Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.

Detection of Mycobacterium bovis infection and production of interleukin-2 by in vitro stimulation of badger lymphocytes.

The Eurasian badger (Meles meles) is considered to be an important wildlife reservoir for Mycobacterium bovis infection of cattle in Ireland and in GB. However, rapid diagnosis of tuberculosis in live badgers has been constrained through a lack of suitable immuno-diagnostic reagents for detection of M. bovis-infected animals. To date, there have been no reports of cytokine activity in badgers that might be associated with specific immune responses to M. bovis infection. In this study, nine badgers were removed from an area with a persistent tuberculosis problem in cattle herds and tuberculosis was confirmed in four of the animals by "post-mortem" examination and M. bovis culture. In preliminary investigations of interleukin-2 (IL-2) activity, we were able to demonstrate that lymphoblasts prepared from badger peripheral blood mononuclear cells (PBMCs) proliferated when cultured in the presence of human recombinant IL-2 (HrIL-2). Supernatants derived from purified protein derivative of tuberculin (PPD-bovine) stimulated PBMC cultures also induced blastogenesis of badger-derived lymphoblasts. The results demonstrate that badger lymphocytes are responsive to HrIL-2 and that PPD-bovine stimulation of badger PBMC results in production of bio-active IL-2.

Immunological responses of Eurasian badgers (Meles meles) vaccinated with Mycobacterium bovis BCG (bacillus calmette guerin).

Wildlife species, such as the badger (Meles meles), may act as maintenance hosts for Mycobacterium bovis and contribute to the spread and persistence of tuberculosis in associated cattle populations. Targeted vaccination of badgers against tuberculosis is an option that, if successfully employed, could directly facilitate the advancement of bovine tuberculosis eradication in affected areas. In this study, the immunological responses of a group of badgers vaccinated subcutaneously with low doses of Mycobacterium bovis bacillus calmette guerin (BCG) were measured in vitro and compared with non-vaccinated control animals over a period of 42 weeks. Peripheral blood mononuclear cells (PBMC) from badgers which had received repeated booster injections of BCG proliferated in response to culture with PPD-bovine (purified protein derivative of tuberculin). The proliferation was significantly greater than that seen in the non-vaccinated control group. In contrast, the proliferative response of PBMC from vaccinated badgers to PPD-avian declined relative to the control group. These results demonstrate that repeated vaccination of badgers with M. bovis BCG induced a population of T-lymphocytes responsive to specific antigens in PPD-bovine. Throughout the course of the study, the sera from all animals were tested (BrockTest) by an enzyme-linked immunosorbent assay (ELISA) system for the presence of antibodies to MPB83, a serodominant antigen whose expression is high in M. bovis, but very low in BCG (Pasteur). No animals at any stage showed seroconversion to the antigen, consistent with the tuberculosis-free status of the badgers under study.

Pathophysiological role of nitric oxide in rat experimental colitis.

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of ulcerative colitis. A 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) colitis model was established to examine the effect of selective iNOS inhibition, by S-(2-aminoethyl) isothiouronium bromide (ITU), on colonic mucosal cell damage and inflammation. Rats, killed 7 days after TNBS, had increased colonic mucosal levels of iNOS and interleukin-8 (IL-8), in addition to severe colonic inflammation which was characterized by significantly increased colon weight, damage score and colonic myeloperoxidase activity (MPO) (a marker of neutrophil influx). TNBS-treated rats had markedly decreased body weight and thymus weight. Administration of colitic rats with ITU significantly inhibited iNOS activity/expression and tended to reduce mucosal levels of IL-8, but no effect on MPO activity was observed. Following ITU therapy, colitic rats had reduced colonic damage and losses in body weight and thymus weight were reversed. Improvement of TNBS colitis by ITU suggested that excess NO, produced by iNOS, may have contributed to the initiation/amplification of colonic disease, by mechanisms including enhancement of IL-8 release. NO-mediated enhancement of pro-inflammatory cytokine release was further investigated in vitro. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) stimulated release of nitrite, lactate dehydrogenase (LDH), TNF alpha, IL-1 beta and IL-8 from rat peritoneal macrophages, all of which were significantly reduced by ITU. This suggests that NO-mediated cell damage enhances pro-inflammatory mediator release from macrophages. In addition, enhancement of IL-8 and TNF alpha release was also partially NO-dependent in activated peritoneal neutrophils. Therefore, the amelioration of TNBS colitis by ITU could include inhibition of NO-mediated pro-inflammatory cytokine release.

alpha 1-Proteinase inhibitor, elastase activity, and lung disease severity in cystic fibrosis.

The potential role of neutrophil elastase in exacerbating pulmonary infection and tissue damage in cystic fibrosis (CF) has led to proposals for treatment of lung disease in CF with the elastase inhibitor, alpha 1-proteinase inhibitor (alpha 1PI). Reports that alpha 1PI is inactivated in the CF lung suggest that the effectiveness of alpha 1PI therapy depends on the quantity of elastase present and the extent of alpha 1PI inactivation, both of which are expected to vary with disease severity. In this study we assessed the elastase-alpha 1PI profile in sputum and plasma from CF patients with various degrees of pulmonary involvement. Levels of active elastase in sputum samples increased with severity of pulmonary disease (F ratio = 5.63, p < 0.01), as did sputum levels of alpha 1PI (F ratio = 4.88, p < 0.01). A positive correlation was observed between sputum levels of active elastase and alpha 1PI (r = 0.68, p < 0.005). Plasma alpha 1PI levels were also elevated in CF patients compared with control subjects (p < 0.005), indicating a compensatory increase in plasma and sputum levels of alpha 1PI in response to increased elastase load. Molar levels of total immunogenic neutrophil elastase were, on average, 12 times higher than alpha 1PI in CF sputum. These results suggest that the major contributor to the elevated levels of active elastase observed in the CF lung is an increase in elastase release rather than inactivation of alpha 1PI.