Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.     

Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain

A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.     

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.     

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.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.     

The effect of the tuberculin test and the consequences of a delay in blood culture on the sensitivity of a gamma-interferon assay for the detection of Mycobacterium bovis infection in cattle

The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. It has been reported that performance of the test can be influenced by various factors including a recent tuberculin skin test and the length of delay between collection and processing of blood samples. In this study, single intradermal comparative tuberculin test (SICTT) reactor and non-reactor cattle were recruited from herds infected with M. bovis and grouped according to their SICTT responses. Group 1 comprised reactor cattle selected on the basis of their SICTT response to PPD-bovine (purified protein derivative of tuberculin) exceeding that of PPD-avian by at least 12mm. Group 2 animals were selected from herds undergoing routine surveillance for bovine tuberculosis and contained standard SICTT reactor cattle (PPD-bovine exceeding that of PPD-avian by at least 4mm) and non-reactors. We investigated the effects of the SICTT on the assay results by measuring the in vitro IFN-gamma responses of Group 1 reactor cattle at time intervals pre- and post-skin test. No significant differences were measured in the IFN-gamma responses of the reactor animals to PPD-bovine and PPD-avian for up to 65 days. To investigate if a delay in processing of blood affected the performance of the assay, we compared results using duplicate blood samples from Group 1 and Group 2 cattle stimulated with PPD antigen at 8h and at 24h after collection. In both groups of animals the mean optical density (OD) values of the assay at 24h post-collection were significantly lower than those at 8h. Our results demonstrated that a delay in processing of the blood samples from cattle subjected to routine surveillance could significantly impact on the outcome of the IFN-gamma assay resulting in a change of the IFN-gamma status of the animals.     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

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.     

The epizootiological significance of positive bacteriological findings on Mycobacterium tuberculosis and Mycobacterium bovis in humans

The relation between the active form of tuberculosis in persons working in agriculture and incidence of tuberculosis in cattle was analyzed in 1974 to 1978, i.e. in the period after the elimination of bovine tuberculosis in Czechoslovakia (in 1968). M. tuberculosis was isolated in 15 cases and M. bovis in four cases of persons employed by the farms on which the Regional Hygienic Station, Brno, was responsible for the microbiological diagnostics of tuberculosis. Direct contact with animals was demonstrated in eight patients; M. tuberculosis was isolated from seven of these patients and M. bovis from one. Seven cattle herds were exposed to spontaneous infection by M. tuberculosis and in one of them tuberculosis was not demonstrated during complex examination. In three herds the examination revealed only a sensitivity of cattle to mammalian tuberculin. In other three herds tuberculosis was detected by allergic tests, patho-anatomic examination and bacteriological examination. M. tuberculosis in cattle was detected in two herds. The occurrence of bovine tuberculosis caused by a cattle tender with a positive finding of M. bovis in sputum was demonstrated in one herd. Virulence for the tested cattle was found in one strain (isolated from a mesenterial lymph node of cattle) of the four strains of M. tuberculosis used for the experimental infection of 17 animals. On the other hand, in three strains of M. tuberculosis, trials with experimental infection demonstrated only allergy to mammalian tuberculin and changes at the sites of subcutaneous inoculation of mycobacteria of regressive nature; these mostly disappeared within 90 days from infection.     

Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques

The early, preclinical stages of bovine TB can be detected in live animals by the use of tests of cellular immunity (the skin, gamma-interferon and lymphocyte transformation tests). Tests of humoral (antibody) immunity, Mycobacterium bovis PCR probes on early tissue cultures or live cattle specimens, and tests based on "electronic nose" technology have been developed more recently. The key measure of diagnostic test accuracy is the relationship between sensitivity and specificity, which determines the false-positive and false-negative proportions. None of the tests currently available for the diagnosis of bovine TB allow a perfectly accurate determination of the M. bovis infection status of cattle. Although various factors can reduce the sensitivity and specificity of the skin tests, these remain the primary ante mortem diagnostic tools for TB in cattle, providing a cost-effective and reliable means of screening entire cattle populations. Despite the inescapable limitations of existing diagnostic tests, bovine TB has been effectively eradicated from many developed countries and regions with the implementation of sound programmes of regular tuberculin skin testing and removal of reactors, coupled with slaughterhouse surveillance for undetected infections, repeat testing and culling of infected herds, cattle movement restrictions to prevent introduction of infected animals and occasional slaughter of entire herds with intractable breakdowns. This is likely to remain the mainstay of bovine TB control programmes for the foreseeable future. Additionally, newer ancillary in vitro diagnostic assays are now available to TB control programme managers to supplement the skin tests in defined circumstances according to the specific disease situation in each country or region. The strategic deployment of ancillary in vitro tests alongside the primary skin tests has enhanced the detection of M. bovis-infected cattle and reduced the number of animals slaughtered as false positives.     

Interference of paratuberculosis with the diagnosis of tuberculosis in a goat flock with a natural mixed infection

Detection of infected animals is a key step in eradication programs of tuberculosis. Paratuberculosis infection has been demonstrated to compromise the specificity of the diagnostic tests. However, its effect on their sensitivity has not been clarified. In the present study, skin tests and the interferon-gamma (IFN-gamma) assay were evaluated in a goat flock (n=177) with a mixed tuberculosis-paratuberculosis infection in order to assess the possible effect of paratuberculosis on their sensitivity. Culture of mycobacteria was performed as the gold standard to determine the true infection status. All techniques showed lower sensitivities than previously described; the single intradermal tuberculin (SIT) test and the IFN-gamma assay detected 71% (62.4-78.6, 95% C.I.) of the infected animals; the single intradermal cervical comparative tuberculin (SICCT) test detected only 42.7% (34.1-51.7, 95% C.I.) of infected animals. The highest level of sensitivity was obtained when SIT test and IFN-gamma assay were combined in parallel (90.8%, 84.5-95.2, 95% C.I.). Sensitivities of the tests were also assessed by comparing animals suffering tuberculosis and animals with a mixed infection; tests were found to be more effective in the former group. Paratuberculosis seems to have a major effect in the sensitivity of the diagnostic tests under study, and therefore must be taken into account; in particular, the use of the SICCT test should be questioned when both tuberculosis and paratuberculosis are present.     

Use of ESAT-6 in the interferon-gamma test for diagnosis of bovine tuberculosis following skin testing

The whole blood interferon-gamma (IFN-gamma) test has proven to be a practical ancillary test for re-testing cattle for bovine tuberculosis 8-28 days following tuberculin skin testing. An improvement in the specificity of the IFN-gamma test could further reduce culling of false positive animals. The primary aim of this study was to evaluate a single mycobacterial antigen, ESAT-6 in the IFN-gamma test for use in skin test-positive cattle. These skin test-positive cattle comprised 51 Mycobacterium bovis-infected animals from tuberculosis-infected herds and 85 non-infected animals from tuberculosis-free herds. The test based on ESAT-6 had a higher specificity than the test based on purified protein derivative (PPD) tuberculin, but this was offset by a small decrease in sensitivity. Use of a lower cut-off in the ESAT-6-based test improved the sensitivity, while still maintaining a very high specificity. A secondary aim in the study was to assess the ESAT-6 and PPD-based tests for detecting bovine tuberculosis in skin test-negative animals from a persistently infected herd. The PPD-based test detected the majority of the lesioned or M. bovis-culture positive animals, while the ESAT-6-based test detected a smaller proportion. The false negatives in the IFN-gamma test from both the skin test-negative and positive groups were predominantly M. bovis-culture positive animals with no visible lesions. The current study has shown that a defined specific antigen such as ESAT-6 can markedly improve the specificity of the IFN-gamma test for re-testing skin test-positive animals. An ESAT-6-based IFN-gamma test could be particularly useful to reduce the false positive rate, yet still maintain an acceptable level of sensitivity.     

Mycobacterial diseases of deer

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.     

Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (Bovigam) assay

The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. When used in parallel with the tuberculin test, it is capable of identifying infected cattle, which might otherwise not be detected until later, if at all. The early detection and removal of these animals reduces the risk that they will become a source of infection for other cattle. When targeted in herds of high prevalence the benefits to the herd owner directly concerned can be considerable as the assay provides a means of shortening the period of restriction for such herds. This serves to generate confidence among herd owners and other stakeholders that effective schemes, based on sound scientific principles, can be developed to eradicate tuberculosis from infected cattle populations.     

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.     

Tuberculosis in goats: assessment of current in vivo cell-mediated and antibody-based diagnostic assays

Tuberculosis in goats, mainly caused by Mycobacterium bovis and M. caprae, is a zoonotic disease with implications for public health, as well as having an economic impact due to decreased goat production, increased mortality rates and costs of diagnosis. There is an increasing need for surveillance of tuberculosis-infected goat herds, particularly in countries that are not officially free of bovine tuberculosis, and goats sharing farms with cattle should be subjected to the official tuberculin test. In Spain, some regions have programmes for the control of tuberculosis in goats, applying the same diagnostic assays that are used for cattle. The objective of tuberculosis eradication in livestock requires adaptation of existing control strategies to include goats. As such, it is necessary to determine whether current diagnostic assays for tuberculosis in cattle will work as efficiently in the goat. This review provides an overview of current in vivo tools for diagnosis of caprine tuberculosis, including estimates of the sensitivity and specificity of tests performed in this species. The number of tested animals and co-infection with Mycobacterium avium subsp. paratuberculosis are also addressed, with the aim of demonstrating the limitations of current assays and the need for further research.     

Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl-Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.     

Evaluation of the long-term immune response in cattle after vaccination against paratuberculosis in two Dutch dairy herds

In the past decades, vaccination against paratuberculosis in cattle was performed in The Netherlands only on a limited scale. Because of its interference with the diagnosis of bovine tuberculosis, vaccination was restricted to herds with a high prevalence of clinical cases of paratuberculosis and was meant to aid in the economical survival of the farm. Recently, a voluntary paratuberculosis certification program has started, based in part on serological screening of cattle of at least 3 years of age. Herds that have been vaccinated against paratuberculosis are, therefore, likely to encounter problems when entering this program.The aim of this study was to evaluate the immune response resulting from vaccination with a heat-killed paratuberculosis vaccine. Over a period of 12-14 years, new-born calves were vaccinated in two herds. The B-cell response was evaluated using both the complement-fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) and the cell-mediated immune response was evaluated using the gamma-interferon assay. Data obtained show a marked and prolonged effect of the vaccination on both cellular and humoral immune responses, in particular to the paratuberculosis antigen but also to the bovine tuberculosis antigen, using the respective tests. These responses were detected rapidly after vaccination. The individual responses were highly variable between animals with respect to both the level and to the duration of the evoked immune response. No relation between the results obtained with the ELISA and the CFT was observed. In conclusion, for a large number of vaccinated cattle, a long lasting interference is to be expected with the presently available immunodiagnostic methods for both bovine tuberculosis and paratuberculosis.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.