Search

Tissues and fecal material were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. Fecal culture indicated that 5 of 14 (35.7%) animals were infected, whereas cultures from tissues detected 12 of 14 (85.7%) animals as infected and 59 of 111 (53.2%) of the tissues as positive for M. paratuberculosis. Polymerase chain reaction analysis identified infection in 14 of 14 (100%) animals and in 91 of 112 (81.2%) tissues. In addition, tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, and immunohistochemical staining. Ziehl-Neelsen and auramine O staining identified 7 of 14 (50%) and 5 of 14 (35.7%) animals as infected and 24 of 112 (21.4%) and 28 of 112 (25%) tissues as positive, respectively. Immunohistochemical analyses of bison tissues, using antisera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and positive tissues (ranging from 28 to 46%). Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more sensitive than bacterial culture or staining, identified infection in all the bison, and detected the greatest number of positive tissues within each animal.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.

A new method (NADC) for isolation of Mycobacterium paratuberculosis from fecal samples is described and evaluated using fecal samples from a known paratuberculosis-infected herd of cattle. The NADC method includes centrifugation of the total fecal sample supernatant and use of a 2-step decontamination protocol. The growth rate of M. paratuberculosis and contamination rate of cultures when using this method are compared to 3 other published methods: sedimentation, centrifugation, and Cornell. Sensitivity was lowest for the Cornell method precluding detection of some low shedders; however, contamination was not observed for this method. Contamination was the most severe in samples processed by the centrifugation method but was also high for the sedimentation method, resulting in unreadable culture tubes for some fecal samples. The NADC method was 10-fold more sensitive for detection of M. paratuberculosis colonies and contamination was significantly reduced compared to other 3 methods.

Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobacterium paratuberculosis and cell-wall proteins of M. paratuberculosis and were evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis (LMp) were inoculated intraperitoneally or intravenously at 10(9)/ml. Heat-killed M. paratuberculosis (HKMp) was prepared by treatment of bacteria at 85 C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. Antibody titers in the terminal blood sample were higher for HKMp and KLH-CWPMp than for LMp rabbits (1:1,024 vs. 1:64). The KLH-CWPMp antibody did not cross-react with M. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues was much higher for the KLH-CWPMp polyclonal antibody. Immunoreactivity of the antibody resulted in staining of bacteria in the cytoplasm of macrophages, mononuclear giant cells, and extracellular bacteria in both intestine and lymph node.

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne's disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratu-berculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for gamma-interferon (gamma-IFN) production. Cells from subclinical cows produced significantly higher levels of gamma-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of gamma-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of gamma-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that gamma-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

Pasteurella multocida toxin depresses weight gain in rats and pigs. It also affects tissues with rapidly dividing cells. In the present study, we investigated the role of this protein toxin on chondrocyte growth in vivo. Rats were divided into a single- or multiple-dose group and were given, respectively, either a single injection (0.15 or 0.6 micrograms/kg toxin subcutaneously) or multiple injections (0.01-0.2 micrograms/kg subcutaneously) of toxin. Bone (humerus) and other selected tissues were stained for bromodeoxyuridine immunoreactivity (BrDU-IR) in order to gauge cell proliferation. Physeal area was measured in rats from the multiple-dose group. Serum from single- and multiple-dose groups were tested for tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) activity using a bioassay system. Decreased weight gain, feed intake, and feed efficiency were observed in single- and multiple-dose groups of rats. Decreased BrDU-IR indices were present in the resting and proliferative zone chondrocytes of the humeral physis in rats from the multiple-dose group, as was decreased physeal area. Increased serum IL-6 bioactivity was present in rats after 24 hours, and no changes in TNF-alpha bioactivity were seen in any group. No alterations in BrDU-IR were seen in rats fed restricted (80% of control) diets. These studies show that sublethal doses of toxin decrease weight gain and affect growth of long bones through suppression of chondrocyte proliferation. These effects may be mediated by direct binding of the toxin to target cells or IL-6 but are not associated with altered feed intake or TNF-induced cachexia.