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PubMed2001

Comparison of two enzyme-linked immunosorbent assays for serologic diagnosis of paratuberculosis (Johne's disease) in cattle using different subspecies strains of Mycobacterium avium.

Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests (z = 0.182).

Animals↗
PubMed2004

A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

Animals↗
PubMed1996

Production of gamma-interferon by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis.

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne's disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratu-berculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for gamma-interferon (gamma-IFN) production. Cells from subclinical cows produced significantly higher levels of gamma-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of gamma-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of gamma-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that gamma-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

Animals↗
PubMed1991

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.

Animals↗
PubMed2005

Comparison of diagnostic detection methods for Mycobacterium avium subsp. paratuberculosis in North American bison.

Tissues and fecal material were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. Fecal culture indicated that 5 of 14 (35.7%) animals were infected, whereas cultures from tissues detected 12 of 14 (85.7%) animals as infected and 59 of 111 (53.2%) of the tissues as positive for M. paratuberculosis. Polymerase chain reaction analysis identified infection in 14 of 14 (100%) animals and in 91 of 112 (81.2%) tissues. In addition, tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, and immunohistochemical staining. Ziehl-Neelsen and auramine O staining identified 7 of 14 (50%) and 5 of 14 (35.7%) animals as infected and 24 of 112 (21.4%) and 28 of 112 (25%) tissues as positive, respectively. Immunohistochemical analyses of bison tissues, using antisera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and positive tissues (ranging from 28 to 46%). Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more sensitive than bacterial culture or staining, identified infection in all the bison, and detected the greatest number of positive tissues within each animal.

Animals↗
PubMed2001

On-farm batch pasteurization destroys Mycobacterium paratuberculosis in waste milk.

A recent dairy survey conducted in 1996 by the National Animal Health Monitoring System suggests between 20 and 40% of dairy herds in the United States have some level of Johne's disease. This figure will continue to increase unless producers implement management regimes that will help control the spread of this disease within their herds. The neonatal calf is the target for infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease. Calves become infected via exposure to the bacterium through contaminated feces, bedding, colostrum, and milk. Shedding of viable M. paratuberculosis has been documented in the colostrum and milk of infected dams. This study evaluated the efficacy of on-farm pasteurization to destroy M. paratuberculosis in waste milk fed to calves to circumvent this mode of transmission. In three replicate experiments, waste milk was experimentally inoculated with M. paratuberculosis and heated at 65.5 degrees C for 30 min. No viable bacteria were recovered after 28 wk of incubation. These results suggest that batch pasteurization of waste milk contaminated with M. paratuberculosis was effective at generating a clean product to feed to young calves.

Animal Feed↗
PubMed2004

Culture and serologic survey for Mycobacterium avium subsp. paratuberculosis infection among southeastern white-tailed deer (Odocoileus virginianus).

From July 1998 through October 2002, radiometric culture (ileocecal lymph node, mesenteric lymph node, and feces) and serologic testing by enzyme-linked immunosorbent assay (ELISA) were used to survey white-tailed deer (Odocoilens virgianus) from the soutlheastern United States for infection by Mycobacterium avium subsp. paratuberculosis (Mptb), the causative agent of paratuberculosis (Johne's disease). Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one of 313 deer (0.3%) originating from 63 populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Six deer (2%), all from different populations, had ELISA results above a 0.25 sample-to-positive cutoff value, but none of the ELISA reactors originated from the population from which the single Mptb isolation was made. These six deer were seronegative when tested by agar gel immunodiffusion (AGID). Collectively, these data indicate that white-tailed deer currently do not constitute a broad regional reservoir for Mptb; however, further study is warranted to clarify the significance, if any, of infected deer to the epizootiology of paratuberculosis on a local scale. Adaptation and validation of an ELISA or another serologic assay for use with deer and other wildlife would markedly enhance Mptb surveillanece among wild populations and would be a powerful tool for gaining information on the role of wild species in epidemiology of paratuberculosis.

Animals↗
PubMed2005

Isolation of Mycobacterium avium subsp paratuberculosis (Map) from feral cats on a dairy farm with Map-infected cattle.

Paratuberculosis is an economically important disease of dairy cattle caused by Mycobacterium avium subsp. paratuberculosis (Map). The role of nonruminant, nondomestic animals in the epidemiology of paratuberculosis in cattle is unclear. To examine nonruminant, nondomestic animals for the presence of Map, 25 feral cats, nine mice (species unknown), eight rabbits (Sylvilagus floridanus), six raccoons (Procyon lotor), and three opossums (Didelphis virginiana) were collected from a mid-western dairy with known Map-infected cattle. Mycobacterium avium subsp. paratuberculosis was isolated from the mesenteric lymph node from seven of 25 (28%) feral cats. Ileum was culture-positive for three of these seven cats, and an isolation of Map was also made from the ileum of one of nine (11%) mice. Tissue samples from other species were negative as determined by Map culture; microscopic lesions consistent with paratuberculosis were not seen in any animal. Restriction fragment polymorphism analysis of isolates from cats and dairy cattle suggest interspecies transmission. The means by which interspecies transmission occurred may be through ingestion of Map-contaminated feces or waste milk or through ingestion of Map-infected prey. Shedding of Map from infected cats was not evaluated. The epidemiologic role of Map-infected feral cats on dairy farms requires further investigation.

Animals↗
PubMed2006

Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the central European Carpathians.

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.

Animals↗
PubMed2000

In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues.

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

Animals↗
PubMed2001

[Modelling of prevalence development in a paratuberculosis control program in a dairy herd].

A modelling approach to calculate the success of a paratuberculosis control programme in dairy herds is presented. The essential parameters of the model are the prevalence at the beginning of the programme, diagnostic sensitivity and specificity of the tests used, discipline in culling test-positive animals, turnover in the herd, percentage of replacement with own stock and paratuberculosis prevalence in animals bought into the herd from outside, and a general hygiene-based factor. Diagnostic measures and time schedule used in the modelling approach are given by the paratuberculosis-control-programme of the local board for infectious disease control in food animals in the state of Lower Saxony. It was found by the model-calculations that in case of a high initial prevalence the anticipated six-year duration of the control programme is justified in order to ensure a lasting improvement of herd health. If hygienic measures are strictly obeyed and all test positive animals are culled a clear reduction on paratuberculosis prevalence can be achieved within the first year. According to the model in the second and third year the prevalence will increase again despite ongoing diagnostic measures in order to decrease again continuously with the beginning of the fourth year. Given an initial prevalence of 10%, 20% or 30% the prevalence after six years is calculated to be at 3%, 5% or 8% when all measures are followed as given in the control programme. The presented programme seems to be appropriate to predict prevalence development in paratuberculosis infected dairy herds if the herds are managed according to the guidelines of the "Tierseuchenkasse Niedersachsen", the local board for infectious disease control in food animals in the state of Lower Saxony, Germany. It becomes apparent that within six years a high decrease of the prevalence in the herds, but not a complete eradication of disease can be achieved by consistently complying with the rules given in these guidelines.

Animals↗
PubMed2002

[Establishment and evaluation of an ELISA for the detection of antibodies in milk against Mycobacterium avium subspecies paratuberculosis].

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is the etiological agent of paratuberculosis (Johne's Disease), a chronic granulomatous enteritis of ruminants occurring worldwide with increasing frequency and leading to growing economic losses. Continuous surveillance of dairy farms would be advisable, particularly with respect to the increasing economic importance of paratuberculosis and the high tenacity of the pathogen, which can persist in the environment for many months. So far, such measures have not been taken as the cost-intensive collection of serum samples would have been required. Based on these considerations, it was the aim of this study to evaluate an economically viable diagnostic method for antibody detection using milk samples. This objective was reached by establishing a milk-ELISA. A commercially available test (Svanovir-ELISA by Svanova, Sweden) was chosen, because this ELISA has an excellent specificity with respect to cultural examination of the ileocaecal lymph node ("Gold-Standard"). The Svanovir-ELISA could be successfully adapted for testing milk for antibodies against M. paratuberculosis. The milk is skimmed by centrifugation and is diluted 1:10 for testing. The inter-assay-variation was 17%. A comparative antibody analysis done in parallel with milk and serum samples from 601 dairy cows using the Svanovir-ELISA showed a significant correlation between the results obtained with both methods. The optimal "cut-off" for the milk-ELISA of 46 EUMS (> 46 EUMS = positive) resulting in a specificity of 94.6% and a sensitivity of 60.9% was confirmed by receiver-operator characteristics (ROC) analysis. In the meantime the Svanovir-ELISA has been licensed for use with milk samples in Germany.

Animals↗
PubMed2002

Adoptive transfer of BALb/c mouse splenocytes reduces lesion severity and induces intestinal pathophysiologic changes in the Mycobacterium avium Subspecies paratuberculosis beige/scid mouse model.

Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with Mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoneally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4+, CD8+, and B220+ lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/ scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation.

Adoptive Transfer↗
PubMedDate not supplied

[Detection of Mycobacterium avium subsp. paratuberculosis from the testicles of a clinically infected breeding animal].

During a post mortem of a six year old simmental bull with severe paratuberculosis infection the testicles were further examined by pathological, histological and microbiological methods. No gross or histological lesions could be observed. Single acid fast organisms were detected in smears taken from sterile testicle tissue. Tissue material was additionally cultured in mycostatin culture media and after 8 weeks of incubation acid fast colonies were demonstrated. Polymerase chain reaction with DNA extracted from cultured bacteria and testicle tissue material resulted in M. avium subsp. paratuberculosis-specific amplicons. The detection of M. avium subsp. paratuberculosis in the testicle of the bull demonstrates the possibility of bacteriemia in the final stage of clinical paratuberculosis infection. The evidence of transmitting paratuberculosis through contaminated semen and its relevance for artificial insemination is being discussed in the presented paper.

Animals↗
PubMed2003

Mycobacterium paratuberculosis and milk.

The possibility that milk from cattle with Johne's disease could be a potential vehicle of transmission of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) to humans has been the focus of a UK government-funded research programme at Queen's University, Belfast since 1993. The main findings of this research programme are reported and practical advice about the most appropriate methods for the isolation/detection of this organism in milk is given. The findings of several milk surveys during which optimised sensitive detection methods were employed (decontamination with 0.75% cetyl pyridinium chloride for 5 h prior to culture and a novel immunomagnetic PCR technique) have revealed that detectable levels of M. paratuberculosis are present in bulked raw cows' milk in the UK at both the farm level and at dairy processing plants prior to pasteurisation. Furthermore, results of three different experimental approaches to assess the effect of pasteurisation time/temperature conditions on the viability of M. paratuberculosis (laboratory pasteurisation studies, a national survey of commercially pasteurised milk, and processing of naturally infected milk through commercial-scale pasteurising plant) provide firm evidence that this organism is capable of surviving commercial milk pasteurisation on occasion. Hence, both raw and pasteurised cows' milk are potential vehicles of transmission of M. paratuberculosis to humans.

Animals↗
PubMed1996

Associations between subclinical paratuberculosis and milk production, milk components, and somatic cell counts in dairy herds.

OBJECTIVE: To determine associations between subclinical Mycobacterium paratuberculosis infection and milk production, milk components, and somatic cell counts of dairy cattle. DESIGN: Cross-sectional epidemiologic survey. ANIMALS: 23 dairy herds in Wisconsin containing 1,653 adult cows were studied. The herds had above average milk production and a history of bovine paratuberculosis in the herd within the previous 12 months. PROCEDURE: All adult cows in the herds were tested for paratuberculosis by use of an absorbed ELISA. Milk yield, fat, protein, and somatic cell count data were retrieved electronically from Dairy Herd Improvement Association records. RESULTS: 147 ELISA-positive and 1,506 ELISA-negative cows were identified. ELISA-positive cows had a mature-equivalent milk production of 376 kg (829 lb)/lactation less than that for ELISA-negative herdmates. Significant difference was not found in lactation average percent-ages of fat and protein, or somatic cell count linear score. When comparing ELISA-positive and -negative cow's current mature equivalent milk with all previous lactations, significant difference was found only from the immediate-preceding lactation. When this difference was examined by parity group, significant difference was confined to cows in the second lactation. CLINICAL IMPLICATIONS: Subclinical paratuberculosis infections, as determined by ELISA, are associated with a 4% reduction in milk yield and add to the already substantial costs of clinical M paratuberculosis infection in the dairy industry.

Analysis of Variance↗
PubMed1996

Analysis of paraffin sections of Crohn's disease for Mycobacterium paratuberculosis using polymerase chain reaction.

Although Mycobacterium paratuberculosis has been reported by both culture and polymerase chain reaction in unfixed intestinal tissue from patients with Crohn's disease, its pathologic significance is unclear. We used polymerase chain reaction to analyze DNA for the presence of M. paratuberculosis from 27 specimens in 23 patients with Crohn's disease. Seventeen of these cases contained granulomata. DNA from unstained paraffin tissue sections was amplified using nested PCR specific for a 264-base pair fragment of IS900, a multicopy insertion element specific to M. paratuberculosis. The sensitivity of this technique was demonstrated by the amplification of small numbers of cultured organisms in a paraffin cell block in the presence of large amounts of added human DNA. M. paratuberculosis was not identified in any of the specimens from patients with Crohn's disease, nor in any of 11 normal colon sections tested. Therefore, we were unable to confirm a role for M. paratuberculosis in Crohn's disease.

Crohn Disease↗
PubMed1998

Interferon-gamma and interleukin 4 gene expression in cows infected with Mycobacterium paratuberculosis.

OBJECTIVE: To determine whether clinical progression of paratuberculosis in cattle was associated with alterations in cytokine gene expression in affected tissues. ANIMALS: 5 uninfected adult Holstein cows, 7 adult Holstein cows naturally infected with Mycobacterium paratuberculosis that did not have clinical signs of disease, and 4 adult Holstein cows naturally infected with M paratuberculosis that had progressive clinical signs of infection. PROCEDURE: Samples of ileum and cecal lymph nodes were obtained from each animal at the time of slaughter. A reverse transcriptase-competitive polymerase chain reaction assay was used to determine mRNA expression of interferon-gamma (IFN-gamma) and interleukin 4 in each sample. RESULTS: Interferon-gamma gene expression was significantly higher in ileum and cecal lymph node samples from subclinically infected cows than from clinically infected cows. CONCLUSIONS AND CLINICAL RELEVANCE: Progression of paratuberculosis to clinical stages is associated with reduced expression of IFN-gamma at site of infection. If immune response to M paratuberculosis can be manipulated so that IFN-gamma expression is increased, resistance to infection in cattle might be enhanced.

Animals↗