Evaluation of specificity of tuberculosis diagnostic assays in caprine flocks under different epidemiological situations

The aim of this study was to evaluate the specificity of the most widely used tuberculosis (TB) diagnostic tests, single intradermal tuberculin (SIT) and single comparative intradermal tuberculin (SCIT) tests and interferon-gamma (IFN-γ) assay in 937 animals from eight TB-free caprine flocks under different epidemiological situations. Maximum specificity was found using SCIT test (99.4-100% depending on the interpretation criteria) while SIT test and IFN-γ assay showed a slightly lower overall specificity (97.6-99.2% and 96.4-98.4% respectively). Specificity of the SIT test in a Corynebacterium pseudotuberculosis infected flock was significantly (P<0.05) lower if a severe interpretation criterion was applied. Similarly, specificity values of SIT test and particularly IFN-γ assay in a paratuberculosis (PTB)-vaccinated flock were lower than those observed in non-vaccinated flocks. Higher proportion of false positive reactors to TB tests (SIT and IFN-γ assay) were observed among animals positive in the PTB-ELISA in PTB vaccinated flock. These results demonstrate that TB diagnostic tests show an adequate specificity when performed in goats from TB-free flocks in most situations. However, certain factors such as C. pseudotuberculosis infection and paratuberculosis vaccination can have a negative impact in the most sensitive tests.     

Evaluation of the gamma interferon test for diagnosis of paratuberculosis in goats

The gamma interferon assay was evaluated for diagnosis of paratuberculosis in goats with special emphasis on false positive reactions. Four categories of herds were tested: (A) herds that had a history of paratuberculosis, had given positive Mycobacterium avium subsp. paratuberculosis fecal samples and were vaccinated against paratuberculosis; (B) herds that had been vaccinated but had never shown clinical signs of paratuberculosis nor given positive M. a. paratuberculosis fecal samples; and (C) non-vaccinated herds without paratuberculosis. To extend the analysis of samples from young goats free of paratuberculosis, animals less than 18 months of age from non-vaccinated herds without paratuberculosis, category D, were included. Heparinized blood was stimulated with purified protein derivate (PPD) from M. a. paratuberculosis for 24 h and plasma was assayed for the presence of gamma interferon. Results were recorded as the difference between OD values of PPD stimulated and control samples. Vaccinated animals from herds with paratuberculosis, category A, showed significant higher gamma interferon responses than animals from vaccinated herds without paratuberculosis, category B. In both these groups the responses were correlated to age with higher responses in younger animals. Some of the vaccinated animals in herds without paratuberculosis had a gamma interferon response lasting for several years, which demonstrate a long lasting interference with diagnostic testing in vaccinated goats. Only three of the 121 non-vaccinated animals free of paratuberculosis in category C had responses against PPD (corrected OD values at 0.2, 0.24 and 0.5), and none of the 255 young animals in category D had corrected OD values exceeding 0.2. This indicates that false positive reactions do not appear to the same extent in young goats as in young cattle. We conclude that the low responses of non-infected goats could make the gamma interferon assay useful in monitoring the paratuberculosis status of non-vaccinated herds. However, more information about the early gamma interferon responses of naturally infected goats and the presence of false negative samples are needed.     

Experimental infection with Mycobacterium caprae in goats and evaluation of immunological status in tuberculosis and paratuberculosis co-infected animals

Tuberculosis in goats (caused by Mycobacterium caprae and M. bovis) has become a significant concern in recent years because of its high prevalence in certain caprine herds in Spain and other European countries, and also due to the potential transmission to other animals and human beings. In the present study, a transthoracic model of tuberculosis infection was performed on goats. Animals were selected based on the serological response used to detect paratuberculosis in goats (negative and positive results). The kinetics of the immune response was evaluated using the interferon-γ (IFN-γ) assay, skin tests and serology of paratuberculosis during nine months post-challenge. At the end of the study the animals were necropsied, tuberculosis-lesions were scored and culture (M. caprae and M. avium subsp. paratuberculosis) was performed to determine the true infection status. Animals were positive to the IFN-γ assay 15 days post-challenge and the values were fluctuating throughout the study. A varied performance of the assay was observed between tuberculosis and tuberculosis-paratuberculosis mixed infection regarding both the number of positive results and the OD values obtained after stimulation with bovine and avian PPDs. Furthermore, the single intradermal comparative cervical tuberculin test did not detect all M. caprae-infected animals. At necropsy, a positive correlation between pathology score and bovine PPD specific IFN-γ response was found.     

Use of the johnin PPD interferon-gamma assay in control of bovine paratuberculosis

Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other issues, this relates to problematic interpretation of the test results and maybe an over-expectation of what can be deducted from this kind of test given the chronic nature and slow development of infection of paratuberculosis. Over a number of years a modified IFN-γ assay with addition of recombinant bovine IL-12 to the PPDj stimulation of blood samples from the heifer group in more than 20 Danish dairy herds which also perform surveillance of MAP antibodies in milk have been performed. The results indicate that IFN-γ assay results are specific for paratuberculosis, but the IFN-γ assay result of an individual animal cannot establish whether the animal is infected or predict the future progression of disease in this animal. The IFN-γ assay should thus be used on a group of animals to test the level of exposure to paratuberculosis bacteria the animals have experienced, and thereby assist in maintaining rational in-herd management procedures and in the establishment of paratuberculosis status of a given herd. Indeed, for any diagnostic test applied in paratuberculosis, both the diagnostic target condition and the purpose of the diagnostic testing must be considered before any meaningful estimates of sensitivity or specificity can be given.     

Use of recombinant proteins in antibody tests for bovine tuberculosis

Tuberculosis (TB) in cattle remains a major zoonotic and economic problem in many countries. Since the standard diagnostic assay, the intradermal test (IDT) with bovine PPD tuberculin, has less than optimal accuracy in all situations, other diagnostic methods such as serological assays have been investigated. Because of fundamental concerns for the low sensitivity and specificity of previous ELISA protocols, a profiling ELISA with nine purified, recombinant proteins of TB complex mycobacteria, was employed on samples from four groups of cattle: (a) naturally Mycobacterium avium-exposed and experimentally Mycobacterium bovis-infected, (b) officially-certified TB-free herds, (c) exposed to M. bovis in two field TB outbreaks and scored as bovine reactors in the gamma-IFN assay for bovine TB, (d) paratuberculosis (para TB)-infected. The described ELISA proved to be highly specific. In fact, the antibody (Ab) response could be consistently detected in 3 out of 3 endotracheally-infected calves and in 1 out of 3 contact-infected calves. There was also a very low prevalence of low-titered, non-specific Ab responses in paraTB-infected animals. As for the animals exposed to field TB outbreaks, 16 out of 28 gamma-IFN positive cattle were also Ab-positive; importantly, 7 out of 12 gamma-IFN positive, IDT-negative cattle showed Ab responses to TB proteins. In general, the profile of the Ab response varied among animals; the reaction to single recombinant antigens was sometimes transient and fluctuating, whereas the panel of antigens on the whole was indeed more effective in Ab detection.     

Evaluation of the specificity of the gamma-interferon test in Italian bovine tuberculosis-free herds

We investigated the specificity of the gamma-interferon test for bovine tuberculosis (TB) in 1,557 cattle in 30 paratuberculosis-free and officially certified TB-free dairy herds, located in three provinces of the Lombardy Region in Northern Italy. The TB-free status of the herds under examination was further confirmed by the tuberculin skin test, by an antibody assay and by post mortem examination of animals culled from the herds during the study period. The specificity of the gamma-interferon tests after a single test and a double sampling scheme were 88.8% and 95.4%, respectively. After a single test, 11.7% of dubious reactors were also detected, while most cattle (47.4%) were shown to be avian reactors, probably due to contamination from infected birds and/or forage. There was strong evidence that the specificity of the test could be related to the animals' interaction with environmental mycobacteria and/or ageing. To reduce the percentage of nonspecific bovine reactors under alleged TB-free conditions, test procedures might involve the use of more specific antigens and/or different reaction thresholds.     

基于结核菌素与CFP10／ESAT6的牛IFN-γ检测法在牛结核病诊断中的比较研究

本研究通过比较三种刺激物（牛结核菌素、禽结核菌素和牛型结核菌特异性抗原CFP10／ESAT6对结核菌素皮内变态反应阳性牛的IFN-γ刺激反应,探讨IFN-γ检测法在我国牛结核病诊断中的应用前景。无菌采集22头结核菌素皮内变态反应阳性牛的血液．肝素抗凝。每1mL全血与1mL RPMI1640完全培养基混合均匀,并加入0.1mL（20μg）PHA（阳性对照孔）、0．1mL（20μg PHA）CFP10／ESAT6融合蛋白、0．1mL（2000U）＆结核菌素（PPD／B）、0．1mL（2500u）禽结核菌素（PPD／A）或等量PBS（无刺激阴性对照）,37℃培养过夜。次日用夹心ELISA法检测各刺激组0．1mL培养上清的IFN-γ,以OD630表示IFN-γ浓度。结果,特异性抗原CFP10／ESAT6刺激组与牛结核菌素（PPD／B）刺激组的IFN-γ反应具有良好的相关性．相关系数为0．84。但CFP10／ESAT6刺激组IFN-γ浓度与牛和禽PPD的比较反应（以两刺激组IFN-γ浓度差值表示）间无相关性,相关系数为-0．11。分析禽PPD组的IFN-γ反应,发现实验牛中有少数牛对禽PPD有反应。以OD630=0．17为阳性反应切割值,牛PPD检出阳性牛21头,CFP10／ESAT6检出20头,牛和禽PPD比较反应（以OD值差值表示）检出14头,禽PPD阳性反应3头。扣除禽PPD阳性反应牛后,牛和禽PPD比较反应与牛PPD刺激组的相关系数增至0．54。结果表明,牛PPD的IFN-γ释放反应检测灵敏度最高。当出现牛型结核菌与环境分枝杆菌混合感染时．应用牛和禽PPD比较反应检测牛结核的准确度低,混合感染牛被误判为结核阴性牛。而基于CFP10／ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性与敏感性。     

Evaluation of cellular and serological diagnostic tests for the detection of Mycobacterium bovis-infected goats

We have evaluated the comparative intradermal skin (CID) test, the interferon gamma (IFN-γ) assay, two ELISAs with bovine PPD antigen, the standard and the anamnestic using sera obtained, respectively, at the time of the tuberculin injection and 15 days later, for the diagnosis of caprine tuberculosis (TB). The sensitivity and specificity results were high for the CID test (83.7%, 100%), the IFN-γ assay (83.7%, 96%) and the anamnestic ELISA (88.6%, 95.8%). In contrast, they were comparatively low for the standard ELISA (54.9%, 88%). However, test results with the standard ELISA were positive in a group of goats with cavitating TB (100%). A combination of the CID test and the IFN-γ assay offered the highest sensitivity, 95.8%, and also high specificity, 96%. In spite of this, the evidence that the serological tests were most sensitive for the detection of goats with severe lesions (100% positivity) suggested that a combination of CID test and anamnestic ELISA may be most useful as part of an eradication campaign against caprine TB.     

Control of paratuberculosis (Johne's disease) in goats by vaccination

After several years of unsuccessful efforts to eradicate paratuberculosis in goats in Norway by conventional methods such as general hygienic precautions and the isolation and slaughtering of clinically affected and serologically positive animals, a vaccination programme was initiated in 1967. The vaccine used consists of two live attenuated strains of Mycobacterium paratuberculosis suspended in a mixture of liquid paraffin, olive oil and pumice powder. The vaccine may be stored at 4 degrees C for two weeks, the dose is 1 ml and the goat kids are vaccinated at the age of two to four weeks. The efficacy of the vaccine has been judged mainly by post mortem examination of vaccinated and unvaccinated goats in the period 1967-82. During this period about 131,000 goats were vaccinated and, based on the post mortem examination of 15,219 goats, the infection rate was reduced from 53 to 1 per cent. Moreover, infection occurred almost exclusively in goats which for some reason or other had not been vaccinated or which had been too old when vaccinated. The results of these examinations showed that the adjuvanted vaccine with live M paratuberculosis bacteria offers a high degree of protection against paratuberculosis in goats.     

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.     

Diagnosis of Mycobacterium bovis infection in calves sensitized by mycobacteria of the avium/intracellulare group

Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.     

Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle

The continued use of purified protein derivative (PPD) tuberculin is considered to be the main factor which limits the specificity of diagnostic tests for bovine tuberculosis (TB). This study evaluated a whole blood interferon-gamma (IFN-gamma) assay and compared the diagnostic potential of PPD with two tuberculosis-specific antigens, ESAT-6 and MPB70. To provide estimates of sensitivity and specificity, responses were measured in 180 skin test-reacting cattle, of which 131 were confirmed as tuberculous, and in 128 cattle from TB-free herds. For the skin test reactors, there was a positive correlation between the IFN-gamma responses to PPD from Mycobacterium bovis (PPDB) and PPD from Mycobacterium avium (PPDA), indicating cross-reactivity between these complex antigens which are the basis of the skin test. In comparisons of the ESAT-6 IFN-gamma test with a PPD IFN-gamma test (using PPDB compared with PPDA), there was a decrease in sensitivity (76.3 per cent vs 89.3 per cent), but a clear increase in specificity (99.2 per cent vs 92.2 per cent). The provision of high specificity, even with lower sensitivity, offers major benefits for testing in areas with a low incidence of TB.     

Low prevalence of bovine tuberculosis in Somali pastoral livestock, southeast Ethiopia

A cross-sectional study of bovine tuberculosis (BTB) detected by the comparative intradermal tuberculin test (CIDT) was conducted in livestock of the Somali region in southeast Ethiopia??in four pastoral associations from January to August 2009. In 94 herds, each of 15 cattle, camels, and goats was tested per herd leading to a total of 1,418 CIDT tested animals, with 421 cattle, 479 camels, and 518 goats. A herd was considered positive if it had at least one reactor. Prevalence per animal species was calculated using a xtgee model for each species. The individual animal prevalence was 2.0% [95% confidence interval (CI), 0.5?C8.4], 0.4% (95% CI, 0.1?C3%), and 0.2% (95% CI, 0.03?C1.3) in cattle, camels, and goats, respectively. Prevalence of avian mycobacterium purified protein derivative (PPD) reactors in cattle, camels, and goats was 0.7% (95% CI, 0.2?C2.0%), 10.0% (95% CI, 7.0?C14.0%), and 1.9 (95% CI, 0.9?C4.0%), respectively, whereby camels had an odds ratio of 16.5 (95% CI, 5.0?C55.0) when compared to cattle. There was no significant difference between livestock species in BTB positivity. In the present study, the prevalence of bovine tuberculosis was low in Somali pastoral livestock in general and in camels and goats in particular. The high proportion of camel reactors to avian PPD needs further investigation of its impact on camel production.     

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis.Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.     

Unexpected high responses to tuberculin skin-test in farmed red deer: implications for tuberculosis control

Tuberculosis (TB) in deer is a serious zoonotic disease of worldwide distribution. Detection of infected animals is usually performed using single or comparative skin-testing (SST/CST), although false responses due to sensitization to other mycobacteria may occur, hampering diagnostic specificity. We describe the evolution of the responses to the SST, CST and to an in-house serological assay in a red deer farm subjected to regular TB testing in southern Spain in an attempt to understand the dynamics of possible non-specific reactions occurring under field conditions. We performed 2288 skin-tests and ELISAs in nine sampling periods between May 2009 and January 2011. In May 2010, a strong increase in skin fold thickness in response to avian purified protein derivative (PPD) (mean=4.0mm, 95% CI=3.5-4.5) and bovine PPD (mean=1.8mm, 95% CI=1.6-2.0) was observed in yearling deer hinds (n=150), compared to values recorded for the same individuals in November 2009 (avian PPD: mean=0.7 mm, 95% CI=0.6-0.8 and bovine PPD: mean=0.7 mm, 95% CI=0.6-0.7) and in January 2011 (avian PPD: mean=2.2mm, 95% CI=1.9-2.4 and bovine PPD: mean=1.1mm, 95% CI=1.0-1.2). Using SST, 54 animals (36%) of the yearlings tested in May 2010 would have been classified as positive reactors, while none of them was positive in the CST. The five animals with highest skin fold increases to mycobacterial antigens were culled and subjected to post-mortem analysis, which confirmed the absence of Mycobacterium tuberculosis complex (MTBC) infection but demonstrated the presence of environmental mycobacteria and closely related bacteria in four out of the five analyzed animals. Our results demonstrated how non-specific responses to mycobacterial antigens can adversely affect the specificity of TB diagnosis based on the SST. Thus, once TB infection has been ruled out using confirmatory techniques, application of comparative diagnostic tests is highly advisable to maximize test specificity and avoid the slaughter of false positive reactors.     

In vitro lymphocyte transformation as a herd survey method for bovine paratuberculosis.

The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.     

Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

Failure of foetal protection after vaccination against an experimental infection with bovine virus diarrhea virus

A protocol is described to measure the protection of the bovine fetus against an experimental bovine virus diarrhea virus (BVDV) infection after vaccination. Two inactivated experimental vaccines were applied twice with a 3 week interval. A mixture of three different Dutch field strains was used as challenge on mainly the 82nd day of gestation to vaccinated and unvaccinated control animals. The challenge was applied 5 months after completion of the two-fold vaccinations. All calves born from unvaccinated control animals were persistently infected. The calves born from dams vaccinated with the two different inactivated BVDV vaccines were persistently infected in 78 and 60%, respectively.     

Application of different methods for the diagnosis of paratuberculosis in a dairy cattle herd in Argentina

Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.