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.     

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.     

Subclinical paratuberculosis in goats following experimental infection: An immunological and microbiological study

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5–8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls.

Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-γ assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15–20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI.

Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-γ assay.

The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of γδ T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.     

Application of a semi-dependent latent model in the Bayesian estimation of the sensitivity and specificity of two faecal culture methods for diagnosis of paratuberculosis in sub-clinically infected Greek dairy sheep and goats

In this study, we compared the frequency of isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from faecal samples grown on Herrold's egg-yolk medium (HEYM) or on Lowenstein-Jensen (LJ) medium and estimated the sensitivity (Se) and specificity (Sp) of the methods separately in sub-clinically infected Greek dairy sheep and goats, using latent-class models and Bayesian estimation procedures. Faecal and blood samples were collected from 400 animals > or =1 year old in April-May 2002. The HEYM supported growth of MAP better than the LJ method and their agreement was very poor (weighted kappa=0.062 (95% CI: -0.098, 0.222)). There was no evidence of dependence between the Ses whereas the Sps were positively correlated. Thus, a semi-dependent model that assumed independence of Ses and accounted for the dependence of Sps was adopted. Under this model, the parallel interpretation of the results of the two methods gave median estimates and 95% credible intervals (CrIs) for Se(par), Sp(par) of 15% (CrIs: 3, 45%), 96% (92, 98%) in sheep and 16% (6, 36%) and 97% (94, 99%) in goats.     

Transabdominal ultrasonographic findings in goats with paratuberculosis

This study describes the transabdominal ultrasonographic findings in 54 goats with confirmed Johne’s disease (JD). Compared with the control group (0.8 ± 0.4 mm thick), the test group presented with mild (2.8 ± 0.2 mm), moderate (4.2 ± 0.4 mm), and severe (6.9 ± 1.1 mm) thickening of the intestinal wall. The most outstanding ultrasonographic findings were pronounced enlargement of the mesenteric lymph nodes in 49 goats. In 36 goats, the enlarged lymph nodes showed a hypoechoic cortex and a hyperechoic medulla. In 7 goats, the cortex and medulla were hypoechoic. In 5 goats, the cortex and the medulla could not be differentiated. In the remaining cases, the cortex and medulla contained small hypoechoic lesions. Necropsy findings included enlarged mesenteric lymph nodes in 52 goats and thickening of the small intestinal wall in 30 goats. Compared with the postmortem results, the antemortem ultrasound sensitivity in detecting intestinal wall thickness and enlarged mesenteric lymph nodes was 80% and 94%, respectively.     

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

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

Progressive immunopathological changes during early stages of experimental infection of goats with Mycobacterium avium subspecies paratuberculosis

A dose of 10(10) Mycobacterium avium subspecies paratuberculosis was administered orally on seven occasions to produce experimental paratuberculosis infection in 10 5-8-week-old goat kids. Bacteriological, immunological, and histopathological changes, their relationships, and the efficacy of the commonly used diagnostic methods were studied during the progressive disease up to 270 days postinfection (DPI). Significant lymphocyte proliferative responses in the peripheral blood of five goats were detected as early as 60 DPI. A lymphoproliferative test was also performed on lymphocytes purified from different compartments of the guts of five infected and five control goats. Significant proliferative responses were observed in lymphocytes of jejunal compartments of all five goats, of which four had also significant lymphocyte proliferation in the blood. The ileal lymphocytes from two goats, one each at 120 and 270 DPI, had significant proliferation. The histological lesions were mainly observed in the gut-associated lymphoid tissues of the ileocecal valve, the ileum, and the terminal jejunum. Acid-fast bacilli were demonstrated in the lesions of two goats at 60 and 210 DPI. Bacterial culture showed poor sensitivity, detecting positive results for only one goat in the fecal and tissue samples at 210 DPI, whereas polymerase chain reaction (PCR) detected one goat in fecal sample at 210 DPI and two goats in tissue samples at 60 and 210 DPIs, respectively. Enzyme-linked immunosorbent assay and agar gel immunodiffusion test were found to be 100% sensitive from 180 and 210 DPI onwards, respectively.     

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.     

Evaluation of different methods for the diagnosis of scabies in swine

Scabies in pigs is still very common in many countries and can be detrimental to the productivity of pigs. However, correct diagnosis of the disease can preclude meaningful comparisons of results. Therefore, the objective of the present study was to determine, on 11 pig farms, the prevalence of scabies by determination of the presence of mites in ear scrapings, the dermatitis score, the SI and the detection of specific serum antibodies. For the latter an indirect ELISA technique was performed using a free-living mite as a source of antigen. A second objective was to compare the value of these different diagnostic tests. Four farms were positive for the presence of mites. Our study indicated that the SI of piglets is not reliable as a diagnostic tool for scabies (all values were below the threshold value of 0.4, even on farms that were positive for mites) but on the two farms with the highest prevalence of mites the SI was above the threshold for the finishers. However, the fact that sows from eight of the 11 farms investigated had a SI>0.4 would indicate that for sows either the SI is not very specific, or that a cut-off level of 0.4 is not relevant for this age group. On three of the four infected farms the ADS was higher than the cut-off value of 0.5, and on the fourth farm, where the ADS was only 0.43, individual carcasses with generalised dermatitis (score 2) were present. However, an ADS>0.5 did not always coincide with the presence of mites. On six farms, ODR values were indicative for the presence of Sarcoptes, and on three of these farms this was confirmed with positive ear scrapings. In conclusion, as determined by the detection of mites in pig ears, especially the results from the dermatitis scores seem to be useful in the diagnosis of scabies. The specificity of the other parameters is not sufficient, and therefore, the detection of mites should still be used to confirm scabies on a farm, in combination with other tools.     

辽宁东南部地区绒山羊副结核病血清学调查

为了摸清我省辽宁绒山羊副结核病的流行情况,给防控工作提供依据,采用ELISA检测方法对辽宁东南部地区的辽宁绒山羊开展了副结核病血清学调查工作。调查结果:该地区绒山羊副结核病平均血清阳性率为12.54%;其中3个规模化养殖场的平均血清阳性率为16.06%,最高阳性率为18.4%,最低为11.82%;5个散养户的平均血清阳性率为7.41%,最高阳性率为10.71%,最低为4.44%;研究结果表明副结核病在该地区辽宁绒山羊中广泛存在,而且感染率较高,规模化养殖场的血清阳性率要明显高于散养户;建议有关部门和广大养殖户应对本病引起足够的重视,加强防控工作。     

Effect of an inactivated paratuberculosis vaccine on the intradermal testing of goats for tuberculosis

The effect of an inactivated paratuberculosis vaccine on the diagnosis of tuberculosis (TB) in goats was investigated in a herd with a history of clinical paratuberculosis but which was free of TB. Cohorts of animals in 2006, 2008 and 2009, were vaccinated once at 1 month of age, and 50% of the 2006 cohort served as unvaccinated controls. The goats were aged 8 months, 20 months and 3.5 years old at the time of the survey. All animals were assessed using a single intradermal injection of bovine tuberculin purified protein derivative (PPD) (SID test), or using both bovine and avian PPD (CID test). An interferon (IFN)-γ assay using both bovine and avian PPD was carried out on the 2006 cohort and was interpreted according to three different 'cut-off' points. No unvaccinated (control) animals tested positive to any of the assays, confirming that the herd was TB-free. The SID test had a low specificity in vaccinated animals at 8 and 20 months of age, whereas the CID test demonstrated 100% specificity in animals ≥20 months-old. The specificity of IFN-γ assay was less than maximal for vaccinated animals 3.5 years old as small numbers of false positives were detected, although this depended on the chosen cut-off point. The study findings demonstrate that the use of an inactivated paratuberculosis vaccine in goats <1 month-old in a TB-free herd does not result in false positives to a CID test for TB when performed in animals ≥20 months-old.     

Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues

The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections.     

Detection methods of Mycobacterium paratuberculosis

Mycobacterium paratuberculosis is receiving increasingly wider interest of scientific groups worldwide. This slow-growing mycobacterium does not only evoke paratuberculosis--an infectious cattle disease that brings huge economic losses--but it is also regarded as a potential cause of human Crohn;s disease. It is very difficult to diagnose precisely this kind of animal infection in its very early stages, as well as to detect occurrence of M. paratuberculosis cells in the environment, including food of animal origin. This paper reviews currently known and employed diagnostic techniques for M. paratuberculosis cell detection and identification.     

Diagnostic testing patterns of natural Mycobacterium paratuberculosis infection in pygmy goats

Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFNγ), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFNγ response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNγ results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats.     

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.     

Bayesian estimation of sensitivity and specificity of serum ELISA and faecal culture for diagnosis of paratuberculosis in Greek dairy sheep and goats

Latent class models were used to estimate the sensitivity (Se) and the specificity (Sp) of a serum ELISA and a faecal culture (FC) method for the diagnosis of paratuberculosis separately, in sheep and goats. The estimates were obtained by a Bayesian method. Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence given the true disease status. ROC analysis for the serum ELISA was also performed and optimized cut-off values based on the misclassification cost term were determined. No evidence of conditional dependence was found. Assuming independence, posterior medians and 95% credible intervals for the Se(ELISA), Sp(ELISA), Se(FC) and Sp(FC), were 63% (42, 93%), 95% (90, 98%), 8% (2, 17%) and 98% (95, 100%) in goats and 37% (10, 80%), 97% (93, 99%), 16% (2, 48%) and 97% (95, 99%) in sheep. AUC was calculated 0.702 for sheep and 0.847 for goats. For the serum ELISA, there is need of species- and purpose-specific cut-off selection. For instance, with 20% prevalence situation and assuming equal and five-fold cost of a false negative to a false positive test result, the optimal cut-off is 0.3 and 0.05 in sheep, respectively, while it is 0.6 and 0.1 in goats, respectively. Serum ELISA performed better in goats than in sheep. Lowering the cut-off, in relation to the one recommended by the manufacturer, improved Se(ELISA) without seriously compromising Sp(ELISA), in either species.     

Agreement between ELISA and complement fixation test used for diagnosing of paratuberculosis in goats

An ELISA with a lipoarabinomannan as an antigen, developed for diagnosis of bovine paratuberculosis, has been adapted for use in goats, and compared with complement fixation test. Kappa value of 0.62 indicated good agreement between CFT and the adapted ELISA and proved that the investigated ELISA may be helpful in diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats. The ELISA has been used to screen a randomly selected representative sample of Polish breeding goat population (21.78% of herds, 21.33% of goats). It has been demonstrated that only 2.42% of animals coming from 15.79% of herds were seropositive. Within-herd seroprevalence varied from 1.69% to 38.10%. Most of the infected animals (67.07%) were 3- 4-years-old. No seropositive cases were found in group up to 1-year-old animals.     

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.