivity, following P. multocida challenge. ELISAs using ApxIVA N terminus antigens were clearly more sensitive than C terminus antigens for detection of App-induced disease. Although affinity-purified ApxIVA-NP antigen detected marginally more diseased pigs than the -N and -NPS ELISAs, these assays only detected 41-47% of 17 pigs with lung lesions and microbiological evidence of App based on sampling up to 4-5 weeks after natural (13 pigs) or 5 weeks after direct App serovar 1 challenge (4 pigs). CONCLUSIONS: The 39-kDa ELISA readily detects App exposure and infection, but is adversely affected by P. multocida infection. ApxIVA-N-based ELISAs can be used to evaluate the App status of commercial herds, but a proportion of infected and diseased animals are seronegative at 1 month after likely exposure to aerosol transmission of App from clinical cases.

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida.

OBJECTIVE: To compare the sensitivity and specificity of six serological enzyme-linked immunosorbent assays (ELISAs) based on serovar-independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross-reactivity in disease-free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. DESIGN: Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. PROCEDURE: A 39-kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N-terminus (ApxIVA-N, ApxIVA-NP, ApxIVA-NPS) or C-terminus (ApxIVA-C, ApxIVA-CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. RESULTS: The 39-kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross-reactivity following P. multocida challenge. ELISAs based on ApxIVA N-terminus antigens were significantly more sensitive than C-terminus antigens for the detection of App-induced disease. Although ApxIVA-N and ApxIVA-NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App-induced disease (90-93%). Affinity purified ApxIVA-NP antigen had marginally better specificity than ApxIVA-N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross-reactivity. In disease-free pigs, the specificity of the ApxIVA-NPS ELISA may be adversely affected by nasal carriage of apparently low-virulence App strains. CONCLUSIONS: ApxIVA-N-based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low-virulence App. The 39-kDa antigen is only of merit in exclusion of App disease by negative serology.

Chemical composition of growing pigs and its relationship with body tissue composition assessed by X-ray-computed tomography.

Ninety hybrid (mainly Large White × Landrace) pigs from 2 experimental replicates were used to study the potential use of computed tomography (CT) as a nondestructive technology for estimating the chemical body composition of growing pigs. Body tissue components (lean, fat, and bone) of 6 live pigs from each sex (boars, gilts, and barrows) were assessed by CT imaging before slaughter at approximately 30, 60, 90, 120, and 150 kg of BW. After slaughter, the empty body components were ground and frozen until analyzed for protein, lipid, ash, and moisture content. Several growth functions were evaluated and the allometric function (Y = aBW(b)), which was evaluated as log(10)chemical component weight = b(0) + b(1)log(10)BW, provided the best fit to the data. For each sex, the allometric coefficient (b(1)) for protein (0.92 to 0.99) was close to but less than 1; for ash (1.03 to 1.12), it was close to but greater than 1; for moisture (0.82 to 0.86), it was less than 1, and for lipid (1.61 to 1.71), it was greater than 1. Deposition rates (change in component weight per unit change in BW) for each chemical component were predicted using derivatives of the function. The mean deposition rates for protein and lipid were 0.141 and 0.286 kg/kg of BW gain, respectively. The deposition rate for protein was generally stable across different BW, whereas that for lipid increased as BW increased. In addition, linear, quadratic, exponential, and logistic functions were fitted to the data to study the relationship between the CT data and chemical components. The linear function was assessed to be the best equation, based on the Bayesian information criterion. The prediction equation for protein (kg) = -1.64 + 0.28 × CT lean (kg), and for lipid (kg) = -0.69 + 1.09 × CT fat (kg), had R(2) values of 0.924 and 0.987, respectively. Sex had no effect (P > 0.05) on the prediction of protein and lipid. The effect of BW was not significant (P > 0.05) for the prediction of lipid, but it was significant (P > 0 0.05) for the prediction of protein. However, the addition of BW to the base prediction equation for protein resulted in an increase of only 0.013 in the R(2) value. It was concluded from this study that CT scanning has great potential as a nondestructive technology for estimating the physical and chemical body composition of pigs. Additional research is required to validate the utility and accuracy of the prediction equations.

Bacterial survival studies to assess the efficacy of static pile composting and above ground burial for disposal of bovine carcases.

AIM: To assess the survival of bacteria during two alternative means of cattle carcase disposal in windrows: static pile composting (SPC) and above ground burial in soil (AGB), under temperate climate conditions on agricultural land, compared to surface disposal as the control method. METHODS AND RESULTS: Bacteriological reference materials (pooled bovine faeces in permeable nylon bags and lyophilized cultures of Escherichia coli in glass ampoules) were positioned above and below each of 33 beef cattle carcases (250-300 kg). Temperatures at these sites were monitored with data loggers, while temperature and CO(2) probes were applied repeatedly at varying depths along the windrows. Aliquots of each reference material were cultured from three randomly selected animals from the SPC and AGB group and from all three control animals on five occasions (at 28, 56, 84, 126 and 182 days). SPC was highly efficacious in the destruction of coliforms in faeces and E. coli in ampoules within 28 days, while AGB was not significantly better than controls until 84 days, and bacteria in reference materials above the AGB carcases were still viable after 182 days. Temperature probes and loggers showed SPC provided sustained temperatures of 55-70°C, while AGB did not reach temperatures of 30°C, and the temperature differences correlated with bacteriological findings. CONCLUSIONS: In relation to emergency disease management, SPC can be successfully applied to eliminate pathogenic bacteria in cattle carcases, but AGB is unsuitable for carcase disposal. SIGNIFICANCE AND IMPACT OF THE STUDY: In emergency, animal disease outbreaks in temperate climates requiring large-scale ruminant carcase disposal, SPC can be successfully applied for the destruction of micro-organisms.

Differential growth and development of pigs as assessed by X-ray computed tomography.

Data from 54 hybrid (mainly Large White x Landrace) pigs (18 boars, 18 gilts, and 18 barrows) were used to quantify and mathematically describe the differential growth and development of body components of live pigs. The pigs were 32.4 +/- 3.2 kg of BW and 70 +/- 1 d of age (mean +/- SD) at the beginning of the study, were individually penned and fed ad libitum, and were weighed weekly. Computed tomography (CT) imaging was used to determine the weights of lean, fat, bone, and skin tissue in the live pig at 30, 60, 90, 120, and 150 kg of BW. For each target BW, the sum of all the weights of the body components, as assessed by CT, was referred to as CT BW. Linear and nonlinear models were developed to evaluate the patterns of growth and development of each body component relative to CT BW. The correlation between the actual BW and CT BW was close to unity (r = 0.99), indicating that CT scanning could accurately predict the BW of pigs. Across sex and castrate status, percentage of fat (fat weight/CT BW) in the pig was least (11.2%) at the 30-kg target BW and continued to increase to 22.6% by the 150-kg target BW. Percentage of lean, however, was greatest (67.2%) at the 30-kg target BW and continued to decrease to 53.4% by the 150-kg target BW. The sex or castrate status x target BW interaction was significant (P < 0.05) for all the body components, indicating that the developmental patterns were different among sex or castrate status. Barrows were fatter relative to gilts, which in turn were fatter than boars. For lean, the observed pattern for sex or castrate status differences was opposite that for fat. To predict responses to management strategies on growth and development in pigs, accurate mathematical models are required, and the results of this study indicate that the nonlinear (e.g., augmented allometric and generalized nonlinear) functions provided better descriptions of the growth and development of most body components of the live pig than did the simpler (e.g., linear and allometric) models.

Serological responses to two serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in Australian commercial pig herds.

OBJECTIVE: To compare serological responses in pig herds classified as low or high risk for disease caused by Actinobacillus pleuropneumoniae, using two ELISA tests based on serovar-independent antigens. PROCEDURE: Cross-sectional sampling was undertaken in 13 commercial herds, the clinical and slaughter histories of which indicated either freedom from (n = 5) or prior confirmed cases of A. pleuropneumoniae (n = 8). In nine herds, approximately 40 pigs each were sampled at 4, 8, 12, 16 and 20 weeks. Three of the remaining four herds were sampled between 6 and 30 weeks of age, and the last was sampled only prior to slaughter, at approximately 24 weeks. Sera were tested in ELISA based on two antigens common among A. pleuropneumoniae serovars: a 39-kDa outer membrane protein and a recombinant ApxIVA-N terminus protein. RESULTS: Sampling of 1 and 5 to 6-month-old pigs provided the most useful information on herd status. The 39-kDa ELISA was sensitive in detecting infected herds, but had evidence of cross-reactivity with high seroreactivity rates in older pigs in some low-risk herds. The ApxIVA-N ELISA was less seroreactive in high-risk herds and had higher specificity in low-risk herds. CONCLUSION: ELISA based on the 39-kDa subunit are of limited use, because of possible cross-reactivity, but a high negative predictive value may be useful for risk assessment in suspect herds. Maternal antibody to ApxIVA-N may be of value in detecting high-risk herds, but 5% of 4-week-old pigs in low-risk herds were also seropositive in this assay.

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle.

OBJECTIVE: To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). METHOD: Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. RESULTS: Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. CONCLUSION: Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.

Salmonella and on-farm risk factors in healthy slaughter-age cattle and sheep in eastern Australia.

OBJECTIVE: To examine healthy slaughter-age cattle and sheep on-farm for the excretion of Salmonella serovars in faeces and to identify possible risk factors using a questionnaire. PROCEDURE: The study involved 215 herds and flocks in the four eastern states of Australia, 56 with prior history of salmonellosis. Production systems examined included pasture beef cattle, feedlot beef cattle, dairy cattle, prime lambs and mutton sheep and animals were all at slaughter age. From each herd or flock, 25 animals were sampled and the samples pooled for Salmonella culture. All Salmonella isolated were serotyped and any Salmonella Typhimurium isolates were phage typed. Questionnaires on each production system, prepared in Epi Info 6.04, were designed to identify risk factors associated with Salmonella spp excretion, with separate questionnaires designed for each production system. RESULTS: Salmonellae were identified in all production systems and were more commonly isolated from dairies and beef feedlots than other systems. Statistical analysis revealed that dairy cattle were significantly more likely to shed Salmonella in faeces than pasture beef cattle, mutton sheep and prime lambs (P<0.05). A wide diversity of Salmonella serovars, all of which have been isolated from humans in Australia, was identified in both cattle and sheep. Analysis of the questionnaires showed access to new arrivals was a significant risk factor for Salmonella excretion on dairy properties. For beef feedlots, the presence of large numbers of flies in the feedlot pens or around stored manure were significant risk factors for Salmonella excretion. CONCLUSION: Dairy cattle pose the highest risk of all the slaughter-age animals tested. Some of the identified risk factors can be overcome by improved management practices, especially in relation to hygiene.

Salmonella Typhimurium persistence in a Hunter Valley dairy herd.

OBJECTIVE: An epidemiological study was undertaken at a Hunter Valley dairy with persistent Salmonella Typhimurium infection. The aim of the study was to identify cattle currently or previously infected with Salmonella, possible sources of the organism, patterns of spread, and husbandry practices that could be improved. METHODOLOGY: Faecal samples, feed, water and environmental samples were cultured for Salmonella and blood samples were tested for antibodies against Salmonella (Dublin and Typhimurium). A questionnaire was designed to identify possible risk factors associated with Salmonella excretion. RESULTS: S Typhimurium was apparently introduced from an old to a new dairy through manure spread as fertiliser. Salmonella apparently persisted in the effluent pond, and the following year clinical cases occurred after pasture, irrigated with water from the pond, was grazed by dry cows, and adult cattle became clinically ill with salmonellosis. The disease spread to other cows and calves. Poor design of calf pens assisted spread of Salmonella from sick to healthy calves. In addition, there was suspected transmission to the dairy farmer's 9-month-old daughter. Salmonellosis on a farm is a potential zoonotic risk to farm workers and their families. There is also the risk that cull cows may carry Salmonella to the abattoir and subsequently into the human food chain. Methods of waste management, and the design of calf pens, were identified as major risk factors that could be improved to minimise the spread of salmonellosis on this property.

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats.

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.

Evaluation of Erysipelothrix rhusiopathiae vaccines in pigs by intradermal challenge and immune responses.

In a vaccine trial, pigs were challenged intradermally with eight E. rhusiopathiae strains of serovars 1a, 1b or 2 given concurrently. The strains were derived from six herds affected with vaccine breakdowns in 1997-1999, one herd without vaccine breakdown and a serovar 2 reference strain. Responses to two commercial bacterins (one implicated in the vaccine breakdowns), and two experimental bacterins (based on field isolates from affected herds) showed distinct differences in protection, particularly in clinical responses measured at 72 h. Less protection was afforded against serovar 1 challenge by the vaccine implicated in the vaccine breakdowns. Antibody and cell-mediated immune (CMI) responses were significantly different between treatments, and highlighted a similar post-vaccinal antibody response was produced against serovar 2 lysate by all vaccines, but only those providing significant protection against serovar 1 [corrected] produced significantly elevated antiserovar I lysate [corrected] antibodies. Vaccination in general significantly reduced CMI responses to the mitogens concanavalin A and phytohaemagglutinin. This experimental pig challenge system was readily able to confirm suboptimal performance of a commercial bacterin that had passed potency tests in mice but was associated with vaccine failure in commercial herds. This vaccine was also the most immunosuppressive to CMI responses associated with E. rhusiopathiae-specific and non-specific stimulation. The best vaccine response was associated with the highest mean serovar 1 antibody response and the highest CMI response (by lymphoproliferation assay) to serovar 2.

Characterisation of Erysipelothrix rhusiopathiae isolates from pigs associated with vaccine breakdowns.

Swine erysipelas vaccines are routinely used to protect pigs against peracute and acute/urticarial forms of Erysipelothrix. Between 1995 and 1998, 34 swine herds across four Australian states experienced vaccine failure. Forty-four isolates of Erysipelothrix rhusiopathiae of serovars 2, 1a, 1b and 1bx21 were recovered from 15 of these 34 vaccine breakdown herds. These isolates were characterised by restriction fragment length polymorphism (RFLP) analyses using RsaI and AluI on whole cell DNA and for the presence of plasmid DNA. Results were compared with those of 20 isolates from 16 herds unaffected by vaccine breakdown and 13 isolates representing 10 reference strains. The majority of breakdown herds possessed isolates of serovar 2 (9/15 herds), followed by serovar 1a (5 herds). No geographic predominance of a single serovar was evident. The identification of 10 RsaI profiles from whole cell DNA among the 44 isolates from 15 breakdown herds indicated that a single, new clonal lineage of E. rhusiopathiae was not responsible for vaccine failure. RsaI RFLP analyses detected a further 14 distinct profiles among 20 field strains unassociated with vaccine breakdowns, and none matched profiles of the 10 serovar reference strains for serovars 1a, 1b, 2 or 21. This technique is recommended for epidemiological studies of E. rhusiopathiae strains.

Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia.

The sensitivities and specificities of an absorbed enzyme-linked immunosorbent assay (ELISA) and an agar-gel immuno-diffusion (AGID) test for the detection of Johne's disease in sheep were estimated using data from six known infected and 12 assumed uninfected sheep flocks. Sensitivities were estimated for all histologically positive sheep, as well as by histological lesion score, lesion type (paucibacillary or multibacillary) and sheep body-condition score, with ELISA sensitivities estimated at 95 and 99% specificity. Logistic-regression analysis was used to test for significant effects of lesion score and condition score, with flock included in the model as a random effect. Estimated specificities were 95% (95% CI: 93.4, 95.6%) and 99% (98.4, 99.4%) for ELISA cut-point ratios of 2.4 and 3.6, respectively, and 100% (99.7, 100.0%) for the AGID. Estimated sensitivities for all infected sheep were 41.5% (35.0, 48.3%), 21.9% (16.6, 27.9%) and 24.6% (19.1, 30.7%) for ELISA cut-point ratios of 2.4 and 3.6 and for AGID, respectively. Sensitivities of all tests and cut-points varied significantly between flocks and between categories of lesion score and condition score. Sensitivity ranged from 25 to 73, 10 to 47 and 9.2 to 63% between flocks, for the ELISA with cut-points of 2.4 and 3.6, and for the AGID, respectively. Sensitivity was highest in thin sheep and in sheep with multibacillary lesions. The effects of lesion type and condition score on test sensitivity were significant in the logistic regressions for the AGID and ELISA at both cut-points and the flock effect was significant for the AGID but not for the ELISA at either cut-point.

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats.

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA.

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.