Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis.

BACKGROUND: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease - Johne's disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups - uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). RESULTS: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. CONCLUSIONS: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer.

Development of the thymus and Peyer's patches in the first year of life in red deer (Cervus elaphus).

In young ruminants, as in most animals, the thymus and Peyer's patches (PP) play a key role in immune function. In sheep and cattle it has been shown that they follow a recognisable pattern of development throughout early life but a study of these tissues in red deer (Cervus elaphus) had not been previously conducted. Red deer are undergoing increasing domestication thus it was important to determine whether there were any significant differences between species. Samples from the thymus, jejunal PP and ileal PP were obtained from deer at 1, 1.5, 2, 3, 6, 9 and 12 months of age. Tissues were measured, processed and comparisons between the equivalent tissues in cattle and sheep were made. The thymus and Peyer's patches of red deer were observed to follow similar patterns of development and involution as in other ruminant species. The thymus was also observed to show a seasonal decrease in weight and cellular composition possibly due to nutritional and environmental stresses. Moreover, evidence for the prevailing idea that the ileal PP acts as primary lymphoid tissue in ruminants was obtained.

Peripheral blood mononuclear cell population changes associated with age and Mycobacterium avium subspecies paratuberculosis infection in red deer (Cervus elaphus).

In this study we aimed to document the maturation of the immune system in red deer and investigate the hypothesis that an immature immune system may predispose young red deer (Cervus elaphus) to an increased susceptibility to Johne's disease. Peripheral blood mononuclear cell populations were analysed using flow cytometry techniques to monitor changes associated with age and severity of infection with Mycobacterium avium subspecies paratuberculosis in red deer. The percentage of cells expressing cell population markers CD4, CD8, WC1, gammadelta TCR, CD14 and B-B4 as well as the cell activation markers CD25, CD44, ACT1 and ACT31 were analysed in relation to age and M. avium subsp. paratuberculosis infection status and disease severity. Significant changes in both cell surface markers and cell activation markers were observed as animals matured irrespective of their response to infection. The levels of CD4 and CD8 increased with age and the levels of B-B4, WC1, CD14 and gammadelta TCR decreased similar to previous studies in cattle. No differences were observed in cell surface markers or cell activation markers in relation to M. avium subsp. paratuberculosis infection status and disease severity. This research demonstrates that peripheral blood cell populations change with age in red deer and suggests that studies of cell surface markers and activation markers in peripheral blood samples do not provide information on the age-related susceptibility to Johne's disease.

Sensitivity, specificity, and confounding factors of novel serological tests used for the rapid diagnosis of bovine tuberculosis in farmed red deer (Cervus elaphus).

In this study, novel serological tests were used to detect tuberculosis (TB) in groups of farmed red deer (Cervus elaphus) varying in disease status or possible confounding factors. Groups of deer naturally or experimentally infected with Mycobacterium bovis and animals vaccinated against paratuberculosis were studied, as were uninfected animals and animals naturally or experimentally infected with Mycobacterium avium subsp. paratuberculosis. Sera were assayed using two rapid lateral-flow tests, Chembio's CervidTB STAT-PAK and DPP VetTB tests, and results were compared to those from tuberculin skin tests. Both serological tests had a high sensitivity, but specificity was adversely affected after animals had received a vaccine against paratuberculosis and were subsequently skin tested. The specificity of the DPP VetTB test was higher than that of the CervidTB STAT-PAK test, with natural infection with M. avium subsp. paratuberculosis adversely affecting the specificity of only the CervidTB STAT-PAK test. The sera from M. avium subsp. paratuberculosis-infected deer that produced false-positive reactions in the CervidTB STAT-PAK test were retested with a multiantigen print immunoassay (MAPIA), and some of these sera were shown to react with the MPB83 antigen. Combining the results from the serological tests and the skin tests showed only a slight increase in the sensitivity of detection of M. bovis-infected animals. It is concluded that both the CervidTB STAT-PAK and DPP VetTB tests offer rapid, convenient, and easy detection of bovine tuberculosis in deer, albeit with significant interference from paratuberculosis vaccination status and subsequent skin testing. The latter finding illustrates one of the limitations of currently available vaccines against paratuberculosis.

Efficacy of novel lipid-formulated whole bacterial cell vaccines against Mycobacterium avium subsp. paratuberculosis in sheep.

Mycobacterium avium subsp. paratuberculosis [MAP], the causative agent of enteric Johne's disease, incurs significant economic losses to the livestock industry. Prophylactic vaccination can be employed as a control means, however mineral oil-based vaccines currently in practice have limited efficacy, produce strong antibody responses that confound serological diagnostic testing, and cause severe injection site reactions. In the present study, the safety and efficacy of a commercial mineral oil-adjuvanted vaccine (Gudair) was compared with novel parenteral-route vaccines in sheep; these comprised live or heat-killed (HK) whole cell preparations of MAP strain 316F, formulated into a food-grade lipid vaccine delivery matrix. Subcutaneous administration of lipid-formulated live or HK 316F-induced significantly fewer adverse injection site reactions than Gudair; adverse injection site reactions were eliminated altogether by intraperitoneal (i.p.) injection of lipid-formulated live 316F. Injections of lipid-formulated 316F-induced significant peripheral blood cell-mediated immune (CMI) responses in the absence of antibody, while Gudair-induced strong antibody and CMI reactivity. Vaccinated and non-vaccinated control sheep were challenged via oral inoculation of a virulent MAP isolate, and disease progress was monitored for 16 months, followed by necropsy. All vaccine regimes reduced the overall pathological grading of biopsied intestinal tract (IT) tissues; among these, only Gudair promoted a significant reduction in the incidence of histopathological IT lesions, while only i.p. injection of lipid-formulated live 316F significantly reduced the incidence of gross IT lesions. All lipid-formulated vaccines (but not Gudair) significantly reduced the incidence of bacteriological culture-confirmed MAP infection. This study identifies a new vaccination strategy against Johne's disease in sheep using conventional MAP vaccine strains formulated in a metabolisable lipid delivery matrix.

Identification of key tissue type for antler regeneration through pedicle periosteum deletion.

Epimorphic regeneration is the "holy grail" of regenerative medicine. Research aimed at investigating the various models of epimorphic regeneration is essential if a fundamental understanding of the factors underpinning this process are to be established. Deer antlers are the only mammalian appendages that are subject to an annual cycle of epimorphic regeneration. In our previous studies, we have reported that histogenesis of antler regeneration relies on cells resident within the pedicle periosteum (PP). The present study elaborates this finding by means of functional studies involving the deletion of PP. Four yearling and four 2-year-old stags were selected for total PP deletion or partial PP deletion experiments. Of the animals in the total PP deletion group, one showed no signs of antler regeneration throughout the antler growth season. Two showed substantial and one showed marginal delays in antler regeneration (at 34, 20 and 7 days, respectively) compared with the corresponding sham-operated sides. Histological investigation revealed that the delayed antlers were derived from regenerated PP. Unexpectedly, the regenerative capacity of the antler from the total periosteum-deleted pedicles depended on antler length at surgery. Of the four deer that had partial PP deletion, two regenerated antlers exclusively from the left-over PP on the pedicle shafts in the absence of participation from the pedicle bone proper. The combined results from the PP deletion experiments convincingly demonstrate that the cells of the PP are responsible for antler regeneration.