RESUMO
The diagnosis and control of Mycobacterium bovis infection (bovine tuberculosis: TB) continues to present huge challenges to the British cattle industry. A clearer understanding of the magnitude and duration of immune response to M. bovis infection in the European badger (Meles meles) - a wildlife maintenance host - may assist with the future development of diagnostic tests, and vaccination and disease management strategies. Here, we analyse 5280 diagnostic test results from 550 live wild badgers from a naturally-infected population to investigate whether one diagnostic test (a gamma interferon release [IFNγ] assay, n = 550 tests) could be used to predict future positive results on two other tests for the same disease (a serological test [n = 2342 tests] and mycobacterial culture [n = 2388 tests]) and hence act as an indicator of likely bacterial excretion or disease progression. Badgers with the highest IFNγ optical density (OD) values were most likely to subsequently test positive on both serological and culture tests, and this effect was detectable for up to 24 months after the IFNγ test. Furthermore, the higher the original IFNγ OD value, the greater the chance that a badger would subsequently test positive using serology. Relationships between IFNγ titres and mycobacterial culture results from different types of clinical sample suggest that the route of infection may affect the magnitude of immune response in badgers. These findings identify further value in the IFNγ test as a useful research tool, as it may help us to target studies at animals and groups that are most likely to succumb to more progressive disease.
Assuntos
Animais Selvagens/microbiologia , Testes de Liberação de Interferon-gama/veterinária , Mustelidae/microbiologia , Mycobacterium bovis , Tuberculose/veterinária , Animais , Progressão da Doença , Feminino , Masculino , Mustelidae/imunologia , Valor Preditivo dos Testes , Tuberculose/imunologia , Tuberculose/microbiologia , Reino UnidoRESUMO
Accurate detection of infection with Mycobacterium bovis in live badgers would enable targeted tuberculosis control. Practical challenges in sampling wild badger populations mean that diagnosis of infection at the group (rather than the individual) level is attractive. We modelled data spanning 7 years containing over 2000 sampling events from a population of wild badgers in southwest England to quantify the ability to correctly identify the infection status of badgers at the group level. We explored the effects of variations in: (1) trapping efficiency; (2) prevalence of M. bovis; (3) using three diagnostic tests singly and in combination with one another; and (4) the number of badgers required to test positive in order to classify groups as infected. No single test was able to reliably identify infected badger groups if 80% sensitive, at least 94% specific, and able to be performed rapidly in the field.
Assuntos
Testes Diagnósticos de Rotina/métodos , Mustelidae , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/veterinária , Medicina Veterinária/métodos , Animais , Inglaterra/epidemiologia , Feminino , Prevalência , Sensibilidade e Especificidade , Tuberculose/epidemiologiaRESUMO
BACKGROUND: Insufficient exposure and poor compliance with anti-tuberculosis (TB) medications are risk factors for treatment failure and the development of drug resistance. Measurement of drugs in biological samples, such as blood and saliva, can be used to assess adherence and make dose adjustments by therapeutic drug monitoring (TDM). Finger sweat testing is a convenient and non-invasive method to monitor patients. OBJECTIVES: To assess the feasibility of finger sweat testing for medication adherence and as a semi-quantitative tool for TDM analysis. METHODS: Ten patients provided finger sweat, blood and saliva samples following a controlled dose of isoniazid. Samples were analysed by liquid chromatography-mass spectrometry. RESULTS: Isoniazid can be detected in finger sweat 1-6 h following administration at typically prescribed dosages. The normalisation of isoniazid to creatinine increases the correlation between finger sweat and serum isoniazid concentration and provides a means to account for inconsistent sample volumes. CONCLUSION: We describe the time-course measurement of isoniazid (or drug-to-creatinine ratio) in finger sweat compared to the pharmacokinetic profile in blood for the first time. This technique, adaptable for other drugs, could reduce the burden on clinics and improve patient experience.
Assuntos
Antituberculosos , Creatinina , Monitoramento de Medicamentos , Isoniazida , Suor , Tuberculose , Humanos , Isoniazida/farmacocinética , Isoniazida/administração & dosagem , Suor/química , Antituberculosos/farmacocinética , Antituberculosos/administração & dosagem , Creatinina/sangue , Monitoramento de Medicamentos/métodos , Masculino , Feminino , Adulto , Tuberculose/tratamento farmacológico , Pessoa de Meia-Idade , Cromatografia Líquida/métodos , Espectrometria de Massas , Adesão à Medicação , Adulto Jovem , Saliva/químicaRESUMO
The behaviour of certain infected individuals within socially structured populations can have a disproportionately large effect on the spatio-temporal distribution of infection. Endemic infection with Mycobacterium bovis in European badgers (Meles meles) in Great Britain and Ireland is an important source of bovine tuberculosis in cattle. Here we quantify the risk of infection in badger cubs in a high-density wild badger population, in relation to the infection status of resident adults. Over a 24-year period, we observed variation in the risk of cub infection, with those born into groups with resident infectious breeding females being over four times as likely to be detected excreting M. bovis than cubs from groups where there was no evidence of infection in adults. We discuss how our findings relate to the persistence of infection at both social group and population level, and the potential implications for disease control strategies.
Assuntos
Transmissão Vertical de Doenças Infecciosas/veterinária , Mustelidae , Mycobacterium bovis , Tuberculose/veterinária , Animais , Inglaterra/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Liberação de Interferon-gama , Modelos Logísticos , Masculino , Mycobacterium bovis/isolamento & purificação , Densidade Demográfica , Risco , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/transmissãoRESUMO
We describe epidemiological trends in Mycobacterium bovis infection in an undisturbed wild badger (Meles meles) population. Data were derived from the capture, clinical sampling and serological testing of 1803 badgers over 9945 capture events spanning 24 years. Incidence and prevalence increased over time, exhibiting no simple relationship with host density. Potential explanations are presented for a marked increase in the frequency of positive serological test results. Transmission rates (R0) estimated from empirical data were consistent with modelled estimates and robust to changes in test sensitivity and the spatial extent of the population at risk. The risk of a positive culture or serological test result increased with badger age, and varied seasonally. Evidence consistent with progressive disease was found in cubs. This study demonstrates the value of long-term data and the repeated application of imperfect diagnostic tests as indices of infection to reveal epidemiological trends in M. bovis infection in badgers.
Assuntos
Mustelidae , Mycobacterium bovis , Tuberculose/veterinária , Animais , Inglaterra/epidemiologia , Feminino , Incidência , Masculino , Modelos Estatísticos , Mycobacterium bovis/isolamento & purificação , Densidade Demográfica , Vigilância da População , Prevalência , Risco , Análise Espacial , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/transmissãoRESUMO
OBJECTIVE: The objective of this study was to determine the repellency and efficacy of a 10% imidacloprid/4.5% flumethrin (Seresto® , Elanco) collar over an 8-month period against the eastern paralysis tick (Ixodes holocyclus) on cats. METHODS: Two non-blinded, open gender, randomised, placebo-controlled pen studies were conducted, with 26 cats enrolled in each study. Prior to inclusion, cats were immunised with I. holocyclus holocyclotoxin. Cats were treated on Day 0 with either an imidacloprid/flumethrin or placebo collar. Tick infestations with 20 unfed adult female eastern paralysis ticks commenced on Day 7, and were repeated monthly for 8 months. Repellency was determined by comparing the mean number of attached ticks on imidacloprid/flumethrin treated cats, to placebo collar treated cats at 6 and 24 h post infestation. Efficacy was determined by comparing the mean number of live ticks on imidacloprid/flumethrin collar treated cats to placebo collar treated cats at 72 h post infestation. RESULTS: Efficacy was 100% (P < 0.001) at 72 h, and repellency was greater than 96% (P < 0.001) at 24 h for every tick challenge in each of the two studies, from Day 7 to the final infestation at 8 months for imidacloprid/flumethrin collar treated cats. CONCLUSIONS: In two pen studies, an imidacloprid/flumethrin collar controlled and repelled the eastern paralysis tick (I. holocyclus) on cats for 8-months. The marked repellency effect in addition to controlling tick paralysis would be beneficial in preventing tick bites and their sequelae.
Assuntos
Doenças do Gato , Doenças do Cão , Ixodes , Infestações por Carrapato , Paralisia por Carrapato , Animais , Doenças do Gato/tratamento farmacológico , Doenças do Gato/prevenção & controle , Gatos , Doenças do Cão/tratamento farmacológico , Cães , Feminino , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neonicotinoides , Nitrocompostos , Paralisia/veterinária , Piretrinas , Infestações por Carrapato/tratamento farmacológico , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Paralisia por Carrapato/veterináriaRESUMO
Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.
Assuntos
Animais Selvagens/microbiologia , Mycobacterium bovis/isolamento & purificação , Testes Sorológicos/veterinária , Tuberculose Bovina/epidemiologia , Animais , Animais Selvagens/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Bovinos , Cervos/sangue , Cervos/microbiologia , Mustelidae/sangue , Mustelidae/microbiologia , Nova Zelândia/epidemiologia , Portugal/epidemiologia , Espanha/epidemiologia , Sus scrofa/sangue , Sus scrofa/microbiologia , Trichosurus/sangue , Trichosurus/microbiologia , Tuberculose Bovina/sangue , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis (M. bovis) in Great Britain (GB) and the Republic of Ireland and therefore constitute a potential source of infection for cattle. Reduction of badger densities in the Republic of Ireland has resulted in an associated reduction in the risk of a herd break-down with bovine tuberculosis and a study to determine whether this is also the case in GB has been running since 1997. If badgers are a significant source of M. bovis infection for cattle, vaccinating badgers with Bacillus Calmette-Guérin (BCG) might prove to be a long term, cost-effective strategy for controlling bovine tuberculosis whilst preserving badger populations. As a first step towards BCG vaccination of wild badgers, it was necessary to demonstrate safety of the vaccine in captive badgers. Therefore, captive badgers were vaccinated with a commercial source of BCG that is already licensed for administration to humans in GB-BCG Danish SSI. Using a protocol prescribed by the Veterinary Medicines Directorate (VMD) of GB, badgers were vaccinated with two consecutive doses of BCG via either the subcutaneous (s.c.) or intra-muscular (i.m.) routes. The first dose was high, ranging from 16 to 22 x 10(7) colony-forming units (CFU), and was followed 15 weeks later by a lower dose in the range of 4-7 x 10(5)CFU. Local reaction at the site of injection and general responses (body temperature, haematology and blood serum chemistry), behaviour and excretion of BCG were monitored for 28 weeks from the time of the first vaccination. The only side-effect observed was the occurrence of localised swelling at the site of BCG injection that disappeared 48 days after i.m. vaccination but persisted longer in the group vaccinated by the s.c. route. Immunological responses were measured at regular intervals. Strong cellular responses were observed 13 days after the first vaccination, which persisted for 76 days. The lower dose induced a weaker and shorter-lived response.
Assuntos
Vacina BCG/farmacologia , Mustelidae/imunologia , Animais , Vacina BCG/efeitos adversos , Vacina BCG/imunologia , Comportamento Animal , Temperatura Corporal , Peso Corporal , Bovinos , Reservatórios de Doenças/microbiologia , Feminino , Masculino , Mustelidae/sangue , Mustelidae/microbiologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Segurança , Tuberculose Bovina/prevenção & controle , Tuberculose Bovina/transmissãoRESUMO
In-bred strains of mice are commonly used to model pathogenic infections due to their cost and utility. In order to understand better the nature of experimental tuberculosis in mice, we infected BALB/c mice with a virulent field isolate of Mycobacterium bovis. Mice were sacrificed at intervals in order to visualise the pathological lesions in major internal organs. Pathological lesions in tissues increased in number and severity over time and replicated many of the salient features observed in badgers and cattle infected with M. bovis. These similarities are discussed. Examination of pathological lesions at terminal stages of infection enabled us to suggest the lethal effects of M. bovis mediated through the host response. We conclude that the mouse is a relevant surrogate species in which to study the virulence of M. bovis, as well as the influence of vaccination on its pathogenicity.
Assuntos
Mycobacterium bovis/fisiologia , Tuberculose/microbiologia , Tuberculose/patologia , Animais , Modelos Animais de Doenças , Coração/microbiologia , Rim/microbiologia , Rim/patologia , Fígado/microbiologia , Fígado/patologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/patogenicidade , Miocárdio/patologia , Baço/microbiologia , Baço/patologiaRESUMO
The sharp rise of bovine tuberculosis (TB) in Great Britain and the continuing problem of wild life reservoirs in countries such as New Zealand and Great Britain have resulted in increased research efforts into the disease. Two of the goals of this research are to develop (1) cattle vaccines against TB and (2) associated diagnostic reagents that can differentiate between vaccinated and infected animals (differential diagnosis). This review summarises recent progress and describes efforts to increase the protective efficacy of the only potential TB vaccine currently available, Mycobacterium bovis BCG, and to develop specific reagents for differential diagnosis. Vaccination strategies based on DNA or protein subunit vaccination, vaccination with live viral vectors as well as heterologous prime-boost scenarios are discussed. In addition, we outline results from studies aimed at developing diagnostic reagents to allow the distinction of vaccinated from infected animals, for example antigens that are not expressed by vaccines like Mycobacterium bovis Bacille-Calmette-Guérin, but recognised strongly in Mycobacterium bovis infected cattle.
Assuntos
Imunização/veterinária , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose Bovina/imunologia , Tuberculose Bovina/prevenção & controle , Animais , Bovinos , Diagnóstico Diferencial , Imunização/métodos , Tuberculose Bovina/diagnóstico , Vacinas Atenuadas/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Today it is generally accepted that the Bacillus Calmette-Guerin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan-tetanus toxoid conjugate (AM-TT), given intransally in a novel Eurocine adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM-TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM-TT vaccines compared to the non-boosted BCG-vaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM-TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM-TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination.
Assuntos
Mananas/administração & dosagem , Toxoide Tetânico/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Administração Intranasal , Animais , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Contagem de Colônia Microbiana/métodos , Feminino , Granuloma/imunologia , Granuloma/patologia , Interferon gama/imunologia , Pulmão/microbiologia , Pulmão/patologia , Mananas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos Bacterianos/administração & dosagem , Polissacarídeos Bacterianos/imunologia , Baço/microbiologia , Toxoide Tetânico/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Vacinação/métodos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
Expression of the interleukin-2 receptor alpha chain (IL-2R alpha) by peripheral blood mononuclear cells (PBMC) from Holstein calves, both experimentally-infected with bovine herpesvirus-l (BHV-l) and controls, was measured by flow cytometry. Expression of IL-2R alpha was 35 percent and 23 percent higher in infected calves than controls, on days 2 and 3 postinfection (PI), respectively. Concurrent with this increase in IL-2R alpha expression, a significant decrease (P < 0.001) was observed in the PHA-induced proliferative responses of PBMC from infected compared with control calves. In vitro treatment with recombinant human (rhu) IL-12 enhanced PHA-induced proliferative responses of PBMC from both infected and control calves. This rhuIL-12 enhancement of mitogen-induced proliferative responses was significant (P < 0.001) in infected calves on day 2 PI and was sufficient to abrogate the decrease observed due to BHV-1 infection. Since the expression of the beta and gamma chains of IL-2R was not measured it is difficult to speculate as to the status of high affinity receptor expression during BHV-1 infection. However results of the present study suggest that the decrease in proliferative responses observed during infection may not be due to a decrease in IL-2R alpha expression but may possibly be due to a selective down-regulation of signal transduction through IL-2R and/or by modulation of the expression of other cytokines involved in lymphocyte activation and proliferation.
Assuntos
Doenças dos Bovinos , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Interleucina-12/farmacologia , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/virologia , Receptores de Interleucina-2/biossíntese , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Infecções por Herpesviridae/imunologia , Humanos , Linfócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Valores de ReferênciaRESUMO
In the UK there has been a sharp rise in the incidence of bovine tuberculosis since the early 1990s and the badger has been identified as an important wildlife reservoir for this infection. Infected badgers can excrete Mycobacterium bovis, putting other badgers and cattle at risk of becoming infected. Vaccination has been proposed as an approach to reducing the excretion of M. bovis by tuberculous badgers. In order to evaluate the efficacy of a badger vaccine it will be necessary to accurately determine the number of badgers excreting M. bovis without removing them for post-mortem evaluation. The existing live tests for tuberculosis in the badger (culture, indirect ELISA, Western blot) have not been assessed for their ability to detect badgers excreting M. bovis. Over the past 18 years, badgers from 31 social groups have been trapped and sampled in a study area of the Cotswold escarpment. We have examined the serological responses of 128 badgers trapped between 1985 and 1998 from social groups where M. bovis infection was endemic. These responses were compared with culture from faeces, urine, tracheal aspirates and bite wound swabs taken from these animals while alive. ELISA was found to be more sensitive than Western blot in detecting badgers excreting M. bovis. The majority of culture-positive badgers excreted M. bovis intermittently over the period of study. As a result, there was only a 27.5% chance of sampling a badger for culture when it was excreting M. bovis. In contrast, a positive ELISA result correctly predicted 68.2% of badgers with a history of excreting M. bovis. In the absence of alternative live tests for the badger, the Brock Test indirect ELISA appears to be more valuable than culture for measuring the effect of vaccination on reducing the number of badgers at risk of transmitting tuberculosis.
Assuntos
Western Blotting/veterinária , Carnívoros , Reservatórios de Doenças/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Mycobacterium bovis/imunologia , Tuberculose/veterinária , Animais , Western Blotting/métodos , Carnívoros/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/microbiologia , Mycobacterium bovis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologia , Reino Unido/epidemiologia , Urina/microbiologiaRESUMO
In order to develop a model of Mycobacterium bovis infection with pathogenetical relevance, a modified version of the Henderson apparatus was used to deliver infectious aerosols directly to the snouts of guinea pigs. Aerosols generated from 10(6), 10(7), 10(8)CFU/ml M. bovis suspensions established disease in every animal, with estimated retained doses of 10, 100, 1000 CFU, respectively. For comparison, other guinea pigs were inoculated with 100 CFU M. bovis intramuscularly (i.m.). Pathology and bacterial colonisation of lungs and spleen varied according to the dose and route of inoculation. Animals inoculated i.m. gave a significant cutaneous tuberculin hypersensitivity reaction earlier after testing than those infected aerogenically. A serological response to M. bovis antigens was detected in all infected animals. Intensity of antigen recognition was dose-dependent and although the range of antigens recognised varied between animals, a 25 kDa antigen present in the cell fraction was serodominant. Thus, a reproducible guinea pig model has been defined that may be suitable for virulence, vaccination, and immunological studies.
Assuntos
Modelos Animais de Doenças , Mycobacterium bovis , Tuberculose Bovina/microbiologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Vacina BCG/imunologia , Western Blotting/veterinária , Bovinos , Feminino , Cobaias , Hipersensibilidade Tardia/microbiologia , Hipersensibilidade Tardia/veterinária , Injeções Intramusculares/veterinária , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Testes Cutâneos/veterinária , Baço/microbiologia , Tuberculose Bovina/imunologiaRESUMO
Attempts to eradicate tuberculosis from cattle and farmed deer in some countries have been frustrated by the existence of wildlife reservoirs of Mycobacterium bovis infection. Possum control programmes in New Zealand using poisons have shown clearly that the brushtail possum is an important source of infection for cattle and farmed deer, and the sum of evidence strongly suggests that badgers serve as a source of infection for cattle in the UK. Bovine tuberculosis can only be eradicated from these countries by controlling M. bovis infection in both wildlife and domestic animals. The most promising options for control of M. bovis infection in wildlife in the longer term include the development of a tuberculosis vaccine for wildlife and a strategy for biological control of possums. The aim of this review is to address the problems and approaches involved in the control of wildlife tuberculosis from an immunological perspective.
Assuntos
Vacina BCG/imunologia , Reservatórios de Doenças/veterinária , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Vacinação/veterinária , Animais , Vacina BCG/normas , Carnívoros , Bovinos , Cervos , Mycobacterium bovis/patogenicidade , Nova Zelândia/epidemiologia , Gambás , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/prevenção & controle , Reino Unido/epidemiologia , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/normasRESUMO
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.
Assuntos
Carnívoros/imunologia , Carnívoros/microbiologia , Reservatórios de Doenças/veterinária , Interferon gama/genética , Linfócitos/imunologia , Mycobacterium bovis/isolamento & purificação , Tuberculose Bovina/diagnóstico , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Carnívoros/sangue , Carnívoros/genética , Bovinos , Clonagem Molecular , Cães , Citometria de Fluxo , Interferon gama/biossíntese , Interferon gama/química , Dados de Sequência Molecular , RNA/química , RNA/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Tuberculose Bovina/microbiologia , Vacinas de DNA/normasRESUMO
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.
Assuntos
Antígenos de Bactérias , Carnívoros , Ativação Linfocitária , Proteínas de Membrana , Mycobacterium bovis/isolamento & purificação , Tuberculose/veterinária , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/imunologia , Bovinos , Reservatórios de Doenças , Ensaio de Imunoadsorção Enzimática/veterinária , Camundongos , Kit de Reagentes para Diagnóstico , Tuberculose/diagnóstico , Tuberculose Bovina/epidemiologia , Reino Unido/epidemiologiaRESUMO
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.
Assuntos
Vacina BCG/imunologia , Carnívoros/imunologia , Mycobacterium bovis/imunologia , Tuberculose/veterinária , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Ativação Linfocitária/imunologia , Masculino , Linfócitos T/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinação/veterináriaRESUMO
Holstein calves given three consecutive i.m. injections of dexamethasone (DEX) (0.04 mg kg-1) showed lymphopenia and neutrophilia with increased numbers of mature neutrophils on post-injection Days 1 and 2, but these values returned to normal levels by post-injection Day 3. Interleukin-2 receptor (IL-2R) expression by peripheral blood mononuclear cells (PBMC) was evaluated by flow cytometry using a monoclonal antibody specific for bovine IL-2R alpha. Treatment with DEX significantly decreased expression of IL-2R alpha in Concanavalin A (Con A)-activated PBMC on Day 1 (P < 0.02) and on Day 2 (P < 0.1). On Day 3, expression of IL-2R alpha by PBMC was similar in control and DEX-treated calves. This decrease in IL-2R alpha expression correlated with decreased proliferative responses of PBMC to the T-cell mitogens, phytohemagglutinin (PHA) and Con A. Following in vitro treatment with recombinant human (rhu) interleukin-12 (IL-12) Con A-induced proliferative responses of PBMC tended to be higher in both groups. However, the rhu IL-12 induced increase of Con A activated proliferative responses were significantly greater in DEX-treated calves than in control calves. IL-2R alpha expression by PBMC was found to be less in calves transported 800 km in a truck as compared to that in PBMC from controls. These data suggest that stress-induced immunosuppression in calves may involve decreased IL-2R alpha expression and decreased IL-12 production. Serum chemistry results indicated a trend toward higher creatine kinase (CK) levels in DEX-treated calves. This may be due to the lysis of corticosteroid sensitive lymphocytes.
Assuntos
Doenças dos Bovinos/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Interleucina-2/biossíntese , Estresse Fisiológico/veterinária , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais , Bovinos , Creatina Quinase/sangue , Dexametasona/farmacologia , Citometria de Fluxo/veterinária , Injeções Intramusculares/veterinária , Interleucina-12/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfopenia/imunologia , Linfopenia/veterinária , Mitógenos , Neutrófilos/imunologia , Estresse Fisiológico/imunologiaRESUMO
An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.