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

Veterinary Immunology and Immunopathology 112 (2006) 24–37 www.elsevier.com/locate/vetimm

The safety and immunogenicity of Bacillus Calmette-Gue´rin (BCG) vaccine in European badgers (Meles meles) S. Lesellier a, S. Palmer a, D.J. Dalley a, D. Dave´ a, L. Johnson b, R.G. Hewinson a, M.A. Chambers a,* b

a TB Research Group, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK Department of Pathology, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK

Abstract 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-Gue´rin (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  107 colony-forming units (CFU), and was followed 15 weeks later by a lower dose in the range of 4–7  105 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. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. Keywords: Badger; BCG; Vaccination; Safety; Immunity

1. Introduction * Corresponding author. Tel.: +44 1932 357494; fax: +44 1932 357260. E-mail address: [email protected] (M.A. Chambers).

The European badger (Meles meles) was first identified as wildlife host for Mycobacterium bovis in Britain in 1971 near a farm where a herd break-down

0165-2427/$ – see front matter. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.03.009

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had just occurred (Muirhead et al., 1974). Since then, epidemiological studies have demonstrated that M. bovis infection is endemic in badger populations (Cheeseman et al., 1989, 1988) and that infected badgers excrete M. bovis (Clifton-Hadley et al., 1993), potentially contaminating areas used by cattle. The same clusters of M. bovis molecular types are found locally in badger and cattle (Woodroffe et al., 2005), and in the Republic of Ireland, removal of infected badgers has been shown to reduce significantly the incidence of tuberculosis in local cattle herds (Griffin et al., 2005). For these reasons, badgers are considered a wildlife reservoir for M. bovis, that might compromise the general scheme of eradication of tuberculosis in cattle herds in the United Kingdom and the Republic of Ireland. If badgers prove to be a significant source of bovine tuberculosis in GB, widespread culling of badgers to control bovine tuberculosis would be very labour intensive, expensive, and require sustained control of populations. Such a strategy is fraught with concerns about the public acceptability and ecological impact of such an approach. Vaccinating badgers is an alternative strategy that may overcome some of these problems (White and Harris, 1995). BCG vaccination has conferred a degree of protection against challenge with M. bovis to most species tested (Suazo et al., 2003), and is currently the only feasible vaccine candidate for badgers. Data on the use of BCG in badgers are understandably limited. However, a BCG vaccination study performed at the Veterinary Laboratories Agency (VLA) in 1985 demonstrated vaccination enhanced cell-mediated immunity, prolonged survival following intradermal M. bovis challenge, and delayed excretion of the organism (Stuart et al., 1988). A recent study performed in the Republic of Ireland demonstrated a protective effect of s.c. BCG vaccination against a more relevant intratracheal challenge with M. bovis (E. Gormley, personal communication). Although these experimental results are promising they require validation under field conditions where the protective efficacy of BCG has been more variable against tuberculosis acquired naturally, especially in cattle and man (Hewinson et al., 2003). Only one study of BCG vaccination in wild badgers has been published (Southey et al., 2001). The objective of that study was to monitor immune responses in an

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isolated population of badgers in the Republic of Ireland known to be free of tuberculosis, following s.c. vaccination with BCG Pasteur. As part of that study, some animals were monitored for signs of local vaccine reactogenicity within the first week of administration. No adverse reactions were observed (E. Gormley, personal communication). In GB, field studies are performed under the authority of an Animal Test Certificate, granted by the VMD following consideration of a safety dossier, compiled from experimental evidence acquired from captive animal studies performed to Good Laboratory Practice (Anonymous, 2004). To expedite this, we have used the Danish strain of BCG produced by the Statens Serum Institute (SSI), Denmark, which is currently the only commercial source of BCG licensed for use in humans in the UK. Following a protocol prescribed by the VMD, a group of captive badgers free of tuberculosis were vaccinated with BCG Danish SSI via the s.c. or i.m. route. An initial high dose of BCG was followed 15 weeks later by administration of a lower dose of the vaccine by the same route. Local reaction at the site of injection and general responses such as body temperature, haematology and blood serum chemistry, general behaviour and excretion of BCG were monitored. Immunological responses were measured throughout the study.

2. Materials and methods 2.1. Badgers Eight badgers were maintained as a confined but free-ranging colony in isolation from wild badgers in the Natural Environmental Centre (NEC) of the VLA. Seven out of the eight badgers were born in the facility and one (A8) originated from the group originally introduced in 1996. Artificial wooden setts were provided and the badgers used these to rest and sleep in. Use of these setts permitted ready access to the badgers during daylight hours. Throughout the study, the badgers received a daily diet of dog food and peanuts. They were also occasionally given eggs obtained from the VLA SPF Unit. The badgers were free to forage in the NEC and so probably supplemented their diet with earthworms and other opportunistic meals. Tap water was given ad libidum.

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For vaccination and/or clinical examination, the badgers were anaesthetised directly in the wooden setts they occupied during the day, by i.m. injection of approximately 10 mg/kg of ketamine (Vetalar1, Pfizer Animal Health, New York, NY, USA) and 100 mg/kg of medetomidine (Domitor1, Pfizer Animal Health), using a thumb controlled pole-syringe (Field Development and Supply, LLC, USA). The badgers were individually identified by unique patterns of hairclipping on both flanks (Stewart and Macdonald, 1997) and by using electronic microchips (IPTT-300 transponders, PLEXX b.v., Elst, Netherlands) programmed with a unique ID, positioned s.c. approximately 10 cm cranially to the tail. The microchips additionally allowed the measurement of body temperature telemetrically without the need for chemical or physical restraint of the animal. Microchips were activated and data read by an external reader, DAS-5002 (PLEXX b.v.). 2.2. BCG vaccines and their administration BCG ‘‘Vaccine’’ SSI and BCG ‘‘Culture’’ SSI were supplied freeze-dried together with Sauton diluent by the Statens Serum Institut, Denmark. The ‘‘Vaccine’’ (used for TB prophylaxis) comprised 2–8  106 CFU BCG Danish strain 1331 when reconstituted and is licensed for administration to humans in the UK. The

‘‘Culture’’ (used for the immunotherapy of superficial bladder cancer) comprised 1–12  106 CFU/mg BCG Danish strain 1331 and was supplied in vials of 30 mg. The vaccines were prepared freshly prior to inoculation. Each vial of BCG ‘‘Culture’’ SSI was reconstituted in 1 ml of Sauton diluent (SSI) to yield a final concentration of 30 mg (approximately 3– 36  107 CFU) per 1 ml and mixed by gently inverting the vial. Between 0.81 and 1 ml was administered per badger as the ‘overdose’ (see Table 1). Each vial of BCG ‘‘Vaccine’’ SSI was reconstituted in 1 ml of Sauton diluent to yield a final concentration of 2–8  106 CFU per 1 ml and mixed by gently inverting the vial. About 0.1 ml was administered to each badger as the repeat, low dose, except in the case of a single animal (B5) that received 0.06 ml. The eight badgers were allocated to two treatment groups of three animals each and one control group of two animals that served as unvaccinated controls. No badger had ever received BCG before the start of the study. The control group was chosen to contain the youngest (B6) and oldest (A8) badgers in the study. The remaining animals were used to make up the two treatment groups, and were allocated so that: (i) both sexes received vaccine via each route of inoculation; (ii) each group contained animals of approximately equal age range. BCG was administered on two

Table 1 Age, sex, and details of BCG vaccination for badgers used in this study Badger ID

Controls A8 B6

Age (years) a

Sex

BCG ‘‘Culture’’

BCG ‘‘Vaccine’’

Volume received

Dose received

b

Route

Volume received

Dose received c

Route

>10 2

M M

None

None

NA

None

None

NA

A13 B4 B5

6 5 5

F F M

0.98 ml 1.0 ml 1.0 ml

6.06  107 21.66  107 8.82  107

s.c. s.c. s.c.

0.1 ml 0.1 ml 0.06 ml

6.73  10 5 6.73  10 5 4.04  10 5

s.c. s.c. s.c.

A14 B2 B3

6 5 5

F M M

1.0 ml 0.81 ml 0.93 ml

5.16  107 5.78  107 13.2  107

i.m. i.m. i.m.

0.1 ml 0.1 ml 0.1 ml

6.73  10 5 6.73  10 5 6.73  10 5

i.m. i.m. i.m.

a

Age at the start of the study based on known year of birth. Badger A8 was an adult when originally brought into the NEC in 1996. The concentration of BCG in each vial of BCG ‘‘Culture’’ used for inoculation was calculated using the residual volume of reconstituted vaccine left in the vial. These concentrations ranged from 5.16  107 to 21.66  107 CFU/ml, which were in the range specified by the manufacturer. c The concentration of BCG ‘‘Vaccine’’ used for administration was calculated using a fresh vial of material from the same batch used for vaccination. This was 6.73  106 CFU/ml, which was in the range specified by the manufacturer. b

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occasions. One group of three badgers (one female, two males) was inoculated i.m. and another group of three (two females, one male), s.c. (see Table 1). Hair was clipped and the skin cleaned with iodine soap and sterile water prior to inoculation. On the first vaccination the badgers were given an overdose, injected at two symmetrical points on the dorsal part of the neck (0.5 ml/site), in the trapezius muscle or more superficially for the s.c. injection. Fifteen weeks later the badgers were given a second single dose (0.1 ml) via the same route but at a point approximately 2 cm caudally to the previous injection. 2.3. Clinical examination, sampling and remote monitoring Clinical examination was performed under anaesthesia every 1–3 weeks for a total of 36 weeks. Badgers were weighed and the extent of any cutaneous reaction produced at the site of BCG injection was measured with an electronic calliper and the site photographed. Rectal temperature was recorded alongside the temperature readings obtained with the implanted microchips. The temperature of badgers when not under anaesthesia was obtained in situ in the wooden setts using the s.c. microchips. This method was employed to obtain readings of body temperature at time points soon after the administration of vaccine. Blood was sampled from the jugular vein for haematology and immunology (into heparinised Vacutainer tubes (BD, Plymouth, UK)), for blood serum chemistry (into Serum Separation Vacutainer tubes), and for measuring glucose levels (in fluorate supplemented Vacutainer tubes). Samples of tracheal aspirate, saliva, urine and faeces were also taken and submitted to culture for BCG. Tracheal aspirates were obtained by inserting a 6FG sterile catheter (BUSTER Cat, KRUUSE UK Ltd., Sherburn in Elmet, UK), connected to a 20 ml syringe, into the trachea with the aid of a laryngoscope fitted with a size two Miller blade (Arnolds Veterinary Products, Shrewsbury, UK) and aspirating tracheal mucous by slowly withdrawing the syringe plunger while gently retrieving the catheter. Saliva was obtained from the mouth using a 1 ml syringe. Both tracheal aspirate and saliva samples were dispensed immediately into 5 ml sterile Middlebrook 7H9 medium (BD Difco, Plymouth, UK). Urine was

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sampled from the bladder using sterile urinary catheters and dispensed directly into sterile tubes. Females were laid in a ventro-dorsal position and a 6FG catheter was introduced in the urethra with the help of a speculum (KRUUSE UK Ltd.). For males, flexible 5GF silicone catheters (Fowley Urethral Catheter, Global Veterinary Products, Waukesha, WI, USA) were used. Faeces were sampled on a swab introduced in the rectum then transferred to 5 ml sterile PBS. Behaviour of the badgers when active at night was observed using CCTV cameras equipped with infrared lamps and recorded onto video cassettes. Each badger could be recognised individually by the pattern of hair-clipping (Stewart and Macdonald, 1997). Badgers activity was observed on several consecutive nights before and after vaccination and compared with normal behaviour observed over the previous 4 years. Seven main activities (feeding, digging, bed making, cuddling, mating, grooming and exploring) were considered as ‘‘normal’’ and six other observations (fighting, excessive shyness, intense scratching, fits, paresis and repetitive behaviour) as ‘‘abnormal’’. A biopsy (approximately 1 cm  0.3 cm) of skin and underlying s.c. tissue from one badger (B5) was taken from a site of high dose s.c. BCG injection 371 days after administration. The tissue sample was placed in 10% neutral buffered-formalin for 7 days and then embedded in paraffin wax. Sections (4 mm) were cut and stained with haematoxylin and eosin and Ziehl-Neelsen method to detect acid-fast bacilli. 2.4. Bacteriology Clinical samples were plated on modified 7H11 medium (Middlebrook 7H11 plus OADC enrichment (BD Difco)) with the addition of 10% inactivated adult bovine serum (Invitrogen Life Technologies, Paisley, UK), 0.5% ovine lysed blood (Tissue Culture Services, VLA), 5 mg/l malachite green (VWR International Ltd., Poole, UK), 100,000 IU/l polymixin B (Sigma–Aldrich, Poole, UK), 10 mg/l trimethoprim lactate (Sigma–Aldrich), 100 mg/l amoxicillin (Sigma–Aldrich), 50 mg/l amphotericin (Fungizone, Bristol-Myers Squibb, New York, NY). Saliva and tracheal aspirates were incubated in Middlebrook 7H9 medium plus ADC enrichment

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(BD Difco) and 0.2% glycerol at 37 8C for 2 weeks in order to enrich the samples for mycobacteria before being plated. Urine and faeces were stored frozen at 20 8C for 2 weeks until plated. PBS containing faecal swabs were left to stand at RT for 6–18 h in order to elute any BCG present in the sample. The tube was then vortexed briefly to resuspend any BCG present and the swab discarded. The sample was decontaminated by the addition of 5 ml 10% oxalic acid and then centrifuged promptly at approximately 1100  g for 10 min. The supernatant was carefully decanted, leaving approximately 1 ml in the tube. A solution of 0.85% saline was added to bring the volume up to 10 ml. The tube was centrifuged a second time and the supernatant decanted, leaving approximately 1 ml in the tube. An additional 2 ml 0.85% saline was added and the tube vortexed briefly before the sample was plated. Plates were incubated at 37 8C for at least 6 weeks and observed every week for the appearance of colonies. A control stock of BCG vaccine was grown on each occasion to confirm the ability of each batch of 7H11 medium to support the growth of BCG. 2.5. Immunology Cellular immune responses were measured in two assays: lymphocyte transformation assay (LTA) and ELISPOT to measure the production of IFNg. Both assays required the isolation of PBMC as per (Dalley et al., 1999). In brief, heparinised blood was overlaid on histopaque 1077 (Sigma–Aldrich) in Accuspin tubes (Sigma–Aldrich) and centrifuged at 840  g for 15 min. The PBMC layer was washed twice in Hank’s Balanced Salt Solution (Invitrogen Life Technologies) and resuspended at 2  106 cells/ml in complete medium: RPMI 1640 (Invitrogen Life Technologies), supplemented with 5% foetal calf serum (Invitrogen Life Technologies), non-essential amino acids (Invitrogen Life Technologies), 5  10 5 M b-mercaptoethanol (Invitrogen Life Technologies) and 100 U penicillin/100 mg streptomycin per ml (Invitrogen Life Technologies). PBMC (2  105 in 200 ml) were stimulated with Purified Protein Derivative from M. bovis (PPD-B) (VLA Weybridge) at 5 mg/ml or medium alone. Concanavalin A (Sigma–Aldrich) (at 5 mg/ml) was used to stimulate PBMC non-specifically as a positive controls for cell viability. The

PBMC were stimulated for approximately 16 h for ELISPOT and for a total of 5 days for the LTA. In the LTA, 1 mCi of tritiated thymidine (TRK120, Amersham) was added per well for the last 18 h and the cells harvested on glass fibre filter plates (Unifilter96, Berthold Technologies (UK) Ltd., Redbourn, UK). Radioactivity in each well was measured by a b counter (TopCount, Berthold Technologies (UK)) and expressed as cpm. ELISPOT was performed using a monoclonal and polyclonal pair. 96-well plates (Millipore multiscreen, Millipore (UK) Ltd., Watford, UK) were coated with capture mAb 11B9 (mouse anti-badger IFNg, VLA) at 10 mg/ml in carbonate/bicarbonate buffer, pH 9.6 overnight at 4 8C. Plates were emptied and blocked with complete medium at 37 8C for 1 h. ConA and antigen at twice the required concentration, were placed in the coated wells 1:1 with PBMCs in duplicate. Following incubation for 18–24 h at 37 8C, 5% CO2, the wells were washed three times in distilled water, followed by three times in PBS, 0.05% Tween20 (v/v), before the addition of rabbit anti-badger IFNg polyclonal detection antibody Rb299 (VLA), diluted 1/400 in PBS, 0.05% Tween-20, 0.1% liquid albumin (v/v, 1 mg/ml Sigma–Aldrich). Plates were incubated for 1 h at 37 8C, washed, and incubated with biotinylated anti-rabbit IgG (Sigma–Aldrich), diluted 1/1000 in PBS, 0.05% Tween-20, 0.1% liquid albumin (v/v). Plates were then washed and incubated with streptavidin-ALP-PQ (Mabtech AB, Hamburg, Germany) diluted 1/4000 in PBS, 0.05% Tween-20, 0.1% liquid albumin (v/v). Between steps the plates were washed three times with PBS, 0.05% Tween-20 (v/v) and incubated at 37 8C. Substrate BCIP-NBT (Sigma– Aldrich) reacted with the alkaline-phosphatase to create spots on the nitrocellulose surface where cells had been producing IFNg. Reactions were stopped by washing copiously with tap water. Wells were counted using an AID reader and software (Autoimmun Diagnostika GmbH, Strassberg, Germany). 2.6. Haematology and serum chemistry Twelve haematological parameters and 13 blood serum chemistry parameters were measured in badger blood by Greendale Veterinary Diagnostics Ltd. (Knaphill, Surrey) using a Sysmex KX21 (Sysmex UK Ltd., Milton Keynes, UK) and a Roche Hitachi

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717 (Roche Diagnostics Ltd., Lewes, UK) Analyser, except for cortisol, which was measured with an Immulite 1000 Analyser (EURO/DPC Limited, Caernarfon, UK). The following parameters were quantified on each occasion the blood was sampled: RBC, total haemoglobin, hematocrit (HT), mean cell volume (MCV), mean cell haemoglobin concentration (MCHC), mean corpuscular haemoglobin, white blood cells, lymphocytes, monocytes, eosinophils, neutrophils, platelets, total protein, albumin, globulin, alanine aminotransferase (ALAT), alkaline-phosphatase (PAL), total bilirubin, total urea, creatinine, gamma-glutamyl transpeptidase (GGT), glucose, calcium, magnesium, and cortisol. For each of these parameters, a range of values considered as normal were established using data from the same eight badgers and five additional badgers (deceased) belonging to the same captive group sampled over the 4 years prior to this study. These ranges were consistent with values reported for other mustelids such as ferrets (Quesenberry, 1997), otters (FournierChambrillon et al., 2003), and mink (FernandezMoran et al., 2001; Weiss et al., 1994), as well as cats and dogs (Meyer and Harvey, 1998).

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specified by the manufacturer. However, no clinical sample obtained during this study yielded a positive culture result for the BCG vaccine. Since, the faecal and urine samples were kept frozen at 20 8C for 2 weeks after harvest, the impact of this on culture sensitivity was determined. Freezing resulted in a 10fold decrease in BCG colony-forming units in urine

2.7. Presentation and analysis of data All immunological results are presented as net values (net cpm for LTA, and net Spot Forming Cells (SFC) per million cells for ELISPOT). Net values were calculated by subtracting the background values obtained with cells stimulated with medium only, from results for specific antigen, all in duplicate. The background values were consistently low (in LTA: average = 1591 cpm, S.D. = 2188 cpm; in ELISPOT: average = 6 SFC/million, S.D. = 9 SFC/million). Statistical analysis was performed using GraphPad InStat version 3.06, 32 bit for Windows, GraphPad Software, San Diego California USA, http://www.graphpad.com.

3. Results 3.1. Bacteriology Table 1 summarises the dose and route of vaccination for each badger in the study. Each vaccinated badger received a dose of BCG within the range

Fig. 1. Local reactions at site of BCG injection. Badgers were vaccinated s.c. (circles) or i.m. (triangles). Measurements were taken using an electronic calliper at the two sites of high dose inoculation (A and B) and at the single site of low dose repeat administration (C).

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samples and a six-fold decrease for faecal samples (data not shown). As a result, culture was determined to be least sensitive from urine, with a limit of detection of 40 CFU/ml. The sensitivity of detection from faeces was 24 and 4 CFU/ml for tracheal aspirate and saliva. 3.2. Local reactions at the site of vaccination Reactions at the site of injection were considered mild. The largest and most prolonged reactions were seen at the sites of vaccine overdose via the s.c. route (Fig. 1A and B). In the case of one badger (B4), there was some suggestion that the swelling at this site was ‘boosted’ by the repeat administration of BCG, albeit at a different site. However, this was not observed in any of the other badgers. Swelling at the site of BCG re-administration was only observed in two s.c. vaccinated badgers (A13 and B4); was transient (i.e. absent 28 days later), with a peak swelling less than observed at the site of overdose (Fig. 1C). It was noted that the reaction at one of the sites of s.c. vaccine overdose in a single badger (B5) was still apparent 371 days after administration. The nature of this reaction was investigated further by skin biopsy. The tissue reaction comprised of focally extensive granulomatous and necrotising cellulitis with moderate, multifocal periadnexal chronic dermatitis (Fig. 2A). Numerous acid-fast bacilli were present (approximately 50–60 per section viewed), generally within macrophages (Fig. 2B).

In contrast to the local reactions observed with s.c. administration, the reactions at the site of i.m. inoculation were minimal, and only observed following the administration of vaccine overdose (and then only in two badgers) (Fig. 1). The peak swelling was 8.46 mm; persisting for a maximum of <48 days postvaccination (badger A14). No ulceration or discharge accompanied any local reaction and there were no marks to the skin that would indicate rubbing of the site, which might be expected if the animal was experiencing itching or irritation at the site of vaccination. 3.3. Body weight and behaviour The badgers stayed clinically healthy for the duration of the study, apart from both control badgers, which had evidence of bite wounding. Food uptake by the group was considered normal and weight variations (Fig. 3) followed a normal seasonal pattern with higher weights at the end of November to early December, just prior to the repeat administration of BCG. Based on experience accumulated over the previous 4 years, no abnormal behaviour was observed on the videos, before or after vaccination. Badgers appeared normally active; interacting, exploring and eating normally. No restriction in the movements of the neck, apathy or intense scratching could be observed at any time. Badgers recovered fully from the anaesthesia accompanying BCG injection, and

Fig. 2. Histological appearance of the site of persistent inflammatory reaction to the injection of high dose BCG in badger B5. Skin biopsy was taken 371 days after s.c. vaccination and stained with haematoxylin and eosin (A), which revealed granulomatous cellulitis (size bar = 200 mm), and Ziehl-Neelsen method (B) to detect acid-fast bacilli (arrows) (size bars = 30 mm).

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Fig. 3. Weights of badgers. Badgers were vaccinated s.c. (circles) or i.m. (triangles). Note seasonal variation in body weights for all animals. Months are indicated by single letter code.

were as active the following night as the night prior to vaccination. 3.4. Body temperature Body temperature was recorded in two ways: (1) by the insertion of a thermometer into the rectum of a badger whilst under anaesthesia (Fig. 4A); (2) by taking a reading from a s.c. transmitting microchip, implanted under the dorsal skin of the badger whilst under anaesthesia prior to commencement of the study (Fig. 4B). Comparison of readings obtained with the rectal thermometer whilst animals were anaesthetised with those obtained from the microchip showed a very significant correlation (r = 0.64, p < 0.0001, Spearman rank). Body temperature (as measured by microchip) was occasionally observed to drop below 30.0 8C. Although these events coincided with reduced rectal temperature, the rectal temperature did not fall below 35.0 8C. This badger appeared healthy: no abnormality was recorded for this badger on clinical examination, in the haematology or in the serum chemistry. For the duration of the study, only one badger (B3) recorded a temperature (rectal) exceeding 40.0 8C. This occurred only once: 22 days prior to the first administration of BCG. Within the limits of what was practical, body temperature was also recorded at time points soon after the administration of vaccine using the microchips. This was as early as four hours after the administration of vaccine overdose (A13) and at frequent time points after that for all badgers up to

Fig. 4. Body temperature of badgers whilst under anaesthesia. Badgers were vaccinated with BCG via the s.c. (circles) or i.m. (triangles) routes. (A) Temperature recorded by insertion of thermometer into the rectum. (B) Temperature recorded from a microchip implanted s.c.

135 h post-overdose. It was not practical to take such intensive readings after the repeat dose of BCG was administered. Nonetheless, readings were taken from 21 to 54 h post-vaccination. Significantly, no vaccinated badger recorded a body temperature exceeding 38.0 8C during the period of intensive observation immediately following vaccination (data not shown). 3.5. Haematology and serum chemistry There was no difference between the animals injected by the i.m. or s.c. route, in terms of the white cell, lymphocyte, monocyte, neutrophil, eosinophil, or platelet counts. The two control badgers (A8 and B6) had values that occasionally exceeded what was regarded as the normal upper limits but this could be explained by occasional minor bite wounds on both the animals (data not shown). Parameters related to RBC were typical of a mild regenerative anaemia,

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with a slight increase in the MCV and a decrease of the MCHC; a physiological response to the regular blood sampling the animals were submitted to for this study. The oldest animal, A8 (at least 10 years of age) was not able to produce as many blood cells as the other badgers and had consistently lower HT and lower RBC counts (data not shown). Total protein, globulin and albumin were generally within the normal ranges for all the vaccinated animals. Control badger A8 occasionally had increased globulin levels, in association with bite wounds found on clinical examination. Values for the parameters representing hepatobiliary function (ALAT, GGT and total bilirubin), renal function (urea and creatinine), as well as glucose, magnesium and calcium stayed within or close to normal limits in all vaccinated animals, with the occasional exception (data not shown). Cortisol levels, reflecting short term stress induced before anaesthetic injection, varied between animals but were generally within the reference limits established previously (data not shown). Serum concentrations of PAL exceeded the normal range in all vaccinated animals at least 5 weeks before and for 11 weeks after BCG injection (Fig. 5). The PAL elevation was not related to BCG vaccination. Levels of PAL had returned to basal values a few weeks before the second dose of BCG was administered and stayed low thereafter. One control badger (A8) had markedly high levels of PAL, ALAT, and on two occasions, of GGT, throughout the period of study.

Fig. 5. Levels of PAL in the serum of badgers (expressed in units per litre). Badgers were vaccinated with BCG via the s.c. (circles) or i.m. (triangles) routes. A high dose was administered on day 44, followed by administration of a low dose via the same route on day 149. Dashed lines indicate the upper and lower 95% confidence intervals around mean values of PAL obtained from badgers during the previous 4 years of captivity.

3.6. Immunology All vaccinated badgers had an increased cellular immune response within 13 days following the injection of the high dose of BCG. High levels of response in both ELISPOT (Fig. 6A) and LTA (Fig. 6B) were maintained in the i.m. group until day 76 following vaccination. The response after s.c. vaccination was maintained to the time of the repeat injection of BCG, 105 days later. The repeat injection of the lower BCG dose induced a response of reduced magnitude and persistence compared with the high dose vaccination, despite prior sensitisation to BCG. Levels of response were similar between the two routes of vaccination, although a boosting effect in the LTA was more apparent in the s.c. group. Reponses in the two negative control animals were consistently low throughout the study, but responded normally to mitogenic stimulation (data not shown).

Fig. 6. Immunological responses to PPD-B of badgers vaccinated with BCG by the s.c. (circles) or i.m. (triangles) routes. A high dose was administered on day 44, followed by administration of a low dose via the same route on day 149. (A) ELISPOT for IFNg. (B) LTA. Mean values and standard deviation for each group are shown.

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4. Discussion The persistence of M. bovis infection in badger populations has been demonstrated since 1976 in the UK (Cheeseman et al., 1989). Badgers can survive for several years after infection, in some cases suffering from very severe clinical disease involving the lungs and other organs when the disease generalises (Gallagher and Clifton-Hadley, 2000). Excretion of M. bovis by tuberculous badgers constitutes an epidemiological threat for other badgers, and a potential source of infection for cattle. BCG vaccine has been administered widely to humans in efforts to control the spread of tuberculosis (Fine, 1995), and has been delivered to badgers (Stuart et al., 1988; Southey et al., 2001) and other animal species under experimental conditions, albeit with variable efficacy (Fine, 1995; Suazo et al., 2003). BCG vaccination has recently been reported to provide some degree of protection against M. bovis infection to feral possums in New Zealand (Corner et al., 2002b). Therefore, BCG is considered the leading candidate for the control of M. bovis infection in badger populations. The Danish strain 1331 of BCG, produced by the SSI was chosen for this study, being the only strain currently licensed for use in humans in the UK and produced to Good Manufacturing Practice. We could find no reports of BCG being intentionally administered i.m., probably because in humans it is considered too painful (Nicoll and Hesby, 2002). However, unpublished studies by us in a guinea pig M. bovis challenge model (Chambers et al., 2001) suggest it to be well tolerated and an effective route of vaccination, just as i.v. and s.c. vaccination with BCG have been demonstrated to induce similar levels of memory Tcell expansion and protection in mice (Palendira et al., 2002). The i.m. route of vaccine administration was chosen in this study for two reasons. First, to determine the safety of BCG administered via this route should a s.c. inoculation be misadministered, and second, to compare the immune response following both routes of administration. An ATC application requires the safety of an overdose and a repeat dose to be assessed for each route of possible administration. Therefore, the experimental work was designed to make maximum use of the badgers available. The ‘dose’ administered s.c. was the dose of BCG administered to adult humans (2–8  105 CFU).

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An ‘overdose’ is defined as at least 10 the expected dose to be used in the field. The overdose in this study ranged between 5.16  107 and 2.17  108 CFU, and was the highest dose that could be obtained from a vial of BCG ‘‘Culture’’; normally used for the immunotherapy of superficial bladder cancer (Ovesen et al., 1993). The culture results from clinical samples indicate that the risk of shedding is minimal when BCG is administered to badgers via either the s.c. or i.m. routes. Control badgers occupied the same living space as vaccinated badgers, and all eight badgers were frequently observed to sleep together where physical contact was prolonged. Under these conditions (that mimic the situation in the wild), the absence of vaccine spread is supported by the lack of an IFNg or LTA response to PPD-B in control badgers, compared with that observed in the vaccinated badgers. The fluctuation in the weights of the badgers during the course of the study were consistent with seasonal weight gains and losses usually seen in badgers (Clifton-Hadley et al., 1993; Neal and Cheeseman, 1996). The weight of the vaccinated badgers followed a similar pattern in this respect to the two control badgers, indicating that BCG vaccine did not affect the feeding behaviour of the vaccinated badgers. Although body temperature fluctuated throughout the study, vaccinated badgers behaved indistinguishably from control animals. Given their s.c. location, it is likely that the microchips were more responsive to reductions in local skin temperature, e.g. caused by the animal lying in close contact with the ground without a layer of insulating bedding material. This is the likely explanation for why body temperature was occasionally observed to drop below 30.0 8C. Nonetheless, temperature readings obtained with the microchips correlated well with rectal temperature, as previously reported for the microchips used in this study (Cilia et al., 1998). Haematological and blood chemistry values obtained for vaccinated badgers stayed within the defined reference ranges on most occasions, with no apparent influence of inoculation route or dose. Our findings are consistent with results of a study in which 11 captive badgers were injected intradermally with 106 CFU BCG Glaxo and haematological results did not differ between control and vaccinated animals

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(Mahmood, 1985). The observed increases in PAL could be related to high levels of cholesterol occurring during the autumnal period of weight increase (Domingo-Roura et al., 2001). Non-pathological hypercholesterolaemia and hyperlipidaemia have been associated with cholestasis, at least in rodents (Smith et al., 1998), and cholestasis can result in elevated levels of plasma PAL in carnivores (Meyer and Harvey, 1998). In support of this hypothesis, the levels of PAL in the study badgers correlated with their body weight (r2 = 0.30, p < 0.0001, by F-test), whereas ALAT levels did not. The high levels of PAL were not observed previously in these badgers. However, they might have occurred in previous years and been missed because of irregular access to badgers; a situation that was resolved for this study. One of the control badgers (A8) presented with markedly elevated PAL values throughout the study, as well as increased ALAT levels and transitory high GGT levels. Taken together, these indicate hepatobiliary disruption in this animal, common in aged animals. No other significant clinical signs or weight loss were observed in this animal. The excellent safety profile of BCG for humans is evidenced by the low number of adverse reactions observed (Lotte et al., 1988) from an estimated administration of over 3 billion doses worldwide, spanning over 80 years (Raviglione et al., 1995). This was confirmed in a recent prospective study carried out between 1997 and 1999 in 9763 South African new-borns vaccinated intradermally with BCG Danish (Jeena et al., 2001). Our results indicate that the BCG Danish vaccine is also safe to badgers when administered via the s.c. or i.m. routes. The usual route of BCG injection in humans is intradermal or percutaneous and is normally followed by local inflammatory reactions, such as erythema, and the formation of a papule, ulcer, and scar occurring between 8 and 16 weeks, depending on the method of vaccination (Brewer et al., 1994; Myint et al., 1985; Rosenthal, 1980). The formation of a granuloma at the site of vaccination, even up to 20 months after administration, would not appear to be uncommon (Gormsen, 1955). The local reactions seen in this study to BCG administration were dependent on the dose and route administered. The size of local papules produced in response to s.c. administration of the low (human) dose of BCG were in the range reported in

children after intradermal administration of fresh BCG Danish, which ranged between 5.7 and 8.7 mm, and decreased in size from 12 weeks after administration (Irvine and Barr, 1960). The larger and persistent local reactions observed in the badgers following the first administration of BCG is a reflection of the unusually high dose used. It has also been observed in humans that the local tissue response is more severe when BCG vaccine is inoculated s.c. than when given intracutaneously (Ustvedt, 1956). When adverse reactions are observed in humans they usually take the form of a local abscess and/or regional lymphadenopathy. In rare cases, musculoskeletal lesions, or nonfatal or fatal disseminated lesions have been reported (Lotte et al., 1988). The skin biopsy taken from the badger with a papule still present 371 days after s.c. inoculation, revealed considerable granulomatous inflammation, including some necrosis, and numerous acid-fast bacilli at the site of injection. The high number of bacilli present and the fact that the lesion was not contained by fibrosis, suggest that dissemination of BCG within this animal was a possibility. However, there was no evidence of excretion of BCG by this, or any other, vaccinated badger. In a comparable study in mice (Acosta et al., 1994), the s.c. injection of 106 BCG Moreau resulted in localised granuloma formation harbouring large numbers of bacilli that persisted for the duration of the study (42 days). The authors described the reaction as ‘‘intense’’, however they could find no evidence of BCG dissemination, despite submitting numerous organs to culture. Vaccination of badgers was associated with the production of IFNg and the proliferation of lymphocytes in the LTA within 13 days of injection of the high dose of BCG. These persisted longer in the s.c. group, but a positive response was still detected by both assays in the i.m. group for 34 days post-vaccination. These responses are the highest observed to-date in badgers vaccinated with BCG. For example, the LTA responses of badgers vaccinated with BCG Pasteur in the Republic of Ireland were low following s.c. administration of 5  104 CFU (Southey et al., 2001), and similarly with 3  105 CFU in a more recent study (E. Gormley, personal communication). This suggests the immune response to BCG in badgers is dose dependent, as observed in cattle and mice (Buddle et al., 1995; Power et al., 1998). However, the

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magnitude of the immune response post-vaccination may not correlate with protection, as doses of BCG ranging from 2  103 to 2  107 CFU provided a similar level of protection against M. tuberculosis challenge in mice while inducing dose-dependant immune responses (Gruppo and Orme, 2002). Ultimately, the efficacy of BCG vaccine for badgers will have to be determined empirically following experimental challenge with M. bovis. However, the difficulties and ethical constraints associated with experimental studies in badgers make it hard to address a number of fundamental questions regarding BCG vaccination, such as the optimum dose and route to use, as well as persistence of the vaccine in the host and the duration of protective immunity. Some of these questions are being addressed experimentally in the Republic of Ireland (E. Gormley, personal communication), including the duration of protection conferred by BCG. In mice, BCG given s.c. can persist in the spleen for up to 30 weeks, although persistence of the vaccine was not required for maintaining protection against M. tuberculosis in the lung, only in the spleen (Olsen et al., 2004; Aldwell et al., 2006). How these studies relate to badgers is unknown, although possums were still protected against M. bovis at least 12 months after i.n. vaccination with BCG, albeit to a lower degree than after 2 months (Corner et al., 2001). The protection conferred in experimental models by a wide range of BCG doses (Gruppo and Orme, 2002) give encouragement that protection may be conferred to badgers by a titre of BCG within the range used in this study. Annual vaccination of badgers may be adequate to maintain protection, within the practical constraints of delivering an injectable vaccine to wild animals. The small number of badgers available for this study did not allow independent injections of high and low doses of BCG, and instead a low dose of BCG was injected in animals already sensitised with the high dose of BCG (as prescribed by the VMD). Only minimal immunological responses were observed following the injection of the low dose of BCG. The most significant boosting response was observed by LTA in the group vaccinated by the s.c. route. In an earlier study, no peripheral LTA response was detected in badgers vaccinated twice with 5  104 CFU BCG; although a response was detected subsequently when the animals were further boosted with 5  105 CFU

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(Southey et al., 2001). Similar low responses to BCG boosting were shown in possums (Corner et al., 2002a) and white-tailed deer (Waters et al., 2004). The poor immunological response of badgers to low doses of BCG and the lack of a strong boosting effect on re-vaccination may be the result of prior sensitisation to environmental mycobacteria, such as M. avium (Brandt et al., 2002). In our experience, wild badgers can respond in the LTA and produce IFNg in response to stimulation with PPD-A. However, the badgers used in this study had low responses to PPD-A prior to vaccination (data not shown), suggesting an alternative explanation. This study has demonstrated the safety of BCG vaccine to badgers and supports the use of the i.m. route for vaccination: there was no adverse reaction to i.m. administration of BCG; the immune response to i.m. vaccination was equivalent to that obtained by the more conventional s.c. route; and i.m. administration of vaccine to wild badgers would be possible without the need for prior anaesthesia, whereas this would be required for s.c. administration.

Acknowledgements This work was funded by the Department for Environment, Food and Rural Affairs (Defra). The authors acknowledge the help of D. Clifford for veterinary advice and other staff from the Animal Services Unit (VLA) for the daily care of the badgers, the staff of the Histopathology Department (VLA), in particular J. Gough, and M. Ilott and M. SpagnuoloWeaver (VMD) for advice in the design of this study. All animal procedures in this study were reviewed by a local Animal Care and Ethics Committee and undertaken in accordance with the UK Animal (Scientific Procedures) Act 1986.

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