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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↗
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↗
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↗
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↗
PubMed1999

Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved.

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.

Animals↗
PubMed1996

Transmission of paratuberculosis.

Infection of the calf soon after birth by oral ingestion of M. paratuberculosis organisms from feces of infected cows is the most important method of transmission of paratuberculosis. The severity and rate of progression of the disease are dependent on the quantity of organisms in the exposure and the age of the animal. Only a small dose of organisms may be required to establish infection in a newborn calf, and overwhelming age-related resistance by introduction of a large dose of organisms to an adult cow is probably possible. Other means of transmission such as transplacental and direct excretion in the milk and colostrum should not be ignored and should be a particular concern primarily in advanced stages of infection when heavy fecal shedding is detected.

Animals↗
PubMed1996

Diagnosis of paratuberculosis.

The currently available and accurate diagnostic tests for paratuberculosis are reviewed. Technical issues concerning test performance are briefly mentioned and the advantages, disadvantages, and comparative accuracy are described. The article concludes with recommendations on which tests to use in eight different situations requiring laboratory diagnostics for paratuberculosis.

Animals↗
PubMed1997

Comparative study of Mycobacterium paratuberculosis strains isolated from Crohn's disease and Johne's disease using restriction fragment length polymorphism and arbitrarily primed polymerase chain reaction.

To obtain insights into the pathogenic mechanisms involving Mycobacterium paratuberculosis in Crohn's disease (CD) we questioned if the strains of M. paratuberculosis isolated from CD are distinguishable from those involved in Johne's disease (JD), a chronic granulomatous enteritis in cattle. Accordingly we compared human and animal strains at the DNA level, both by the analysis of restriction fragment length polymorphism (RFLP) in and around the insertion sequence IS 900 and by the arbitrarily primed chain reaction (AP-PCR). Results are in favour of a common clonal origin for the 4 strains isolated from CD and for 8 of the 11 strains isolated from cattle and sheep JD.

Animals↗
PubMed1993

Use of rifabutin in treatment of systemic Mycobacterium paratuberculosis infection in mice.

BALB/c mice were infected intraperitoneally with Mycobacterium paratuberculosis and, after allowing the infection to progress for 30 days, were treated with rifabutin at 0, 12.5, 25, and 50 mg/kg of body weight. Rifabutin was administered in drinking water under conditions of water deprivation, whereby the entire daily dose was delivered within a 1-h period. Animals were killed at biweekly intervals from time zero of treatment to 180 days. Spleens and livers from each animal were examined by quantitative bacteriologic culture and histopathology. Restricted water availability was found to be a viable alternative to daily gavage for single-dose bolus administration. Infection, as assessed by bacterial counts, was reduced only in animals that received 50 mg of rifabutin per kg. In these animals, bacterial counts in the liver and spleen were reduced from 7.2 x 10(5) +/- 4.1 x 10(4) and 6.5 x 10(5) +/- 4.1 x 10(4) to 3.0 x 10(3) +/- 1.8 x 10(2) and 3.1 x 10(3) +/- 2.2 x 10(2), respectively, over the 6-month treatment period. Rifabutin may be an appropriate chemotherapeutic drug for long-term treatment of M. paratuberculosis infection and should be considered in any multidrug regimen.

Animals↗
PubMed1995

Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.

Animals↗
PubMed1998

Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures.

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

Animals↗
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↗
PubMed1992

Effects of mycobactin J and lactoferrin supplementation of drinking water on the in vivo multiplication of Mycobacterium paratuberculosis in gnotobiotic mice.

In this study the effect of supplementation of drinking water with mycobactin J or lactoferrin on the multiplication of Mycobacterium paratuberculosis in gnotobiotic mice was investigated. The results indicated that neither mycobactin J nor lactoferrin, at the doses used, appeared to have any effect on fecal shedding or tissue burdens of M. paratuberculosis.

Administration, Oral↗
PubMed1991

Associations between dairy production indices and lipoarabinomannan enzyme-immunoassay results for paratuberculosis.

Data from an epidemiological study in Ontario, involving 304 dairy herds, were used to identify associations between selected production indices and lipoarabinomannan antigen serological test results for paratuberculosis (LAM-ELISA). Analyses were conducted at both the herd and individual cow levels of organization. After analytically controlling for management and cow factors in the respective regression models, positive serological paratuberculosis status (as defined by the LAM-ELISA test), was associated with higher milk somatic cell counts at both the herd average (p less than 0.01), and individual cow levels of organization (p less than 0.0001). In contrast, LAM-ELISA test results were consistently not associated with calving intervals in either the herd average or individual cow level analyses. Associations between LAM-ELISA results and milk production were inconsistent. No associations were found at the herd level of organization, and LAM-ELISA results were not associated with a change in breed class average (BCA) for milk, between the previous and the most recent lactations of individual cattle. However, at the individual cow level, LAM-ELISA results were positively associated with higher milk production as measured by the current BCA (p less than 0.05), and individual cow average kg of milk produced per year of life since two years of age (p less than 0.0001).

Animals↗
PubMed1994

Mycobacterium paratuberculosis infection in two llamas.

Mycobacterium paratuberculosis was diagnosed in 2 llamas by bacteriologic culturing and DNA probe testing of fecal and intestinal specimens. Diarrhea and weight loss were observed in both animals. Lesions in the gastrointestinal tracts of the 2 llamas were similar to pathologic changes reported in cattle infected with M paratuberculosis.

Animals↗
PubMed1994

Herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in Wisconsin.

A random sample of Wisconsin dairy herds, stratified by herd size, were tested for paratuberculosis by use of an absorbed ELISA procedure. The ELISA was optimized for overall accuracy by means of receiver operating characteristic curve analysis, and had a sensitivity and specificity of 50.9 and 94.9%, respectively. Herd prevalence was analyzed for correlation with responses to a management practices questionnaire completed by the herd owners. One hundred and fifty-eight herds and 4,990 cattle were tested. Of these, 50% of herds and 7.29% of cattle had positive test results. Calculation of true prevalence from the apparent prevalence indicated that 4.79% of cattle and 34% of the Wisconsin dairy herds tested had serologic evidence of paratuberculosis. Among the 54 herds classified as positive on the basis of true prevalence estimation, the mean number of test positive cattle was 20.3%. The geographic distribution of herds with positive results was not uniform. More infected herds were found in the southern and western districts of Wisconsin than in the eastern district. The west-central district had a larger number of infected herds than did other districts. By use of chi 2 analysis, the only management factor found to be significantly associated with herd prevalence was housing of calves after weaning (P = 0.03). Specifically, in herds with higher prevalence, calves were separated after weaning into calf barns and hutches rather than into pens in the cow barn more often than in herds with lower prevalence. This factor was also considered significant by use of logistic regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Husbandry↗