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PubMed1977

Distribution of paratuberculosis in Wisconsin, by soil regions.

A study was undertaken to determine the relationship between concentration of paratuberculosis-infected herds and soil types in Wisconsin. Locations of farms on which paratuberculosis had occurred between 1971 and 1975 were obtained from the Wisconsin Department of Agriculture. The disease occurred mainly in the rolling and undulating areas. Fifty percent of the infection herds were in the southwestern "driftless area" and the southeast uplands; 43% occurred in the western part of the northern silty and loamy uplands; the other 7% were scattered in the east and east-central part of the state. The disease did not necessarily occur in the areas of high cattle concentration. It was concluded that the disease persisted in regions with acid soils but not in regions with alkaline, calcereous soils.

Animals↗
PubMed1990

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe.

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated 'A', 'B', 'C' and 'E') were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern 'A', was found in 46 isolates (75%). The second, pattern 'E', included 8 isolates (13%), while the third, pattern 'B', included 6 isolates (10%). Pattern 'C' was found for only one isolate. All of the deer isolates were classified as pattern 'A', while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern 'A', was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns 'B', 'C' and 'E', were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.

Animals↗
PubMed2002

Cloning and characterization of the genes coding for antigen 85A, 85B and 85C of Mycobacterium avium subsp. paratuberculosis.

Three genes encoding the secreted proteins (antigen 85-A, B, and C) of Mycobacterium avium subsp. paratuberculosis were cloned, sequenced and studied. The complete sequences of these three 85-complex proteins revealed their similarity with 85-complex proteins of other mycobacterial species. Specifically, these sequences showed 99% homology with M. avium 85-complex protein sequences. The multiple homology analysis of these sequences revealed that variations occur at only certain amino acid positions and this is true with all other 85-complex protein sequences of mycobacteria. However, the proposed three conserved regions involved in fibronectin binding in other mycobacteria were observed in N-terminal regions 85A, B and C of M. avium subsp. paratuberculosis.

Amino Acid Sequence↗
PubMed1989

Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of a putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis.

A putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis was isolated from a lambda gt11 genomic expression library by screening with serum from a naturally infected sheep. The gene was contained in two overlapping clones, which were shown by antibody elution to encode a protein of 34 kDa in M. a. paratuberculosis. The clones were sequenced and database searches detected a motif identical to the active serine site in trypsin, and 30% homology to the putative serine proteases (HtrA proteins) of Escherichia coli, Salmonella typhimurium, Brucella abortus and Rochalimaea henselae.

Amino Acid Sequence↗
PubMed2005

Opsonization effects on Mycobacterium avium subsp. paratuberculosis--macrophage interactions.

High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis.

Animals↗
PubMed2006

Relationship between Crohn's disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients.

AIM: To study the association between Crohn's disease (CD), Mycobacterium avium subspecies paratuberculosis (MAP), and genetic factors by examining the role of natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (now SLC11A1) in Sardinian patients with CD and controls. METHODS: Thirty-seven CD patients and 34 controls with no inflammatory bowel disease (IBD) were recruited at the University of Sassari after giving written consent. Six SCL11A1 polymorphisms previously reported to be the most significantly associated with IBD were searched. M. paratuberculosis was identified by IS900 PCR and sequencing. Logistic regression was used to calculate odds ratios (OR) for the associations among CD, presence of MAP, and 6 loci described above. RESULTS: For the first time, a strong association was observed between polymorphisms at NRAMP1 locus 823C/T and CD. While CD was strongly associated with both NRAMP1 and MAP, NRAMP1 polymorphisms and MAP themselves were not correlated. CONCLUSION: Combined with previous work on the NOD2/CARD15 gene, it is clear that the interplay of genetic, infectious, and immunologic factors in the etiology of CD is complex.

Case-Control Studies↗
PubMed2000

Quality control and optimized procedure of hybridization capture-PCR for the identification of Mycobacterium avium subsp. paratuberculosis in faeces.

Nucleic acid sequence capture techniques are used to improve both the sensitivity and specificity of PCR for the diagnosis of plant, animal and human diseases. Hybridization capture-PCR (HC-PCR) was first reported as a method for the detection of Mycobacterium avium subsp. paratuberculosis in 1995 and was successfully trialed on a small number of faecal samples from cattle with Johne's disease. A locally optimized HC-PCR method was evaluated on faeces from infected and non-infected animals. However, sample to sample cross contamination during the DNA purification step highlighted that the original format of the test was unsuitable for routine diagnostic use. Here, we report modifications and optimization of HC-PCR, particularly with respect to DNA purification from faeces, hybridization and capture steps. We also identified procedurally sensitive critical points in the test during capture and washing of magnetic beads. Southern blotting was omitted from the protocol to preserve specificity but this resulted in analytical sensitivity of 5000 organisms per 200 mg faecal sample. Nevertheless, HC-PCR detected M.paratuberculosis in pellets from infected sheep diluted at rates of up to 1 in 100 in normal faeces, suggesting that the technique should be evaluated further for low-cost diagnosis in flocks/herds using pooled samples.

Animals↗
PubMed2001

Progress towards a rapid polymerase chain reaction diagnostic test for the identification of Mycobacterium avium subsp. paratuberculosis in faeces.

Hybridization-capture polymerase chain reaction (HC-PCR), a nucleic acid sequence capture technique, was evaluated on faecal samples pooled from 50 sheep and individual faecal samples as a rapid diagnostic test for Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The status of each of the faecal samples was determined by radiometric culture. A simpler direct-PCR technique was evaluated on the same samples and was found to be more sensitive than HC-PCR. The lack of sensitivity of HC-PCR was neither due to location nor length of capture probe on IS 900 nor deterioration of the probe but was associated with inefficiencies in liquid phase hybridization and solid phase magnetic bead capture. Direct-PCR using primers from the 5' region of IS 900 was evaluated in a blind trial on 502 pooled faecal samples which were concurrently examined by culture. Twenty-one (64%) of the 33 culture positive pools were detected by direct PCR, representing 11 (79%) of the 14 farms with infected sheep. Direct-PCR was also more sensitive than immunomagnetic bead capture-PCR. Using individual faecal samples, 74% of culture positive samples were detected with direct-PCR compared to 44% with immunomagnetic bead capture-PCR. Direct-PCR from faeces can be used as a rapid means of screening pooled faecal samples for flock diagnosis of Johne's disease in sheep.

Animals↗
PubMed1996

Study on immune response of goats vaccinated with a live strain of Mycobacterium paratuberculosis.

A study of the immune response of goats vaccinated with a live strain (316-F) of Mycobacterium paratuberculosis has been performed, taking into consideration both cellular and serological responses using a lymphocyte transformation assay (LTA) and a counterimmunoelectrophoresis test (CIET). The analyses made at three monthly intervals reveal an in vitro proliferative response of blood lymphocytes to specific antigen and lymphocytes T mitogen over a year of post-vaccination, but no humoral response was observed when CIET was used.

Animals↗
PubMed1994

The effect of vaccination on the prevalence of paratuberculosis in large dairy herds.

Eight hundred and sixty-six one-month-old female calves were vaccinated using heat-killed Mycobacterium paratuberculosis in oil during a five-year period. Seven hundred and twenty-one bull calves and 379 female calves served as nonvaccinated controls. Two large herds were used in the study. The calves were tested by faecal culture and by serologic tests on the day of vaccination, at 3, 6, 9 and 12 months of age, at breeding and on the day of parturition. The results were evaluated by trend analyses. Following vaccination, the faecal shedding of bacteria was greatly reduced as determined by annual faecal microscopic tests. During the last 6 months of the experiment as few as 9 samples from 612 cattle proved positive by microscopy and/or culture. The number of seropositive animals and the antibody titres (CFT and AGID) increased during the first three years, then declined.

Animals↗
PubMed2005

Design and development of an internal control plasmid for the detection of Mycobacterium avium subsp. paratuberculosis using real-time PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants. The hspX gene and insertion sequence IS900 can be used to diagnose Johne's with PCR. Generally, a single PCR tube containing the DNA sequence of interest is run as a positive control with each set of reactions. Single reactions within a PCR run can fail while the positive control does not. Thus, a single positive control tube does not determine if all PCR reactions worked properly. Our objective was to construct a plasmid to use as an internal control in each reaction. A plasmid containing an insert of M. bovis-hspX-M. bovis DNA was modified to remove a portion of the hspX insert used by the reverse hspX primer. The remaining insert was ligated back together and transformed into competent cells. Sequencing confirmed removal of 71 bp. PCR reactions using three primers (TB/M. bovis reverse, hspX forward and reverse) for hspX gene detection and four primers (IS900 forward and reverse, hspX forward, and TB/M. bovis reverse) for IS900 detection were optimized by titrating various amounts of plasmid against varied amounts of MAP genomic DNA. Plasmid insert amplification confirms a successful PCR reaction and identifies true positives and negatives within each individual reaction. The optimal plasmid amounts are 10 fg/reaction (hspX detection) and 1 fg/reaction (IS900 detection).

Animals↗
PubMed2006

Paratuberculosis and Type I diabetes: is this the trigger?

Type 1 diabetes mellitus (T1DM) is an autoimmune disease. The etiology of T1DM is incompletely understood but environmental agent(s) are thought to trigger T1DM in the genetically at risk. Exposure to cow's milk early in life is a recognized risk factor in the development of T1DM. Mycobacterium avium ss. paratuberculosis (MAP) is the cause of bovine Johne's disease and also is thought to act as an immune antigen in Crohn's disease and other granulomatous diseases. MAP is shed in cow's milk and has been shown to survive pasteurization. Genetic susceptibilities, epitope homologies and epidemiologic studies are presented that support MAP as a causative agent of T1DM in the genetically at risk.

Animals↗
PubMed2005

Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays.

Sensitive real-time sequence detection methods based on two different chemistries were developed for Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease in cattle. One is based on the detection of SYBR Green bound to PCR products and the second method is more specific, detecting the cleavage of a fluorogenic (TaqMan) probe bound to a target sequence during primer extension phase. Novel primers and probes that amplify small fragments (<80 bp) of the Map specific insertion sequence, IS900 were designed. Both the SYBR Green and TaqMan assays are sensitive, able to detect 4 fg of DNA extracted from Map strain ATCC19698. This amount of DNA corresponds to the detection of 0.8 cells. Map cells were quantified directly from 7H9 broth using the SYBR Green assay and compared to dilutions of DNA extracted from an equivalent number of cells. The SYBR Green assay of 7H9 broth resulted in a minimum detectable limit of 0.07 cells (equivalent to 0.34 fg of DNA). Media ingredients were not observed to interfere with the assay. Since no extraction step was necessary in the direct cell measurements, direct detection was ten-fold more sensitive than detection of extracted DNA. Both SYBR Green and TaqMan assays are highly specific for the detection of Map. They did not detect any closely related members of the avium complex, other species of mycobacteria, or related genera that are likely to be present in environmental samples. No reporter signal was detected during TaqMan assays performed with 100 pg of template DNA from the non-Map organisms.

Animals↗
PubMed2004

Relationship between antibodies against Mycobacterium avium subsp. paratuberculosis in milk and shape of lactation curves.

To analyze how infection with Mycobacterium avium subsp. paratuberculosis (MAP) affects the shape of lactation curves, a three-level hierarchical test-day model was set up with fat-corrected test-day milk yield (FCTM) as response. Milk samples from 6955 cows in 108 Danish dairy herds were tested with ELISA to detect antibodies against MAP. Optical densities (ODs) recorded on a continuous scale were standardized according to parity and stage of lactation. In addition to standardized ODs (stOD), seven fixed covariates, quadratic terms and first-order interactions were included in the model. Cow and cow nested in herd were included as random effects. Cows of first, second and higher parities were analyzed separately. The lactation curves after peak yield were significantly less persistent in young infected cows, where an increase of one stOD unit was associated with a depression of the milk yield per day through day 305 of 3.7 kg FCTM in first parity and 2.7 kg FCTM in second parity. In second-parity cows, the lactation curve also was both depressed through the entire lactation and more steep after 60 days in milk (DIM). In third and older parities, a significant effect of the quadratic term of stOD indicated exponentially increased losses with increased ODs.

Animals↗
PubMed2005

Cow-level evaluation of a kinetics ELISA with multiple cutoff values to detect fecal shedding of Mycobacterium avium subspecies paratuberculosis in New York State dairy cows.

In control programs for Mycobacterium avium subsp. paratuberculosis (Map), the infection status of the cows in a herd is often obtained by testing (a sample of) the herd with an ELISA that may lack some sensitivity and specificity but that is fast and inexpensive. In New York State (NYS), an unabsorbed kinetics ELISA (KELA) has been used extensively for Map control. The objective of this study was to determine the relative sensitivity and specificity of the KELA for detection of fecal shedding of Map for the NYS dairy cow population, taking into account possible confounders such as different antigen batches and Map prevalence in a herd. The data for the study consisted of all serum samples from NYS dairy cows with concurrent fecal culture results submitted to the NY Animal Health Diagnostic Laboratory (NYAHDL) between 1991 and 1996 (n=10,562). The data represented cows with different levels of fecal shedding from herds with different within-herd Map prevalence, including herds that were whole herd fecal culture negative on repeated testing. The cutoff values were based on the predictive value for fecal shedding obtained with a multiple logistic regression model that included variables for the three antigen batches and the Map prevalence in the herd. The KELA could not distinguish between non-shedders and low shedders ( 30 TCFU) was modeled. The three cutoff values of 65, 135 and 170 were based on low (<0.2), moderate (<0.80) and high (>0.95) probabilities for moderate to heavy fecal shedding. The sensitivity and specificity values relative to culture were 67% and 95.2%, 31% and 99.7%, and 11% and 99.9% for the three cutoff values, respectively. Cutoff values for the KELA decreased for herds with increasing within-herd Map prevalence. For the best positive predictive value of a KELA for moderate to heavy fecal shedding, the cutoff values should be determined based on the apparent within-herd prevalence in a herd.

Animals↗
PubMed2006

Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle.

Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4cfu per tube less than placebo treated cows (P=0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P=0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR=0.27; P=0.03). Monensin sodium administered to infected animals at 335mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown.

Animals↗