Disseminated Mycobacterium paratuberculosis infection in a cow

A cow with chronic diarrhea and weight loss caused by localization of Mycobacterium paratuberculosis in the intestinal tract (Johne's disease) had gross and microscopic changes indicative of a disseminated infection. A direct association between the remote lesions and the intestinal infection was shown by isolation of M paratuberculosis from renal tissue, detection of intracellular M paratuberculosis antigen(s), using an indirect immunoperoxidase method, and by the characteristic granulomatous nature of the lesions. This case illustrates the potential for extra-intestinal lesions in M paratuberculosis infection of cattle and should cause veterinarians to consider mycobacterial disease when confronted with multinodular lesions of the bovine kidney. The immunoperoxidase method was useful in determining the cause of the inflammatory lesion in which intact organisms were not evident.     

Bovine fetal infection with Mycobacterium paratuberculosis

In utero transmission of Mycobacterium paratuberculosis, the causative agent of paratuberculosis in cattle, has been suggested. Tissue specimens were obtained at a packing plant from pregnant dairy cows and their fetuses and from cows with clinical signs of paratuberculosis and from their fetuses. Specimens were processed according to methods described for isolating M paratuberculosis from bovine tissues and were incubated on Herrold egg yolk medium for 16 weeks. Presumed positive specimens were confirmed to be M paratuberculosis, using acid-fast staining and subculturing. Of 407 lymph nodes from cows, 34 (8.4%) were culture positive for M paratuberculosis; 9 of 34 (26.4%) of these culture-positive cows had fetuses from which specimens were also culture positive. The results estimated the risk of fetal infection with M paratuberculosis to be 26.4% (95% confidence interval between 11.3 and 40.7%).     

Progressive immunopathological changes during early stages of experimental infection of goats with Mycobacterium avium subspecies paratuberculosis

A dose of 10(10) Mycobacterium avium subspecies paratuberculosis was administered orally on seven occasions to produce experimental paratuberculosis infection in 10 5-8-week-old goat kids. Bacteriological, immunological, and histopathological changes, their relationships, and the efficacy of the commonly used diagnostic methods were studied during the progressive disease up to 270 days postinfection (DPI). Significant lymphocyte proliferative responses in the peripheral blood of five goats were detected as early as 60 DPI. A lymphoproliferative test was also performed on lymphocytes purified from different compartments of the guts of five infected and five control goats. Significant proliferative responses were observed in lymphocytes of jejunal compartments of all five goats, of which four had also significant lymphocyte proliferation in the blood. The ileal lymphocytes from two goats, one each at 120 and 270 DPI, had significant proliferation. The histological lesions were mainly observed in the gut-associated lymphoid tissues of the ileocecal valve, the ileum, and the terminal jejunum. Acid-fast bacilli were demonstrated in the lesions of two goats at 60 and 210 DPI. Bacterial culture showed poor sensitivity, detecting positive results for only one goat in the fecal and tissue samples at 210 DPI, whereas polymerase chain reaction (PCR) detected one goat in fecal sample at 210 DPI and two goats in tissue samples at 60 and 210 DPIs, respectively. Enzyme-linked immunosorbent assay and agar gel immunodiffusion test were found to be 100% sensitive from 180 and 210 DPI onwards, respectively.     

Treatment of Mycobacterium paratuberculosis infection in ruminants.

Paratuberculosis is a chronic, debilitating, fatal condition that usually is clinically undetectable until the onset of copious diarrhea. Paratuberculosis is caused by an acid-fast organism, M. paratuberculosis. Successful eradication of paratuberculosis depends on the early detection of infected animals, thereby allowing removal of carrier animals from the herd. Treatment for paratuberculosis is therefore rarely indicated or undertaken; however, treatment may be considered for animals of exceptional genetic value or companion animals. Antimicrobials reviewed in this article for the treatment of paratuberculosis include isoniazid, rifampin, streptomycin, amikacin, clofazimine, and dapsone. Treatment of paratuberculosis requires daily medication for extended periods and results in palliation of the disease rather than a definitive cure. The treatment for paratuberculosis recommended by the authors is isoniazid at 20 mg/kg administered orally every 24 hours for the rest of the animal's life. When the animal has acute onset of diarrhea, rifampin at 20 mg/kg every 24 hours is also administered orally. In severe, imminently life-threatening cases, an aminoglycoside should be administered concurrently for 3 to 8 weeks. This protocol (isoniazid, rifampin, and an aminoglycoside) will help ensure that Mycobacteria organisms are sensitive to at least two of the antibiotics. Rifampin treatment can be discontinued if clinical signs of paratuberculosis disappear and the cost of therapy is judged excessive. The combined therapeutic approach has been used in three animals, and the results are presented in this article. Because isoniazid, rifampin, and some aminoglycosides are not approved for use in food animals in the United States of America, the meat or milk from treated animals should not be used for human consumption.     

Experimental infection with Mycobacterium caprae in goats and evaluation of immunological status in tuberculosis and paratuberculosis co-infected animals

Tuberculosis in goats (caused by Mycobacterium caprae and M. bovis) has become a significant concern in recent years because of its high prevalence in certain caprine herds in Spain and other European countries, and also due to the potential transmission to other animals and human beings. In the present study, a transthoracic model of tuberculosis infection was performed on goats. Animals were selected based on the serological response used to detect paratuberculosis in goats (negative and positive results). The kinetics of the immune response was evaluated using the interferon-γ (IFN-γ) assay, skin tests and serology of paratuberculosis during nine months post-challenge. At the end of the study the animals were necropsied, tuberculosis-lesions were scored and culture (M. caprae and M. avium subsp. paratuberculosis) was performed to determine the true infection status. Animals were positive to the IFN-γ assay 15 days post-challenge and the values were fluctuating throughout the study. A varied performance of the assay was observed between tuberculosis and tuberculosis-paratuberculosis mixed infection regarding both the number of positive results and the OD values obtained after stimulation with bovine and avian PPDs. Furthermore, the single intradermal comparative cervical tuberculin test did not detect all M. caprae-infected animals. At necropsy, a positive correlation between pathology score and bovine PPD specific IFN-γ response was found.     

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.     

Cultural and biochemical characteristics of Mycobacterium paratuberculosis isolated from goats in Norway.

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant.The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria.Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.     

Hematologic values in normal pygmy goats

Pygmy goat blood was analyzed to determine hematologic base-line values. Male, female, and castrated male goats were used in this investigation. Influence of age on these variables was also studied. Most of the hematologic values of pygmy goats were similar to the reference values reported for several other ruminant species and for man. Values significantly different from reference values in man were as follows: Erythrocyte counts were slightly higher than for human beings. The relative lymphocyte count was comparable to that in infants, but significantly greater than values in adult human beings. Also, although not of statistical significance, hemoglobin levels in goats were slightly lower than in both infants and adult human beings, and the leukocyte count was just slightly higher than the usual values for infants and adult human beings. Thus, of 9 hematologic variables, only 1 was significantly different from those in man, and 2 were slightly different. The fact that pygmy goats have hematologic responses similar to those of man, and the similarity of hematologic values to reference values in man, further enhances the pygmy goat for use as an experimental animal.     

Serum biochemistry values in normal pygmy goats

Blood serum of pygmy goats (both sexes, and castrated males) was analyzed to establish biochemical reference values. Influence of age on reference values was also studied. Serum biochemical analyses were made for urea nitrogen, creatinin, bilirubin, lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase, glucose, uric acid, and total lipids. These serum values for pygmy goats were similar to those reported for man, except as follows: Aspartate aminotransferase activities were slightly higher than those reported for man. Glucose concentrations in pygmy goats were slightly lower than in human beings, and uric acid levels were significantly lower than the values for man. Female and castrated male goats had lower total lipid concentrations than did human beings, whereas intact males had higher concentrations. Thus, of the 9 measured variables for pygmy goats, 5 were comparable to human values. This, together with other attributes, including the small size which conduces to economics of maintenance and enhances the desirability of using pygmy goats in research.     

Differences in intermittent and continuous fecal shedding patterns between natural and experimental Mycobacterium avium subspecies paratuberculosis infections in cattle

The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.     

Diagnostic detection methods for Mycobacterium avium subsp. paratuberculosis in white-tailed deer

This study compares the results and suitability of serological testing, microscopic examination, deoxyribonucleic acid (DNA) detection, and bacterial culture for detecting Mycobacterium avium subsp. paratuberculosis (Map) infection in asymptomatic farmed white-tailed deer (WTD) (Odocoileus virginianus). Deer were classified as infected if culture slants from their feces, lymph nodes, or ileum were positive, or if a polymerase chain reaction (PCR) assay detected Map DNA in any of its tissues. Deer identified as positive by agar gel immunodiffusion (AGID) testing or enzyme-linked immunosorbent assay (ELISA) but not by bacterial culture, Ziehl-Neelsen staining, or PCR assay were classified as suspect. Culture of tissues classified 10/16 (62.5%), histopathologic examination 1/16 (6.3%), tissue smears 4/16 (25%), culture slant (CS)-PCR on feces 12/15 (80%), CS-PCR on tissue 13/16 (81.3%), and direct PCR on uncultured tissues 5/16 (31.3%) deer as infected. The ELISA classified 2/15 (13.3%) deer as positive and therefore suspect. The AGID test was negative for all deer. Fifteen of 16 deer were positive by 1 or more tests; only 1 deer was negative on all 11 assays. The CS-PCR gave superior results on antemortem fecal testing as well as postmortem tissue testing and can be recommended for improving the detection of Map in WTD at every stage of infection.     

Field evaluation of tracer sheep for the detection of early natural infection with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To determine whether tracer sheep could be used to detect S strain Mycobacterium avium subsp paratuberculosis on pasture, and to provide further insight into the early stages of infection. DESIGN: A field study on two farms in an endemic area for ovine Johne's disease in New South Wales. Procedure Lambs, weaners and adult ewes were introduced to pasture with varying amounts of M. a. paratuberculosis contamination and monitored using skin tests, gamma interferon assay, faecal culture and serial necropsy of small groups for up to 15 months after first exposure. RESULTS: Culture from tissues was the most sensitive method for detecting early infection in sheep after natural exposure to S strain M. a. paratuberculosis. The organism was detected in at least one introduced sheep from every exposed group, 6 to 12 months after first exposure. Histopathological lesions were detected in only 17% of culture-positive sheep, and only after at least 8 months of exposure. Similarly, antemortem diagnostic tests had low sensitivity during the early stages of naturally acquired infection. There was no evidence of any differences in infection rate between sheep first exposed as neonates, as weaners or as adults. A higher proportion of lambs born to ewes from an infected flock were infected than lambs suckling uninfected ewes introduced to the same infected environment, and infection was detected earlier in these 'resident' lambs. CONCLUSION: These findings indicate that groups of unexposed 'tracer' sheep, tested by culture of tissues at slaughter 6 to 12 months after first exposure, might be a useful way to assess pasture infectivity in control programs for ovine Johne's disease.     

Strain-specific sensitivity estimates of Mycobacterium avium subsp. paratuberculosis culture in Greek sheep and goats

The requirements for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) may be related to the strain-type [sheep (S)- or cattle (C)-type] and not to the host. The objective of this paper was to estimate and compare strain- and biological sample (faeces or pooled-tissue)--specific sensitivities (Ses) of two solid culture media, Herrold's egg yolk medium (HEYM) and Lowenstein-Jensen (LJ) medium, for the isolation of Map from Greek dairy sheep and goats. From 400 faecal samples collected from sub-clinically infected sheep and goats of four flocks and from 214 pooled-tissue samples (142 from sheep and 72 from goats) collected, at the abattoir, from >1-year-old routinely slaughtered animals, with gross pathology suggestive of paratuberculosis, we isolated 34 Map strains. Of those, by the IS1311 PCR, 18 were categorized into the C-type and nine into the S-type; seven were not typed. We used a Bayesian approach to estimate the strain-specific Ses. SeHEYM-C-faecal=17% (95% credible interval: 7, 40) was higher than SeHEYM-S-faecal=2% (0.3, 11). Also, SeHEYM-C-faecal was higher than SeLJ-C-faecal=4% (1, 12). In pooled-tissue samples, the strain-specific Ses did not differ between the two media.     

Seroprevalence of Mycobacterium avium subspecies paratuberculosis in Korean black goats (Capra hircus aegagrus)

In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (chi=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.