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PubMed2001

Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle.

OBJECTIVE: To evaluate sensitivity and specificity of a new ELISA for antibodies against Mycobacterium avium subsp paratuberculosis. DESIGN: Cross-sectional observational survey. SAMPLE POPULATION: Serum samples from 590 cattle that were infected with M avium subsp paratuberculosis and 723 cattle that were not infected. PROCEDURE: Serum samples were tested by use of an ELISA for antibodies against M avium subsp paratuberculosis. RESULTS: Sensitivity of the test varied from 15.4 to 88.1%, depending on the clinical stage and bacterial shedding status of the cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

Animals↗
PubMed2006

Genetic variation of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins.

The objective of this study was to estimate genetic variability of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. Blood and fecal samples were collected primarily from daughters of 12 bulls in their second or third lactation. Routine disease testing of the sires documented that they were not infected. Herds without a "suspect" or positive ELISA (sample/positive ratio > or = 0.10) or positive fecal culture test were deleted from the data set. The remaining 4,603 cows from 238 herds and 46 sires were used to estimate heritability of M. paratuberculosis infection. Heritability was estimated with 3 Johne's disease diagnostic tests: 1) fecal culture alone, 2) serum antibody ELISA alone, and 3) both tests (combined) with a positive animal defined as all animals with either a positive fecal culture or ELISA test. Four statistical models were used to estimate heritability: 1) linear (ELISA), 2) threshold (fecal culture and combined), 3) ordered threshold (ELISA), and 4) bivariate linear-threshold (ELISA-fecal culture). A sire model and Bayesian approach using Markov chain Monte Carlo methods were used in each case. Heritability of infection based on the fecal culture test was 0.153 [posterior standard deviation (PSD) = 0.115]. Heritability with the ELISA was 0.159 (PSD = 0.090) with a linear model and 0.091 (PSD = 0.053) with an ordered threshold model. Heritability of the combined tests was 0.102 (PSD = 0.066). Heritability estimates of fecal culture and ELISA with the bivariate model varied slightly from estimates obtained with the univariate models (0.125 and 0.183, respectively), with a corresponding increase in precision (PSD = 0.096 and 0.082, respectively). This study demonstrates that exploitable genetic variation exists in dairy cattle for M. paratuberculosis infection susceptibility.

Animals↗
PubMed2006

Age at occurrence of Mycobacterium avium subspecies paratuberculosis in naturally infected dairy cows.

Paratuberculosis is a chronic infection of ruminants and other species caused by Mycobacterium avium ssp. paratuberculosis (Map). Establishing test strategies for paratuberculosis will require insight into the temporal aspects of certainty with a given test. In this study, the age at which cows tested positive by ELISA and fecal culture (FC) was investigated by use of time-to-event analyses. The effects of herd, parity, and shedding group were evaluated at the age of test-positive ELISA and FC, respectively. Finally, the test frequency was investigated for the probability of cows being tested ELISA-positive. Milk and fecal samples were collected repeatedly over a 3-yr period from 1,776 Danish dairy cows from 8 herds. The milk samples were tested for the presence of antibodies by using an ELISA, and an FC test was used for detection of Map. Repeated ELISA testing detected 98 and 95% of cows classified as high and low shedders, respectively, suggesting that most infected cows develop antibodies. Among the high shedders, 50% were positive before 4.3 yr of age (quartiles 1 to 3: 3.4 to 5.7 yr of age). Repeated FC detected only 72% of the cows that were ELISA-positive, and 50% of the ELISA-positive cows were detected by FC at 7.6 yr of age. The age with the highest probability of testing positive was determined as the interval with the steepest slope in the survival probability plots. The highest probability of testing positive by ELISA was from 2.5 to 4.5 yr of age. The highest probability of testing positive by FC was from 2.5 to 5.5 yr of age. For both ELISA and FC, testing positive was highest in the first 300 d in milk. For cows younger than 4 yr of age, monthly testing with ELISA, compared with testing every 2 yr, could increase the probability of detecting cows with antibodies by 19%. In older cows, there were no apparent differences in the probability of testing positive by monthly sampling compared with sampling every second year. Therefore, for older animals the effect of more frequent sampling would be for early detection rather than to obtain additional information. Cows shedding high numbers of Map will produce antibodies, although not necessarily concomitantly with the shedding. These antibodies can be detected by ELISA with a test strategy that is different for younger and older cows. We suggest testing younger cows more frequently than older cows and that testing should be done prior to 350 d in milk.

Age Distribution↗
PubMed2002

Paratuberculosis in free-ranging fallow deer in Spain.

Paratuberculosis was diagnosed in a population of approximately 1,000 free-ranging fallow deer (Dama dama) sampled from 1997-98 in the Regional Hunting Reserve of El Sueve (Asturias, Spain). Five of eight animals observed with diarrhea were diagnosed as having paratuberculosis on the basis of gross lesions at postmortem examination and histopathology. In two deer, Mycobacterium avium subsp. paratuberculosis was cultured and identified by polymerase chain reaction. Indirect enzyme-linked immunosorbent assay and immunodiffusion tests were used to evaluate sera from 33 adult deer from this population. All fallow deer tested were seronegative.

Animals↗
PubMed2003

Testing for Mycobacterium avium subsp. paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd.

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johne's disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.

Animals↗
PubMed2000

[First description of concomitant infection with tuberculosis and paratuberculosis in dairy sheep in Argentina].

The authors present the results of a comparative intradermal tuberculin test performed on dairy sheep to determine rates of reaction to bovine and avian purified protein derivative (PPD). Test reactions were interpreted by palpation and measurement with a calliper. Results were analysed by plotting the values obtained. Agar gel immunodiffusion tests, growth in culture and histopathological examinations were also performed to allow differential diagnosis between tuberculosis and paratuberculosis. In the first tuberculin test, performed on 288 sheep, 74 animals showed a positive reaction to avian PPD (25.69%), two animals showed a positive reaction to bovine PPD (0.69%), and one animal was positive with respect to both tuberculins (0.34%). In the second test, involving 247 sheep, 55 demonstrated a positive reaction to avian PPD (22.26%), whilst none reacted to bovine PPD. The presence of Mycobacterium avium subsp. paratuberculosis was confirmed by culture and serological testing. In addition, lesions compatible with those caused by M. bovis were found by histopathological examination. These findings highlight the importance of the accurate diagnosis of tuberculosis and paratuberculosis for the effective control of both diseases.

Animals↗
PubMed2002

[Paratuberculosis: the pathogen and routes of infection].

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is a chronic and incurable enteritis of ruminants with economic importance worldwide. The infectious agent is an acid-fast rod defined solely based on its mycobactin-dependent growth in vitro and the presence of insertion element IS900. The bacterium, which is difficult to culture primarily due to its extremely slow growth, occurs not only in cattle but also in other ruminant. In addition, it has been isolated from non-ruminant species. Despite its wide spectrum of potential hosts the contact between adult cattle and calves is the predominant route of infection within a herd as well as among herds. To interrupt this route of infection hygienic measures, primarily for the housing and feeding of calves, as well as diagnostic measures prior to trading of cattle are urgently required.

Animals↗
PubMed2002

[The importance of allergic skin test with Johnin, antibody ELISA, cultural fecal test as well as vaccination for the sanitation of three chronically paratuberculosis-infected dairy herds in Rhineland-Palatinate].

Three chronically paratuberculosis infected herds were tested for six years twice a year (intradermal Johnin test, antibody ELISA (IDEXX Corp.), microbial culture) according to a sanitary program. Culling of shedding animals and vaccination of calves with NEOPARASEC (Merial Corp.) were part of the program. In course of experiment, 1015 samples of 228 non vaccinated cows and 1502 samples of 293 vaccinated cattle have been tested. 3.8% of the vaccinated animals proved positive in microbial culture. Nearly all vaccinated calves developed granulomas sized from hazelnut to loaf at the injection site. Positive reactions in intradermal test as well as in antibody ELISA were found in very young calves. 24.3%, 33.7%, 25.9%, respectively of the non vaccinated animals were identified as shedders of M. avium subsp. paratuberculosis (MAP) by microbial culture. In the first and in the second herd most shedders of MAP were found in the first herd examination (66.7%, 42.9%, respectively), whereas in the third herd they were detected in the fifth examination (31.0%). At the beginning, 17.9% of non vaccinated animals proved positive in intradermal test, 14.4% in antibody ELISA. Afterwards, the number of positive test results decreased but increased again towards the end of the experiment. 48.5% of the 66 shedders showed positive reactions in intradermal test, 57.6% in antibody ELISA, 77.3% in at least one of these both tests. Antibodies in ELISA were found in rising frequency from two years before the time of shedding. 50.0% of the shedders reacted positive in ELISA at the time of shedding. In selected shedders first positive results were found at the age of about two years. Unfortunately, only incomplete hygienic measures were realized by the farmers. Under field conditions the realisation of attending sanitary programs is difficult. MAP is spread mainly by buying of animals, therefore a certification program for paratuberculosis free herds is urgently necessary as well as an improvement of diagnostic methods.

Animals↗
PubMed1992

Isolation of Mycobacterium paratuberculosis after oral inoculation in uninfected cattle.

Feces from cows naturally infected with Mycobacterium paratuberculosis was given to 6 uninfected heifers by orogastric intubation, to determine whether ingested organisms could be passively excreted and detected by bacteriologic culture of feces (ie, false-positive result). Heifers were paired, and each pair received a different dose of feces on days 1 and 2. Fecal samples were collected from the heifers 3 times daily. Mycobacterium paratuberculosis was detected in fecal samples of all heifers within 18 hours of being given the first dose of feces. The number of colony-forming units peaked on days 3 or 4, and organisms were no longer detected by day 7. The number of colony-forming units in fecal samples from the heifers was approximately proportional to the dose given. On days 15 and 16, the experiment was repeated with feces from a second infected cow. Results were similar to those in the first experiment. All heifers remained seronegative (agar-gel immunodiffusion test and ELISA) and had negative results to the intradermal johnin test throughout the experiment. Lymph node and intestinal tissues were obtained from all 6 heifers at slaughter on day 28. Mycobacterium paratuberculosis was not isolated from mesenteric lymph nodes from the ileocecal valve region, but was isolated from ileal mucosal samples from each heifer.

Animals↗
PubMed1992

[Experimental paratuberculosis in sheep after intravenous or oral inoculation: pathogenicity and biologic diagnosis].

An experimental paratuberculosis study was performed in sheep. One group of 6 lambs was inoculated intravenously with the equivalent of 50 mg (wet weight) of live bacilli, another group of 6 lambs was inoculated orally by placing 500 mg (wet weight) of live organisms in milk feed and a group of 3 lambs was used as controls. The degree of cellular immunity was followed by examining delayed hypersensitivity using 3 allergens (bovine tuberculin PPD, avian tuberculin PPD and johnine PPD) and that of humoral immunity using complement fixation test, agar gel immunodiffusion test and ELISA. The elimination of bacilli in the faeces was examined simultaneously. After 2 years no macroscopic or microscopic lesion was observed in intravenously inoculated lambs and in those exposed orally to M paratuberculosis; cultures were negative. It appears that domestic sheep were able to control the infection. Nevertheless, most of them developed cellular and humoral immunity against paratuberculosis antigen. The best results were obtained in intravenously inoculated lambs.

Animals↗
PubMedDate not supplied

Comparison between a commercial and an in-house ELISA or anti-M. avium paratuberculosis antibodies detection in dairy herds in Rio de Janeiro, Brazil.

Paratuberculosis (Johne's disease) is a chronic enteritis that affects ruminants and is caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease is worldwide spread and causes important economic losses. In Brazil, Map has already been isolated, but there are no enough epidemiological studies about its prevalence. In this study, 179 sera of dairy cows originated from Rio de Janeiro State, Brazil, were tested for the presence of anti-Map antibodies using two ELISAs. First one was an "in house" ELISA using a protoplasmic paratuberculosis antigen (PPA - Allied Monitor) as capture antigen and a monoclonal anti-bovine IgG conjugated to alkaline phosphatase (Sigma) and the other was "HerdChek Mpt" (IDEXX), used as standard method in this study. Fifty-nine (32.9%) samples were reactive in the PPA-ELISA. Sensitivity of ELISA-PPA in relation to HerdCheck was 47.5% and specificity was 86.8%, with 54/103 of false negatives and 10/76 of false positives. Positive predictive value of PPA-ELISA was calculated as 86% and efficiency as 64.2%. PPA-ELISA and HerdChek results were also compared by kappa test (k) and concordance index was 0.320. PPA-ELISA can be useful as a collective herd test and can be recommended as a screening test for identification of infected herds and foci of Johne's disease.

Animals↗
PubMed1991

Serologic enzyme-linked immunosorbent assay responses of calves vaccinated with a killed Mycobacterium paratuberculosis vaccine.

The purpose of this study was to document the effect of calfhood vaccination for Mycobacterium paratuberculosis on a serologic ELISA. Fifteen calves vaccinated with a killed paratuberculosis vaccine and 5 unvaccinated control calves were tested from the first through the fifteenth month of life. Age of vaccination ranged from 5 to 40 days. Blood samples were collected prior to vaccination and periodically thereafter. Serum antibody was analyzed by use of the ELISA. All calves were ELISA-negative prior to vaccination. Thirteen of 15 vaccinated calves became ELISA-positive between 2 and 6 months after vaccination. The unvaccinated cohort remained ELISA-negative. Wide-spread use of vaccine may interfere with diagnosis of paratuberculosis and with control programs that are based on serologic tests that measure humoral antibody.

Animals↗
PubMed1995

[The prevalence of paratuberculosis in the Plateau de Diesse region].

We analysed 595 cows in 20 herds for serological evidence of paratuberculosis with an ELISA procedure for which we had recently determined a sensitivity of 50% and a specificity of 98%, and we obtained an overall calculated real prevalence of 5.99%. In two herds with clinical cases of paratuberculosis, the calculated true prevalence of seroreactors was 21.47%. In 9 herds with clinical suspicion of Johne's disease, the rate was 4.23%, 9 herds without any clinical suspicion showed a mean prevalence of 0%. Whereas the trend was clear for groups of herds, the observed prevalence in individual herds varied from 0% to 17.9%, without obvious association with manifest clinical problems of paratuberculosis.

Animals↗
PubMed1996

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.

Animals↗
PubMed1995

Mycobacterium paratuberculosis and Crohn's disease.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has been suggested that Crohn's disease, a gastrointestinal disease in humans with similar clinical symptoms and histopathological findings, is caused by the same bacteria. Epidemiological data support Crohn's disease possibly being caused by an infectious agent. Recently, with improved culture techniques and the advent of polymerase chain reaction, M. paratuberculosis has been identified from tissues of increasing numbers of patients with Crohn's disease. However, conflicting data still occur and the case for M. paratuberculosis as the etiological agent of Crohn's disease can be neither proved nor disproved. The development of new diagnostic tests and comprehensive well-defined clinical studies will hopefully provide us with information about this association in the future.

Antibodies, Bacterial↗
PubMed1996

Polymerase chain reaction analysis of TNF-alpha and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis.

Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using the polymerase chain reaction (PCR) and specific bovine oligonucleotide cytokine primers and probes, we examined the expression of messenger RNA for these cytokines in whole blood from M. paratuberculosis infected and uninfected cattle. Cytokine mRNA levels were examined before and after in vitro incubation with E.coli lipopolysaccharide (LPS) and lipoarabinomannan (LAM) purified from M. paratuberculosis. Uninfected calves, experimentally infected calves, and naturally infected cattle all displayed similar cytokine mRNA expression patterns. However, individual animals demonstrated variability in the levels of IL-6 and TNF-alpha mRNA expression as determined by a semiquantitative PCR method using 32P-labelled oligonucleotide probes.

Animals↗
PubMedDate not supplied

Comparison of the absorbed ELISA and agar gel immunodiffusion test with clinicopathological findings in ovine clinical paratuberculosis.

Thirty-two sheep with clinical paratuberculosis and 43 normal, healthy control animals were tested for serum antibodies to Mycobacterium avium subspecies paratuberculosis with the absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test. All the sheep were necropsied and the diseased cases were categorised as having either multibacillary ('lepromatous') or paucibacillary ('tuberculoid') intestinal lesions. The ELISA and AGID test were highly sensitive when testing the multibacillary group (86.4 per cent and 100 per cent, respectively) but the sensitivity of the tests in the paucibacillary group was significantly lower (10 to 50 per cent and 30 per cent, respectively). These findings were related to the ELISA optical density readings, with the multibacillary samples having values significantly greater than those of the paucibacillary and control groups, and the optical density values appearing to correlate with the numbers of mycobacteria present in the intestinal lesions. These results indicate both the usefulness of serological testing in the diagnosis of the multibacillary form of paratuberculosis and the difficulty in identifying animals with the paucibacillary form of the disease.

Animals↗
PubMedDate not supplied

Cost-benefit analysis of vaccination against paratuberculosis in dairy cattle.

Paratuberculosis is an infectious and incurable disease which causes considerable economic losses in dairy cattle, due mainly to premature disposal and losses of milk production. In 1984 the Animal Health Service North-Netherlands started a vaccination trial in which young calves were vaccinated once, to test whether vaccination reduced the production losses and whether the overall costs of vaccination were outweighed by the benefits. Vaccination against paratuberculosis reduced the number of clinically infected animals by almost 90 per cent. It also reduced the numbers of subclinically infected animals and animals with a positive histological and/or bacteriological test result. Although vaccination did not prevent losses in milk production, it reduced the infection pressure and the clinical signs of the disease. Partial budgeting showed that vaccination against paratuberculosis was highly profitable. The costs of vaccination were US$15 per cow and the benefits (total returns minus costs) were US$142 per cow.

Animals↗