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.     

The sensitivity and specificity of a modified ELISA for the diagnosis of Johne's disease from a field trial in cattle

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycobacterium paratuberculosis in cattle was evaluated in three herds known to have Johne's disease. Prior to testing, the plasma was absorbed with dried Mycobacterium phlei in order to remove cross-reacting antibody specificities. The sensitivity and specificity of the ELISA were calculated after repeatedly testing 327 cattle in the infected herds. Of these, 53 animals had one or more positive faecal cultures or had post-mortem histopathological evidence of infection. The other 274 had three or more negative faecal culture results, and were regarded as non-infected for the purpose of evaluation of the ELISA. Using these criteria for the presence or absence of infection, the M. phlei-absorbed ELISA under field conditions had a 57% sensitivity and a 98.9% specificity. The sensitivity of the absorbed ELISA depended on the stage of disease of the animal under test. In general, it appeared that animals in the more advanced stages of disease were absorbed ELISA positive, whereas those in the early stages of infection were not detected. These results indicate that the M. phlei-absorbed ELISA has an important role as a test for the diagnosis and control of Johne's disease in cattle.     

Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne''s disease in cattle

An absorbed enzyme immunoassay (EIA) test for Johne's disease in cattle was developed in which absorption of cross-reacting antibodies occurred as a rapid reaction in solution rather than overnight with whole organisms and a subsequent centrifugation step. Total test time was reduced to less than 2 h with a minimum of manipulations. The test was evaluated in cattle herds from Johne's disease-endemic and Johne's disease-free regions of Australia. Specificity was 99.8%. Calculations of sensitivity were affected by the history of the herd under test. However, the EIA detected in excess of 80% of animals before onset of clinical disease and 65% of faecal shedders were EIA positive on, or before, first detection of Mycobacterium paratuberculosis in their faeces. The test should aid epidemiological studies and be a useful tool in the management and control of Johne's disease.     

Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.     

Analysis of repeated tests for interferon-gamma (IFN-gamma) response and faecal excretion for diagnosis of subclinical paratuberculosis in Danish cattle

A total of 315 cattle were tested for infection with Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) at three consecutive samplings, using the interferon-gamma (IFN-gamma) test on whole blood and bacteriological culture of faecal samples. Of 205 cattle from 10 infected herds 99 (48%) were positive in the IFN-gamma test on at least one sampling using "IDEXX-criteria" for interpretation, and of 110 cattle from five non-infected herds three (3%) were positive. Forty-four animals from infected and one from non-infected herds tested positive at all three samplings. Although support for the specificity of the IFN-gamma test was provided by these results, they also indicate problems with false positives. Approximately half of the positive animals did not give the same result at all three samplings, indicating that repeated testing increases the chance of detecting reactors. Changing, or fluctuating, IFN-gamma test results occurred most frequently in animals younger than 1 year, indicating that the IFN-gamma test should be applied only to animals 1 year and older. M. paratuberculosis was isolated from 16 (4%) of 371 cattle, all from infected herds. Fifteen culture-positive cattle tested positive at least once in the IFN-gamma test. It was not possible to predict from the IFN-gamma test result the number of animals that would eventually develop disease. However, the test may be useful to detect animals that have been exposed to M. paratuberculosis earlier in their lives, and the testing of young cattle could be included in a control program to check for the effectiveness of preventing transmission of infection to calves and to identify animals at risk of developing disease later in their lives.     

Effect of a test and control program for Johne's disease in Victorian beef herds 1992-2002

OBJECTIVE: To report on progress in Johne's disease (JD) control in infected beef herds participating in the Victorian Johne's disease test and control program (TCP). PROCEDURE: Clinical histories and JD testing data recorded by the Department of Primary Industries were analysed for 18 beef herds participating in the TCP. The herds were required to conduct annual whole herd tests with an absorbed ELISA, cull reactors and control the grazing of young cattle to minimise infection. RESULTS: Testing of over 11,000 animals identified 68 reactors giving an average prevalence of reactors at the first whole-herd test round (T1) of 0.77%. There had been 20 clinical cases detected in the 7 years before the TCP started and two cases detected in the 10 years after TCP started. Most reactors and all clinical cases were born before the TCP started. Of 34 reactors necropsied, 25 (74%) were confirmed to have JD by histology or culture of tissues. The modal age of reactors and clinical cases was 5 and 6 years respectively. Six herds completed the program by achieving three successive negative whole herd tests, four herds dropped out and eight continued to test. There were 18 reactors detected at T1 and 33 reactors that were negative at T1 but detected at subsequent tests. CONCLUSION: The TCP was associated with a marked decline in clinical cases. The similar age distributions of clinical cases and reactors probably meant that testing detected animals for which clinical disease was imminent. Whether the measures used in the TCP were adequate to control the disease in beef herds could not be determined because of the long incubation period of the disease. The relatively high proportion of the reactors investigated that were confirmed as infected provided confidence that the test was continuing to operate at a high specificity. If all unconfirmed reactors were presumed to be uninfected, the minimum specificity of the ELISA was 99.83%. The sensitivity of the ELISA appeared to be very low because of the large number of reactors that were negative at T1 but positive at later tests.     

Sensitivity of test strategies used in the Voluntary Johne's Disease Herd Status Program for detection of Mycobacterium paratuberculosis infection in dairy cattle herds

OBJECTIVE: To evaluate sensitivities at the herd level of test strategies used in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and alternative test strategies for detecting dairy cattle herds infected with Mycobacterium paratuberculosis. DESIGN: Nonrandom cross-sectional study. SAMPLE POPULATION: 64 dairy herds from Pennsylvania, Minnesota, Colorado, Ohio, and Wisconsin. Fifty-six herds had at least 1 cow shedding M. paratuberculosis in feces; the other 8 herds were free from paratuberculosis. PROCEDURE: For all adult cows in each herd, serum samples were tested for antibodies to M. paratuberculosis with an ELISA, and fecal samples were submitted for bacterial culture for M. paratuberculosis. Sensitivities at the herd level (probability of detecting infected herd) of various testing strategies were then evaluated. RESULTS: Sensitivity at the herd level of the testing strategy used in level 1 of the VJDHSP (use of the ELISA to test samples from 30 cows followed by confirmatory bacterial culture of feces from cows with positive ELISA result) ranged from 33 to 84% for infected herds, depending on percentage of cows in the herd with positive bacterial culture results. If follow-up bacterial culture was not used to confirm positive ELISA results, sensitivity ranged from 70 to 93%, but probability of identifying uninfected herds as infected was 89%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the testing strategy used in the VJDHSP will fail to identify as infected most dairy herds with a low prevalence of paratuberculosis. A higher percentage of infected herds was detected if follow-up bacterial culture was not used, but this test strategy was associated with a high probability of misclassifying uninfected herds.     

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.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.     

Effect of a test and control program for bovine Johne's disease in Victorian dairy herds 1992 - 2002

OBJECTIVE: To report on progress in Johne's disease (JD) control in infected dairy herds participating in the Victorian Johne's disease Test and Control Program (TCP). PROCEDURE: Clinical histories and JD testing data recorded by the Department of Natural Resources and Environment (now called Department of Primary Industries) were analysed for 542 dairy herds participating in the TCP. The herds were required to conduct annual herd tests of cattle 2 years old and older with an enzyme linked immunosorbent assay (ELISA), cull the reactors and manage the younger cattle to minimise infection. RESULTS: Testing of over 680,000 animals identified over 10,000 reactors giving an average prevalence of reactors at the first whole-herd test (T1) of 1.78%. There was a relatively rapid increase in the incidence of clinical disease before the TCP started and then it markedly declined. There was a slow and interrupted decline in reactor prevalence, with a marked peak occurring at the fourth herd test (T4). The average age of reactors and clinical cases was 5.7 and 5.9 years, respectively. Of the reactors and clinical cases detected during the TCP, 87% and 95% respectively, were born before the TCP started. Thirty herds completed the program by achieving three successive negative whole herd tests and 91 herds dropped out because of inability to comply with the agreed requirements of the program. There were no home-bred reactors born after the start of the program in 253 (47%) herds and of the 522 herds that were tested more than once, there were 319 (61%) herds in which no home-bred reactors were detected after the first year of testing. The number of ELISA positive animals detected at T1 appeared to be only about 26% of the animals from that round that subsequently became positive or developed clinical disease at later test rounds. CONCLUSION: The TCP caused a marked decline in the number of clinical cases, probably because animals in which clinical disease was imminent were detected by testing and removed. A reduction in prevalence of reactors occurred only when most herd members were born after the TCP started. The sensitivity of the ELISA appears to be low based on the large number of reactors that were negative at T1 but were positive at later tests. Low sensitivity of diagnostic tests and the long incubation period of the disease limits meaningful analysis of the program until it has continued for some years. Measures adopted in the TCP have not broken the cycle of infection in many participating herds. It is unsure if this was because of poor compliance with control recommendations or a poor understanding of methods of transmission by scientists. Eradication is not feasible in the short-term.     

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.     

Comparison of two enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subsp. paratuberculosis.

Enzyme-linked immunosorbent assays (ELISAs) are used in Johne's disease (JD) control programs as a first screening for presence of the disease in a herd. A high sensitivity of the ELISA is therefore important, yet the commonly used ELISAs have relatively low sensitivity. The inclusion of an absorption phase, although improving specificity, potentially decreases sensitivity. Sera and feces of 383 adult dairy cows in 8 herds were used to compare the test characteristics of an absorbed and a nonabsorbed indirect ELISA for the detection of JD. The absorbed ELISA is based on a protoplasmic antigen, whereas the nonabsorbed uses a lipoarabinomannan-based antigen. The potential advantage of the nonabsorbed ELISA is that it may be less specific and more sensitive. Two herds certified free of JD were used to compare the specificity of the ELISAs. The other herds used to compare sensitivity were either infected with Mycobacterium avium subsp. paratuberculosis or had unknown status. Using fecal culture as a gold standard, the diagnostic specificity for the absorbed and nonabsorbed ELISAs were 98.4% and 87.9%, respectively. The diagnostic sensitivity was 72.4% and 65.5% for the absorbed and the nonabsorbed ELISA, respectively. Furthermore, a comparison using a fecal DNA probe as the comparison standard resulted in both ELISAs having a sensitivity of 61.9%. Agreement between the 2 ELISAs was moderate, with a kappa statistic of 0.58. The nonabsorbed ELISA did not have a higher sensitivity and had a lower specificity than the absorbed ELISA. Therefore, in this population, there was no advantage gained with using the nonabsorbed ELISA.     

An estimated prevalence of Johne's disease in a subpopulation of Alabama beef cattle.

The objective of this study was to estimate the overall prevalence of animals that were infected with Mycobacterium avium ssp. paratuberculosis in a subpopulation of Alabama beef cattle. This was determined using a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of M. avium ssp. paratuberculosis-specific antibodies in serum. Serum was collected from 79 herds that were participating in the Alabama Brucellosis Certification program. A total of 2,073 beef cattle were randomly tested by selecting 30 animals per herd in herds greater than 30 and selecting all animals in herds 30 and less for testing. It has been estimated that the commercial ELISA test used has a 60% sensitivity and a 97% specificity. Of the 79 herds tested, 29 herds were seronegative, 24 herds had 1-2 positive animals, and 26 herds had 3 or more seropositive animals. The average number of infected animals per positive herd was 3.3. In addition, a calculated minimum of 53.5% of the herds were identified as Johne's positive herds with a 95% confidence level. Of the total number of animals tested, 8.0% (166/2,073) of them were positive by the ELISA. After adjustments for test sensitivity and specificity and the proportion of animals sampled per herd, the true prevalence was calculated to be 8.75%. These data suggest that approximately 50% of the herds are infected with M. avium ssp. Paratuberculosis, and the overall prevalence of infection in Alabama beef cattle is approximately 8%, which correlates with other previously published regional estimates.     

Comparative sensitivity of various faecal culture methods and ELISA in dairy cattle herds with endemic Johne's disease

In three New South Wales dairy cattle herds with endemic Johne's disease, prevalence rates by faecal culture were determined to be 12, 18 and 22%, respectively. Whole herd faecal culture was shown to detect markedly more infected cattle than whole herd testing by the EMAI absorbed ELISA, particularly in the two herds with greatest prevalence. In the three study herds, five methods for whole herd faecal culture were compared in each. These included two methods based on primary culture on Herrold's egg yolk medium with mycobactin J (HEYM): (1) conventional decontamination with sedimentation and primary culture on HEYM; (2) Whitlock decontamination and culture on HEYM. The remaining three methods were based on radiometric (BACTEC) culture: (3) decontamination and filtration to BACTEC medium; (4) modified Whitlock decontamination to BACTEC medium and (5) Whitlock decontamination to BACTEC medium. For BACTEC cultures, two methods were compared as confirmatory tests for Mycobacterium paratuberculosis: mycobactin dependence on conventional subculture to HEYM and IS900 PCR analysis of radiometric media.Among 179 cattle tested simultaneously by all five culture methods, 38 cattle were confirmed to be shedding M. paratuberculosis. In identifying shedder cattle, method 5 was the most sensitive, followed by methods 2, 4, 1, and 3 was the least sensitive. The number of BACTEC cultures confirmed by mycobactin dependence or PCR was similar.     

ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method

The sensitivity and specificity of the ELISA and fecal culture tests for paratuberculosis in dairy cattle are examined. ELISA and fecal culture data from seven dairy herds where both fecal cultures and ELISA testing was done concurrently are included. A cohort of 954 cattle including 697 parturient adults, cultured every 6 months from 10 herds followed over 4 years served as the basis to determine fecal culture sensitivity. The fecal culture technique utilized a 2g sample with centrifugation and double incubation. Of the 954 cattle cohort of all ages (calf to adult) that were fecal sampled on the first herd visit, 79 were culture positive. An additional 131 animals were detected as culture positive over the next seven tests at 6-month intervals. The sensitivity of fecal culture to detect infected cattle on the first sampling was 38%. Of the 697 parturient cattle cohort, 67 were positive on the first fecal culture, while an additional 91 adult cattle were culture positive over the next seven tests, resulting in a sensitivity of 42% on the first culture of the total animals identified as culture positive. Animals culled from the herds prior to being detected as infected and animals always fecal culture negative with culture positive tissues at slaughter are not included in the calculations. Both groups of infected cattle will lower the apparent sensitivity of fecal culture. Infected dairy herds tested concurrently with both fecal culture and ELISA usually resulted in more than twofold positive animals by culture compared to ELISA.The classification of infected cattle by the extent of shedding of Mycobacterium paratuberculosis in the feces helps define the relative proportion of cattle in each group and therefore the likelihood of detection by the ELISA test. ELISA has a higher sensitivity in animals with a heavier bacterial load, i.e. high shedders (75%) compared to low shedders (15%). Repeated testing of infected herds identifies a higher proportion of low shedders which are more likely to be ELISA negative. Thus, the sensitivity of the ELISA test decreases with repeated herd testing over time, since heavy shedders will be culled first from the herds.     

A long-term bacteriological and immunological study in Holstein-Friesian cattle experimentally infected with Mycobacterium avium subsp. paratuberculosis and necropsy culture results for Holstein-Friesian cattle, Merino sheep and Angora goats

The aims were to longitudinally evaluate the interferon-gamma (IFN-gamma) test in comparison to faecal culture and the absorbed ELISA in a cattle infection model for Johne's disease and to determine the adult infection status, by necropsy and tissue culture, of sheep, goats and cattle infected as young animals. Clinical disease, faecal culture results and immunological responses for Merino sheep [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2004. A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 104, 165-178] and Angora goats [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2006. A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 113, 13-24], in the same experiments as the Holstein-Friesian cattle, have been described. Two longitudinal experiments involving Holstein-Friesian cattle challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the IFN-gamma test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Cell-mediated (CMI) responses were substantially higher for the bovine Map strain during the 42-month period following dosing but then declined in the remaining 12 months. However, for the ovine Map challenge and control groups, CMI responses were not significantly different from each other. None of the cattle developed clinical disease and only one of the cattle in the bovine Map gut mucosal tissue challenged group was a persistent faecal shedder and also an ELISA antibody responder which developed after shedding commenced. Culture of tissues, following necropsy at the completion of the experiments, showed no evidence of infection in any of the challenged cattle and sheep for either the bovine or ovine Map strain in contrast to positive cultures for challenged goats in the same experiments. The tissues from the control cattle, sheep and goats were culture negative. The cattle were less susceptible to the bovine and ovine Map strains than goats and sheep with the goats being the least naturally resistant.     

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.     

Effects of prevalence and testing by enzyme-linked immunosorbent assay and fecal culture on the risk of introduction of Mycobacterium avium subsp. paratuberculosis-infected cows into dairy herds.

A stochastic simulation model was developed to assess the risk of introduction of Mycobacterium avium subsp. paratuberculosis infection into a dairy herd through purchase of female replacement cattle. The effects of infection prevalence in the source herd(s), number of females purchased, and testing by enzyme-linked immunosorbent assay (ELISA) alone or ELISA and fecal culture as risk mitigation strategies were evaluated. Decisions about negative test results were made on a lot and individual basis. A hypothetical dairy herd, free from M. a. paratuberculosis, which replaced 1 lot (10, 30, or 100) of cows per year, was considered. Probability distributions were specified for the sensitivities and specificities of ELISA and fecal culture, the proportion of infected herds and within-herd prevalence for randomly selected replacement source herds (high prevalence) and herds in level 2 (medium prevalence) and level 3 (low prevalence) of the Voluntary Johne's Disease Herd Status Program (VJDHSP). Simulation results predicted that 1-56% of the lots had at least 1 M. a. paratuberculosis-infected cow. Assuming that ELISA sensitivity was 25%, simulation results showed on a lot basis that between 0.4% and 18% and between 0.1% and 9% were predicted to have at least 1 infected cow not detected by ELISA and by a combination of ELISA and fecal culture, respectively. On an individual cow basis, between 0.1% and 8.3% of ELISA-negative cattle in ELISA-positive lots were estimated to be infected. In both the lot and individual analyses, the probability of nondetection increased with larger lot sizes and greater prevalence. Sensitivity analysis indicated that the effect of a lower ELISA sensitivity (10%) was a variable decrease in mean detection probabilities for all combinations of prevalence and lot size. The benefit of testing introduced cattle with ELISA alone or in combination with fecal culture was found to be minimal if cows were purchased from known, low-prevalence (level 3) herds. The value of testing by ELISA alone or in combination with fecal culture was greatest in high-prevalence herds for all lot sizes. Testing of random-source cattle, bought as herd replacements, can partially mitigate the risk of introduction of M. a. paratuberculosis but not as well as by using low-prevalence source herds (level-3 VJDHSP), with or without testing.     

Estimate of the sensitivity of an ELISA used to detect Johne's disease in Victorian dairy cattle herds

OBJECTIVE: To estimate the sensitivity of the ELISA used in dairy cattle herds participating in the Victorian Bovine Johne's Disease Test and Control Program (TCP). PROCEDURE: The percentage of ELISA reactors in age and test cohorts was estimated from age-specific test data derived from TCP herds with long testing histories. Age-distribution data from production-tested herds enabled estimation of reactor rates in animals that were culled or died. RESULTS: ELISA sensitivities at the first test round in herds achieving five, six and seven annual herd tests were 16.1, 14.9 and 13.5% respectively. The ELISA sensitivity in 2, 3 and 4-year-old animals at the first test round in herds testing seven times was 1.2, 8.9 and 11.6% respectively but remained between 20 and 30% in older age-groups. CONCLUSION: The sensitivity of the ELISA is considerably lower than previous estimates, probably because previous estimates were predominantly measured against faecal culture, which has subsequently been shown to have low sensitivity itself, and did not appreciate the long period that appears to precede detectable faecal excretion in most animals.     

Prevalence of serum antibodies to Mycobacterium avium subsp. paratuberculosis in cattle in Galicia (northwest Spain)

Prior to establishing a control and prevention program for Johne's disease in cattle in Galicia (northwest Spain), a survey was conducted to estimate the prevalence of the disease. For this survey, 61,069 animals of at least 1-year of age from 2735 randomly selected herds were bled and samples analyzed with a commercial ELISA. The estimated true individual-level prevalences – assuming the manufacturer's reported test sensitivity of 48.5% and specificity of 98.9% – were 3.02% in dairy cattle, 1.03% in beef cattle and 2.83% in animals from farms with both dairy and beef cattle. True herd prevalences (with herds declared positive if one or more animals tested positive) were 10.69% for dairy herds, 0% for beef herds and 2.71% for mixed herds. When herds were declared positive if at least two animals tested positive, true herd prevalences were 14.75% for dairy herds, 1.47% for beef herds and 12.01% for mixed herds. Assuming a higher specificity of 99.4%, true individual-level prevalences increased to 4.03% in dairy herds, 2.07% in beef herds and 3.84% in mixed herds. Herd prevalences were 27.77%/18.79%, 2.78%/2.40% and 5.70%/12.24% (using the one/two-animal cut-offs) in dairy, beef and mixed herds, respectively. In conclusion, these results seem to indicate that a small percentage of cows and a rather high percentage of dairy herds in this region are MAP-seropositive.