Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep.
Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media. (+info
Antigen-specific B-cell unresponsiveness induced by chronic Mycobacterium avium subsp. paratuberculosis infection of cattle.
Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne's disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne's disease in cattle. (+info
Epidemiological study of paratuberculosis in wild rabbits in Scotland.
A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease. (+info
Factors influencing the isolation of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples.
A modified procedure was used for culture of Mycobacterium paratuberculosis (Mptb) from bovine feces. Bovine fecal samples were decontaminated with NaOH, exposed to a mixture of oxalic acid and malachite green, incubated in a mixture of neomycin and amphotericin B. Decontaminated specimens were inoculated onto modified Lowenstein-Jensen medium. Specimens processed by high-speed centrifugation showed growth earlier than specimens prepared by low-speed centrifugation. However, the overall number of positive cultures at 16 weeks was not different for the 2 methods. When infected dairy herds were sampled 4 times at 6-month intervals and culture-positive cows were culled, the prevalence of infected cattle declined over time. After selective culling, the cattle left in the herds shed low numbers of Mptb, which explains why it took longer for cultures to become positive. No heifers younger than 11 months were culture positive, but heifers 13-14 months of age were more frequently culture positive than were heifers of any other age. The 16-week culture period is needed with this method to detect cattle shedding low numbers of Mptb. High-speed centrifugation of samples does not increase the efficiency of identification of animals shedding Mptb. (+info
Bacterial isolation, immunological response, and histopathological lesions during the early subclinical phase of experimental infection of goat kids with Mycobacterium avium subsp. paratuberculosis.
The diagnosis of Mycobacterium avium subsp. paratuberculosis infection is difficult, especially in the early stages of disease. This is due to the long incubation period, the variable lag phase associated with bacterial proliferation, and the multifocal distribution of slowly developing lesions. There are few previous studies of the early stages of experimental paratuberculosis in goats. In the present study, the ability of conventional diagnostic methods to detect M. a. paratuberculosis infection during the early stages of infection was assessed. Eight goat kids were experimentally infected with M. a. paratuberculosis and subjected to a series of immunological and bacteriological tests before being euthanatized at various times postinfection. At postmortem examination, the ages of the kids ranged from 1 1/2 to 12 months. Of the eight goats infected, three had histopathological evidence of paratuberculosis. Two of these goats were positive with bacteriology, but only one was also positive with all immunological tests. One animal had a positive immunological response, but infection could not be demonstrated by bacteriologic or histopathologic examination. Histopathologic lesions were found in the jejunum, in the ileum, and in one mesenteric lymph node, but only the mesenteric lymph nodes and one retropharyngeal lymph node gave positive results following bacteriologic culture. The disparity between the localization of histopathologic lesions and bacteriologic results emphasizes the need for exhaustive sampling to confirm a diagnosis during the early phase of an infection. It also highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the immune system of the host. (+info
Use of pooled fecal culture for sensitive and economic detection of mycobacterium avium subsp. paratuberculosis infection in flocks of sheep.
Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997, M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P<0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting > or =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp. paratuberculosis infection at the flock level. (+info
Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.
An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture. (+info
Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.
We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex. (+info