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PubMed1993

Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease.

A species-specific recombinant clone (F57) was obtained from a genomic library of Mycobacterium paratuberculosis in the transcription vector pGem 3Z. This clone proved to be specific for all mycobacteria tested, including M. avium, and was able to recognize all of the tested M. paratuberculosis strains isolated from animals and humans (patients with Crohn's disease). The F57 insert was sequenced and a segment of 620 bp with a G + C content of 58.9% was identified. Comparison of the sequence with sequences in the EMBL and UGEN data banks revealed the uniqueness of the F57 sequence, which had no resemblance to other known genes.

Animals↗
PubMed2000

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.

Animals↗
PubMed2005

Mycobacterium avium subsp. paratuberculosis, genetic susceptibility to Crohn's disease, and Sardinians: the way ahead.

The present study was performed to determine what proportion of people in Sardinia with or without Crohn's disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod 2, an intracellular protein involved in Crohn's disease susceptibility. Genetic analysis of the alleles of the NOD 2/CARD 15 gene (ins C 3020, G 908 R, and R 702 W alleles), linked to susceptibility or genetic predisposition to Crohn's disease in humans, was carried out on specimens from 37 Crohn's disease patients and 34 patients without Crohn's disease. Our results show that more than 70 percent of people in Sardinia with Crohn's disease carry at least one of the susceptibility-associated NOD 2/CARD 15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis.

Crohn Disease↗
PubMed2004

Factors affecting the herd level of antibodies against Mycobacterium avium subspecies paratuberculosis in dairy cattle.

A case-control study was made of Norwegian dairy herds with high and low herd levels of antibodies against Mycobacterium avium subspecies paratuberculosis. A high proportion of the herds had a considerable number of seropositive cows, and environmental and management factors were examined for possible associations with the high serological levels of antibodies. The most important appeared to be: geographical location, red deer (Cervus elaphus) gaining access to the pastures for cattle, the observation of wild birds in the feed storage, and herds sharing common pasture with other herds of cattle. However, diagnostic tests showed that none of the animals in the case herds was infected with M a paratuberculosis.

Animal Feed↗
PubMed2003

Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

Animals↗
PubMed2006

Evaluation of the gamma interferon ELISA in sheep subclinically infected with Mycobacterium avium subspecies paratuberculosis using a whole-cell sonicate or a johnin purified-protein derivative.

The aim of the study reported here was to estimate the sensitivity and specificity of the gamma interferon (IFN-gamma) ELISA for paratuberculosis in sheep using receiver-operating characteristic analysis. Bacteriologic culture of tissues was used to define the reference positive population (n = 33). Two reference negative populations were used: culture-negative sheep from infected flocks (n = 77), and sheep from noninfected flocks (n = 358). We also evaluated the accuracy of 2 Mycobacterium avium subspecies paratuberculosis (MAP) antigen preparations, a whole-cell sonicate (MpS) and a johnin purified-protein derivative (PPDj). The source of the reference negative sheep used in the analysis affected overall accuracy of the IFN-gamma ELISA. The area under the curve was 0.683 (95% confidence interval 0.574-0.787), using culture-negative sheep from infected flocks, was 0.831 (0.764-0.889), using sheep from noninfected flocks for the MpS, and was 0.809 (0.726-0.881) and 0.897 (0.862-0.925) for the PPDj, respectively. Using the MpS, the cut point that classified the most sheep correctly was an optical density reading of 0.20, for sensitivity of 40.7% (19.4-57.6) and specificity of 88.7% (77.0-95.7) or 97.6% (93.04-99.5), depending on the reference negative population used. Using the PPDj, the cut point that classified the most sheep correctly was 0.25 for sensitivity of 66.7% (47.2-82.7) and specificity of 93.5% (85.5-97.9) or 98.3% (96.4-99.4), respectively. The PPDj was more accurate at identifying MAP-infected sheep than was the MpS (P = 0.034).

Animals↗
PubMed2006

Use of age and milk production data to improve the ability of enzyme-linked immunosorbent assay test results to predict Mycobacterium avium ssp. paratuberculosis fecal culture status.

Cows from 2 California dairies were tested for paratuberculosis at the end of lactation by using fecal culture and a commercially available serum enzyme-linked immunosorbent assay (ELISA) test kit. Individual cow characteristics and production variables were evaluated along with ELISA testing results as predictors of fecal culture status. In multivariable logistic regression analysis, age and a herd-standardized version of 305-day mature equivalent (305 ME) milk production were significant predictors of fecal culture status after adjusting for herd, quarter of the study year, and ELISA sample-to-positive (S/P) ratio. The area under a nonparametric receiver operating characteristic curve was significantly greater for a multivariable model that included age and the level of milk production when compared with a model without these covariates. In conclusion, consideration of cow-level covariates was useful as an aid in predicting Mycobacterium avium ssp. paratuberculosis (MAP) fecal culture status. For a given ELISA S/P ratio, older cows and those with lower 305 ME milk production relative to other cows in the herd were significantly more likely to be shedding MAP in their feces at the end of lactation.

Age Factors↗
PubMed2004

Sequential development of histologic lesions and their relationship with bacterial isolation, fecal shedding, and immune responses during progressive stages of experimental infection of lambs with Mycobacterium avium subsp. paratuberculosis.

Understanding pathogenesis during progressive stages of infection by Mycobacterium avium subsp. paratuberculosis (MAP) and finding suitable methods for its diagnosis are key to the control of Johne's disease in animals. Paratuberculosis was experimentally produced in 20 crossbred lambs by oral administration of MAP to study the sequential development of lesions between 10 and 330 days postinfection and to assess commonly used diagnostic methods such as bacterial culture, lymphocyte stimulation test (LST), and enzyme-linked immunosorbent assay (ELISA) during progressive stages of infection. Histologic lesions were classified into four grades from grade 1 (least severe) to grade 4 (most severe) on the basis of location of granulomatous lesions in different regions and layers of intestines, their association with intestinal lymphoid tissues, pattern and distribution of lesions, types of cellular infiltration, and presence of acid-fast bacilli. It is evident that infection first establishes in lymphoid tissues of the small intestine, possibly at multiple sites, producing segmental lesions and from there spreads to lamina propria and local lymph nodes. Wide variability in the histologic lesions in relation to postinfection periods and initial tropism of MAP to the intestinal lymphoid tissues (Peyer's patches) suggests a differential susceptibility of young animals, possibly because of compositional phenotypic variation of Peyer's patches influencing subsequent course of infection. Histopathology was found to be a better indicator of paratuberculous infection than bacteriology in sheep. The LST (reflecting the cellular immune response) and ELISA (reflecting the humoral immune response) had overall sensitivities of 65% (11 of 17) and 42% (8 of 19), respectively, in sheep with different types of pathology but when employed together could detect about 88% of infected animals.

Animals↗
PubMed2005

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.

Animals↗
PubMed2001

Mycobacterium avium subsp. paratuberculosis infection in an addax (Addax nasomaculatus).

Mycobacterium avium subsp. paratuberculosis was cultured from a single fecal sample collected from a 10-yr-old, captive-bred male addax (Addax nasomaculatus). Attempts to confirm infection with additional fecal cultures, serology, semen culture, and tissue biopsy were unsuccessful. There were no gross lesions on necropsy. On histopathology there were neither acid-fast organisms nor microscopic changes suggestive of active or clinical Johne's disease. Mycobacterium avium subsp. paratuberculosis was isolated from four organ tissues: ileum, jejunum, colon, and mesenteric lymph node.

Animals↗
PubMed2001

Mycobacterium avium subspecies paratuberculosis: a review of current knowledge.

Although Mycobacterium avium subsp. paratuberculosis infection has had its greatest effect on domestic agricultural animal species, it can also have a significant impact on wildlife species. More cases of infection are being reported, and because of its ability to elude immunologic control and to persist in the environment, M. paratuberculosis may spread within and among captive and free-ranging wildlife populations in the absence of organized control programs. Studies to improve our ability to detect the organism in biologic samples such as milk, blood, and manure through immunomagnetic separation, automated culture methods, and improved polymerase chain reaction procedures are underway in several countries. Studies of the organism's genetic components, virulence factors, and antigens support the development of new diagnostic tools and vaccines.

Animals↗
PubMed2005

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.

Animals↗
PubMed2005

Risk of removal and effects on milk production associated with paratuberculosis status in dairy cows.

OBJECTIVE: To determine effects on production and risk of removal related to Mycobacterium avium subsp paratuberculosis (MAP) infection at the individual animal level in dairy cattle. DESIGN: Longitudinal study. ANIMALS: 7,879 dairy cows from 38 herds in 16 states. PROCEDURE: A subset of dairy cattle operations that participated in the National Animal Health Monitoring System Dairy 2002 study was evaluated via a serum ELISA for antibodies against MAP and categorized according to ELISA score. Dairy Herd Improvement Association records were obtained to collect current and historical lactation data and removal (ie, culling) information. Production variables were evaluated on the basis of serum ELISA category. RESULTS: Cows with strong positive results had mature equivalent (ME) 305-day milk production, ME 305-day maximum milk production, and total lifetime milk production that were significantly lower than cows in other categories. No differences were observed for ME 305-day fat and protein percentages, age, lactation, and lactation mean linear somatic cell count score between cows with strong positive results and those with negative results. After accounting for lactation number and relative herd-level milk production, cows with strong positive results were significantly more likely to have been removed by 1 year after testing. CONCLUSIONS AND CLINICAL RELEVANCE: Without management changes designed to reduce the farm-level prevalence of MAP infection, paratuberculosis will continue to reduce farm income by decreasing milk production and potentially increasing premature removal from the herd.

Animals↗
PubMed2006

Age-specific characteristics of ELISA and fecal culture for purpose-specific testing for paratuberculosis.

Paratuberculosis is a chronic infection, and animals are not equally affected by it. Therefore, diagnostic tests that are able to detect different stages of the infection are needed for objective decision making. A longitudinal study was carried out to describe the ability of 2 tests to predict 2 conditions in dairy cattle: "infection" and "infectious," exemplifying 2 different purposes of testing. "Infection" is the term of choice for certification and eradication purposes, and "infectious" is more relevant for control purposes. In the study period of 3 yr, repeated sampling of milk (n = 23,219) and feces (n = 8,832) was performed. A total of 1,985 Danish dairy cows provided material for the study. Milk samples were analyzed for antibodies using an ELISA, and fecal samples were analyzed for mycobacteria by culture. A reference test to correctly classify cattle antemortem does not exist; thus, "infection" and "infectious" were defined by repeated testing using one test as the condition to be detected by the other test. Fecal culture responses were evaluated against antibody status, and ELISA responses were evaluated against detected bacterial shedding. The results of this study indicate that the ability of both tests to detect "infection" increases almost linearly from 2 to 5 yr of age, whereas the ability of both tests to detect "infectious" is not affected by age. Purpose-specific tests are required to appropriately interpret and use test results for management of paratuberculosis, and relevant covariates, such as age, should be included when possible.

Aging↗
PubMed2006

Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies.

Environmental samples collected from each of 3 locations on 23 large California dairies were cultured to evaluate the utility of this approach for identifying herds infected with Mycobacterium avium ssp. paratuberculosis. Results were compared with concurrent ELISA testing of > or = 60 animals in each herd, and with previously performed individual and pooled fecal cultures of 60 animals. The estimated proportions of infected herds did not differ significantly among the testing methods (environmental sampling, 74%; previous fecal culture, 70%; and concurrent ELISA testing, 65%). Measures of agreement between environmental sampling and the results of previous fecal cultures were 70% (observed agreement), 85% (positive agreement), 62% (negative agreement), and 0.47 (kappa), whereas agreement between environmental sampling and concurrent ELISA testing was 65, 75, and 43%, and 0.19, for the same measures, respectively. The proportion of positive environmental samples on each farm was significantly correlated with the proportion of seropositive animals (r = 0.53), suggesting that environmental sampling may also provide a qualitative estimate of within-herd prevalence. Of the sampling locations that were evaluated, samples of lagoon water (15/23; 65%) were significantly more likely to yield a positive result than were composite manure samples (8/22; 36%) collected from the sick/fresh cow pen or from the alleyway (9/23; 39%) where cows exited from the milking parlor. Environmental sampling was an effective and inexpensive method of identifying herds infected with Mycobacterium avium ssp. paratuberculosis.

Animals↗
PubMed2006

Analysis of the seroprevalence of bovine paratuberculosis and the application of modified absorbed ELISA to field sample testing in Korea.

Paratuberculosis (PTB) is a major disease problem worldwide, and causes major economic losses in the dairy industry. Although PTB has been reported in Korea, no studies have been conducted to determine its prevalence and no program has been developed to control the disease. In this study, the sera of beef (n = 1,056) and dairy cattle (n = 1,105) from all provinces in Korea were tested to determine the prevalence of PTB using two different ELISA: an 'in house' modified absorbed ELISA (P-ELISA) based on sonicated antigen from Mycobacterium avium subsp. paratuberculosis ATCC 19698, and a commercial ELISA (C-ELISA). Receiver operating characteristic analysis was used to determine the cutoff point for P-ELISA. Based on C-ELISA results, the area under the curve for P-ELISA was 0.913 (95% CI, 0.883 to 0.943). Using a cutoff point of 0.100, P-ELISA showed a sensitivity of 62.0% and a specificity of 93.7%. The kappa value and the percent agreement between the two ELISAs were 0.322 and 92.5%, respectively. Both ELISAs showed a significant correlation between age and seropositivity (p < 0.01). According to C-ELISA, 71 of 2,161 sera (3.3%, 95 CI, 2.6% to 4.1%) were test-positive. The national true prevalence of PTB was estimated to be 7.1%. The findings suggest that a control program should be implemented to limit the spread of this disease, and that P-ELISA could be used as a screening test that produces results similar to C-ELISA.

Animals↗
PubMed2001

Identification, cloning and expression of sodC from an alkaline phosphatase gene fusion library of Mycobacterium avium subspecies paratuberculosis.

The phoA gene technology was used to investigate secreted proteins of the intracellular pathogen Mycobacteriumn avium subspecies paratuberculosis. This led to the identification of sodC, a gene which codes for a copper and zinc cofactored superoxide dismutase (Cu,ZnSOD) which has been implicated as a virulence factor for some pathogens. The predicted protein possessed a 76% identity with Cu,ZnSOD of Mycobacterium tuberculosis. To characterize Cu,ZnSOD from M. avium subspecies paratuberculosis, the gene was cloned and overexpressed in Escherichia coli. The renatured, affinity-purified recombinant protein possessed enzymatic activity that was inhibited by the presence of cyanide, which is characteristic of a Cu,ZnSOD.

Alkaline Phosphatase↗
PubMed2002

[Pathogenesis and immune reactions of paratuberculosis].

Mycobacterium avium subspecies paratuberculosis (M. ptb) is known as the cause of paratuberculosis for over a century but the knowledge on biology of the organism and pathogenesis of the disease is still limited. There are several reasons for the present lack of progress, these are (i) the extremely slow growth of the bacterium, a feature which has also protected the organism against researchers, (ii) confusion over its taxonomy and identification, (iii) limited possibilities for the application of molecular biology techniques, and (iv) the extremely long incubation period in natural infection for which no suitable laboratory model exists. Despite these discouraging facts, recent research efforts have led to important findings, which have shown that a better understanding of the disease may contribute to the improvement of control strategies. This presentation focuses mainly on the unique nature of M. ptb within the mycobacteria and the central role of the macrophage in pathogenesis and immune response. More details can be found in a number of excellent recent reviews (see list of references).

Animals↗