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Papers & Articles Genotyping of Mycobacterium bovis isolates from badgers in four areas of the Republic of Ireland by restriction fragment length polymorphism analysis E. Costello, O. Flynn, F. Quigley, D. O’Grady, J. Griffin, T. Clegg, G. McGrath An analysis of the molecular epidemiology of Mycobacterium bovis in badgers was made in four selected areas of the Republic of Ireland in which an intensive badger removal programme was being carried out over a period of five years. Tissue samples from 2310 badgers were cultured. Restriction fragment length polymorphism (RFLP) analysis with IS6110, polymorphic GC-rich sequence (PGRS) and direct repeat sequence (DR) probes was applied to the isolates from 398 badgers, and 52 different RFLP types were identified. Most of the isolates belonged to seven predominant types, and the other 45 types were represented by few isolates. An analysis suggests that some of these 45 types may have been introduced by the inward migration of badgers and others may have been the result of genetic changes to one of the prevalent types. The badgers were divided into groups on the basis of the sett at which they were captured, and RFLP typing was applied to isolates from two or more badgers from 85 groups. Multiple RFLP types were identified among isolates from 50 of these groups, suggesting that badgers probably moved frequently between group territories.

Veterinary Record (2006) 159, 619-623 E. Costello, MVB, MVM, MRCVS, O. Flynn, MPhil, F. Quigley, FIBMS, D. O’Grady, MSc, Central Veterinary Research Laboratory, Abbotstown, Dublin 15, Republic of Ireland J. Griffin, MVB, MSc(Edin), MSc(Gu), PhD, MRCVS, T. Clegg, BSc, MSc, G. McGrath, BA(Mod), MSc, Centre for Veterinary Epidemiology and Risk Analysis, Faculty of Veterinary Medicine, University College Dublin, Bellfield, Dublin 4, Republic of Ireland

Mycobacterium bovis has a wide host range and has become established in wildlife populations in several countries (O’Reilly and Daborn 1995). The epidemiology of the disease in wildlife may be influenced by factors such as the range of species affected, its pathogenesis and methods of transmission, the survival of the organism in the environment and the ecology of its hosts (Morris and others 1994). The DNA fingerprinting of strains has been widely used to study the dynamics of tuberculosis in wildlife and to investigate possible links between infections in farmed and wild species (Skuce and Neill 2001, Haddad and others 2004). The genotyping of 452 isolates of M bovis by restriction fragment length polymorphism (RFLP) analysis in the Republic of Ireland revealed that the most prevalent RFLP types were widely distributed and present in both cattle and badgers (Costello and others 1999). M bovis-infected badgers are considered to be a factor in the persistence of tuberculosis in cattle in the Republic of Ireland (Gormley and Collins 2000). In 1997, a five-year study of the epidemiological relationships between the infection of cattle and badgers was begun in four areas (Griffin and others 2005), the major objective of which was to assess the effect of the removal of badgers on the incidence of the infection in cattle. An intensive culling programme was started to try to remove as many badgers as possible. Tissues from 98 per cent of the culled badgers were cultured for M bovis and RFLP typing was applied to 92 per cent of the isolates. Lesions from cattle herds in the four areas were also cultured and the isolates were genotyped, providing an opportunity for a detailed study of the molecular epidemiology of the disease in both species. The relationships between the strains isolated from cattle and badgers in these areas and the extent to which the strains clustered have been described by Olea-Popelka and others (2005). The same range of RFLP types was common to the isolates from cattle and badgers, but no spatial clusters common to both species were found. There was only limited spatial clustering of strains within each species. It was suggested that the low level of spatial clustering may have been due to badgers moving over long distances. This paper describes the differentiation of the strains of M bovis isolated from the badgers in the four areas by RFLP The Veterinary Record, November 4, 2006

typing and discusses the evolutionary relationships among the RFLP types. Badgers have a social structure and usually live in territorial groups; the badgers were divided into groups on the basis of the setts at which they were captured, and the distribution of RFLP types within these groups during the five years of the study was investigated. MATERIALS AND METHODS Study areas The four areas were located in counties Cork, Kilkenny, Monaghan and Donegal, and were 307 km2, 313 km2, 368 km2 and 226 km2 in area respectively. They corresponded to the removal and buffer areas described by Griffin and others (2005). For the most part they consisted of pasture land, with the exception of the area in Donegal, where two-thirds was mountain and moorland. The main criterion for selecting the areas was a higher-than-average prevalence of tuberculosis in the cattle populations in them. Badger removal A detailed survey of badger setts was carried out and their location was recorded on a custom-designed geographical information system (Griffin and others 2005). The badgers were captured by using multistrand wire restraints which were positioned close to each of the setts and normally left in position for 11 consecutive nights on two or three occasions each year during the five years of the study (Griffin and others 2005). Arrangement of the badger population into a group structure It was not feasible to identify and map the badgers’ social group territories in the large areas, and they were therefore assigned to groups on the basis of the sett at which they were captured; these were termed sett-associated groups. Postmortem examination The skin of each badger was examined for evidence of bite wounds. The prescapular, popliteal, retropharyngeal,


Papers & Articles

TABLE 1: Numbers of isolates of different restriction fragment length polymorphism (RFLP) types identified in Mycobacterium bovis strains isolated from badgers in Cork in successive years of the study RFLP type C1H1J A1A3A A2A1B A1A1A C1H7J B2A3Y A2A2B A1B1D A1L2D A1A5A A1B3D

Other*

1

2

Year 3

48 34 6 5 5 2 2

25 16 6 1

6 4 3 1

2 1 1

1

2 2 1 3

1 1 2

4

5

Total

5 3

3 1 1

87 58 16 8 5 5 3 3 2 2 2 8

1

1

2

1

TABLE 2: Numbers of isolates of different restriction fragment length polymorphism (RFLP) types identified in Mycobacterium bovis strains isolated from badgers in Monaghan in successive years of the study RFLP type B1C1C A1A1A A1E2A A1A5A B1C3C B1C2C D2C1L A1T3A B1C1R T1A5A

Other*

1

2

27 10 1 1 2 1 2 1 1

6 5 5 2 1 1

1

1

Year 3 9

4

5

Total

2 3

5 1

49 19 6 4 3 3 2 2 2 2 6

1 1

1 1 1 4

1

* RFLP types represented by only one isolate * RFLP types represented by only one isolate

submandibular, parotid, bronchial, mediastinal, hepatic and mesenteric lymph nodes were incised and examined. The lungs, kidneys, spleen and liver were removed and examined. All the lesions were examined histopathologically, and a composite sample of the lesions was cultured. If there were no tuberculous lesions a composite sample of the retropharyngeal, bronchial, mediastinal and mesenteric lymph nodes together with approximately 2 g of kidney tissue and 2 g of lung tissue was cultured. The samples were decontaminated with 5 per cent oxalic acid and cultured on four tubes of Stonebrinks medium with added pyruvate (Costello and others 1998). Seventy-eight per cent of the samples were also cultured on Bactec 12B liquid medium (Collins and others 1997), which was not available at the start of the study. The isolates were identified presumptively on the basis of their colony morphology, growth rate, pigmentation and cording characteristics (Collins and others 1997). RFLP method The extraction of DNA, its cleavage with restriction endonucleases, electrophoresis and Southern blotting have been described by Costello and others (1999). A probe consisting of the entire IS6110 insertion sequence was hybridised to membrane bound PvuII-digested DNA. Polymorphic GCrich sequence (PGRS) and direct repeat sequence (DR) probes were hybridised to membrane bound AluI-digested DNA. The detection of the probes and sizing of the fragments has been described by Costello and others (1999). RFLP nomenclature The RFLP types were identified in the order IS6110, PGRS and DR. A letter followed by a single digit number was used to name the IS6110 and PGRS types and a letter was used to name the DR types; the names assigned by Costello and others (1999) and Olea-Popelka and others (2005) have been retained. Spoligotyping The isolates were spoligotyped by the method described by Kamerbeek and others (1997). This analysis resulted in only a limited differentiation of RFLP types and was therefore applied to only approximately 50 per cent of the isolates. It was used to validate the isolate identification procedure and to corroborate data on the phylogeny of the RFLP types. RESULTS Postmortem examination In total, 2360 badgers were culled, of which 2310 were examined postmortem and had tissues cultured. Isolates obtained

from tissue samples from 431 badgers were identified as M bovis on the basis of their phenotypic characteristics; they were obtained from 202 badgers from Cork, 107 from Monaghan, 77 from Kilkenny and 45 from Donegal. An analysis of a sample of 214 isolates by spoligotyping, showed that all of them had a characteristic M bovis profile, on the basis of the absence of spacers 39 to 43 (Kamerbeek and others 1997). Sett-associated group structure of the infected badger population The population of 2360 badgers was distributed among 802 setts. The majority of the setts contained few badgers, the median number per sett being two, with a range from one to 19. The 431 infected badgers were distributed among 297 setts, the median number per sett being one, with a range from one to six. Only one infected badger was captured at 205 of the setts, 131 of which were categorised as main setts and 74 as subsidiary setts. Two infected badgers were captured at 67 setts, 54 of which were categorised as main setts and 13 as subsidiary setts. Three or more infected badgers were captured at 25 setts, 22 of which were categorised as main setts. Description of RFLP types RFLP typing was applied to 398 of the 431 M bovis isolates, and 52 different RFLP types were identified. However, 73 per cent of the isolates belonged to seven predominant RFLP types, which were identified in isolates from 10 or more badgers in at least one of the areas (Tables 1, 2, 3, 4); three of them were identified in more than one of the areas. The majority of the strains with these RFLP types were isolated from badgers culled in the first two years, and the numbers then declined, as did the overall number of infected badgers.

TABLE 3: Numbers of isolates of different restriction fragment length polymorphism (RFLP) types identified in Mycobacterium bovis strains isolated from badgers in Kilkenny in successive years of the study RFLP type

Year 3

1

2

C1H1J A4A1H A1A1A A3D1G A1A8A O4D4A

10 7 6

1 1 1 5

3

2 4 3 1 1

Other*

3

3

1

4

5

Total

2 1

15 13 11 7 4 2 10

1 1 1 1

1 2

* RFLP types represented by only one isolate

The Veterinary Record, November 4, 2006


Papers & Articles

TABLE 5: Numbers of sett-associated groups in each of the four areas from which either one or more than one restriction fragment length polymorphism (RFLP) type was isolated

TABLE 4: Numbers of isolates of different restriction fragment length polymorphism (RFLP) types identified in Mycobacterium bovis strains isolated from badgers in Donegal in successive years of the study RFLP type

1

2

A1A5A A1T2A A1A1A D1H3I

19 4 4

3 2

Other*

1

Year 3

4

5

Total

1

23 6 5 2 3

1 1

2 1

Area

1 RFLP type

>1 RFLP type

20 8 2 5 35

29 9 9 3 50

Cork Monaghan Kilkenny Donegal Total

* RFLP types represented by only one isolate

were removed in different years. In 19 of these 46 groups the isolates from both badgers were of the same RFLP type, but the isolates from the two badgers in the other 27 groups had different RFLP types. Isolates from three or more badgers from 20 groups were typed. In six of these groups, all the badgers had the same RFLP type (Fig 1); in eight of the other 14 groups isolates from two or more badgers were of the same RFLP type, but an isolate from one badger was of a different type. In one group, isolates from four badgers were equally divided between the two RFLP types. Three different RFLP types were identified among the isolates from the remaining five groups.

Forty-five of the RFLP types were each represented by fewer than 10 isolates, and 27 of them by only one isolate (Tables 1, 2, 3, 4). Fourteen strains had RFLP profiles that closely resembled the profile of a predominant RFLP type in the same area. Nine had a PGRS band pattern with one or two minor band differences from a predominant type and IS6110, DR and spoligotype patterns identical to the predominant type. Similarly, three had a DR band pattern with a single band difference from a predominant type and IS6110, PGRS and spoligotype patterns identical to the predominant type. Two had an identical PGRS pattern to a predominant type and slightly different DR, IS6110 and spoligotype patterns. A further 13 types carried between two and five copies of the insertion sequence IS6110. Twenty-seven of the 45 low-prevalence types were identified primarily in isolates from badgers culled during the first two years, and the other 18 types were mainly identified in isolates from badgers culled later during the study (Tables 1, 2, 3, 4).

DISCUSSION These results describe the distribution of M bovis RFLP types in the badger population of four widely separated areas covering 1214 km2, approximately 2·4 per cent of the agricultural land area of the Republic of Ireland. Seventy-three per cent of the 398 isolates that were typed belonged to seven RFLP types and the other 27 per cent were distributed among 45 RFLP types. In each of the areas in Cork, Kilkenny and Monaghan there were two or three of the predominant types and between 13 and 16 of the low-prevalence types. In the area in Donegal that contained the smallest number of badgers, there was one predominant type and six low-prevalence types. Three of the seven predominant types were present in more than one of the four areas; type A1A1A occurred frequently in two and less frequently in the other two, type A1A5A was the predominant type in Donegal and was less common in the other three areas, and C1H1J was the type most frequently identified in isolates from Cork and Kilkenny. Previous work has shown that the predominant RFLP types identified are either widely

Distribution of RFLP types within sett-associated groups Isolates from two or more badgers from 85 sett-associated groups were RFLP-typed, and multiple types were identified in 50 of these groups (Table 5). Isolates from two badgers from 65 groups were typed; in 19 of these groups the two infected badgers were removed during the same trapping period, and all but one of the 19 groups were removed during the first two years; in 10 of these 19 groups the isolates from both badgers were of the same RFLP type. The two infected badgers in the other 46 groups were removed during different trapping periods; 12 of the groups were removed during the same year, and the two badgers in each of the other 34 groups

FIG 1: Distribution of Mycobacterium bovis restriction fragment length polymorphism (RFLP) types in the setts from which isolates from three or more badgers were typed. * Badgers captured during the same trapping period

Area Cork Cork Cork Cork Cork Cork Cork Cork Cork Cork Cork Cork Cork Monaghan Monaghan Monaghan Monaghan Monaghan Monaghan Kilkenny

Sett Year 1 5738403 * * 5739302 5740401 5810202 5811404 * * 5811405 5883402 5944318 * * * 5947401 5999204 6000201 * * * 6044205 * * * 6045209 1039301 1154101 1155202 * * 9829402 9832102 9872014 4892401 * *


Year 2 *

Year 3

Year 4

Year 5

A1A1A A1A3A A1A5A A2A2B E8D4A A2A1B A1B1D B1C1C B1C3C B2A3Y C1H1J C1H7J C4T6X D2C1L A1E2A F1A1C G6O1J N3H8Z

*

*

*

*

*

*

*

*

*

*

*

*

RFLP type

*

*

*


Papers & Articles

distributed throughout the country or are established in large areas covering several counties (Costello and others 1999). There were 45 RFLP types that were represented by fewer than 10 isolates; 14 of them had RFLP band patterns that closely resembled the pattern of a predominant RFLP type present in the same area, and in most of them the only difference was in the PGRS band pattern. Almost all the isolates of these 14 types were cultured from badgers culled during the first two years of the project, suggesting that the strains were probably indigenous to the areas. A further 13 RFLP types had more than one copy of the insertion sequence IS6110, but there were only 16 isolates of these multi-copy types. The majority of M bovis strains in Ireland have only one copy of this insertion sequence (Skuce and others 1996, Costello and others 1999). Five multi-copy strains from Kilkenny had exactly matching PGRS and DR patterns and identical spoligotypes; four of them had three IS6110 copies with three different RFLP patterns and the other had five IS6110 copies. The IS6110 patterns of all five isolates had four bands in common, suggesting that they may have been genetically related strains that might have evolved as a result of an increased rate of IS6110 transposition. This may have occurred if one of the IS6110 sequences was located in a transcriptionally active site (Wall and others 1999). Eighteen low-prevalence types were confined to isolates obtained from badgers culled in the last three years of the study and 14 of them were represented by only one isolate. Approximately 70 per cent of these badgers were captured at setts within 3 km of the perimeter of the particular area, suggesting that badgers may have migrated into the area after the resident population had been substantially reduced. Multiple RFLP types were identified among isolates from 50 of the 85 groups from which two or more infected badgers were culled; strains with very different RFLP profiles were frequently present in a group, indicating that for the most part this diversity was not caused by the evolution of strains within the groups. In some cases, it is possible that multiple strains may have been introduced into a group by the acquisition of the infection through contact with different outside sources. However, it is likely that there was a minimal risk of infection from other species. There were no wild or domestic deer in the area in Cork and Donegal, and in Kilkenny and Monaghan wild deer were present in only one district, which constituted less than 10 per cent of each area (D. Butler, M. Duggan, J. Murphy, F. O’Shea, personal communication). There have been very few reports of M bovis infection in wild animals in the Republic of Ireland apart from deer and badgers, a situation similar to that in the UK where there is no evidence of a significant reservoir of the infection in wild mammals other than badgers (Delahay and others 2002). The annual tuberculin testing of the cattle population should have substantially reduced the risk of infection from this species by removing animals while they were still in the early stages of the disease. Multiple strains may have been introduced into some groups by contact with badgers from neighbouring groups at territorial boundaries, but it is questionable whether this could have occurred frequently enough for the level of within-group diversity observed. The authors suggest that the most plausible explanation for the diversity of RFLP types within groups may have been the movement of badgers between group territories. For example, isolates from two badgers culled from sett 1155202 in the first year were type D2C1L, but the isolates from another two badgers culled from the same sett the following year were type A1E2A (Fig 1). The isolate from the only infected badger removed from sett 5811405 for the first year was type A2A2B, but in the following year the isolate from another badger was type C1H1J, and in the third year all the isolates from three infected badgers culled from the same sett were type A1A3A (Fig 1); these three badgers may have migrated as a group from another territory.

Such group movements from one territory to another have been recorded (Woodroffe and others 1993, Christian 1994, Rogers and others 1998, Roper and others 2003). Owing to the large size of the areas studied no attempt was made to identify and map social group territories, and the badgers were assigned to groups on the basis of the sett at which they were captured. It is likely that some social groups were subdivided into different groups, and, as a result, the diversity of strains within social group territories may have been underestimated. The spatial distribution of the RFLP types was consistent with the hypothesis that there may have been much extraterritorial movement by the badgers. An analysis to detect spatial clusters revealed a limited degree of clustering of RFLP types (Olea-Popelka and others 2005). Different RFLP types were often identified in isolates from badgers captured at contiguous setts (data not shown). Furthermore, the spatial distribution of the types changed continually during the study. For example, in the first year two main setts (5811404 and 5811405), located in the area in Cork and separated by a distance of 1·2 km, had two badgers infected with type C1H1J and one badger infected with type A2A2B respectively (Fig 1). In the second year, set 5811404 had one badger infected with type C1H1J and one badger infected with type A1A3A, and sett 5811405 had one badger infected with type C1H1J. In the third year, sett 5811405 had three badgers infected with type A1A3A. Studies in Great Britain (Cheeseman and others 1988, Woodroffe and others 1993) and Northern Ireland (Feore and Montgomery 1999) have shown that high-density badger populations that have not been disrupted by human interference exist in stable social groups, with well-defined territories. Permanent dispersal movements in such groups are considered to be rare, although temporary movements are more common (Woodroffe and others 1993, Rogers and others 1998). Short-term extraterritorial movements to the sett of another social group may vary from a brief foray in which the visitor might investigate only the outside of the sett to visits lasting several days (Christian 1994). In this study, the traps were set in the immediate vicinity of the setts, so that it is possible that the sett-associated groups included some badgers that were trapped while making a brief exploratory visit. Field studies, using bait-marking and radio tracking procedures, suggest that there is a less stable social structure and increased extraterritorial movement in low density populations and during culling operations (Sleeman 1992, O’Corry-Crowe and others 1993, Tuyttens and others 2000). Before this study began the population density of all four areas was low in comparison with the highdensity populations described in some areas of Great Britain (Rogers and others 1998, Griffin and others 2005). In addition, some badgers were removed from the areas in Kilkenny and Monaghan during the two years before the study began (Griffin and others 2005). These factors, combined with the disruption caused by the start of culling, may have resulted in some extraterritorial movements, which could have been responsible for much of the within-group diversity of RFLP types observed in the first year. More than one RFLP type was identified in nine of 11 groups from the area in Kilkenny, a higher proportion than in the other areas. In Kilkenny, there were also more types represented by only one isolate than in any other area, possibly owing to the high degree of movement associated with the removal of up to 300 badgers in the two years before the study began (Griffin and others 2005). In all four areas almost half the badgers culled were culled during the first year. The increasing variation of RFLP types observed within groups as the study progressed suggests that the decrease in population density and the disruption caused by the regular culling operations may have resulted in an increase in extraterritorial movements. The Veterinary Record, November 4, 2006


Papers & Articles

The results of the RFLP analyses support the hypothesis that there was a considerable level of extraterritorial movement by the badgers in all four areas. To understand the epidemiological implications more fully more information is required about the influence of habitat, region and population density on the movements of badgers in undisturbed populations and the extent to which culling operations affect extraterritorial movements in different populations. The possible diversity of strains of M bovis both within and between groups of badgers needs to be considered when genotyping is used in epidemiological investigations. ACKNOWLEDGEMENTS The authors acknowledge the contribution of staff at Abbotstown, Cork, Kilkenny and Sligo Regional Veterinary Laboratories and The Irish Equine Centre, Naas, who examined the badgers postmortem, the staff of the Mycobacteriology Division, Central Veterinary Research Laboratory, Abbotstown, who assisted with the culture of samples, and the staff at Cork, Kilkenny, Monaghan and Donegal District Veterinary Offices for field work. They are grateful to Dr Leigh Corner and Mr James O’Keefe for advice and critical reviews of the manuscript. References CHEESEMAN, C. L., CRESSWELL, W. J., HARRIS, S. & MALLINSON, P. J. (1988) Comparison of dispersal and other movements in two badger (Meles meles) populations. Mammal Review 18, 51-59 CHRISTIAN, S. F. (1994) Dispersal and other inter-group movements in badgers, Meles meles. Zeitschrift fur Saugetierkunde 59, 218-223 COLLINS, C. H., GRANGE, J. M. & YATES, M. D. (1997) Cultural methods. In Tuberculosis Bacteriology, Organisation and Practice. 2nd edn. Oxford, Butterworth-Heinemann. pp 57-68 COSTELLO, E., O’GRADY, D., FLYNN, O., O’BRIEN, R., ROGERS, M., QUIGLEY, F., EGAN, J. & GRIFFIN, J. (1999) Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection. Journal of Clinical Microbiology 37, 3217-3222 COSTELLO, E., QUIGLEY, F., FLYNN, O., GOGARTY, A., MCGUIRK, J., MURPHY, A. & DOLAN, L. (1998) Laboratory examination of suspect tuberculous lesions detected on abattoir post mortem examination of cattle from non-reactor herds. Irish Veterinary Journal 51, 248-250 DELAHAY, R. J., DE LEEUW, A. N. S., BARLOW, A. M., CLIFTON-HADLEY, R. S. & CHEESEMAN, C. L. (2002) The status of Mycobacterium bovis infection in UK wild mammals: a review. Veterinary Journal 164, 90-105 FEORE, S. & MONTGOMERY, W. I. (1999) Habitat effects on the spatial ecol-


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Genotyping of Mycobacterium bovis isolates from badgers in four areas of the Republic of Ireland by restriction fragment length polymorphism analysis E. Costello, O. Flynn, F. Quigley, et al. Veterinary Record 2006 159: 619-623

doi: 10.1136/vr.159.19.619

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