A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction.
(73/464)
Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g. (+info)
Effects of prevalence and testing by enzyme-linked immunosorbent assay and fecal culture on the risk of introduction of Mycobacterium avium subsp. paratuberculosis-infected cows into dairy herds.
(74/464)
A stochastic simulation model was developed to assess the risk of introduction of Mycobacterium avium subsp. paratuberculosis infection into a dairy herd through purchase of female replacement cattle. The effects of infection prevalence in the source herd(s), number of females purchased, and testing by enzyme-linked immunosorbent assay (ELISA) alone or ELISA and fecal culture as risk mitigation strategies were evaluated. Decisions about negative test results were made on a lot and individual basis. A hypothetical dairy herd, free from M. a. paratuberculosis, which replaced 1 lot (10, 30, or 100) of cows per year, was considered. Probability distributions were specified for the sensitivities and specificities of ELISA and fecal culture, the proportion of infected herds and within-herd prevalence for randomly selected replacement source herds (high prevalence) and herds in level 2 (medium prevalence) and level 3 (low prevalence) of the Voluntary Johne's Disease Herd Status Program (VJDHSP). Simulation results predicted that 1-56% of the lots had at least 1 M. a. paratuberculosis-infected cow. Assuming that ELISA sensitivity was 25%, simulation results showed on a lot basis that between 0.4% and 18% and between 0.1% and 9% were predicted to have at least 1 infected cow not detected by ELISA and by a combination of ELISA and fecal culture, respectively. On an individual cow basis, between 0.1% and 8.3% of ELISA-negative cattle in ELISA-positive lots were estimated to be infected. In both the lot and individual analyses, the probability of nondetection increased with larger lot sizes and greater prevalence. Sensitivity analysis indicated that the effect of a lower ELISA sensitivity (10%) was a variable decrease in mean detection probabilities for all combinations of prevalence and lot size. The benefit of testing introduced cattle with ELISA alone or in combination with fecal culture was found to be minimal if cows were purchased from known, low-prevalence (level 3) herds. The value of testing by ELISA alone or in combination with fecal culture was greatest in high-prevalence herds for all lot sizes. Testing of random-source cattle, bought as herd replacements, can partially mitigate the risk of introduction of M. a. paratuberculosis but not as well as by using low-prevalence source herds (level-3 VJDHSP), with or without testing. (+info)
A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.
(75/464)
We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. (+info)
Cytokine gene expression in peripheral blood mononuclear cells and tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis: evidence for an inherent proinflammatory gene expression pattern.
(76/464)
In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johne's disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1alpha (IL-1alpha), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-gamma), transforming growth factor beta (TGF-beta), and tumor necrosis factor alpha (TNF-alpha), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-gamma, TGF-beta, TNF-alpha, IL-1alpha, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended to reduce the differential expression observed in infected and uninfected cows for genes encoding IFN-gamma, IL-1alpha, and IL-6. Only IL-10 gene expression was consistently enhanced by M. avium subsp. paratuberculosis stimulation of PBMCs from subclinically infected cattle. In ileal tissues from M. avium subsp. paratuberculosis-infected cattle, expression of the genes encoding IFN-gamma, TGF-beta, IL-5, and IL-8 was greater than the expression in comparable tissues from control uninfected cattle, while expression of the gene encoding IL-16 was lower in tissues from infected cattle than in control tissues. Mesenteric lymph nodes draining sites of M. avium subsp. paratuberculosis infection expressed higher levels of IL-1alpha, IL-8, IL-2, and IL-10 mRNA than similar tissues from control uninfected cattle expressed. In contrast, the genes encoding TGF-beta and IL-16 were expressed at lower levels in lymph nodes from infected cattle than in tissues from uninfected cattle. Taken together, our results suggest that cells or other mechanisms capable of limiting proinflammatory responses to M. avium subsp. paratuberculosis develop in infected cattle and that a likely place for development and expansion of these cell populations is the mesenteric lymph nodes draining sites of infection. (+info)
Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis.
(77/464)
There is renewed enthusiasm for exploring the possibility that Mycobacterium paratuberculosis may be causative in Crohn's disease (CD). We aimed to determine whether CD subjects are more likely to be M. paratuberculosis seropositive than controls. Using our population-based University of Manitoba Inflammatory Bowel Disease Research Registry, we recruited CD and ulcerative colitis (UC) subjects between 18 and 50 years of age for a study involving detailed questionnaires and venipuncture. We accessed the population-based databases of Manitoba Health (single provincial health insurer) to get age-, gender-, and geography-matched controls to our inflammatory bowel disease (IBD) population. We asked enrolling IBD subjects for potential nonaffected sibling controls. We used an enzyme-linked immunosorbent assay (ELISA) for serum antibodies to M. paratuberculosis initially developed for cattle but adapted for human use. The rate of positive ELISA results, based on previously published interpretation criteria, was significantly higher for all study groups. There was no difference in M. paratuberculosis seropositivity rate among CD patients (37.8%; n = 283), UC patients (34.7%; n = 144), healthy controls (33.6%; n = 402), and nonaffected siblings (34.1%; n = 138). For siblings, there was no correlation between M. paratuberculosis serological status and that of the corresponding IBD affected sibling. None of the demographic or questionnaire variables studied were predictive of M. paratuberculosis status. Subjects with CD and UC were less likely to have ingested unpasteurized milk and less likely to have had a non-tap water source as a primary water source. In conclusion, in this population-based case control study, the M. paratuberculosis seropositivity rate was approximately 35% for all groups and there was no difference in rates between CD patients, UC patients, healthy controls, or nonaffected siblings. The much higher rate of seropositivity for subjects from Manitoba, Canada, than for those from Denmark or Wisconsin cannot be obviously explained. While these data seem to refute any association of CD with M. paratuberculosis, the high seroprevalence in Manitobans raises the possibility that the high rates of CD in Manitoba could be related to high exposure rates for M. paratuberculosis. Hence, the possibility of an association between M. paratuberculosis and CD remains inconclusive. (+info)
Effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of Mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle.
(78/464)
Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis. (+info)
Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis.
(79/464)
Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-gamma) secretion 23-fold and also increased IFN-gamma secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle. (+info)
Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.
(80/464)
We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses. (+info)