Eight-month repellency and efficacy of an imidacloprid/flumethrin (Seresto® ) collar against the eastern paralysis tick (Ixodes holocyclus) on cats.

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.

Quantitative interferon-gamma responses predict future disease progression in badgers naturally infected with Mycobacterium bovis.

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.

Diagnosis of tuberculosis in groups of badgers: an exploration of the impact of trapping efficiency, infection prevalence and the use of multiple tests.

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.

Vaccination against tuberculosis in badgers and cattle: an overview of the challenges, developments and current research priorities in Great Britain.

Bovine tuberculosis (TB) is a significant threat to the cattle industry in England and Wales. It is widely acknowledged that a combination of measures targeting both cattle and wildlife will be required to eradicate bovine TB or reduce its prevalence until European official freedom status is achieved. Vaccination of cattle and/or badgers could contribute to bovine TB control in Great Britain, although there are significant gaps in our knowledge regarding the impact that vaccination would actually have on bovine TB incidence. Laboratory studies have demonstrated that vaccination with BCG can reduce the progression and severity of TB in both badgers and cattle. This is encouraging in terms of the prospect of a sustained vaccination programme achieving reductions in disease prevalence; however, developing vaccines for tackling the problem of bovine TB is challenging, time-consuming and resource-intensive, as this review article sets out to explain.

Review of the diagnosis of tuberculosis in non-bovid wildlife species using immunological methods--an update of published work since 2009.

The infection of both captive and free-ranging wildlife species with pathogenic mycobacteria (including Mycobacterium tuberculosis) poses a zoonotic risk and continues to cause challenges for the livestock industry, zoos and governments around the world. Central to the management and control of tuberculosis is timely and accurate diagnosis. In many cases, bacterial culture is insufficiently sensitive and confirmation of TB post-mortem is neither feasible nor desirable. In this context, there is still considerable research interest in, and need for, immunological methods for diagnosis. Reviews on this topic were published in 2005 and 2009, but since then veterinarians and other researchers have continued to evaluate immunodiagnostic approaches to TB. These include serological tests such as lateral-flow devices, and enzyme-linked immunosorbent assay (ELISA) and those based on evaluation of cell-mediated immunity, such as the tuberculin skin test and interferon-gamma release assay (IGRA). Since 2009, the range of publications on this topic has been extended to a number of new species, including South American camelids, black rhinoceros, lions and non-human primates. Therefore, it seemed appropriate to review the literature in the 3 years since 2009 and provide an overview of progress.

Sex-related heterogeneity in the life-history correlates of Mycobacterium bovis infection in European badgers (Meles meles).

Heterogeneity in the progression of disease amongst individual wild animals may impact on both pathogen and host dynamics at the population level, through differential effects on transmission, mortality and reproductive output. The role of the European badger (Meles meles) as a reservoir host for Mycobacterium bovis infection in the UK and Ireland has been the focus of intense research for many years. Here, we investigate life-history correlates of infection in a high-density undisturbed badger population naturally infected with M. bovis. We found no evidence of a significant impact of M. bovis infection on female reproductive activity or success, with evidence of reproduction continuing successfully for several years in the face of M. bovis excretion. We also found evidence to support the hypothesis that female badgers are more resilient to established M. bovis infection than male badgers, with longer survival times following the detection of bacterial excretion. We discuss the importance of infectious breeding females in the persistence of M. bovis in badger populations, and how our findings in male badgers are consistent with testosterone-induced immunosuppression. In addition, we found significant weight loss in badgers with evidence of disseminated infection, based on the culture of M. bovis from body systems other than the respiratory tract. For females, there was a gradual loss of weight as infection progressed, whereas males only experienced substantial weight loss when infection had progressed to the point of dissemination. We discuss how these differences may be explained in terms of resource allocation and physiological trade-offs.

Insulin stimulation regulates AS160 and TBC1D1 phosphorylation sites in human skeletal muscle.

INTRODUCTION: Individuals with obesity and type 2 diabetes (T2D) are typically insulin resistant, exhibiting impaired skeletal muscle glucose uptake. Animal and cell culture experiments have shown that site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 and TBC1D1 is critical for GLUT4 translocation facilitating glucose uptake, but their regulation in human skeletal muscle is not well understood. METHODS: Here, lean, obese and T2D subjects underwent a euglycemic-hyperinsulinemic clamp, and vastus lateralis muscle biopsies were obtained before, and at 30 and 180 min post insulin infusion. RESULTS: Obese and T2D subjects had higher body mass indexes and fasting insulin concentrations, and T2D subjects showed insulin resistance. Consistent with the clamp findings, T2D subjects had impaired insulin-stimulated phosphorylation of AS160 Thr(642), a site previously shown to be important in glucose uptake in rodents. Interestingly, insulin-stimulated phosphorylation of TBC1D1 Thr(590), a site shown to be regulated by insulin in rodents, was only increased in T2D subjects, although the functional significance of this difference is unknown. CONCLUSION: These data show that insulin differentially regulates AS160 and TBC1D1 phosphorylation in human skeletal muscle. Impaired insulin-stimulated glucose uptake in T2D subjects is accompanied by dysregulation of AS160 and TBC1D1 phosphorylation in skeletal muscle, suggesting that these proteins may regulate glucose uptake in humans.

Heterogeneity in the risk of Mycobacterium bovis infection in European badger (Meles meles) cubs.

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.

Long-term temporal trends and estimated transmission rates for Mycobacterium bovis infection in an undisturbed high-density badger (Meles meles) population.

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.

Pelioid hepatocellular carcinoma in an adult Eurasian badger (Meles meles).

A mass was identified within the left lateral lobe of the liver of a 10-year-old Eurasian badger (Meles meles). The mass was friable and multilobulated, with blood-filled spaces between the lobules. Microscopically, the lesion consisted of sheets and trabeculae of neoplastic hepatocytes often forming cystic spaces containing erythrocytes, fibrin and necrotic debris. The histological appearance was consistent with hepatocellular carcinoma (HCC). Immunohistochemically, the neoplastic cells expressed cytokeratin 18 but not von Willebrand factor. Multiple intranuclear (amphophilic or acidophilic) inclusion bodies were observed in hepatocytes at the junction between the tumour and normal hepatic tissue. HCCs have also been reported in other domestic and wild animals. As hepadnavirus infection has been associated with HCC in woodchucks, further histochemical and transmission electron microscopical studies were performed; however, these demonstrated that the inclusions consisted of lipid droplets and not viral particles. To our knowledge, this is the first report of a naturally occurring HCC in a Eurasian badger.

Evaluation of the CervidTB STAT-PAK for the detection of Mycobacterium bovis infection in wild deer in Great Britain.

Deer are acknowledged as hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), and determining the prevalence of infection in deer species is one of the key steps in understanding the epidemiological role played by cervids in the transmission and maintenance of bTB in the United Kingdom. This study evaluated a rapid lateral-flow test for the detection of bTB in samples from wild deer species in the United Kingdom. Fallow deer (Dama dama), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) from areas in Wales, the Cotswolds, and southwestern England were necropsied for a bTB survey. Serum samples from individual deer were tested with the CervidTB STAT-PAK, and the results were evaluated against the culture of M. bovis from tissues (n = 432). Sensitivity and specificity were 85.7% (95% confidence interval [CI], 42.1 to 99.6%) and 94.8% (95% CI, 92.3 to 96.7%), respectively, with an odds ratio of 109.9 (95% CI, 12.7 to 953.6%) for a positive STAT-PAK result among culture-positive deer. The low prevalence of infection (3.8%, n = 860) affected the confidence of the sensitivity estimate of the test, but all culture-positive fallow deer (n = 6) were detected by the test. In addition, antibodies to M. bovis could be detected in poor-quality serum samples. The results suggest that the CervidTB STAT-PAK could be deployed as a field test for further evaluation.

Review of the diagnosis and study of tuberculosis in non-bovine wildlife species using immunological methods.

The infection of a variety of free-ranging wildlife species with Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), can cause problems for biodiversity and species conservation. In some notable cases, particular species act as a reservoir of infection that can spill over into domestic livestock with economic and zoonotic consequences. Immunological methods for the detection of TB infection in wildlife are important for diagnostic and research purposes, especially where post-mortem examination is neither feasible nor desirable. In this review, the approaches taken to the immunological study of TB in wildlife species are summarized, with particular emphasis on their suitability for different applications and their applicability to different species. Different approaches to improving diagnostic sensitivity are discussed together with factors that can confound the use of tests in certain situations. Caution in the interpretation of test results for TB in wildlife is encouraged, especially where it has not been possible to confirm the accuracy of the test.

A review of infection of wildlife hosts with Mycobacterium bovis and the diagnostic difficulties of the 'no visible lesion' presentation.

The pathology, frequency and diagnostic implications of 'no visible lesion' (NVL) tuberculosis (Tb), i.e. infection with Mycobacterium bovis in the absence of macroscopic lesions, are described in a wide taxonomic range of wildlife hosts. Information collected and evaluated on the definition and occurrence of NVL Tb, histopathological characteristics, post-mortem techniques to detect minimal lesions, and diagnostic difficulties revealed most Tb-infected individuals with NVL had minute tuberculous lesions, which were difficult to see by eye. Acid-fast organisms (AFO) were sometimes detected in the lesions. Ideally, mycobacterial culture of pools of lymph nodes and/or oropharyngeal tonsils is necessary for the accurate diagnosis of Tb in the absence of macroscopic lesions. At a very minimum, the diagnostic methods applied for studying the prevalence of Tb in the population should be clearly described, to allow comparison between studies.

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife.

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.

Development and evaluation of a test for tuberculosis in live European badgers (Meles meles) based on measurement of gamma interferon mRNA by real-time PCR.

A real-time PCR assay for the measurement of gamma interferon (IFN-gamma) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN-gamma mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN-gamma mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN-gamma mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.

The safety and immunogenicity of Bacillus Calmette-Guérin (BCG) vaccine in European badgers (Meles meles).

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.

Progress in the development of vaccines and diagnostic reagents to control tuberculosis in cattle.

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.

Histopathogenesis of experimental Mycobacterium bovis infection in mice.

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.

Use of an electronic nose to diagnose Mycobacterium bovis infection in badgers and cattle.

It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.

Nasal boost with adjuvanted heat-killed BCG or arabinomannan-protein conjugate improves primary BCG-induced protection in C57BL/6 mice.

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.