Cloning and sequencing of badger (Meles meles) interferon gamma and its detection in badger lymphocytes

The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 × 10⁴ colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 × 10⁴ colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.     

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian badger (Meles meles).

The Eurasian badger (Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.     

Evidence of canine distemper and suggestion of preceding parvovirus-myocarditis in a Eurasian badger (Meles meles).

An approximately 1.5-yr-old free-ranging male Eurasian badger (Meles meles) from the eastern part of Austria had macroscopic and microscopic lesions consistent with canine distemper virus infection, including nonsuppurative meningoencephalitis, interstitial pneumonia with accumulation of macrophages in alveoli that contained intranuclear inclusion bodies, vesicular exanthema of the ventral abdomen, and atrophy of lymphoid tissues. Canine distemper virus-antigen was demonstrable in a variety of organs by using immunohistology. In addition, there were widespread areas of fibrosis in the myocardium that were rich in collagen and paucicellular. Because such changes are comparable with sequelae of the acute cardiac form of canine parvovirus (CPV) infection in dogs, it was speculated that this badger may have experienced CPV myocarditis as a cub but that the corresponding antigen or DNA was not detectable due to resolution of the disease.     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

Experimental infection of dogs with Brucella abortus

Thirteen female dogs, which included eight principals that were fed approximately 4.4 X 10(10) colony forming units (cfu) of Brucella abortus strain 2308 and five sentinels that were housed with the principals, were examined for serologic responses, blood culture, tissue distribution of the organisms and pathologic lesions. Serum samples from each dog were tested on the day of exposure and on post exposure days 5, 7, 10, 14, 21, 28, 35, 42 and 49 for antibodies to B. abortus, using the brucellosis card (BC), standard tube agglutination (STA), 2-mercaptoethanol (ME) and rivanol (RIV) tests. Antibodies were detected in the principals by day 5 and increased through day 21. The STA test was the first to become positive, followed by the BC, ME and RIV tests. After 28 days, the serologic titers receded. From day 14 through day 42, all principals had greater than or equal to 1:50 STA titers. On day 49, seven principals had greater than or equal to 1:50 STA titers and one had a 1:25 STA titer. The sentinels were negative for all tests, except sentinel number 9 which had STA titers ranging from 1:25 to 1:50 on day 14 through day 35. Blood cultures that were obtained from each principal at intervals from one hour after exposure through 49 days were negative. Brucella abortus was isolated from various lymph nodes of the eight principals and from sentinel number 9, which was apparently infected by ingesting brucellae contaminated feces from the principals. Microscopic lesions were not observed in the culture-positive tissues examined.     

Experimental infection of sheep with Brucella abortus

A case of brodifacoum poisoning is described in a six-year-old male Kelpie cross working dog. The clinical features were severe exercise intolerance, haemorrhage from the oral and nasal cavities, dyspnoea and pale mucous membranes. Diagnosis was confirmed by demonstrating an abnormally long whole blood clotting time. The dog was treated successfully by administering 1 litre of whole blood intravenously, intramuscular vitamin K₁ and a three week course of oral vitamin K₃.

Experience at the Massey University Small Animal Clinic and Hospital has indicated that poisoning of dogs with the newer long acting anticoagulant rodenticides is becoming more common.     

Resistance of preimplantation bovine embryos to infection with Brucella abortus

Preimplantation bovine embryos were exposed in vitro to Brucella abortus to determine if the bacteria would adhere to zona pellucida (ZP)-intact embryos or adhere to or infect ZP-free embryos. Brucella abortus was not isolated from ZP-intact or ZP-free groups of embryos after 10 sequential antibiotic-free washings. Brucella abortus was isolated from all groups containing ZP-defective embryos after the exposure period and washing. Detrimental effects on healthy in vitro development of embryos were not observed.     

Immunological responses following experimental endobronchial infection of badgers (Meles meles) with different doses of Mycobacterium bovis

The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.     

狗獾的人工养殖技术

狗獾（Ｍｅｌｅｓ　ｍｅｌｅｓ）是一种皮、毛、肉、药兼用的野生经济动物。獾皮可供制裘、制革，毛可制高级胡刷和油画笔，肉可供食用，油脂入药能医治各种烫伤。过去因其对农业生产有害，大肆捕杀，而导致其种质资源衰退，数量锐减，人工养殖势在必行。１狗獾的形态特征狗獾身体肥胖、头扁、鼻端尖、耳短、眼小、颈短粗、尾短。背部被毛粗硬而致密，背毛基部呈灰白色，毛尖白色，中段为黑褐色。体侧白色被毛较多。头部有三条白色纵纹，颜面两侧由嘴经耳基部至头后各有一条，中间一条由鼻尖至额部。耳缘呈白色。下颌、喉、腹部和四肢均为棕…