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PubMed1992

Diagnostic accuracy of a Mycobacterium phlei-absorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows.

The purpose of this study was to describe the responses of sera from five groups of cattle to an enzyme-linked immunosorbent assay (ELISA) for paratuberculosis by using serum absorbed with Mycobacterium phlei at a single working dilution. The infection status of the cattle was determined by fecal culture. Cattle with different levels of exposure (high versus low prevalence and test negative) and disease manifestation (clinically suspect infection versus subclinical infection) were examined, as follows: (i) two paratuberculosis-negative herds; (ii) a fecal culture-confirmed, clinically suspect cases of paratuberculosis; (iii) cows from a paratuberculosis-infected herd with a high infection rate, as determined by fecal culture, but with no clinical cases at the time of sampling; (iv) cows from three paratuberculosis-infected herds known to have paratuberculosis diagnosed on the farm (low infection rate determined by fecal culture); and (v) one fecal culture-negative herd with known serologically positive cattle. Results generally showed a decreased ELISA response when absorbed rather than nonabsorbed serum from each animal was used. The results of the fecal culture confirmed clinically suspect cases, which were analyzed in relation to the amount of colonies isolated from the animals on fecal culture (0, +, ++,+++ , ++++, and above). There was a significant increase in the ELISA response for animals with heavy Mycobacterium paratuberculosis shedding ( ++++ or above), when both unabsorbed and absorbed sera were used, compared with the response in animals that were fecal culture negative or that shed M. paratuberculosis at lower levels (less than +) (P less than 0.05). The effects on sensitivity and specificity by using different cutoff points for the five groups of cattle with different levels of exposure is described, since sera were not discretely segregated into distinct groups of positive and negative samples. The specificity of the ELISA in the two fecal culture-negative herds was 100% at an ELISA cutoff of an optical density (OD) of 0.1 and above for absorbed serum. For unabsorbed serum the specificity was 62.9% at a similar cutoff value. Similarly, the specificity of the fecal culture-negative, serologically positive herd increased from 37.5 to 72.2 at an ELISA cutoff value of 0.1 to 0.2 (OD) by using absorbed versus unabsorbed serum from 75.0 to 94.4 at an ELISA cutoff value of 0.2 to 0.3 (OD).

Animals↗

PubMed1992

Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Evaluation of four serological tests for bovine paratuberculosis.

The standard complement fixation (CF) test, a commercial agarose gel immunodiffusion (AGID) test (ImmuCell Corporation, Portland, Maine), and two commercial enzyme-linked immunosorbent assays (ELISAs; Allied Laboratories, Glenwood Springs, Colo. [Allied ELISA], and the CSL, Limited, [Parkville, Victoria, Australia] enzyme immunoassay [CSL ELISA]) were evaluated by using sera from the Repository for Paratuberculosis Specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of dairy cattle without diarrhea or weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. None of the cattle evaluated had been vaccinated for paratuberculosis. Among 177 M. paratuberculosis-infected cattle, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA had test sensitivities of 38.4, 26.6, 58.8, and 43.4%, respectively, and specificities of 99.0, 100.0, 95.4, and 99.0%, respectively. Only 108 of the infected cattle were confirmed by culture or by a commercial DNA probe (IDEXX Corporation, Portland, Maine) to be shedding the organism in fecal samples at the time of serological testing. Among the 108 M. paratuberculosis fecal shedders, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA were positive for 54.6, 40.7, 65.7, and 56.5% of the cows, respectively; and among the 69 cows that were nonshedders, the tests were positive for 14.5, 4.3, 47.8, and 24.6% of the cows, respectively. There was a significant difference (P < 0.05) in the rate of positive test results for all four tests between these two groups of animals. The CF test performed well when it was compared with the other serological tests if a titer of >/- 1.8 was classified as a positive test result.

Animals↗

PubMed1992

Comparison of a commercial DNA probe test and three cultivation procedures for detection of Mycobacterium paratuberculosis in bovine feces.

Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 10(4) M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.

Animals↗

PubMed1975

Analysis of the effect of inapparent bovine paratuberculosis.

Inapparent paratuberculosis was studied in a Guernsey herd with a history of paratuberculosis (Johne's disease); the herd was maintained at between 900 and 950 cattle. Fecal specimens and intestinal tissue specimens from any of these that were slaughtered were examined culturally for the presence of Mycobacterium paratuberculosis. The reasons given for culling and slaughtering of cows from this herd were compared with infection status determined by culturing. Less than one-third of the culturally confirmed paratuberculous cattle were culled because of clinically apparent paratuberculosis. Mastitis was the reason for culling 3.6% of the non-infected cattle and 22.6% of the cattle with inapparent paratuberculosis. Infertility was also significantly higher in cows with inapparent paratuberculosis than in noninfected cows in the same herd. Separation of parturient dams and calves from the rest of the herd was shown to materially reduce the level of infection and incidence of clinical paratuberculosis.

Animals↗

PubMed1992

Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis.

TMA (thermostable macromolecular antigens) are major mycobacterial complexes present in all mycobacteria. We have purified A36, the TMA complex of M. paratuberculosis, the etiological agent of paratuberculosis (Johne's disease), and shown by the immune electron microscopy approach its presentation at the cell surface. The immunodominance of the A36 complex in Johne's disease was suggested by comparative ELISA analysis of infected bovine sera, using either A36 or M. paratuberculosis total soluble sonicate as antigens. The cross-reactivity of TMA complexes from different mycobacteria was evaluated by immunoenzymometric measurements. Percentage of shared epitopes was high for the couple M. paratuberculosis-M. avium, and somewhat lower for the couple M. paratuberculosis.-M. bovis. Immunological kinship between M. paratuberculosis and M. leprae was suggested by the finding that out of eleven anti-M. leprae monoclonals, four cross-reacted with A36 proteins. The specificity missing at the level of the whole A36 complex was sought at the level of its protein components. Comparative immunoblot analysis of electrophoresed A36 proteins indicated three of them to contain epitopes not shared by M. bovis proteins, and one of them to contain epitopes specific with respect to M. avium, M. bovis and M. phlei. The latter component, a 34-kDa protein, could be an ideal reagent for a serological test for Johne's disease, being immunodominant in infected cattle and endowed with species-specific epitopes.

Animals↗

PubMed1992

Mycobacterium paratuberculosis DNA in Crohn's disease tissue.

Crohn's disease has long been suspected of having a mycobacterial cause. Mycobacterium paratuberculosis is a known cause of chronic enteritis in animals, including primates, but may be very difficult to detect by culture. IS900 is a multicopy genomic DNA insertion element highly specific for M paratuberculosis. A polymerase chain reaction (PCR) based on the 5' region of IS900 and capable of the specific detection of a single M paratuberculosis genome was developed. This was applied to DNA extracts of full thickness samples of intestine removed at surgery from 40 patients with Crohn's disease, 23 patients with ulcerative colitis, and 40 control patients without inflammatory bowel disease. Stringent precautions were taken that excluded contamination artefact. M paratuberculosis was identified in 26 of 40 (65%) Crohn's disease, in 1 of 23 (4.3%) ulcerative colitis, and in 5 of 40 (12.5%) control tissues. Positive samples from Crohn's disease were from both the small intestine and colon, those from control tissues were from the colon those from control tissues were from the colon only. All PCR internal control reactions were negative. The presence of M paratuberculosis in a small proportion of apparently normal colonic samples is consistent with a previously unsuspected alimentary prevalence in humans. The presence in two thirds of Crohn's disease tissues but in less than 5% of ulcerative colitis tissues is consistent with an aetiological role for M paratuberculosis in Crohn's disease.

Adult↗

PubMed1992

Paratuberculosis in saiga antelope (Saiga tatarica) and experimental transmission to domestic sheep.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.

Animals↗

PubMed1992

Comparison of subjective and objective test evaluations for use of Mycobacterium phlei-adsorbed serum in a dot-enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in cattle.

Use of a dot-ELISA with serum adsorbed with Mycobacterium phlei or with nonadsorbed serum was compared. In addition, results attained using visual observation were compared with those obtained using a densitometer. Infection status of cattle was determined by results of culture of feces from a number of cattle with various degrees of exposure (low prevalence and test-negative) and disease manifestation (clinical suspect vs subclinical infection). Two paratuberculosis-negative herds, fecal culture-confirmed clinically suspect cases of paratuberculosis, and cows from 2 paratuberculosis-infected herds with diagnosis confirmed on the farm (low infection rate) were tested. Significant (P less than 0.05) increase in the dot-ELISA response was found in cattle with heavy M paratuberculosis shedding when nonadsorbed and adsorbed sera were used, compared with the response in cattle that were fecal culture-negative or were shedding M paratuberculosis at lower amounts. Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture-positive, clinically suspect cattle when nonadsorbed serum was used. Results of the visual test were negative in 85 of 93 (91.4%) of the fecal culture-negative cattle when nonadsorbed serum was used. However, when using M phlei-adsorbed serum, the sensitivity of the visual determination decreased to 34.1% (15/44), and the specificity increased to 97.8% (91/93).(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMedDate not supplied

Mycobacterium paratuberculosis. Factors that influence mycobactin dependence.

Mycobacterium paratuberculosis does not produce any detectable mycobactin, an iron-binding compound that is synthesized by most Mycobacterium spp. and necessary for the growth of all mycobacteria. This study examined the influence of various culture conditions on mycobactin dependence in M. paratuberculosis. Using a radiometric growth assay, we found the minimal concentration of mycobactin J necessary for growth of M. paratuberculosis to be 0.006 microM, whereas 1.2 microM (1 microgram/ml) was required for optimal growth. In media without mycobactin at iron concentrations less than or equal to 100 microM, growth of M. paratuberculosis occurred at pH 5.0, but not pH 6.8. Iron concentrations greater than 100 microM did not significantly increase growth at pH 5.0, but at pH 6.8 the growth rate increased with increasing amounts of iron reaching a rate equal to control cultures containing mycobactin. Mycobacterium paratuberculosis appeared to lose mycobactin dependence when subcultured; however, this was subsequently shown to be a result of mycobactin carried over from primary medium. Removal of this contaminating cell-wall-associated mycobactin reestablished mycobactin dependence. We conclude that mycobactin dependence must be carefully determined because it is a key test used in identification of M. paratuberculosis and may be easily influenced by media pH, iron concentration, and mycobactin carryover from primary media.

Animals↗

PubMed1992

Detection of Mycobacterium paratuberculosis antigen with colloidal immunogold in naturally infected sheep.

The anti-Mycobacterium paratuberculosis polyclonal serum is proved useful for labelling Mycobacterium paratuberculosis in glutaraldehyde-osmium-fixed and epon-embedded intestinal samples from sheep with clinical symptoms of paratuberculosis. M. paratuberculosis marked with antibody-coated colloidal gold stain was seen in macrophages, epithelioid cells, giant cells and neutrophils throughout intestinal mucosa. In large macrophages with a low lysosomal content, a great number of intact mycobacteria was seen within phagosomes. In macrophages with average lysosomal content, very few intact mycobacteria or mycobacterial debris were present and lysosome-phagosome fusions were observed. Mycobacteria within neutrophils were scanty. These results show the usefulness of colloidal immunogold techniques for studies of the pathogenesis of paratuberculosis.

Animals↗

PubMed1992

The cellular immunology of bovine paratuberculosis: the predominant response is mediated by cytotoxic gamma/delta T lymphocytes which prevent CD4+ activity.

Peripheral blood T-cell subsets were obtained from an experimentally sensitized bovine and from nine bovines naturally infected with Mycobacterium paratuberculosis and tested for their ability to respond to antigen. It was determined that following antigen challenge, proliferative responses followed a biphasic pattern: an initial CD4+ proliferative response, a period of anergy, and a final response governed by gamma/delta T lymphocytes. The anergic phase was characterized by a dramatic drop in peripheral blood CD4+ cells; the nature of the non-responsiveness could not be determined. The anergic phase was followed by increased proliferative responses of non-MHC restricted gamma/delta T lymphocytes. Although CD4+ cells had the ability to proliferate in response to M. paratuberculosis antigens in the absence of gamma/delta T cells, antigen-primed CD4+ lymphocytes failed to incorporate [3H]-thymidine in the presence of gamma/delta T cells and M. paratuberculosis antigen. It was concluded that M. paratuberculosis-specific gamma/delta T lymphocytes have immunoregulatory function and exhibit cytotoxic activity against antigen-primed CD4+ helper cells. The data suggest that the inability of effector cell populations to prevent intracellular proliferation of M. paratuberculosis may be a result of cytotoxic killing of the T helper lymphocyte population required for macrophage activation.

Animals↗

PubMed1992

Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin.

Severe combined immunodeficient beige mice were inoculated orally and intraperitoneally with a bovine strain of Mycobacterium paratuberculosis to explore their potential as laboratory animal models in the study of paratuberculosis (Johne's disease). Control animals were similarly inoculated with heat-killed M. paratuberculosis. In the mice inoculated intraperitoneally, focal lesions and acid-fast bacilli were first detected in the livers (4 weeks postinfection) and later in the spleens and intestines of the test but not the control animals. No bacteria were seen in the hearts, kidneys, or lungs. At 12 weeks postinfection, all test mice had significant losses in body weight compared with those in controls (P less than 0.05), a characteristic sign of bovine paratuberculosis. Tumor necrosis factor alpha was not detected in the serum. Histologic lesions were seen in the intestines, livers, and spleens of the animals in the orally inoculated test group after 26 weeks of infection. Our results suggest that the severe combined immunodeficient beige mouse may be a useful model for the investigation of paratuberculosis and cachexia and the evaluation of antimycobacterial drugs.

Animals↗

PubMed1992

Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue.

Mycobacterium paratuberculosis is known to cause Johne's disease, a granulomatous ileitis in ruminants, and may be involved in some cases of Crohn's disease. Like M. paratuberculosis, the wood pigeon strain of Mycobacterium avium may also show mycobactin dependence on primary isolation that is attenuated on further subculturing. A wood pigeon strain, M. avium restriction fragment length polymorphism (RFLP) type A/I, is also capable of causing granulomatous ileitis in experimental animal models but is not known to cause disease in humans. M. avium RFLP type A is associated with disease in immunocompromised hosts. Three DNA probes, pMB22 and the two subclones pMB22/S4 and pMB/S12, were found to be capable of distinguishing among M. paratuberculosis, M. avium type A, and M. avium type A/I (wood pigeon strain) on the basis of RFLPs. These DNA probes were used to identify two mycobacterial isolates (M. paratuberculosis and M. avium type A/I, wood pigeon strain) derived from the intestinal tissues of two patients with Crohn's disease. In addition, the wood pigeon strain of M. avium was identified from a patient with ulcerative colitis, and M. avium RFLP type A was identified from a patient with colonic carcinoma. This is the first time that M. avium A/I (wood pigeon strain) is known to have been isolated from human tissue. There are too few isolates to speculate about the etiological significance of mycobacteria and inflammatory bowel disease, but it is reasonable to conjecture that M. paratuberculosis may be responsible for some cases of Crohn's disease and that the wood pigeon strain of M. avium may also be an inflammatory bowel disease pathogen in humans.

Crohn Disease↗

PubMed1979

Paratuberculosis in cattle and free-living exotic deer.

Paratuberculosis was studied among dairy cows and exotic deer that shared grazing areas at Point Reyes National Seashore, California. Of the 10 dairy herds tested, 5 (50%) were infected with Mycobacterium paratuberculosis (based on results of fecal culture). Mycobacterium paratuberculosis was cultured from 9 (8.7%) of the 103 bovine fecal samples and from 4 (3.9%) of the 103 bovine rectal mucosa scapings tested. Of 89 fecal samples from 52 axis deer (Axis axis) and 37 fallow deer (Dama dama), 5 (9.6%) and 3 (8.1%), respectively, contained M paratuberculosis. Culture of intestinal necropsy samples from the same deer indicated that 3 (5.8%) of the axis deer and 2 (5.4%) of the fallow deer were infected with M paratuberculosis. The cows were tested for serum antibodies by the complement-fixation test and by radioimmunoassay. Of 95 sera tested by complement fixation, 15 (15.8%) were positive, as were 15 (14.7%) of 102 sera tested by radioimmunoassay. Culture results and serologic test results were compared on a herd basis.

Animals↗

PubMed1992

Mycobacterium paratuberculosis isolated from fetuses of infected cows not manifesting signs of the disease.

Fetuses were obtained from 58 cows that were fecal culture-positive for Mycobacterium paratuberculosis, but were not manifesting signs of paratuberculosis. Fetal tissues from 5 of 58 cows were culture-positive for M paratuberculosis. All 5 culture-positive fetuses were from cows that were classified as heavy fecal shedders (5/28; 17.8%). Difference in prevalence of fetal infection between light (less than 70 colonies/tube) and heavy fecal shedders was significant (Fisher's exact test, P less than 0.05). Association was not evident between serologic status of the dam and prevalence of fetal infection. In infected cows without signs of paratuberculosis, fetal infection develops with lower frequency than previously reported for cows with clinical signs of the disease. In this study, fetal infection was found only in cows that were heavy fecal shedders.

Animals↗

PubMed1992

[Bacterial studies on the occurrence of Mycobacterium paratuberculosis in fecal samples of zoo ruminants].

Mycobacterium (M.) paratuberculosis was isolated from fecal samples of 3 (21.4%) from 14 mouflons, of 10 (20.4%) from 49 dwarf goats, of 5 (14.3%) from 35 Cameroon sheep and of 1 (9.1%) from 11 alpine ibex. M. paratuberculosis could not found by cultural method in fecal samples of 22 Pinzgauer goats, of 15 bantengs, of 9 wild goats, of 9 skuddens, of 6 four-horned sheep, of 3 red-head sheep, and of 1 chamois. From all 19 animals with cultural positive fecal samples complement binding antibodies against M. paratuberculosis could not be found in the corresponding serum samples. The results confirm that M. paratuberculosis is more frequently in small zoo ruminants than up to now was suspected. The cultural examination of fecal samples has been proved to be a better method for detecting animal excretors than serological investigations by means of the complement fixation test.

Animals↗

PubMed1992

Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol.

Radiometric MICs of clarithromycin, a new macrolide drug, were determined against five mycobactin-dependent strains of Mycobacterium paratuberculosis (including two Crohn's disease clinical isolates) and compared with those of other drugs which included rifampin, ethambutol, amikacin, ofloxacin, ciprofloxacin, and sparfloxacin. Among the drugs screened, clarithromycin was the drug for which MICs were lowest against the five strains tested. As MICs were significantly below the reported Cmax levels (about 4 micrograms/ml), the intracellular activity of clarithromycin against the type strain of M. paratuberculosis maintained in cultured macrophages was screened. Clarithromycin was able to kill the initial inoculum by more than 1 log within 7 days, and this activity was further potentiated by ethambutol. Extracellular drug combination screened by using sublethal concentrations of the drugs showed that ethambutol was able to enhance clarithromycin activity in three out of four M. paratuberculosis strains instead of only one out of four strains (or none in the case of ofloxacin) when associated with other drugs. These results suggest that clarithromycin may be fruitful to treat human disease in which M. paratuberculosis may be etiologically involved.

Animals↗