Herd management practices and the transmission of Johne's disease within infected dairy herds in Victoria, Australia.

A retrospective cohort study involving 137 dairy herds randomly selected from all 390 participating in the Victorian Test and Control Program for bovine Johne's disease was undertaken to gain insight into the relationships between calf rearing practices and the occurrence of bovine Johne's disease on infected dairy farms. Each study farm was visited between July 2005 and January 2006 and a structured survey examining herd management and calf rearing practices was completed. The resultant data, along with information from annual herd testing for Johne's disease and records of clinical Johne's disease diagnosed in the herd, from May 1990 to March 2008, were analysed. Factors associated with time to the birth of the animal that was the first home-bred clinical case of Johne's disease or ELISA positive animal born after the second annual whole herd test in the herd were investigated using survival analysis methods. The publicly-subsidised Test and Control Program commenced in 1996. On the 1st of July 2003 the program was modified with more rigorous and externally audited calf rearing requirements introduced for all participants. The more stringent calf rearing requirements introduced in July 2003 appear to have translated into significantly reduced disease transmission within the infected study herds.

Inter-laboratory comparison of radiometric culture for Mycobacterium avium subsp. paratuberculosis using raw milk from known infected herds and individual dairy cattle in Victoria.

OBJECTIVE: To compare the results of radiometric culture conducted in three Australian laboratories for Mycobacterium avium subsp. paratuberculosis (Mptb) using bulk vat and individual animal milk samples. PROCEDURE: Milk samples were collected from 15 cows exhibiting clinical signs of Johne's disease, and subsequently confirmed as infected with Mptb, and from the bulk milk vats on 91 farms running herds known to be infected with Mptb. Each milk sample was divided into three equivalent samples and one of each of the replicates was forwarded to the three participating laboratories. The identity and nature of the samples was protected from the study collaborators. The laboratories processed the samples and undertook radiometric culture for Mptb using their standard method. Results of testing were provided to the principal investigator for collation and analysis. RESULTS: In total, 2 (2.2%) of 91 vat-milk samples and 8 (53.3%) of 15 individual cows' milk samples returned positive radiometric milk culture results. Only one sample, from a clinical case of Johne's disease, was identified as positive by more than one laboratory. There were differences in the absolute frequency with which Mptb was identified in the milk samples by the collaborating laboratories. CONCLUSIONS: Mptb was cultured from a very small percentage of Australian raw bulk milk samples sourced from known infected herds. By contrast, Mptb was successfully cultured from half of the milk samples collected from clinically affected cows. There was no statistical difference between laboratories in the proportion of vat samples or individual animal milk samples in which Mptb was detected.

A comparison of two ELISAs for the detection of antibodies to bovine leucosis virus in bulk-milk.

OBJECTIVE: To estimate the sensitivity, specificity and detection limits for two bulk-milk enzyme-linked immunosorbent assays, the Svanovir BLV-gp51-Ab and the Lactelisa BLV Ab Bi indirect tank 250, for the detection of antibody to bovine leucosis virus in milk. PROCEDURE: Milk samples from 27 cows known to have enzootic bovine leucosis (EBL) were serially diluted with milk from a herd known to be free from the disease. The dilution at which antibodies could no longer be detected by each test was determined. A total of 1959 bulk-milk samples submitted to a laboratory for the Victorian (EBL) eradication program were tested with both the Svanovir and the Lactelisa assays. A Bayesian approach was used to calculate maximum-likelihood estimates of test sensitivity and specificity. An additional 660 bulk-milk samples were tested with both the Svanovir and the Lactelisa assays. Herds that had positive results on either or both of the assays were subjected to blood or milk testing of individual cattle. RESULTS: The dilution of milk at which the Svanovir assay failed to detect enzootic bovine leucosis antibody in half of the samples was 1 in 40, whereas the comparable value for the Lactelisa was 1 in 200. Computer modeling of the operating characteristics of the Svanovir assay indicated that the sensitivity of that assay would be considerably lower than that for the Lactelisa, and the specificity was estimated to be higher. Evaluation of the assays using 660 bulk-milk samples showed that the Lactelisa assay detected four infected herds that were not detected by the Svanovir test. No false positive results were recorded for either assay. CONCLUSION: Use of the Lactelisa assay in the Victorian EBL eradication program will enhance disease detection and eradication, but may also result in an increased frequency of false positive bulk-milk test results.

Effect of compliance with recommended calf-rearing practices on control of bovine Johne's disease.

OBJECTIVE: To assess the degree of compliance with recommended management procedures for the control of bovine Johne's disease and study the relationship between aspects of calf management and testing/disease outcomes in the herds. PROCEDURE: Fifty-four south Gippsland dairy herds participating in the Victorian bovine Johne's disease test and control program were visited between July and November 2002 and an audit of calf rearing practices was conducted. The results of testing completed under the program were analysed for each of the herds. Twenty seven management factors were examined for a relationship with the presence of clinical cases of Johne's disease or cattle with positive ELISA test results that were born after the completion of the second whole herd test. Logistic regression was used to examine the strength of relationships between the management practices and the frequency with which new cases of Johne's disease arose. RESULTS AND CONCULSIONS: Calves were removed from their dams within 12 hours of birth in only 17 (31.5%) of the herds. However, in all but one herd the calves were removed within 24 hours of birth. In 42 herds (77.8%) calf rearing facilities were adequately separated from adult cattle and the faeces from adult cattle. In 41 herds (75.9%) calves up to the age of 12 months were grazed on paddocks that were free of manure or effluent from adult cattle. However, in only 10 (18.5%) of the herds were all three of these calf management practices applied. Feeding whole milk containing antibiotic residues, or providing water for calves from birth, were found to have statistically significant associations with an increased occurrence of cases of bovine Johne's disease in the study herds. The practice of allowing cows to calve in a paddock was found to be associated with reduced occurrence of bovine Johne's disease. These associations were still found after analysis that included herd size, the number of clinical cases that had occurred in the herds before the start of testing, the number of animals with positive ELISA tests that were detected at the first test and the number of years of participation in the test and cull program. Early separation of newborn calves from cows and grazing calves under 12 months of age in areas free of adult cattle were not found to be protective against Johne's disease.

Evaluation of diagnostic tests for Johne's disease in young cattle.

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.

Johne's disease in alpacas (Lama pacos) in Australia.

Johne's disease was diagnosed in 10 alpacas (Lama pacos) in Australia between February 1993 and May 1994. Eight of the animals were between 12 and 24 months of age, one was a 6-year-old female, and one was a 4-year-old male. Five, including the 6-year-old and the 4-year-old alpacas, showed weight loss and diarrhoea before death or slaughter. The other cases showed no clinical signs of Johne's disease but 4 gave a positive result on faecal culture and one gave a positive result on testing with the caprine AGID assay and had acid-fast organisms in its faeces. At necropsy, all cases had grossly enlarged mesenteric lymph nodes. Johne's disease was diagnosed after histological examination of the lymph nodes with conventional culture and polymerase chain reaction testing of tissue samples. This report outlines the clinical, epidemiological, and pathological findings in these cases.

Determination of the optimal cutoff value for a serological assay: an example using the Johne's Absorbed EIA.

Traditionally, in order to improve diagnostic accuracy, existing tests have been replaced with newly developed diagnostic tests with superior sensitivity and specificity. However, it is possible to improve existing tests by altering the cutoff value chosen to distinguish infected individuals from uninfected individuals. This paper uses data obtained from an investigation of the operating characteristics of the Johne's Absorbed EIA to demonstrate a method of determining a preferred cutoff value from several potentially useful cutoff settings. A method of determining the financial gain from using the preferred rather than the current cutoff value and a decision analysis method to assist in determining the optimal cutoff value when critical population parameters are not known with certainty are demonstrated. The results of this study indicate that the currently recommended cutoff value for the Johne's Absorbed EIA is only close to optimal when the disease prevalence is very low and false-positive test results are deemed to be very costly. In other situations, there were considerable financial advantages to using cutoff values calculated to maximize the benefit of testing. It is probable that the current cutoff values for other diagnostic tests may not be the most appropriate for every testing situation. This paper offers methods for identifying the cutoff value that maximizes the benefit of medical and veterinary diagnostic tests.

Cultivation of Mycobacterium paratuberculosis from bovine fecal samples by using elements of the Roche MB Check system.

Components of a commercially available, nonradiometric, biphasic (liquid medium then solid medium) system for the detection of Mycobacterial species, Roche MB Check, were adapted for the isolation of Mycobacterium paratuberculosis from bovine fecal specimens. A two-stage culture procedure was developed in which processed fecal samples were incubated in modified commercial liquid medium and then subcultured onto Herrold's egg yolk medium with mycobactin. By using known culture-positive samples and/or samples from animals clinically affected with paratuberculosis, it was found that visible colonial growth on solid media could be obtained after 4 weeks of incubation in liquid medium containing egg yolk and mycobactin followed by 8 weeks of incubation on Herrold's egg yolk medium. In the second part of the study, conventional fecal culture (sample sedimentation in hexadecylcetylpyridinium chloride followed by incubation on Herrold's egg yolk medium) was compared with the two-stage system using a two-step centrifugation technique for sample preparation. One hundred fecal samples from clinically normal but absorbed-enzyme immunoassay-positive cattle were used for the comparison. Conventional culture yielded a sensitivity of 16.5%, whereas the sensitivity of the two-stage system was 29.4%. When used in parallel, the tests detected 36.5% of the samples. There was no significant difference between the two methods in the time taken to obtain visible colonies. These results indicate that the two-stage method is a sensitive method for isolation of M. paratuberculosis from fecal samples obtained from cattle with clinical paratuberculosis. In addition, the two-stage system is more sensitive than conventional culture for the isolation of M. paratuberculosis from subclinically infected cattle.

Comparison of the Johne's absorbed EIA and the complement-fixation test for the diagnosis of Johne's disease in cattle.

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.