MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title The Potential of Ticks as Vectors of Mycobacterium bovis

MAFF project code SE3012

Contractor organisation Dept. Zoology and location Oxford University OX1 3PS

Total MAFF project costs £ 41,817.32

Project start date 02/08/00 Project end date 02/08/01

Executive summary (maximum 2 sides A4)

Published data from credible but obscure sources, coupled with evidence collected by scientists at VLA Starcross, strongly suggested that ticks, including the British Ixodids are capable of acquiring and transmitting Mycobacterium bovis by bite.

To investigate this further, transmission experiments were conducted to infect cohorts of larvae and nymphs of the ubiquitous British tick Ixodes ricinus with M. bovis by feeding them on experimentally infected guinea pigs (GPs). Post mortem and culture demonstrated tuberculosis in the experimentally infected GPs. However, three lines of evidence suggest that no transmission of M. bovis to ticks occurred: (i) that there was no identifiable infiltration of infected macrophages to the site of feeding; (ii) that culture of fed ticks shortly after feeding was negative for M. bovis; and (iii) culture of ticks post-moult was also negative for M. bovis.

A small follow-on transmission experiment was conducted against the possibility that M. bovis in ticks is in a viable but non-culturable state. A cohort of ticks fed on and to separate GPs resulted in no demonstrable tuberculosis in the GPs at post-mortem after 10 weeks.

Simultaneous with the laboratory experiments, ticks were collected from badgers from Woodchester Park, including culture and/or BrockTest ELISA positive badgers. Three species of tick were recovered totalling 393 nymphs and adults. In order of abundance they were I. canisuga, I. ricinus and I. hexagonus. Attempts to culture M. bovis from these ticks were negative.

We conclude that it is highly unlikely that British Ixodid ticks play a significant role in the transmission of M. bovis from badgers.

CSG 15 (Rev. 12/99) 1 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

Scientific report (maximum 20 sides A4)

Purpose

Based on the pathology of bovine tuberculosis in cattle, it is generally accepted that the majority of within-herd transmission of Mycobacterium bovis is by the aerosol route. However, there is still doubt as to the major route (or routes) by which herds initially acquire infection (known as herd breakdown). In the U.K., several species of wild animal have been found infected with M. bovis, of which badgers appear to be the major reservoir. The European badger, Meles meles, is known to forage for earthworms on cattle pasture where infected individuals can shed M. bovis in urine and sputum. It has been suggested that cattle feeding on pasture infected by discharge from badgers may therefore acquire infection, and in the 1980s a highly artificial experimental study showed that infected badgers could transmit M. bovis to cattle when kept in continual close proximity over several months.1 However, it is generally accepted that, though strongly suggestive, this evidence does not demonstrate causation.2 We proposed to test a second, not necessarily exclusive hypothesis, that the European tick Ixodes ricinus is competent to act as a vector for M. bovis. If proven, the implications for the epidemiology and control of M. bovis would be considerable, as discussed below. This constituted a strong case for MAFF funding. There is scattered empirical evidence that ticks fulfil most of the criteria necessary to be implicated as vectors (see next section). There is also the following theoretical evidence in support of this idea. Although many different species of animals, most of which act as hosts to this catholic tick, have been trapped and examined for M. bovis infection, relatively few are thought to be competent reservoir hosts because they show no evidence of disease or dissemination of the bacillus.3 Two features of the transmission of tick-borne bacteria, however, recently revealed by researchers in the Oxford Tick Research Group (OTRG), render this evidence unreliable. First, Lyme borreliosis spirochaetes are sequestered in the deep organs of their vertebrate hosts, from where, although not regularly detectable in the skin or peripheral blood, they can nevertheless infect their tick vectors as they feed.4 Secondly, hosts which do not develop systemic infections of Lyme spirochaetes (e.g. sheep), nevertheless permit transmission from infected to uninfected ticks co-feeding in close proximity on them.5 This transmission route alone is sufficient to sustain natural endemic cycles of Lyme borreliosis. We now understand that host species not previously thought capable of acting as reservoirs of pathogens, can in fact amplify the prevalence of infection in a tick population.5,6 Currently, bovine TB is controlled principally through focal culling of badgers in response to herd breakdown. This is labour intensive (and therefore expensive) and also both ethically and scientifically questionable. If tick mediation in herd breakdown were established, it would offer a better understanding of the spatial and temporal patterns of M. bovis infection, and therefore greater powers of prediction and strategic control planning. Ideally, an intervention against the putative tick vector, aimed either at cattle or badgers, would limit or even interrupt the rate of herd breakdown without the need for badger culling. The theoretical tools for such intervention are already being developed in the OTRG. A recently developed population model for the African tick vector of East Coast Fever, driven by simple climatological factors, has unprecedented power in predicting tick abundance in time and space.7,8,9 A similar model is currently being developed for I. ricinus. Epidemiological models show that the seasonal dynamics of ticks have the greatest quantitative impact on the force of infection of tick-borne diseases,10 and predictive tick population models offer the best tool for carefully targeted programmes of control.

Scientific Context

In Europe, the Eurasian badger, Meles meles, is thought to be the major wild animal reservoir of Mycobacterium bovis, agent of bovine tuberculosis. However, there is considerable uncertainty as to the principal route by which cattle herds initially acquire infection. Here we present empirical evidence and theoretical arguments which suggest that ticks may play a role in transmission from badger to cow.

CSG 15 (1/00) 2 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

Ticks typically pass through three life stages, from larva to nymph to adult. Each stage may last several weeks, or months, including an overwintering phase. A single bloodmeal lasting several days is taken at each stage, after which the tick drops from the host and finds a suitable spot for digestion, growth and moulting to the next stage. After moulting, the tick begins questing for a new host, usually by climbing vegetation, from which vantage it can attach itself to passing animals and re-feed. Three criteria must be satisfied if ticks are to act as vectors of M. bovis. Firstly, they must be capable of acquiring the bacillus when feeding. Secondly, since ticks feed only once per life stage infection must be supported transstadially. Thirdly, ticks must be able to transmit infection to a healthy host. Evidence that ticks can acquire infection when feeding comes from two separate sources. In 1995, researchers at VLA Starcross conducted a post-mortem on a badger diagnosed with acute tuberculous pneumonia (Brian Preece & J. Chanter, pers. comm.). The animal was heavily infested with hard ticks, identified as Ixodes hexagonus, known to parasitise both cattle and badgers.11 Smears prepared from two of the engorged ticks using Ziehl-Neelsen (ZN) stain showed large numbers of acid-fast bacilli within the blood meal. Two ticks were fixed in 10% formol saline solution, sectioned in wax and ZN stained to reveal acid-fast bacilli within the tick gut. A further six ticks, when homogenised and inoculated onto selective media, gave a very strong growth of Mycobacterium species, identified by growth rate and colony morphology as M. bovis. Unfortunately, MAFF did not commission spoligotyping, and no further material was collected for transmission experiments. The work was never published. In a much earlier piece of work, but one which has remained obscure to the Western literature, Russian researchers demonstrated that the soft tick Ornithodoros lahorensis acquired M. bovis when fed on experimentally infected guinea pigs.12 The Russian study went much further. It found that O. lahorensis could maintain its infection over several months. Critically, having maintained infected ticks transstadially, the Russian study then showed that infection could be induced in healthy guinea pigs by feeding them macerates of infected ticks, thus satisfying all of the biological criteria necessary for vector competence. A small number of active transmission experiments was also attempted, but without success. O. lahorensis is in the family Argasidae, while ticks of the genus Ixodes, including I. hexagonus and I. ricinus, that parasitise badgers and cattle in the UK, are members of the Ixodidae. Thus, although there is no evidence that the two families differ in ways which would affect their vector competence for M. bovis, it might be argued that they are not directly comparable. However, cockroaches (Blatta orientalis) that had fed on M. tuberculosis sputum samples shed viable bacilli for several months afterwards,13 suggesting that the ability to maintain Mycobacterium infection is widespread among the arthropoda. Additionally, the tick Argas persicus was successfully infected with Mycobacterium avium and M. tuberculosis.14 We concluded that there is a significant possibility that ticks are biologically capable of acting as vectors of M. bovis in the UK. Herd breakdown may be achieved by the dissemination of infected ticks from badgers. Once these ticks have moulted and begun questing for a new host, they might readily be ingested by browsing cattle or grooming badgers. The possibility that successful tick feeding on cattle might also induce infection should not be ruled out, but passive transmission alone could well be sufficient: an entire genus of blood parasites – Hepatozoon spp. – propagate entirely by this route and have a global distribution, including very high prevalence in Great Tits in the UK (>60%, D. Kelly, unpublished observations) where they are transmitted by mites.15 Very recently, tick-borne louping-ill virus has also been shown to infect grouse chicks via this passive route. Notwithstanding their obscurity, perhaps the main reason why the Russian and the MAFF studies have not been followed up is the absence of a credible theory for the transmission of infections from host to tick. Now, recent advances in the study of another tick-borne infection – Tick-Borne Encephalitis (TBE) virus – provide us with just such a theory. Unlike most vector-borne disease agents, M. bovis parasitises and thrives in macrophages, the vast majority of which are sequestered in the lymph nodes.16 At face value, the low and transient levels of M. bovis in peripheral blood make it unlikely that ectoparasites will acquire an infection. However, ticks are not typical arthropod vectors.10 They have relatively long periods of attachment and feeding on the host (ca.10 days for adult I.

3 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

hexagonus, 4 days for larvae and nymphs), accompanied by a strong host immune response to the tick's saliva.17 Like M. bovis, TBE virus infects and multiplies in the host's immune cells. During the immune response to tick feeding, infected lymphocytes migrate from the lymph nodes to the feeding site,18 delivering infected cells to the tick. Areas of skin without ticks have no detectable virus and the presence or absence of viraemia has no effect on infectiousness.19,20 Such an immune route for M. bovis transmission would explain both the known absence of M. bovis from transiently feeding ectoparasites of badgers such as fleas and lice,21 and the observed presence of M. bovis in the ticks I. hexagonus and O. lahorensis taken from infected hosts. If tick-mediated infection of cattle were established as a route of dissemination of M. bovis, it would have a significant impact on the understanding of spatial and temporal patterns of M. bovis infection, and therefore on the design of strategic control. Transmission cycles of tick-borne parasites depend crucially on geographically variable patterns of seasonal tick population dynamics.10,22 Furthermore, it might be possible to limit the rate of herd breakdown by targeting intervention against the putative tick vector. Clearly, we needed to reproduce the results of Postoian and Agabalov using the Ixodes spp. ticks which commonly parasitise badgers and cattle, as a critical first step to teasing out the role of ticks as vectors of M. bovis. Correlative epidemiological studies alone cannot confirm the role of ticks: alternative explanations for apparent relationships can almost always be adduced.

Scientific Objectives

The key scientific objectives of the proposed research were:

(1) To test whether the tick Ixodes ricinus is competent to transmit Mycobacterium bovis between guinea pigs.

Specifically, arising from the foregoing discussion, two ‘transmission’ routes were to be investigated concurrently, each with a number of associated questions:

A. Host-Tick-Host Transmission

(i) Can I. ricinus acquire M. bovis from infected hosts? (ii) Which life stages – larvae and/or nymphs - are capable of acquiring and transmitting infection? (iii) Is the probability of transmission from host to tick correlated with the intensity of bacteraemia or pattern of dissemination to other organs in the host? (iv) Is the density of infected macrophages in the host skin elevated around the feeding site? (v) Does M. bovis disseminate to the tick haemocoel and salivary glands? (vi) Do infected I. ricinus shed M. bovis into the environment? Over what time period? (vii) Can M. bovis infection in I. ricinus be maintained transstadially? (viii) Can infected I. ricinus transmit M. bovis to uninfected hosts by their bite? (ix) Can infected I. ricinus transmit M. bovis when ingested?

B. Co-feeding & Tick-tick Transmission – non-systemic infections in the host

(i) Can a tick become infected by co-feeding with an infected tick on an uninfected host? (ii) Does the probability that an uninfected tick becomes infected through co-feeding decrease with distance from the infected tick?

4 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

(2) To test whether ticks can acquire M. bovis from infected badgers and transmit infection to guinea pigs.

Specifically, we asked a subset of the questions in ‘1A’, limited by the practicalities of tick acquisition from badgers:

A. Transmission from Badgers

(i) What is the background prevalence of infection of unfed ticks removed from badgers? (ii) Can Ixodes spp. acquire M. bovis from infected badgers? (iii) Which life stages – larvae, nymphs and/or adults - are capable of acquiring and/or transmitting infection? (iv) Is the probability of transmission from badger to tick correlated with the intensity of bacteraemia or pattern of dissemination to other organs in the host? (v) Can M. bovis infection in Ixodes spp. be maintained transstadially? (vi) Does M. bovis disseminate to the tick haemocoel and salivary glands? (vii) Can infected Ixodes spp. transmit M. bovis to uninfected GPs by their bite? (viii) Can infected Ixodes spp. transmit M. bovis when ingested by GPs?

Materials and Methods

Project Overview

Two lines of investigation addressed the question of whether ticks are competent to transmit M. bovis. The first was a detailed experimental study using artificial infections in GPs (objective 1 above). The laboratory GP model has been used successfully to achieve transmission of M. bovis with O. lahorensis.12 GPs are routinely and readily infected by VLA Weybridge to produce large numbers of infected macrophages23 and are known to support co-feeding transmission of tick-borne viruses.24 Equally importantly, they do not mount so strong an immune response to feeding ticks that the tick is prevented from engorging fully.25 They were therefore our ‘gold standard’ with which to test the possibility of tick-borne transmission.

Work from artificial infections in the controlled conditions of the laboratory allowed us to ask carefully controlled and detailed questions about the existence of mechanisms of acquisition (co-feeding; macrophage migration to the feeding site) which would not have been possible with naturally feeding ticks from field material. The large number of ticks which we used also allowed us to sacrifice some at intermediate stages in the transmission cycle to investigate the progress of infection in the tick (maintenance of infection transstadially; bacterial invasion of the haemocoel and salivary glands).

The second approach exploited material generated from the live-trapping programme at Woodchester Park (objective 2 above). Ticks –I. hexagonus, I. canisuga and I. ricinus – could be recovered from badgers as part of the routine trapping programme. Observations of transmission from badgers to ticks constitute the most natural possible system without involving deliberate infection of badgers. To this extent they give strong support to the findings from the GP model.

Experimental Facilities & Study Timing

All experimental work and the maintenance of infectious material (GPs, ticks and bacterial cultures) was carried out in Category 3 containment facilities at VLA Weybridge. ‘Week 1’ (inoculation of source GPs) began 2nd August 2000, when animal licenses were finally obtained.

5 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

Artificial Guinea Pig Infections, Culture Strain & Post-Mortem

Artificial infections were produced in outbred GPs by injection of 100 colony forming units (CFU) of Mycobacterium bovis, intramuscularly, to produce disseminated tuberculosis within 3-4 weeks. The VLA Weybridge challenge strain was 7449/97 (spoligotype 9) isolated from a badger. Post-mortem was conducted by Dr. Mark Chambers of VLA Weybridge. Post-mortem data were compiled in compliance with the standard VLA Weybridge pro-forma.

Material taken post mortem:

1. Blood by cardiac puncture. 2. Two explants of full-thickness skin from each guinea pig – one from the site of tick feeding and one from a neighbouring site where ticks had not fed. 3. Lymph node(s) draining the site of tick feeding – principally the accessory axillary node. 4. Spleen.

Processing of material:

1. Whole blood plated on Middlebrook 7H10 medium with supplements (pyruvate and OADC). 2. Half of each skin explant homogenised and plated as for blood. Remaining half of explant placed in formalin. Following fixation, the tissue was sectioned, stained with H&E and ZN and examined for the presence of inflammation and acid-fast bacilli (AFB). 3. Lymph nodes and spleen were homogenised separately and plated as for blood for quantitation of the bacterial load.

Tick Culture & Feeding

Primary cultures of I. ricinus were previously established from field collections of questing ticks by blanket dragging at the field site of Wimborne St. Giles, Dorset, regularly used by us as a source of Ixodes ricinus.4, 26 This material was added to over the project, and used to establish cultures to provide uninfected larvae and nymphs.

Ticks were fed on individual host animals using neoprene feeding chambers. The chambers were made of 1cm diameter neoprene rings covered with gauze and glued to the flank of each GP (after Labuda et al.19) and used successfully by OTRG. 5, 26,

Potentially infected ticks were handled with entomological forceps and brushes and maintained on the bench in the Category 3 facilities at VLA Weybridge, at 19-210C, 85% relative humidity, in individual culture vessels in an air-tight glass cabinet (dessicator), further isolated with an oil trap.

Mycobacterium bovis Culture & Enumeration

On advice from VLA Weybridge, we used the more sensitive culture technique, rather than PCR, to follow the course of infections in GPs and ticks. Critically, this also allowed us to confirm or reject the presence of viable bacteria in the ticks, rather than DNA contamination.

6 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

Standard VLA Weybridge culture protocols were followed, using Middlebrook’s 7H10 solid medium plates plus OADC supplement and pyruvate. To enumerate bacterial load in GP organs and ticks the tissue was first homogenised in sterile water, then serially diluted in sterile water plus 0.05% Tween-80 to avoid bacterial clumping.

Tick Collection from Badgers

The original plan was to have obtained ticks from badgers at VLA Starcross, as part of the on-going culling trial. A brief synopsis of this work is presented below. At Starcross 196 badgers were examined, only 50 of these yielded ticks (26%) and only 219 ticks were collected in total. Order of abundance I. canisuga (202), I. ricinus (15) and I. hexagonus (2). Only 7 of the badgers (14%) from which the ticks were collected were mycobacterium positive by culture:-

? 4 of the badgers (8%) were M. bovis positive by culture and yielded only 14 ticks, ? 3 of the badgers (6%) were M. avium positive by culture and yielded only 33 ticks.

Of the ticks from the M. bovis positive badgers, 2 sets had died before the culture result of the badgers was known, a further set of only 2 nymphs also sufferred one death. These ticks were cultured and were all negative. The last set of ticks from an M. bovis positive badger consisted of 2 live adult female I. ricinus and 2 I. canisuga nymphs, one of which was dead. The two I. ricinus ticks were cultured together and were negative. The dead nymph was cultured, also negative. The live nymph was put up to moult 23/10/00 and still had not moulted by 30/01/01. Later found dead, not cultured. Of the ticks from the M. avium positive badgers, 1 set consisted of only 1 dead nymph which was cultured and found to be negative. The remaining 2 sets contained many live nymphs which were put up to moult. A few of these did moult and cultures of dead nymphs and newly emerged adults were negative.

Therefore it can be seen that only a very limited number of suitable ticks were available for this experiment. This sparsity of cull material throughout 2000 led us to seek an alternative source of badgers. We are very grateful to Dr. Chris Cheeseman and the staff of the Wildlife Diseases Unit at Woodchester Park for their generous cooperation in providing alternative material. An intensive live trapping programme is conducted annually on the badger population at Woodchester. A range of samples and fitness measures are taken on each badger, including assay for M. bovis. Thus the current and past TB status of each badger is known. We collected live nymphs and adult ticks from anaesthetised badgers using a Trix? tick remover (InnoTech, Sweden). Larval ticks were too small to be removed by this technique.

Research Plan

1A. Host-tick-host Transmission & Transstadial Transmission The basic experimental design paralleled that of Labuda et al.19 and Ogden et al.5 Six cohorts of I. ricinus were fed on six GPs that had been inoculated with M. bovis 3-4 weeks earlier. After feeding to repletion and detaching from the host (approximately 4 days), all engorged ticks were held in separate culture vessels. Immediately after tick detachment, two skin samples were taken from each infected GP: at the site of, and immediately adjacent to, the feeding chamber. The presence of M. bovis from each sample was assayed by culture and histology to test for recruitment of M. bovis to the site of tick feeding (question 1Aiv, above). Cardiac blood was taken by syringe post mortem and each animal examined for the form and intensity of infection. Spleen and lymph node(s) draining the site of tick feeding were obtained for culture.

7 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

After one week (pre-moult), 25% of ticks fed on each host were macerated and cultured for the presence of M. bovis to test for transmission of M. bovis from host to tick (1Ai). The infection rate in ticks was to be correlated with level and pattern of infection in the source GPs (Aiii). At monthly intervals, the culture vessels from a sample of individuals from each tick group were swabbed and cultured to test whether ticks were shedding viable M. bovis (1Aiv). After six months, when all surviving ticks had moulted, the remaining ticks were split into two groups. One group was allowed to re-feed on a healthy tuberculosis-free GP. The other group of ticks was pooled and macerated and fed to uninfected GPs, to test for the potential for tick-borne oral transmission (1Aix). These GPs were held and monitored for weight loss for 10 weeks, after which they were sacrificed, post-mortemed and cultured as before to test for bite-transmission of M. bovis (1Aviii). One group of ticks was assayed for M. bovis abundance to test for transstadial transmission (1Avii). In order to assess the vectorial capacity of each life stage (1Aii), the experiment was run with starting cohorts of larval and nymphal I. ricinus with a view to yielding approximately 10 nymphs and 5 adult females respectively for re-feeding.

1B. Co-feeding & Tick-Tick Transmission Were infected ticks generated, twelve groups of ten uninfected nymphal I. ricinus were to have been restrained in a feeding chamber and each group fed on the left flank of a separate, uninfected GP. Each group would also contain a pair of adult I. ricinus infected by feeding on artificially infected GPs. For five of the ten procedures, a second feeding chamber containing five uninfected nymphs would have been placed on the right flank of each GP. After engorgement and detachment from the host, ticks would have been held for 2 weeks and then sacrificed and cultured for the presence of M. bovis, in order to assess whether infection can be transmitted by co-feeding (1Bi), and whether the probability of this decreases with distance from the infected ticks (1Bii), as previously described for other tick-borne pathogens.

2A. Transmission from Badgers All ticks collected from badgers at Woodchester Park were categorised by species, age and size of bloodmeal, for correlation with infectious status. Collected ticks were macerated and cultured for the presence of M. bovis (2Ai-iv). The terms of the animal license required us to demonstrate infection in initial collections, prior to conducting transmission experiments to GPs using such ticks (2Av-viii).

Results & Discussion

1A. Host-tick-host Transmission & Transstadial Transmission

The laboratory component of the project encountered early delays as a result of new internal procedures at VLA Weybridge for ethical clearance of animal work, but otherwise ran smoothly.

Tuberculosis was generated in all source GPs. All guinea pigs had lesions visible in their spleen post mortem, typical of tuberculosis. Consistent with the gross appearance, all spleens yielded variable amounts of M. bovis on culture:

Animal # 1 2 3 4 5 6 7 8 9 10 11 Total CFU: 1256 37 7 640 196 784 70 216 274 1376 664

8 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

Only one guinea pig (#11) yielded a positive culture from the lymph node (2752 CFU).

Bacterial cells from the lymph node culture and from the spleen culture of animal #9 were spoligotyped and found to have the pattern consistent with M. bovis strain 7449/97.

The overall percentage of fed nymphs and larval ticks recovered were 68% and 49% respectively. There was no evidence of M. bovis in GP skin samples by culture and no evidence of AFB by histology. Histology sections taken through an attached tick were also negative for AFB. A large amount of excreted bloodmeal was deposited on the skin within the feeding chambers. Culture of this material was also negative for M. bovis. Ticks that had died within the feeding chambers, all well fed and presumably dead from dehydration, were also cultured with negative results.

There was no evidence of M. bovis in the fed ticks, by culture, either pre- or post-moult, or from the swabs taken in the tick culture vessels. A small number of culture samples did produce colonies that resembled M. bovis by growth rate and morphology. However, when typed, these proved to be Candida tropicalis. Otherwise, negligible fungal growth or extraneous bacterial contamination was observed, such that any M. bovis present in the samples would have been free to grow unhindered.

As agreed in the revised animal license procedure for this work, in the absence of evidence for transmission from GPs to ticks, the transmission attempt from ticks to GPs was scaled back. A small transmission experiment was, nevertheless, thought worthwhile, allowing for the possibility that M. bovis in the tick was viable but non-culturable, a phenomenon occasionally seen for this organism in other circumstances. Thus a single cohort of ticks was fed on a healthy GP and another was fed to a healthy GP.

Animals showed no clinical signs of tuberculosis (e.g. weight loss) throughout the 10 week period and were subsequently killed by peritoneal overdose of sodium pentobarbitone (Euthatal, Rhone Merieux). Examination was carried out immediately after death. External assessment of body condition was followed by gross internal examination of the neck region, and thoracic and abdominal cavities. In particular, the lungs, spleen and liver were carefully examined for signs of gross lesions.

No gross abnormalities were observed in any organ or tissue. There was no evidence to suggest any animal had contracted tuberculosis. Culture results were negative.

1B. Co-feeding & Tick-Tick Transmission

This phase of the laboratory transmission study could not be conducted, since no infected ticks were generated.

2A. Transmission from Badgers

The original intention was to collect ticks from badgers sent to VLA Starcross for post-mortem, generated primarily from the cull experiments. Unfortunately, the culling work was severely disrupted through the summer of 2000, and negligible numbers of ticks were collected. For this reason, we are extremely grateful to Dr. Chris Cheeseman and the staff of the Central Science Laboratories Wildlife Disease Unit at Woodchester Park for allowing us to collect ticks from the badgers routinely trapped at their study site.

Three sampling sessions were conducted on 25/05/00, 4/07/00 and 5/07/00. A total of 393 ticks comprising three species were collected from 86 badgers: Ixodes canisuga (known as the ‘fox tick’), I. ricinus (known as the ‘common sheep tick’) and I. hexagonus (known as the ‘hedgehog tick’), all identified according to Hillyard

9 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

(1996 The Ticks of North-West Europe. Field Studies Council, UK). The number of ticks collected for each species and life stage are shown in the table below:

No. Adult No. Adult No. Nymphal Tick Species Female Ticks Male Ticks Ticks

I. canisuga 133 2 234 I. ricinus 10 1 1 I. hexagonus 5 0 7

Thirteen (15%) of badgers had no detectable infestation. The remainder had nymphs and adult females widely distributed, most commonly attached between the toes of fore and hind limbs, on the ears and muzzle, and the axillary and inguinal regions, genitals and anus. Ticks were frequently seen, but at lower density, on the chest and abdomen and on the limbs. Hair clipping also revealed ticks attached at low density under the coat of the back and flanks. Free-roaming males were rarely encountered.

At the May sampling session, all ticks recovered were I. canisuga, and this species continued to be by far the most abundant, and appears to be the primary tick infesting badgers, as reported by Hillyard (1996). I. ricinus always proved extremely difficult to remove, and all bar one adult were killed in the process. Also, many adult I. ricinus were found dead on the badger – possibly immune-mediated.

No ticks collected from the badgers at Woodchester Park were culture positive for M. bovis, despite the fact that 14 of the badgers had a history of M. bovis infection (ELISA positive, culture positive, or both). These 14 badgers gave: 27 adult females and 67 nymphs of I. canisuga; 2 adult females and 3 nymphs of I. hexagonus; and 3 adult females and 1 nymph of I. ricinus. Negligible fungal growth or extraneous bacterial contamination was observed, such that any M. bovis present in the samples would have been free to grow unhindered.

Given the lack of culture positive ticks collected from badgers, it was not possible to conduct transmission experiments to GPs.

Conclusion

We conclude that it is highly unlikely that British Ixodid ticks play a significant role in the transmission of M. bovis from badgers.

References

1. Little, T.W.A., Naylor, P.F. & Wilesmith, J.W. (1982) Laboratory study of Mycobacterium bovis infection in badgers and calves. Veterinary Record 111, 550 2. Krebs, J.R., Anderson, R.M., Clutton-Brock T., Donnelly, C.A., Frost, S., Morrison, W.I., Woodroffe, R. & Young D. (1998) Badgers and bovine TB: conflicts between conservation and health. Science 279, 817-818. 3. Hardie, R.M. & Watson, J.M. (1992) Mycobacterium bovis in England and Wales: Past, present and future. Epidemiology & Infection 109, 23-33. 4. Kurtenbach, K., Peacey, M.F., Rijpkema, S.G.T., Hoodless, A.N., Nuttall, P.A. & Randolph, S.E. (1998) Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England. Applied and Environmental Microbiology 64, 1169-1174. 5. Ogden, N.H., Nuttall, P.A. & Randolph, S.E (1997) Natural Lyme disease cycles maintained via sheep by co-feeding ticks. Parasitology 115, 591-599.

10 Project The Potential of Ticks as Vectors of Mycobacterium bovis MAFF SE3012 title project code

6. Randolph, S.E., Gern, L. & Nuttall, P.A. (1996) Co-feeding ticks: epidemiological significance for tick-borne pathogen transmission. Parasitology Today 12, 472-479. 7. Randolph, S.E. (1994) Population dynamics and density-dependent seasonal mortality indices of the tick Rhipicephalus appendiculatus in eastern and southern Africa. Medical and Veterinary Entomology 8, 351-368. 8. Randolph, S.E. (1997) Abiotic and biotic determinants of the seasonal dynamics of the tick Rhipicephalus appendiculatus in South Africa Medical and Veterinary Entomology 11, 25-37. 9. Randolph, S.E. & Rogers, D.J. (1997) A generic population model for the African tick Rhipicephalus appendiculatus. Parasitology 115, 265-279. 10. Randolph, S.E. (1998) Ticks are not insects: consequences of contrasting vector biology for transmission potential. Parasitology Today 14, 186-192. 11. Arthur, D.R. (1953) The host relationship of Ixodes hexagonus Leach in Britain. Parasitology 43, 227-238. 12. Postoyan, S.R. & Agabalov, G.P. (1971) Notes on the role of Ornithodoros lahorensis Neumann in preserving mycobacteria of tuberculosis and transmitting them from diseased to healthy animals. Academia de Ciencias de Cuba – Biologica 34, 9-12. 13. Allen, B.W. (1987) Excretion of viable tubercle bacilli by Blatta orientalis (the oriental cockroach) following ingestion of heat- fixed sputum smears: a laboratory investigation. Transactions of the Royals Society of Tropical Medicine & Hygiene 81, 98-99. 14. Blagodarnyi, Ya.A., Blekhman, I.M., Lepyko, V.N., Larin, S.A. & Yakunin, M.P. (1971) The role of ticks in the transmission of the mycobacteria of tuberculosis. Veterinariya 7, 48-49. 15. Smith-Todd, G. (1996) The genus Hepatozoon (Apicomplexa: Adeleina). Journal of Parasitology 82, 565-585. 16. Fanning, E.A. (1994) Mycobacterium bovis infection in animals and humans. In: Clinical Tuberculosis (Ed. P.D.O. Davies). Chapman & Hall, London. 17. Wikel, S.K. (1996) The Immunology of Host-Ectoparasitic Arthropod Relationships. CABI, Oxon. 18. Nuttall, P.A. (1998) Displaced tick-parasite interactions at the host interface. Parasitology 116, S65-S72. 19. Labuda, M., Nuttall, P.A., Kozuch, O., Eleckova, E., Williams, T., Zuffova, E. & Sabo, A. (1993) Non-viraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature. Experientia 49, 802-805. 20. Labuda, M., Austyn, J.M., Zuffova, E., Kozuch, O., Fuchsberger, N., Lysy, J. & Nuttall, P.A. (1996) Importance of localised skin infection in tick-borne encephalitis virus transmission. Virology 219, 357-366. 21. Barrow, P.A. & Gallagher, J. (1981) Aspects of the epidemiology of bovine tuberculosis in badgers and cattle. I. The prevalence of infection in two wild animal populations in south-west England. Journal of Hygiene 86, 237-245. 22. Randolph, S.E., Miklisová, D., Lysy, J., Rogers, D.J. & Labuda, M. (1999) Incidence from coincidence: patterns of tick infestations on rodents facilitate transmission of tick-borne encephalitis virus. Parasitology 118, 177-186 23. Smith, D.W. & Wiegeshaus, E.H. (1989). What animal models can teach us about the pathogenesis of tuberculosis in humans. Review of Infectious Diseases 11, S385-393. 24. Jones, L.D., Davies, C.R., Steele, G.M. & Nuttall, P.A. (1987) A novel mode of arbovirus transmission involving a nonviraemic host. Science 237, 775-777. 25. Jones, L.D., Davies, C.R., Steele, G.M. & Nuttall, P.A. (1988) The rearing and maintenance of ixodid and argasid ticks in the laboratory. Animal Technology 39, 99-106. 26. Kurtenbach, K., Carey, D., Hoodless, A.N., Nuttall, P.A. & Randolph, S.E. (1998) Competence of pheasants as reservoirs for Lyme disease spirochetes. Journal of Medical Entomology 35, 77-81.

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