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

Veterinary Immunology and Immunopathology 79 (2001) 197±207

Immunological responses of Eurasian badgers (Meles meles) vaccinated with Mycobacterium bovis BCG (bacillus calmette guerin) A. Southeya, D.P.S. Sleemanb, K. Lloydc, D. Dalleyc, M.A. Chambersc, R.G. Hewinsonc, E. Gormleya,* a

Large Animal Clinical Studies, Faculty of Veterinary Medicine, UCD, Dublin 4, Ireland b Department. of Zoology, University College Cork, Cork, Ireland c Department of Bacterial Diseases, Veterinary Laboratories, Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK Received 27 September 2000; received in revised form 28 February 2001; accepted 28 February 2001

Abstract 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 (puri®ed protein derivative of tuberculin). The proliferation was signi®cantly 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 speci®c 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. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Badger; Meles meles; Mycobacterium bovis BCG; Tuberculosis; Vaccine; Immunity Abbreviations: LTA, lymphocyte transformation assay; SI, stimulation index; CMI, cell-mediated immunity Corresponding author. Tel.: ‡353-1-6687988/ext. 2552; fax: ‡353-1-6671460. E-mail address: [email protected] (E. Gormley). *

0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 2 6 8 - 9

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1. Introduction In Ireland and Great Britain, the badger is thought to act as a reservoir host for Mycobacterium bovis and contributes to the spread and persistence of tuberculosis in associated cattle populations (Krebs et al., 1997; O'Boyle, 1998). Clinical and postmortem studies on badgers suggest that infection commonly spreads via the respiratory tract or by bite wounds within and between social groups (Gallagher et al., 1976; Pritchard et al., 1986) with more than 40% of lesions most often being found in bronchial and mediastinal lymph nodes and lung tissue (Fagan, 1993; Nolan and Wilesmith, 1994; O'Boyle, 1998). Evidence from a longitudinal study of natural M. bovis infection in badgers has indicated that individual badgers can shed bacilli intermittently over a number of years (Newell et al., 1997). The application of strain identi®cation using molecular typing techniques has demonstrated that badgers and cattle in a locality may harbour the same strains of M. bovis in some of the cases investigated, adding further support to the suggestion that badgers are a source of infection in cattle (Collins et al., 1994; Costello et al., 1999). Epidemiological evidence demonstrates a high prevalence of tuberculosis in badgers and controlled studies in Ireland involving comprehensive badger removal have shown that this strategy can serve to reduce cattle reactor rates signi®cantly in the targeted areas (O'Mairtin et al., 1998). However, since the badger is an ecologically important and protected wildlife species, alternative strategies are required to combat the disease. 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 by breaking the chain of infection between infected and susceptible animals. The sole existing vaccine for tuberculosis, M. bovis BCG, was developed in the early 1900s from a virulent M. bovis isolate and has since been widely used to control tuberculosis in humans, with varying degrees of success (Fine, 1989). Vaccination studies carried out in New Zealand with cattle and deer (Cervus elaphus) have demonstrated that the BCG vaccine, when delivered at appropriate doses, can generate signi®cant protective immunity against experimental challenge with virulent M. bovis (Buddle et al., 1995; Grif®n et al., 1999). In similar investigations with possums (Trichosurus vulpecula), ferrets (Mustela fero) and badgers (Meles meles) vaccination with BCG, although not preventing establishment of infection following virulent challenge, did lead to a reduction in the severity of the disease (Aldwell et al., 1995; Buddle et al., 1997; Qureshi et al., 1999; Stuart et al., 1988). Until recently there were few reagents for evaluating badger immune responses directed against tuberculosis. An enzyme-linked immunosorbent assay (ELISA) system (BrockTest) has since been developed which measures antibody responses to MPB83, a glycosylated lipoprotein which appears to be a major target of the antibody response in M. bovis infected badgers (Goodger et al., 1994). In contrast, other studies have shown that there is comparatively low expression of this protein in some strains of M. bovis BCG (Hewinson et al., 1996; Wiker et al., 1996) suggesting that it could form the basis of a diagnostic tool to distinguish M. bovis BCG-vaccinated from M. bovis-infected hosts. A comparative lymphocyte transformation assay (LTA) has also been developed using bovine and avian tuberculin as the source of antigens to detect cell mediated responses in

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infected badgers (Dalley et al., 1999). With the availability of such tests, the present study was carried out to determine the immune responses of a population of badgers vaccinated with M. bovis BCG and compare these responses with a control non-vaccinated population. We consider that a knowledge of these responses will help our understanding of immune parameters associated with protective immunity and will lead to the development of a vaccine suitable for use in badgers. 2. Materials and methods 2.1. Animals and anaesthesia The badgers used in this study were routinely cage-trapped on an island in southeast Ireland containing a high density isolated badger population. The badgers were distributed among 4±6 main setts. Prior to trapping, cages were pre-baited intensively with peanuts. The trapped badgers were anaesthetised with Ketamine hydrochloride1 (0.1 ml/kg) and Domitor1 (0.1 ml/kg), administered by intramuscular injection. Once unconscious, the animals were removed from the cage and were weighed, tattooed, eartagged, checked for parasites (¯eas, ticks, lice) and other signs of disease or injury (bite wounds, trap wounds, etc.). The sex, approximate age by tooth wear (Hancox, 1988) and body length, were also recorded. Blood samples (20 ml) were drawn from the jugular vein of each badger into heparinised vacutainer tubes. A separate blood sample (2 ml) was also drawn into non-heparinised tubes to provide sera. During the ®rst visit to the badger study area, tracheal aspirate samples were taken from all badgers and subsequently cultured to test for the presence of Mycobacterium sp. Following blood sampling, the animals were returned to the cage and allowed to recover from effects of anaesthesia. After at least 1 h of visual monitoring the badgers were released near to the sett entrances. Animals were trapped in weeks, 0, 10, 18, 23, 30, 37 and 42. 2.2. Vaccine preparation and delivery Nine badgers were vaccinated subcutaneously with M. bovis BCG (5  104 colony forming units (CFU) in 0.5 ml PBS) at week 0. The M. bovis BCG (Pasteur) strain was provided by Veterinary Laboratories Agency (UK) as a frozen stock of predetermined titre of CFU. The stock was thawed on site and diluted in PBS to the desired concentration. The vaccination site, approximately 1.0 cm above the right ear, was lightly shaved and swabbed with ethanol (95%) prior to injection. Six non-vaccinated control badgers were injected subcutaneously with 0.5 ml of PBS. At week 18, the nine animals received booster vaccinations with 5  104 CFU of M. bovis BCG. A second boost of 5  105 CFU M. bovis BCG was delivered at 30 weeks. 2.3. Lymphocyte transformation assay (LTA) Heparinised blood samples were processed within 8 h as described previously (Dalley et al., 1999) except residual red blood cells were lysed with 0.25% ice-cold NaCl.

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PPD-bovine and PPD-avian (Institute of Animal Science and Health, Lelystad, The Netherlands) were used as antigens at 1.25 mg/ml. The mitogen, ConA, was included as a positive control at 5 mg/ml. PBMC were incubated for 5 days at 378C in a humidi®ed atmosphere with 5% CO2. Proliferation was assessed by pulsing each well with 0.5 mCi of methyl-3 H-thymidine (Amersham) for the ®nal 16 h. Cells were harvested on to glass ®bre ®ltermats (Packard Instrument Co., UK) using a cell harvester (Micromate 196, Packard Instrument Co., USA) and the incorporated radioactivity was measured in a beta scintillation counter (Packard Instrument Co., UK). Results are expressed as stimulation indices (SIs) where SI ˆ

mean counts per minute …cpm† of stimulated wells mean cpm of non-stimulated wells

Cut-off values for SI ratios of PPD-bovine/PPD-avian were derived from the mean values of control badgers ‡ 3:29 times the standard deviation (S.D.). Statistical analysis of untransformed data was carried out using paired t-tests for matched values derived from animals throughout the study. The Mann±Whitney unpaired t-test was used to test signi®cance of data between groups. 2.4. Indirect ELISA for the detection of M. bovis antigen MPB83 (the BrockTest) Serum IgG antibodies speci®c for puri®ed 25 kDa M. bovis antigen (MPB83) were detected using an indirect ELISA as described previously (Goodger et al., 1994). Brie¯y, the antigen was adsorbed onto 96-well plates and incubated with badger sera (diluted 1:10). Badger IgG antibodies attached to the antigen were detected with mouse monoclonal anti-badger IgG coupled to peroxidase. A sample was considered to contain antibodies when the optical density was greater than the mean ‡ 3:29  S:E:M: (99.9% con®dence limit) of values given by sera from the control PBS-vaccinated badgers at all time points and the study population prior to vaccination. In all ELISA assays, a pool of sera from con®rmed tuberculosis diseased badgers that had previously tested positive by BrockTest ELISA was used as a positive control. 3. Results 3.1. Health status of badgers Fifteen badgers were cage-trapped at three different setts during the ®rst capture period. As judged by extent of tooth-wear the ages of animals ranged from juvenile (<1year-old) to mature adult (>3-year-old). Equal numbers of males and females were represented overall. Gross examination of the animals indicated a healthy population, though many had high numbers of ecto-parasites (lice, ticks, ¯eas) and several had bite wounds. Tracheal aspirate and urine samples were taken and cultured to test of presence of M. bovis. No positive cultures were recorded. During the course of the study, postmortem analyses were carried out on three (non-study) badgers which had died on the island from natural causes. No evidence of tuberculosis infection was found on the basis

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of histopathology and culture. Prior to vaccination, serum samples were taken from all badgers and tested by the BrockTest ELISA for the presence of antibodies to MPB83, a serodominant antigen of M. bovis, the recognition of which is associated with exposure to M. bovis infection (Clifton-Hadley et al., 1995). No recognition was measured with any of the animals consistent with the bacteriological results, indicating that the badger population was free of tuberculosis. 3.2. T-lymphocyte responses of badgers vaccinated with M. bovis BCG The study was carried out to measure the immune responses of nine badgers vaccinated subcutaneously with M. bovis BCG (5  104 CFU) and to compare the immune responses of these badgers with six non-vaccinated control animals. Booster vaccinations were delivered at week 18 (5  104 CFU) and at week 30 (5  105 CFU). Throughout the course of the study, animals were captured and blood samples taken on seven occasions. Not every badger was captured on each occasion, yet the overall recapture rate of vaccinates and control groups was high (Table 1). The immune responses of both groups of badgers were routinely monitored by measuring proliferation by T-lymphocytes cultured with PPD-bovine and PPD-avian. The mitogen, ConA, was also included as a positive control to monitor for cell survival. Lymphocytes were considered to be responding to the ConA when the proliferative SI was greater than 3 (SI > 3). Although the individual responses of all animals varied over time, T-lymphocytes incubated with ConA consistently proliferated to SIs ranging from 3 to 400 with a median value of 36.53. There were no signi®cant differences between the ConA responses of vaccinates and controls (data not shown) nor with the background mean cpm of unstimulated PBMC (Table 1). Following the primary vaccination and ®rst boost, the lymphocyte responses of both vaccinated and control groups of badgers to PPD-bovine remained low relative to prevaccination responses and were not signi®cantly different from each other (Fig. 1A). However, after receiving a second boost at 30 weeks, the mean SI of the vaccinated group started to increase relative to the control group, reaching a peak at 37 weeks. The responses of the vaccinated animals to PPD-bovine at 37 weeks were variable but signi®cantly greater than those measured in the control group (P ˆ 0:005). The Table 1 Numbers of badgers captured at each time point and mean cpm of unstimulated PBMC Week

Number of vaccinates

Unstimulated cpm

Number of controls

Unstimulated cpm

0a 10 18a 23 30a 37 42

9 9 9 9 9 7 6

632.8 574.7 681.9 911.5 608.5 1055.6 1161.1

5 4 6 6 4 6 4

621.8 2165.7 581.1 577.6 1058.7 1518.6 665.2

a

BCG delivered to vaccinates at these time points.

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Fig. 1. Comparison of peripheral blood lymphocyte responses of BCG-vaccinated (&) and control nonvaccinated (~) badgers to PPD-bovine (A) and PPD-avian (B). Values are expressed as mean stimulation indices …SIs†  S:E:M. M. bovis BCG was delivered at time points indicated by arrows.

maximum response of any individual vaccinated badger to PPD-bovine was recorded as SI ˆ 5:67. Although the mean response declined at 42 weeks, it was still signi®cantly greater at this time point compared to the control animals (P ˆ 0:0056). In order to examine the speci®city of the LTA response, the proliferation of PBMC to PPD-avian was compared for both groups of animals (Fig. 1B). With the vaccinated

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group no signi®cant differences were observed in the PPD-avian responses during the ®rst 18 weeks of study. However, following delivery of the ®rst boost of BCG, there was a signi®cant decrease in the responses to PPD-avian between weeks 18 and 23 (P ˆ 0:0485). Thereafter, there were no signi®cant changes in the avian-type responses, although there was a decline in the mean values following delivery of the second BCG boost at week 30. In the control group, the mean responses to PPD-avian declined over the ®rst 18 weeks of study relative to the vaccinated group. Between weeks 18 and 23 there was an increase in the responses of control animals, although this was not determined to be signi®cant (P ˆ 0:315). For the remainder of the study, the avian responses of the control group were comparatively higher than those observed with the vaccinated animals. The PPD-bovine to PPD-avian SI ratio values from the control nonvaccinated animals were used to de®ne a positive `cut off' point for the LTA of 1.6 (at the 99.9% con®dence limit). Five of the vaccinated animals tested at 42 weeks (n ˆ 6) were considered positive by this criterion with the maximum PPD-bovine/PPD-avian ratio of any individual badger responding to PPD-bovine recorded as 13.2. The results indicated that repeated vaccination of badgers resulted in a switch of responses from recognition of M. avium-type antigens to M. bovis antigens. 3.3. BrockTest ELISA tests of serum samples from vaccinated and control badgers Serum samples taken from both groups of badgers were tested by the BrockTest ELISA for the presence of antibodies to MPB83. No badger was seropositive by BrockTest ELISA at any point prior to or after BCG vaccination (data not shown). This indicated that repeated BCG (Pasteur) vaccination of badgers does not elicit a potent speci®c antibody response against MPB83. 4. Discussion Many studies have been carried out to elucidate the cellular basis for generation of a protective immune response, following vaccination with M. bovis BCG (Buddle et al., 1995; Carpenter et al., 1997; Grif®n et al., 1999). It has been demonstrated that development of acquired immunity to tuberculosis is mediated through a speci®c T-cellmediated immune (CMI) response in which the co-operative action of antigen-speci®c Tlymphocytes and macrophages ultimately control infection by inhibiting intracellular growth of the tubercle bacilli (Orme et al., 1993). The proliferation of T-lymphocytes is generally considered to be an indicator of an antigen-speci®c CMI response against M. tuberculosis/M. bovis antigens(Gulle et al., 1995; Pal and Horwitz, 1992). In contrast, the presence of high titres of antibody recognising M. bovis antigens is usually associated with chronic or progressive disease (Fi®s et al., 1994). Recent work has demonstrated that badgers infected naturally with M. bovis do elicit a speci®c T-lymphocyte response which can be measured in a blood-based assay (Dalley et al., 1999). The objectives of this study were to determine if M. bovis BCG vaccination of badgers could induce similar measurable responses and also determine the logistics involved in carrying out future badger vaccine trials.

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The initial dose of BCG (5  104 CFU) used in this study was chosen because this dosage level has been shown to confer signi®cant protection against challenge with virulent M. bovis in other animal species (Buddle et al., 1995; Grif®n et al., 1999). Throughout the period of study an enhanced T-lymphocyte response from the vaccinated animals to PPD-bovine was only recorded following three subcutaneous injections of M. bovis BCG. This is in contrast to the considerably higher T-lymphocyte responses observed in other studies with badgers infected naturally with M. bovis (Dalley et al., 1999). It is possible that the low proliferation was directly related to the dose of BCG delivered as it has been shown in previous BCG vaccine studies with deer and cattle that levels of proliferation do correlate directly with the dose of BCG (Buddle et al., 1995; Grif®n et al., 1999). However, even when similar doses and boosters were given subcutaneously to cattle, the T-lymphocyte activity was considerably greater than that observed in the current study with badgers, over a similar time period (Carpenter et al., 1997). This would suggest that the relatively low badger T-lymphocyte proliferation to PPD-bovine is a feature characteristic of the badger response (Stuart et al., 1988). Alternatively, the badger may exhibit a strong and rapid innate response to subcutaneous vaccination and the BCG is cleared before large numbers of T-lymphocytes are primed. Culturing of lymph node samples from recent naturally deceased vaccinated badgers has failed to grow M. bovis BCG (data not shown). Thus, the optimum dose of BCG determined for one species cannot be extrapolated directly to another species, such as the badger. Further, empirical work is, therefore, required to determine the optimal BCG dose for badgers. The speci®city of the LTA was evaluated by considering the responses to PPD-avian. Throughout the study, the responses of the control group were consistently higher towards PPD-avian compared to PPD-bovine. It is likely that this results from the frequent exposure of badgers to M. avium or environmental mycobacterial antigens which are cross-reactive with PPD-avian antigens. In the vaccinated group, the responses to PPDavian antigens declined throughout the study. It appeared that successive exposures to M. bovis BCG antigens, provided by the vaccination and boosting, switched the response to speci®c recognition of PPD-bovine antigens. How this underlying response to M. avium antigens might affect the ef®cacy of protective immunity, generated through M. bovis BCG vaccination, remains to be determined for badgers. However, studies in guinea pigs suggest that infection with environmental mycobacteria (M. simiae or M. avium-intracellulare) does not interfere with the induction of a protective response in animals subsequently vaccinated with BCG (Edwards et al., 1982; Herbert et al., 1994; Smith et al., 1985). The results obtained in this study using the BrockTest ELISA test on serum samples from both groups of badgers indicated that antibody recognition of MPB83 was not signi®cantly enhanced by vaccination with BCG (Pasteur). In contrast, MPB83 is one of the ®rst antigens recognised by badgers infected with M. bovis (Goodger et al., 1994). In M. bovis, MPB83 is expressed constitutively at high levels but in BCG Pasteur little expression of the antigen can be detected (Hewinson et al., 1996; Wiker et al., 1996) which may explain the lack of antibody response observed against MPB83 in badgers vaccinated with BCG Pasteur. These results suggest that it may be possible to use the BrockTest or other tests based on an immunological response to MPB83 to differentiate between badgers infected with M. bovis and those vaccinated with BCG.

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In this paper we describe the ®rst ®eld evaluation of the immune responses stimulated by BCG vaccination in badgers. In a previous study of BCG vaccination in captive badgers (Stuart et al., 1988) the protective effect of intradermally inoculated BCG vaccine was investigated in twelve badgers. An intradermal inoculation of BCG (Glaxo) was found to be non-pathogenic, was not excreted by the badgers and was not transmitted to in-contact animals. There was an increase in LTA response but no antibody increase to PPD-bovine in all twelve vaccinated badgers. When seven of these badgers were subsequently challenged intradermally with M. bovis, between 5 and 25 months after vaccination, the LTA response tended to fall and the antibody response rose. The vaccinated badgers were shown to live longer, shed fewer tubercle bacilli and their inoculation sites healed more rapidly after challenge than a group of three infected control badgers. Thus, although only small numbers of badgers were involved, CMI did seem to be enhanced by BCG vaccination, leading to prolonged survival of the badgers and delayed excretion of tubercle bacilli. Although the vaccination of badgers in this study used subcutaneous injection it is likely that a vaccine designed for ®eld-use will be based on an oral delivery system. Given the complexities of progression of disease and the immune responses directed to counteract virulent in vivo growth of M. bovis in the host, there are many challenges to developing an effective vaccine strategy to prevent transmission of the bacterium from a population of badgers. Chief among these would be issues relating to (a) de®ning the type and formulation of the vaccine to be used (b) assessing a large number of target host factors relevant to the generation of protective immunity in a population and (c) achieving the desired level of protection. As a long term control measure, the aim of vaccination will be to decrease the incidence of infection in susceptible hosts or reduce development of disease pathology in infected individuals to the extent that the disease will eventually be eliminated in badger populations. It is not expected that vaccination would signi®cantly affect the density or social structure of the badger population. The risk to cattle then posed by such badgers would be expected to be less than that at present. By removing this additional source of infection, the ef®ciency of the tuberculin testing program, for controlling cattle to cattle spread, would be signi®cantly improved and this major constraint to the eradication of tuberculosis will have been addressed. Acknowledgements This study was funded by the Department of Agriculture, Food and Rural Development (Republic of Ireland). The work performed at VLA Weybridge was funded by the Ministry of Agriculture, Fisheries and Food (MAFF), UK. All work with badgers was carried out under licences issued by Duchas (Wildlife Service) and the Department of Health. The authors wish to thank Tom Partridge for his assistance with the study. They also acknowledge the help and support provided by Ian O'Boyle, Michael Sheridan, Margaret Good, Prof. Dan Collins and Prof. Joe Quinn. The help and assistance of the Wildlife Service and all those others who participated in the study is greatly appreciated.

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