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PubMed1981

Lack of intracellular degradation of Mycobacterium paratuberculosis by bovine macrophages infected in vitro and in vivo: light microscopic and electron microscopic observations.

Blood-derived macrophages from clinically healthy cows and cows infected with Mycobacterium paratuberculosis were cultured in vitro and inoculated with heat-killed M paratuberculosis. Five weeks after infection, acid-fast bacteria continued to be demonstrated in the macrophages. Macrophage cultures also were infected with living M paratuberculosis and were examined by electron microscopy. Four weeks after infection, intact bacteria were found in all cultures. Electron microscopic examinations of intestinal macrophages from infected cows also demonstrated intracellular intact bacteria.

Animals↗
PubMed1993

The cellular immunology of bovine paratuberculosis: immunity may be regulated by CD4+ helper and CD8+ immunoregulatory T lymphocytes which down-regulate gamma/delta+ T-cell cytotoxicity.

In a previous investigation, we obtained evidence that the major histocompatibility complex (MHC)-restricted proliferative response of CD4+ lymphocytes to Mycobacterium paratuberculosis antigens was depressed in naturally infected and immunized animals. Findings suggested that depression of the response was attributable to an abrogation in the ability of CD4+ cells to respond to specific antigens and/or the actual loss of antigen-reactive cells. In vitro cell experiments indicated that the depression was associated with the presence of gamma/delta+ T cells that modulated CD4+ cell function. Examination of additional animals confirmed and extended these observations and showed that the ability of gamma/delta+ T cells to regulate CD4+ responses were blocked by the presence of CD8+ cells. CD4+ T cells from some exposed animals incorporated [3H]-thymidine in the presence of CD8+, gamma/delta+ cells and/or antigen and antigen-presenting cells, but CD4+ cell proliferation was abrogated when CD8+ were excluded from the assays. Likewise, gamma/delta+ T-cell proliferation was abrogated when CD8+ cells were present. The mechanism by which CD8+ cells blocked gamma/delta+ T-cell responses could not be determined, however, the observed effect resembled the veto cell phenomenon. The data suggest that the development of protective immunity against M. paratuberculosis may be dependent on the capacity of CD8+ cells to modulate the regulatory activity of gamma/delta+ T populations.

Animals↗
PubMed2000

Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries.

Mycobacterium paratuberculosis causes Johne's disease, a common wasting disease in ruminants. As a first step in studying virulence mechanisms, libraries of random mutants were produced in two M. paratuberculosis strains by using the conditionally replicating shuttle phasmid phAE94 which contains the transposon Tn5367. Two thousand mutants were screened for auxotrophy, carbon source preference, and altered cell wall. Genes interrupted by insertion were identified for seven mutants isolated from the screening process. Two mutants had insertions in putative genes involved in synthesis of the cell wall.

Animals↗
PubMed1993

Comparison of polymerase chain reaction tests and faecal culture for detecting Mycobacterium paratuberculosis in bovine faeces.

A polymerase chain reaction (PCR) test for M. paratuberculosis was developed based on a 218 bp segment of a DNA insertion sequence, IS900, that is specific for this organism. The method involved two consecutive amplification reactions, with the second set of primers being nested inside the first set. The method reliably detected 50 organisms/g faeces. This PCR test was applied to 32 bovine faecal specimens containing high, moderate or low numbers of M. paratuberculosis organisms as determined by culture. The PCR test detected all specimens containing > or = 1600 colony forming units (cfu)/g faeces, six of ten specimens with 160-480 cfu/g faeces but only two of 13 specimens containing < or = 112 cfu/g faeces. The sensitivity of this test was better than that of a commercial PCR test which was carried out on the same faecal specimens.

Animals↗
PubMed1994

Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep.

A complement fixation test for paratuberculosis, a gel diffusion test and two enzyme-linked immunosorbent assays (ELISA) were evaluated using sera from Mycobacterium paratuberculosis infected and non-infected sheep. Gross pathology and histopathology were used as parameters of infection. The two ELISAs, one of which is commercially available for testing cattle, were used before and after sera had been absorbed with a soluble sonicate of Mycobacterium phlei. Differences between the various tests and between ELISAs before and after absorption were non-significant (P > 0.05) in non-infected sheep or in animals with gross or histopathological lesions. The specificity of all the tests was at least 97%. Sensitivity in histopathologically positive sheep was at least 98%. Sheep from infected flocks but without histopathological lesions showed serological results which were poorly correlated between the various tests.

Animals↗
PubMed2006

Mycobacterium avium subspecies paratuberculosis infection of cattle does not diminish peripheral blood-derived macrophage mycobactericidal activity.

Ruminants infected with Mycobacterium avium subspecies paratuberculosis consistently develop a multibacillary form of disease that is centered on the ileum. Mechanisms responsible for failure of macrophage function during multibacillary disease are incompletely characterized. Our data suggest that mycobactericidal functions are present, and potentially enhanced, in monocyte-derived macrophages from M. avium subsp. paratuberculosis infected cattle. Addition of CD4(+) T cells from infected animals to autologous in vitro infected macrophages did not increase bacterial killing. In contrast, CD4(+) T cells from non-infected animals did increase bacterial killing in autologous macrophages. In macrophages from both infected and non-infected cattle, bacterial killing appeared to be independent of interferon-gamma (IFN-gamma) and nitric oxide production.

Animals↗
PubMed2004

Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.

Adaptation, Physiological↗
PubMed2006

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity.

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.

Animals↗
PubMed2005

Tissue predilection sites and effect of dose on Mycobacterium avium subs. paratuberculosis organism recovery in a short-term bovine experimental oral infection model.

The objective of this study was to develop a short-term experimental infection model for Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, using small oral doses of organisms. Specifically, the effect of dose size was evaluated, as well as specific tissue predilection sites for recovery of MAP. Oral doses as low as 1.5 x 10(6) CFU reliably produced infection that could be detected 3 weeks following infection. Detection of infection required culture of multiple intestinal samples (jejunum and ileum) for MAP. Histological examination did not permit detection at this early stage. Results from this study suggest intestinal mucosa, rather than tonsil, as the primary portal of entry for MAP. The experimental infection model described here is useful for studying the early effects of preventive and therapeutic interventions for paratuberculosis in cattle.

Animals↗
PubMed2006

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.

Animals↗
PubMed2004

Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis.

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.

Administration, Oral↗
PubMed1995

Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis.

The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.

Administration, Oral↗
PubMed1998

Changes in lymphocyte subsets in the intestine and mesenteric lymph nodes in caprine paratuberculosis.

Changes in the number and distribution of lymphocyte subsets were investigated in the intestinal mucosa and mesenteric lymph nodes of three goats with natural paratuberculosis, comparisons being made with a single uninfected control animal. Lesions in the naturally infected goats varied from small granulomata with scarce epithelioid or multinucleated giant cells, containing few or no bacilli, in the intestine (tuberculoid type) to an extensive, diffuse epithelioid cell infiltrate containing numerous bacilli in the gut and mesenteric lymph nodes (lepromatous type). The number and distribution of lymphocyte subsets in the control were consistent with data reported from other non-infected goats. However, in the goats with paratuberculosis, significant changes were observed in the number and distribution of CD4+ and CD8+ T lymphocytes, the changes being related to the severity of the lesions. In the intestinal mucosa of the goat with tuberculoid lesions no significant changes were observed, but in the cortical area of mesenteric lymph nodes the number of CD4+ T lymphocytes decreased and the number of CD8+ T lymphocytes increased. In the two goats with lepromatous lesions, there was a decrease in the CD4+ T subpopulation and an increase of CD8+ T lymphocytes in the lamina propria of the intestine and particularly in the cortical area of the mesenteric lymph nodes, the CD4:CD8 ratio (< 1) being the opposite of that observed in healthy goats. Because of the small numbers of animals, further studies including additional animals are needed to confirm these preliminary results, which suggest that the progression of paratuberculous lesions may be due to an ineffective host immune response attributable to the CD8+ T lymphocyte subset that "downregulates" the activity of the CD4+ T lymphocytes required for macrophage activation.

Animals↗
PubMedDate not supplied

Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease.

BACKGROUND: Crohn's disease, a form of inflammatory bowel disease, resembles some aspects of tuberculosis, leprosy, and paratuberculosis. The role of Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease is controversial. METHODS: We tested for MAP by PCR and culture in buffy coat preparations from 28 individuals with Crohn's disease, nine with ulcerative colitis, and 15 without inflammatory bowel disease. FINDINGS: MAP DNA in uncultured buffy coats was identified by PCR in 13 (46%) individuals with Crohn's disease, four (45%) with ulcerative colitis, and three (20%) without inflammatory bowel disease. Viable MAP was cultured from the blood of 14 (50%) patients with Crohn's disease, two (22%) with ulcerative colitis, and none of the individuals without inflammatory bowel disease. Current use of immunosuppressive medication did not correlate with a positive MAP culture. Sequencing of PCR products from MAP cultures confirmed the presence of the MAP-specific IS900 fragment. Among 11 MAP isolates assessed, we identified nine strains that were not identical. INTERPRETATION: We detected viable MAP in peripheral blood in a higher proportion of individuals with Crohn's disease than in controls. These data contribute to the evidence that MAP might be a cause of Crohn's disease.

Adolescent↗
PubMed1996

Phenotypic characterisation of intestinal lymphocytes in ovine paratuberculosis by immunohistochemistry.

Characterisation of the T-cell subsets in intestinal lesions in sheep with paratuberculosis may contribute to our understanding of the pathogenesis of this disease. To determine the phenotype and distribution of lymphocytes in the normal sheep intestinal mucosa and in Mycobacterium avium subspecies paratuberculosis infected sheep, immunohistochemistry was performed on 12 normal sheep and 18 naturally infected, clinically diseased sheep of which 12 showed lepromatous and six tuberculoid forms of the disease. Immunoperoxidase staining was carried out on frozen sections of ileum using monoclonal antibodies against ovine CD4, CD8, and gamma delta T-cell receptor (TCR) markers. In all three sample groups, cells appeared to be non-randomly distributed throughout the lamina propria. Higher densities of lymphocytes were present in villus than in crypt areas. CD8+ cells were located principally around the epithelial basement membrane, whereas CD4+ cells were localised towards the central villus area of the lamina propria. Lymphocytes bearing the gamma delta T-cell receptor were more widely distributed, both in epithelial and lamina propria compartments. Ileum with tuberculoid lesions had higher densities of CD4 and gamma delta T-cell subsets while lepromatous lesions had lower densities of CD4 and CD8 cells compared with normal tissues. The median relative percentage of CD4+ cells was increased and that of CD8+ cells decreased in tuberculoid cases, with a corresponding increase in the CD4:CD8 ratio, while the relative percentage of gamma delta + cells was increased in lepromatous cases.

Animals↗
PubMed2000

A cross-sectional study of paratuberculosis in 1155 Danish dairy cows.

In a cross-sectional study on milk samples from 1155 cows from 22 Danish dairy herds, selected risk factors for paratuberculosis were identified. The diagnostic procedure used was an indirect enzyme-linked immuno-sorbent assay (ELISA) to detect antibodies against Mycobacterium avium subsp. paratuberculosis. A sample was considered test-positive if it had a corrected optical density >/=0. 025 (test sensitivity 71.4% and test specificity 89.7%). Of the 1155 samples, 8.8% (102/1155) were test-positive, and 19 out of the 22 dairy herds had >/=1 test-positive cows. The significant risk factors in a multiple logistic regression analysis were: Jersey versus large breeds, high parity versus low parity, the first month after calving versus other months of lactation, and a large herd size compared to a small herd size. The highest probability (37-38%) of a positive test was observed among older cows (parity >4) and tested within the first month after calving (irrespective of breed). The lowest probability (2%) of a positive test-result was observed among first parity, large-breed cows tested before calving or later than one month after.

Animals↗
PubMed2002

The Mycobacterium avium subsp. paratuberculosis ELISA response by parity and stage of lactation.

Two cross-sectional studies were carried out to determine the enzyme-linked immunosorbent assay (ELISA) response to Mycobacterium avium subsp. paratuberculosis (Map) by cow characteristics and stage of lactation. One of the studies (referred to as "milk-group") used milk samples from all lactating cows (n=7994) in 108 Danish dairy herds. The other study (referred to as "serum-group") used serum samples collected from all cows (n=5323) in a subset of 72 herds from the 108 herds. These samples were analysed using a similar ELISA for detection of antibodies. The results from the ELISAs were interpreted with two cut-off values as the optimal cut-off value is not known, and as several levels are recommended to be used in practice. The results showed that the probability of being ELISA-positive was two to three times lower for cows in parity 1 relative to cows in other parities using both milk and serum ELISA. At the beginning of the lactation the probability of being positive was highest in the milk ELISA. In the serum ELISA the odds of being positive was highest at the end of lactation. The findings are important in the interpretation of ELISA results at cow level with a subsequent tentative diagnosis and correction for parity and stage of lactation should be considered when providing a diagnosis of paratuberculosis. Some issues related to the pathogenesis are also discussed.

Animals↗
PubMed1997

Estimated prevalence of paratuberculosis in Missouri, USA cattle.

An absorbed ELISA for detection of antibodies against Mycobacterium paratuberculosis was performed on serum samples obtained from the Missouri Animal Health Laboratory. Samples from 1954 Missouri cattle representing 89 herds were randomly selected from samples submitted for brucellosis testing. The apparent seroprevalence of paratuberculosis in dairy cattle (8 +/- 3%) was similar to that in beef cattle (5 +/- 2%). When herds were classified as dairy or beef, 74% (14 of 19) of dairy herds and 40% (27 of 68) of beef herds were positive.

Animals↗