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Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain W.L. García-Jiménez a,∗ , P. Fernández-Llario a , J.M. Benítez-Medina a , R. Cerrato a , J. Cuesta c , A. García-Sánchez b , P. Gonc¸alves a , R. Martínez a , D. Risco a , F.J. Salguero d , E. Serrano e,f , L. Gómez c , J. Hermoso-de-Mendoza a a

Red de Grupos de Investigación Recursos Faunísticos, Facultad de Veterinaria, Universidad de Extremadura, E-10003 Cáceres, Spain Producción Animal, Centro de Investigación “Finca La Orden Valdesequera”, E-06187 Badajoz, Spain Unidad de Anatomía Patológica, Departamento de Medicina Animal, Facultad de Veterinaria, Universidad de Extremadura, E-10003 Cáceres, Spain d Pathology Unit, Animal Health and Veterinary Laboratories Agency, AHVLA-Weybridge, KT15 3NB, Addlestone, Surrey, United Kingdom e Servei d’Ecopatologia de Fauna Salvatge, Departament de Medicina i Cirurgia Animals, Universitat Autónoma de Barcelona, E-08193 Bellaterra, Spain f Estadística i Investigació Operativa, Departament de Matemàtica, Universitat de Lleida, E-25001 Lleida, Spain b c

a r t i c l e

i n f o

Article history: Received 18 September 2012 Received in revised form 8 February 2013 Accepted 13 February 2013 Keywords: Mycobacterium bovis Wild boar Fallow deer Disease management Population density reduction

a b s t r a c t Research on management of bovine tuberculosis (bTB) in wildlife reservoir hosts is crucial for the implementation of effective disease control measures and the generation of practical bTB management recommendations. Among the management methods carried out on wild species to reduce bTB prevalence, the control of population density has been frequently used, with hunting pressure a practical strategy to reduce bTB prevalence. However, despite the number of articles about population density control in different bTB wildlife reservoirs, there is little information regarding the application of such measures on the Eurasian wild boar (Sus scrofa), which is considered the main bTB wildlife reservoir within Mediterranean ecosystems. This study shows the effects of a management measure leading to a radical decrease in wild boar population density at a large hunting estate in Central Spain, in order to assess the evolution of bTB prevalence in both the wild boar population and the sympatric fallow deer population. The evolution of bTB prevalence was monitored in populations of the two wild ungulate species over a 5-year study period (2007–2012). The results showed that bTB prevalence decreased in fallow deer, corresponding to an important reduction in the wild boar population. However, this decrease was not homogeneous: in the last season of study there was an increase in bTB-infected male animals. Moreover, bTB prevalence remained high in the remnant wild boar population. © 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +34 927 257114; fax: +34 927 257110. E-mail address: [email protected] (W.L. García-Jiménez). 0167-5877/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

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1. Introduction Tuberculosis is a chronic granulomatous infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC). Mycobacterium bovis (M. bovis), the etiological agent of bovine tuberculosis (bTB), and its close relative Mycobacterium caprae (M. caprae), can infect a wide range of domestic and wild animals. The infection of domestic animals presents important economic, environmental and human health risks (de la Rua-Domenech, 2006; Good et al., 2011). The disease is of particular importance in countries where eradication programs, with the application of strict testing and slaughter measures on cattle, have substantially reduced the incidence of bTB. Nevertheless, there are areas where the disease persists and new outbreaks occur frequently. In these countries, studies have described the important role of wild animals in the maintenance and spread of bTB infection to livestock, representing the greatest economic impact of the disease in Europe (Corner, 2006). The management of bTB in wildlife is of global concern and several reviews summarize the current status of bTB in wildlife worldwide, e.g. in Africa (Michel et al., 2006; Renwick et al., 2007), Europe (Delahay et al., 2002; Delahay, 2006; Gortázar et al., 2007), North America (Conner et al., 2008) and New Zealand (Ryan et al., 2006). In general, these studies emphasize the complexity of multi-host epidemiological systems which sustain bTB, and the importance of generating practical bTB management recommendations, rather than reporting local epidemiology or outbreak descriptions. Research on the management of bTB in wildlife and understanding the risk factors associated with bTB infection in wildlife reservoir hosts (Corner, 2006; Thirgood, 2009) are crucial for the implementation of effective disease control measures. Among the management methods implemented on wild species to reduce bTB prevalence, the control of population density has been one of the most commonly used. In areas where bTB is known to be density-dependent, the reduction of wildlife population densities through hunting is a recognized strategy to reduce bTB prevalence given the risk of lateral transmission of bTB. The prevalence decreases because the probabilities of infectious contacts are diminished (Schmitt et al., 1997; de Lisle et al., 2002; O’Brien et al., 2006, 2011). Furthermore, an even more intense method of reducing wildlife densities is severe culling, a tool that has been implemented in different wildlife bTB reservoirs to control or prevent the spread of bTB (O’Brien et al., 2006; Radunz, 2006; Livingstone et al., 2009), but the results have been dissimilar depending on the multiple factors involved (Carter et al., 2007; Carstensen et al., 2011). However, despite the important number of scientific works about population density control in different bTB wildlife reservoirs, there is little information regarding the application of such measures on the Eurasian wild boar (Sus scrofa), which is considered the main bTB wildlife reservoir in the Iberian Peninsula (Parra et al., 2003; Hermoso de Mendoza et al., 2006; Naranjo et al., 2008).

To our knowledge, there is only one recent report on this topic (Boadella et al., 2012). Our study is focused on the management of bTB in wildlife on a large hunting estate in Central Spain. It shows the evolution of bTB prevalence over a 5-year study period (2007–2012) in two wild ungulate populations (wild boar and fallow deer). Epidemiological data was obtained before and after the implementation of a management measure resulting in a radical decrease in wild boar population density, in order to assess the evolution of the bTB prevalence in both species. Additionally, information about the lesion patterns found in both species and the spoligotype profiles isolated are provided. 2. Materials and methods 2.1. Study area The study was carried out in a fenced estate of 3028 ha, near Madrid, Central Spain. The estate consists of a typical Mediterranean forest in which holm oak (Quercus ilex) is the dominant tree species. Approximately 46% of the land area (1400 ha) is formed by dense Mediterranean scrubland (mainly Cistus spp., Erica spp., Phillerea spp. and Pistacia spp.), which is an ideal habitat for the hiding and resting of wild boar and the most suitable for hunting. The rest of the area (1628 ha) is “dehesa”, a scrubland-free area with scattered holm oak trees (Q. ilex). There is abundant scientific literature reporting that the home range of different sex and age classes of wild boar in diverse Mediterranean countries is less than that of the estate under study (Boitani et al., 1994; Russo et al., 1997; Calenge et al., 2002; Keuling et al., 2008). Annual rainfall is variable, ranging from 350 to 600 mm, and the climate is typical Mediterranean. The periods of greatest rainfall are autumn and spring months, with the dry season (from June to September) being a period of limited food and water resources for ungulates. Some water streams cross the estate and there are artificial ponds dispersed throughout the estate. A herd of free-ranging domestic cattle shares pastures with wild ungulates for most of the year, except during the hunting season (October to February) in order to prevent damage to livestock by dogs participating in the hunting activities. 2.2. Relative density and changes in the wild boar population: methodology and values before and after the measurement of population reduction Wild boar population density was estimated by analysis of the results of hunting activities, a common and widely used method to obtain reliable estimates of the relative population density in large areas (Fernández-Llario et al., 2003; Fernández-Llario and Mateos-Quesada, 2003; Bosch et al., 2012), and even about bTB prevalence (Hermoso de Mendoza et al., 2006). In this estate, given the interest of the estate owners in the elimination of wild boar as a sanitary risk for the population of fallow deer, the hunters were informed and committed to hunting as many wild boar as possible. Thus, every season two hunting events were conducted within

Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

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Table 1 Classification of hunted wild boar by age, sex and study season in a large hunting estate in Central Spain. Agea

Sex

Weaners

Male Female Male Female Male Female

Juveniles Adults Total a

Study season 2007–2008

2008–2009

6 10 7 5 4 5

16 17 14 21 3 20

14 15 18 18 28 20

6 12 4 9 1 4

4 3 4 4 2 1

37

91

111

36

18

2009–2010

2010–2011

2011–2012

Age groups based on classification by Saez-Royuela et al. (1989).

the estate. The first always occurred in October involving 20 hunters and 10 “rehalas” (“rehala” = a group of approximately 25 hunting-dogs) establishing a hunting quota of two male fallow deer per hunter and no restrictions on the number, sex or age of wild boar hunted. The second hunting event occurred in February with the same conditions, (20 hunters and 10 rehalas), but with the aim of hunting fallow deer females with a quota of 5 females per hunter and no restrictions on wild boar. Under these conditions, the hunted wild boar can be considered a valid sample to be used as a relative density measure of the wild boar population present on the estate (Bosch et al., 2012). According to the population density estimate method used, at the beginning of the study (2007–08 hunting season), the wild boar population was low (37 individuals hunted, 1.22 wild boar hunted/100 ha) in comparison to other Mediterranean populations, where the reported wild boar density extraction is around 3 individuals hunted/100 ha (Fernández-Llario et al., 2003; Bosch et al., 2012). Since that season the number of wild boar increased, with 91 wild boar hunted the following season (3 wild boar hunted/100 ha). At the beginning of the third season (2009–10), an increase in hunting pressure was implemented by carrying out one more hunting events in attempt to reduce the density of wild boar. On that occasion, 30 hunters and 15 rehalas participated, with the support of hunting guards to increase the likelihood of hunting more wild boar and with the result of 111 animals hunted. In the next two seasons, the same hunting pressure was maintained and the number of wild boar hunted decreased, dropping in the last season to only 18 animals hunted (Table 1). 2.3. Fallow deer (Dama dama) density and population evolution The methodology described below is the standard census estimation method for cervids in large game estates of Mediterranean areas. Fallow deer census estimation was carried out during the first days of October, each year. This is the best time of year for counts because in early autumn, when the availability of natural food is scarce, a common practice in large estates is to feed cervids with supplementary food, and the supply sites are very useful for reliable population counts. In this estate, the feeding sites consisted of a few streaks of alfalfa (Medicago sativa) with a length of nearly 500 m and good visibility. Taking advantage of the

dusk, three independent counts were simultaneously performed (to avoid the double counting of fallow deer) along the feeding sites where most of the animals were eating. In each count, animals were recorded and classified into four categories: bucks (over two years), young males (animals between one and two years), females (all ages) and juveniles (animals less than one year). Sex was not assessed in juveniles given the limited relevance of this information for the study and the difficult distinction of sex in cervids at these ages. The census estimation results showed a very important increase in the number of animals throughout the 5 years of study. Between 2008 and 2011 there was a population increase of almost 250% (Table 2). 2.4. Animal sampling Regarding wild boar, a systematic collection of morphometry, sex and age data (based on tooth eruption patterns) of all hunted wild boar was taken at each hunting event. Wild boar between 6 and 12 months were classified as weaners, those between 12 and 30 months as juvenile, and those more than 30 months old as adults (Saez-Royuela et al., 1989). Fallow deer were classified by age and sex as described above (bucks, young male, female and juveniles). For bTB inspection in wild boar, at each hunting event when the number of hunted animals was less than 25, all animals were inspected (as describe below), with the exception of the last two seasons, when some animals were severely damaged by dogs and were excluded (4 and 6 animals, respectively). Nevertheless, when the number of hunted wild boar was greater than 25, only 25 wild boar were randomly selected and attempts were made to sample similar numbers of males and females, including all age groups. The number was limited due to the simultaneous Table 2 Fallow deer census estimation in a large hunting estate in Central Spain. Study year

Bucks Young males Females Juveniles Total

2008

2009

2010

2011

256 80 465 219

518 126 932 302

527 167 1059 498

564 178 1129 532

1020

1878

2251

2403

Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

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Table 3 Summary of wild boar hunted, analyzed and showing visible lesions typical of bovine tuberculosis (VLTB) classified by sex and study season in a large hunting estate in Central Spain. Study season

Hunted wild boar

Analyzed wild boar

Males

Females

2007–2008 2008–2009 2009–2010a 2010–2011 2011–2012

37 91 111 36 18

17 21 20 8 7

20 29 30 24 5

37 50 50 32 12

7 14 18 7 6

10 25 27 21 4

17 39 45 28 10

Total

293

73

108

181

57

87

139

Males

a

Wild boar with VLTB

Females

Total

Total

Study season in which the measure of reducing wild boar population density was implemented.

collection of samples for complementary studies (including many other tissue types necessary for other research activities) and our inability to stop the work of butchers on the carcasses during these commercial hunting events. Necropsy examination on selected animals (Tables 3 and 4) was performed in the field with detailed macroscopic inspection of lymph nodes, and abdominal and thoracic organs. This routine examination included the incision of retropharyngeal and mandibular lymph nodes, the incision of the tracheobronchial and mediastinal lymph nodes, the palpation of the lungs, the incision of hepatic and mesenteric lymph nodes and finally the inspection of the liver and spleen. All mandibular lymph nodes of selected wild boar with and without visible lesions typical of TB (VLTB and NVLTB) were carefully collected in sterile containers. If any wild boar had VLTB in other organs, these were collected in a different container. Selected samples for microbiological isolation were not sectioned in the field to avoid cross contamination. For fallow deer, all hunted animals were inspected without restrictions, following the same methodology described above. 2.5. bTB prevalence estimation bTB prevalence was estimated based on the number of animals showing VLTB in the detailed macroscopic inspection conducted in each hunting event. This kind of inspection is the official method used by the Spanish Sanitary Authorities to decide if carcasses can continue in the food chain or must be condemned and is the basis for estimating the prevalence of bTB in hunted animals, as each decision and its reason are systematically recorded. In the scientific literature there are numerous reports that use

gross lesions to estimate bTB prevalence in wild boar in enzootic areas (Parra et al., 2006; Vicente et al., 2006, 2007), assuming that an underestimation error may be present (Corner, 1994). 2.6. Bacteriology Samples from animals inspected at the different hunting events were analyzed to characterize mycobacterial species responsible for the infection. For economic reasons and sometimes due to the inability to stop the processing of carcasses in such commercial hunting activities, a representative portion of the inspected animals were subjected to these studies (see Tables 5 and 6). For bacterial culture, tissue samples were sectioned and dissected, trimming the fat and connective tissue, using sterile scissors and forceps for each individual sample. Each tissue sample from the same animal with lesions in different organs was separately processed and cultured. Two grams of tissue were homogenized in 10 ml of sterile water with 0.2% albumin (Albumin from bovine serum Sigma, St Louis, MO, USA) for 4 min in a mechanic homogenizer (Smasher; AES Laboratories, Montreal, QC, Canada). The homogenized material was then decontaminated by the hexadecyl pyridinium chloride method (Corner and Trajstman, 1988). Finally, two Lowenstein–Jensen slants, with pyruvate and without glycerol, were inoculated in parallel and incubated for 6–8 weeks. Genomic DNA was extracted from suspect colonies by “heat-treatment technique”, which is described as follows: one to three colonies with similar phenotypic characteristics of each isolate were scraped from the surface of Löwenstein–Jensen medium and suspended in 150 ␮l of distilled water. When colonies with different phenotypic

Table 4 Summary of fallow deer hunted, analyzed and showing visible lesions typical of bovine tuberculosis (VLTB) classified by sex and study season in a large hunting estate in Central Spain. Study season

Hunted fallow deer Males

2007–2008 2008–2009 2009–2010a 2010–2011 2011–2012 Total a

Analyzed fallow deer

Females

Total

25 28 36 29 18

5 22 43 83 221

30 50 79 112 239

136

374

510

Males

Fallow deer with bTB

Females

Total

Males

Females

Total

25 28 36 29 18

5 22 43 83 221

30 50 79 112 239

10 17 29 11 12

1 4 7 14 22

11 21 36 25 34

136

374

510

79

48

127

Study season in which the measure of reducing wild boar population density was implemented.

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Table 5 Presence of visible lesions typical of bovine tuberculosis (VLTB), bacterial growth and Mycobacterium tuberculosis Complex-PCR results in wild boar samples from a large hunting estate in Central Spain. Season

Gross lesion

2007–2008c

VLTB NVLTB VLTB NVLTB VLTB NVLTB VLTB NVLTB VLTB NVLTB

2008–2009 2009–2010d 2010–2011 2011–2012 Total

Culture/PCR+a

Culture/PCR−b Total

Males

Total

Males

Females

2 – 12 2 15 – 6 1 6 –

6 – 23 1 14 – 15 – 4 –

8 – 35 3 29 – 21 1 10 –

1 2 2 4 – 2 1 – – 1

Females – 4 2 4 2 2 6 3 – 1

Total 1 6 4 8 2 4 7 3 – 2

9 6 39 11 31 4 28 4 10 2

44

63

107

13

24

37

144

a

Presence of growth and positive Mycobacterium tuberculosis Complex-PCR result. b Absence of growth or presence of growth but negative Mycobacterium tuberculosis Complex-PCR result. c The samples from the 22 wild boar hunted in the second hunting event of this season were not cultured (4 males and 4 females with lesions typical of bovine tuberculosis and 8 males and 6 females without lesions typical of bovine tuberculosis). d The samples from the 15 wild boar hunted in the third hunting event of this season (increase of hunting pressure) were not cultured (3 males and 11 females with lesions typical of bovine tuberculosis and 1 female without lesions typical of bovine tuberculosis).

characteristics were observed, these were inactivated separately. In generalized processes with more than one culture, colonies were inactivated separately depending on the organ from which they were isolated. The suspension was heated at 99 ◦ C for 10 min and centrifuged at 10,400 × g for 5 min. The supernatant was stored at −20 ◦ C until use.

The spoligotyping technique was applied according to Kamerbeek et al. (1997). DNA from M. bovis BCG Pasteur II and M. tuberculosis H37Rv were used as reference isolates and positive controls in this study, and water as a negative control. International names were assigned to spoligotype patterns using http://www.mbovis.org (Smith and Upton, 2012).

2.7. Molecular identification and spoligotyping 2.8. Statistical modeling Identification of the M. tuberculosis complex was carried out by PCR following standard methods, as amplification of Mycobacterium genus-specific 16S rRNA fragment and MPB70 sequences (Wilton and Cousins, 1992) with a slight modification in the primer concentrations (Liébana et al., 1996).

To study whether wild boar population density reduction was a suitable strategy for reducing bTB prevalence in the wild boar itself and in cervids (fallow deer in our case), we fitted several generalized linear models (GLM) in which bTB probability of infection (0 for healthy animals

Table 6 Presence of visible lesions typical of bovine tuberculosis (VLTB), bacterial growth and Mycobacterium tuberculosis Complex-PCR results in fallow deer samples from a large hunting estate in Central Spain. Season

Gross lesion

2007–2008c

VLTB NVLTB VLTB NVLTB VLTB NVLTB VLTB NVLTB VLTB NVLTB

2008–2009 2009–2010 2010–2011d 2011–2012e Total

Culture/PCR+a

Culture/PCR−b

Total

Males

Females

Total

Males

7 – 15 – 28 – 7 – 10 –

1 – 3 – 7 – 5 – 10 –

8 – 18 – 35 – 12 – 20 –

3 15 2 11 1 7 – 13 2 –

Females – – 1 18 – 36 1 34 – 38

Total 3 15 3 29 1 43 1 47 2 38

11 15 21 29 36 43 13 47 22 38

67

26

93

54

128

182

275

a

Presence of growth and positive Mycobacterium tuberculosis Complex-PCR result. b Absence of growth or presence of growth but negative Mycobacterium tuberculosis Complex-PCR result. c Only the female with VLTB from the second hunting event was sampled and cultured (4 females without lesions typical of bovine tuberculosis were excluded). The last two seasons some fallow deer were excluded from sampling, given the large number of animals hunted: d 3 males and 9 females with lesions typical of bovine tuberculosis and 6 males and 34 females without lesions typical of bovine tuberculosis were excluded. e 2 males and 10 females with lesions typical of bovine tuberculosis VLTB and 4 males and 163 females without lesions typical of bovine tuberculosis were excluded.

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and 1 for infected) was explained by the single effects of sex, disease management (pre-management: for game seasons 2007–08, 2008–09 and 2009–10 and post-management for 2010–11 and 2011–12) and their interaction. Models were fitted using a binomial distribution and the logit link function, since no over dispersion (residual deviance greater than the residual degrees of freedom, Zuur et al., 2009) was detected in our analyses. Typically, this kind of biological analysis would have been undertaken using generalized linear mixed models GLMM (e.g., several game seasons under the same disease management strategy, Zuur et al., 2009), however preliminary modeling showed that the inclusion of game season as random term did not improve the fit of our models (results not shown). Knowing that bTB infection in wild boar is a chronic disease, the effects of wild boar population density reduction were explored only in weaners (animals between 6 and 12 months) with the aim of evaluating recent infections. bTB prevalence during our study period did not differ among age classes: 26.5% (12.88–44.36 CI 95%) for weaners, 22.5% (13.9–33.21 CI 95%) for juveniles and 22.3% (13.11–34.22 CI 95%) for adult wild boar (Chi2 = 0.2509, df = 2, p-value = 0.8821), and thus the results based on weaners should be considered representative for the whole population. For all the statistical models, we performed a model selection procedure based on the information-theoretic approach and Akaike’s Information Criterion corrected for small sample sizes (AICc) (see Burnham and Anderson, 2002 as reference book but Johnson and Omland, 2004 for a short review). In short, competing models are ranked in relation to the difference between their Akaike scores and those for the best model (i ), which has the lowest AICc. Models with i < 2 units have substantial support for explaining the observed variability in the variable of interest. Subsequently, we estimated the Akaike weight (wi ,), defined as the relative probability that a given model is the best model amongst those being compared. Once the best model had been selected, the explained deviance (ED) was calculated as a measure of explained variability for each response variable (Zuur et al., 2007). All statistical analyses were performed using R software version 2.15.1 (R Development Core Team, 2013), including prevalences estimated by the “epiR” version 0.9-43 package (Stevenson et al., 2012). 3. Results 3.1. Wild boar and fallow deer population dynamics during the study period and consequences of the increase in wild boar hunting pressure on bTB prevalence During the study period, a total of 293 wild boar were hunted, 128 before and 165 after the increase in hunting pressure (Table 1). In the second game season (2008–09), the increase of 54 wild boar with respect to the previous period (2007–08) suggested a wild boar population density increase. However, the increase in 20 animals in 2009–10 and especially the dramatic fall in the number of hunted animals (75 wild boar less) in 2010–11 can be attributed to the management and hence would indicate a population

Table 7 Temporal trend of bovine tuberculosis prevalence at 95% confidence interval in weaners (6–12 months), juveniles (12–30) and adults (over 30) wild boar hunted in a large hunting estate in Central Spain. Study season

Weaners

Juveniles

Adults

Total

2007–2008

40 (16.3–67.7) 83.3 (62.6–95.3) 91.3 (71.9–98.9) 81.3 (54.4–95.9) 100 (15.8–100)

50 (21.1–78.9) 73.3 (44.8–92.2) 90 (68.3–98.7) 91 (58.7–99.7) 77.7 (39.9–97.2)

50 (18.7–81.3) 72.7 (39–93.9) 85.7 (42.1–99.6) 100 (47.8–100) 100 (2.5–100)

45.9 (29–63.1) 78 (64–88.5) 90 (78.2–96.7) 87.5 (71–96.5) 83.3 (51.6–97.9)

2008–2009 2009–2010a 2010–2011 2011–2012

a Study season in which the measure of reducing wild boar population density was implemented.

reduction. Concerning fallow deer, no measures were supported to control the population density until the last study year and censuses showed that the number of fallow deer increased by 250% throughout the 5-year study period (from 2008 to 2011, Table 2). The bTB descriptive prevalence (based on VLTB), and their associated 95% confidence interval, for wild boar and fallow deer are shown in Tables 7 and 8, respectively. Considering the whole study period, bTB prevalence in both ungulate species peaked (90% for wild boar and 45.5% for fallow deer) during the third study season, when the disease management plan started. However, bTB prevalence trends in the two species during the study period was uncorrelated (R2 = 2.5, F1,3 = 0.08, p-value = 0.79), mainly because of the reduction of bTB prevalence in fallow deer after the implementation of the disease management plan. Concerning wild boar, our model selection procedure indicated that the population density reduction did not influence bTB probability of infection in wild boar weaners (wNull model = 0.38, Table 9) which would reflect recent infections. bTB prevalence before the application of the management measure (75%, 63.2–85.7, 95% CI) was slightly lower, yet not significantly different, than after management measures (83.3%, 58.6–96.4, 95% CI). In fallow deer, however, wild boar population density reduction was associated with bTB prevalence (wManagement + Sex = 0.15, Table 9) and 17.6% of the observed Table 8 Temporal trend of bovine tuberculosis prevalence at 95% confidence interval in adult (over 2 years) males and females fallow deer hunted in a large hunting estate in Central Spain. Study season

Males

Females

Total

2007–2008

40 (21.1–61.3) 60.7 (40.5–78.5) 80.4 (63.9–91.9) 37.9 (20.7–57.7) 66.6 (40.9–86.6)

20 (0.5–71.6) 18.2 (5.2–40.3) 16.3 (6.8–30.7) 16.8 (9.53–26.7) 9.9 (6.3–14.7)

36.6 (19.9–56.2) 42 (28.2–56.8) 45.5 (34.3–57.2) 22.3 (14.9–31.2) 14.2 (10.1–19.3)

2008–2009 2009–2010a 2010–2011 2011–2012

a Study season in which the measure of reducing wild boar population density was implemented.

Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

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Table 9 Model selection based on generalized linear modeling for exploring the effects of intensive wild boar population density reduction on the probability of bovine tuberculosis infection in 70 males and females weaners (6–12 months) wild boar and 519 fallow deer cohabiting in a large hunting estate in Central Spain. Biological models Wild boar Mo Sex Management Management + Sex Management × Sex Fallow deer Management + Sex Management × Sex Sex Management Mo

K

AICc

i

wi

1 2 2 3 4

87.31 87.70 88.83 89.37 91.07

0.00 0.52 1.64 2.37 4.32

0.38 0.29 0.17 0.12 0.04

3 4 2 2 1

477.92 479.96 480.07 539.00 574.49

0 2.03 2.14 61.08 96.56

0.59 0.21 0.20 0 0

K = number of parameters, AICc = Akaike Information Criterion corrected for small sample sizes, AICc = difference of AICc with respect to the best model, wi = Akaike weight, Mo = null model only with the constant term. In bold the best model for explaining the observed variability on bovine tuberculosis probability of infection.

probability of bTB infection was explained by this model indicating that during the entire period the probability of bTB infection in males was higher than in females (ˇmales = 1.96, SE = 0.25, Z = 7.7) and that the management of the wild boar population reduced the bTB probability of infection in fallow deer (ˇpre-management = 0.52, SE = 0.25, Z = 2.1). bTB prevalence before (42.7%, 39.9–50.8 at 95% CI) the disease management plan was 2.2 times higher than after (19.4%, 15.3–23.9 at 95% CI). 3.2. Gross pathology The bTB lesion patterns found in the wild boar inspected, classified by hunting season, sex and age group are summarized in Table 10. During the first two seasons of study, only localized lesions in mandibular lymph nodes were found (Fig. 1A) and macroscopic lesions in other organs were not encountered. Generalized patterns were identified beginning in the third season and were grouped into three categories: Score 1 (mandibular lymph node and mesenteric lymph node with VLTB, Fig. 1B), Score 2 (mandibular lymph node, lung and mesenteric lymph node with VLTB) and Score 3 (more than three organs with VLTB). Regarding fallow deer, bTB lesion patterns found are summarized in Table 11. Generalized patterns were found throughout the study, but were more frequent in the last seasons. Generalized lesion patterns were grouped into three categories: Score 1 (mandibular lymph node and lung with VLTB, Fig. 1C), Score 2 (mandibular lymph node, lung and mesenteric lymph node with VLTB, Fig. 1D) and Score 3 (more than 3 organs with VLTB). 3.3. Spoligotyping patterns Three different patterns from wild boar were characterized by spoligotyping technique (SB0121, SB0339 and SB1142) showing different frequencies each season. However, the most prevalent pattern for the whole period of

7

study was SB1142 (58.8%). Five animals were co-infected with two different M. bovis spoligotyping profiles, four of them being females. In these four cases the different patterns were isolated from the same sample (mandibular lymph nodes); however, the fifth case was a male yearling, in which profile SB0339 was isolated from mandibular lymph nodes and lungs and profile SB1142 from mesenteric lymph nodes. In the case of fallow deer, five different spoligotype patterns were found (SB0121, SB0339, SB1142, SB1627 and SB1970). The three most frequent profiles (SB0121, SB0339, and SB1142) were shared with wild boar. Regarding the other two profiles, SB1970 was isolated from two animals, one buck (season 2009–10) and one adult female (season 2011–12) and SB1627 was isolated from the mandibular lymph nodes and lungs of one buck, which also showed a co-infection with SB1142, isolated from mesenteric lymph nodes. Another four animals suffered co-infection with different isolates: two bucks with SB0121 and SB1142 (in one case, both isolates were obtained from lungs and in the other case SB0121 was obtained from lungs and SB1142 from mesenteric lymph nodes); one adult female with SB1142 and SB0339 profiles (isolated from lungs and mesenteric lymph nodes, respectively); and one adult female with SB0121, SB0339 and SB1142 spoligotype patterns (SB0121 and SB0339 from lungs and SB1142 from mesenteric lymph nodes). 4. Discussion Due to the recognized role of wildlife as bTB reservoirs (Corner, 2006), research on wildlife management and the understanding of the risk factors associated with this disease is crucial for the implementation of effective disease control measures (Thirgood, 2009). This is the first study that monitored and evaluated the effects of the reduction of the Eurasian wild boar population density on bTB prevalence in the same wild boar population and in the sympatric fallow deer population in Central Spain over a 5-year period. The positive effect of reducing the population density of the main bTB reservoir has been previously demonstrated and usually results in a significant reduction in the prevalence of bTB in other susceptible species sharing the same habitat. Eurasian badgers (Meles meles) as a source of infection of cattle bTB has been investigated in the United Kingdom (UK) and Republic of Ireland (RoI). In these cases, the reduction of density has been reported to reduce bTB prevalence in cattle herds (Clifton-Hadley et al., 1995; Eves, 1999). However, there have also been some instances in the UK in which the reduction of badger population density has not been effective in reducing the bTB prevalence in cattle, probably due to migration of infected badgers to the depopulated area (Jenkins et al., 2007). Consistent with this idea and knowing the role of wild boar as the main bTB wild reservoir and spreader in Mediterranean ecosystems (Parra et al., 2003; Hermoso de Mendoza et al., 2006; Naranjo et al., 2008), our study was carried out to evaluate the effects of implementing such methods on a wild boar population. The study estate, although enclosed by fences, is 3028 ha, making it large

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Table 10 Summary of bovine tuberculosis lesion patterns found in wild boar in a large hunting estate in Central Spain. Study season

Age

Sex

Localized

2007–2008

Weaners

Male Female Male Female Male Female

1 5 4 2 2 3 17

Male Female Male Female Male Female

9 11 3 8 2 6 39

Male Female Male Female Male Female

9 12 8 10 1 5 44

Male Female Male Female Male Female

3 9 – 6 1 3 22

Male Female Male Female Male Female

– – 2 1 – – 3

Juveniles Adults Total 2008–2009

Weaners Juveniles Adults Total

2009–2010

Weaners Juveniles Adults Total

2010–2011

Weaners Juveniles Adults Total

2011–2012

Weaners Juveniles Adults Total

Score 1

Score 2

Score 3

1

1 1 1 1

1

1

1 3

1

2

1 1 3 1 1 7

Localized: “n” of wild boar with single visible lesions typical of bovine tuberculosis localized in mandibular lymph node. Score 1: “n” of wild boar with visible lesions typical of bovine tuberculosis in mandibular lymph node and mesenteric lymph node. Score 2: “n” of wild boar with visible lesions typical of bovine tuberculosis in mandibular lymph node, lung and mesenteric lymph node. Score 3: “n” of wild boar with visible lesions typical of bovine tuberculosis in more than three organs. Table 11 Summary of bovine tuberculosis lesion patterns found in fallow deer in a large hunting estate in Central Spain. Study season

Age

Sex

2007–2008

Adult

Male Female Male

Young Total 2008–2009

Adult

Male Female

Total 2009–2010

Adult

Male Female

Total 2010–2011

Adult

Adult Total

Male Female

Score 1

Score 2

Score 3

9 1 10

1 1

2 1 3

15 2 17

1 1 2

27

1 1

27

1 6 7

8

3 5 16

10 6 1

2 2

1 7 8

11 13 28

Male Female

Total 2011–2012

Localized

1

Localized: “n” of fallow deer with single visible lesions typical of bovine tuberculosis localized in mandibular lymph node. Score 1: “n” of fallow deer with visible lesions typical of bovine tuberculosis in mandibular lymph node and lung. Score 2: “n” of fallow deer with visible lesions typical of bovine tuberculosis in mandibular lymph node, lung and mesenteric lymph node. Score 3: “n” of fallow deer with visible lesions typical of bovine tuberculosis in more than three organs.

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Fig. 1. (A) Wild boar mandibular lymph node with typical bovine tuberculosis lesion. (B) Detail of the bovine tuberculosis generalized process affecting mesenteric lymph nodes in a wild boar. These lesions could be related with the fact of scavenging carcasses. (C) Fallow deer lung with multiples and homogeneous (0.5 cm diameter) bovine tuberculosis granulomas. (D) Fallow deer bovine tuberculosis generalized process affecting mesenteric lymph nodes.

enough to draw valid comparisons with natural areas, with the advantage of not being biased by the entrance of external animals. The wild boar population in our study began with a low density compared to other populations living in Mediterranean ecosystems (Fernández-Llario et al., 2003; Bosch et al., 2012) and moderate bTB prevalence. Density increased in the following hunting seasons as did bTB prevalence, which reached 90%. Along with the increasing prevalence in wild boar, bTB significantly increased in the fallow deer population (especially in males), along with an increase in mortality rate. Cervids are considered highly susceptible to bTB (Rhyan and Saari, 1995; Jaroso et al., 2010). Given this scenario, it was decided that the wild boar population should be reduced through increased hunting pressure, a measure easily implemented due to the ecological characteristics of the estate. After the significant reduction of the wild boar population beginning in the 2009–10 season, disease prevalence decreased in the fallow deer population. Nevertheless, the remnant wild boar population persisted with high bTB prevalence (87.5%) without sex and age differences and not showing any mortality in the field attributable to the disease. It is possible that factors such as the gregarious behavior of this species

(Fernández-Llario et al., 1996) contribute to disease persistence. These results disagree with data presented by other authors (Boadella et al., 2012), who estimated that culling of at least 50% of the wild boar population would allow a reduction in bTB prevalence by 21–48% in high prevalence sites. Despite the positive association between the wild boar density reduction and bTB prevalence in fallow deer, the prevalence of VLTB did not decrease continually: an increase of VLTB prevalence affecting male fallow deer was detected in the last study season, reaching values similar to that of the third season. This could be explained by the fact that only males older than 5 years are hunted, because they are wanted as trophies, and these may had been infected several years before. In the case of females the decrease was more continual, although their population density had been steadily increasing over the study period. The females are hunted at any age and are usually younger than males. Thus, the results of the sampled females may reflect recent infections, better indicating the effect of the action implemented. In the last season 221 female fallow deer were hunted as a complementary measure to the reduction of the wild boar population density, with the aim of discerning the efficacy of this measure in subsequent studies.

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Regarding lesion patterns found in wild boar, an increase in the generalized lesions was observed during the final study seasons, despite population density reduction. Some scientific studies consider the presence of generalized patterns as rare in wild boar and show that the most common tissues affected are the head lymph nodes (Bollo et al., 2000; Zanella et al., 2008). Nevertheless, other scientific studies have reported that generalized patterns are more frequent in areas with a high density of wild boar intensively managed with food supplementation (Gortazar et al., 2003; Martín-Hernando et al., 2007). In our study, the wild boar density was low and without any intensive management, but contacts between wild boar and fallow deer (whose density is much higher) were possibly frequent. Other factors that could explain the appearance of generalized bTB lesions are the gregarious behavior of this species as well as easy access to other sources of infection, such as the carcasses of dead fallow deer with bTB. This could explain the presence of lesions in the digestive tract. The importance of scavenging habits have been described previously by other authors (Gortazar et al., 2008), as well as the increased presence of generalized processes in younger animals (Martín-Hernando et al., 2007). Regarding the high number of generalized lesions observed in the fallow deer population, similar situations have been described by other authors as a result of the susceptibility of this species to bTB infection (Johnson et al., 2008; Martín-Hernando et al., 2010). In fallow deer, digestive lesions could be attributed to the swallowing of bacilli released from respiratory tract infections. The most prevalent spoligotype was SB1142 in both ungulate species. This profile as well as the other two most common profiles (SB0121 and SB0339), were isolated from wild boar and fallow deer, evidence of inter-species transmission. In addition, since the 2009–10 hunting season, two new profiles (SB1627 and SB1970) were isolated only from fallow deer. The infection of fallow deer with these profiles could be from contacts with other bTB reservoir species such as the cattle herd that lives on the estate (the bTB status of the cattle herd is unknown), because the entry of foreign wild boar or fallow deer is practically impossible due to the types of fences used around the estate. Another possibility could be a single deletion in the more common spoligotype patterns, but this can be absolutely ruled out in the case of SB1970, which showed an important number of differences from the other spoligotypes and it would be unlikely in the case of SB1627, which showed two deletions compared to SB0121. Evidence of co-infection by different M. bovis types in a single host has been previously described (Gortazar et al., 2011) in wildlife. In this study, co-infections in both species were found, being predominant in wild boar females (4/5), which could be a result of the especially gregarious behavior of females (Fernández-Llario et al., 1996). The fifth case, a young male, differed from the females’ co-infections in that isolates were from different organs. Young males are under high-stress conditions, are highly mobile and in constant search of food, with scavenging and poor body condition common risk factors for this age group (GarcíaSánchez et al., 2007).

Regarding co-infections in fallow deer, five cases were found affecting three bucks and two adult females. This could also be related to gregarious behavior typical of this species and to the high fallow deer population density (Apollonio et al., 1992), which favors the possibility of bTB lateral transmission. 5. Conclusion The reduction in wild boar population density living in contact with cervids such as fallow deer seems to improve the prevalence rate in the sympatric population. Nevertheless, in this study the prevalence remained high in the wild boar population, with a high risk of a rise in tuberculosis prevalence persisting in the sympatric cervid population, if complementary control measures are not implemented. Conflict of interest statement The authors have not declared any conflict of interest. Acknowledgments This study was supported by Ministerio de Ciencia e ˜ Innovación (Gobierno de Espana) PS0900513, Junta de Extremadura (PDT09A046 and GRU10142), and by the European Community‘s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 228394 (NADIR). W.L. García-Jiménez and J.M. Benítez-Medina acknowledges the Junta de Extremadura and FSE for his research fellowships (PRE07024 and PRE08042) and E. Serrano is supported by the Beatriu de Pinós programme (BP-DGR 2011) of the Catalan Science and Technology System, Spain. References Apollonio, M., Festa-bianchet, M., Mari, F., Mattioli, S., Sarno, B., 1992. To lek or not to lek: mating strategies of male fallow deer. Behav. Ecol. 3, 25–31. Boadella, M., Vicente, J., Ruiz-Fons, F., de la Fuente, J., Gortazar, C., 2012. Effects of culling Eurasian wild boar on the prevalence of Mycobacterium bovis and Aujeszky’s disease virus. Prev. Vet. Med., http://dx.doi.org/10.1016/j.prevetmed.2012.06.001. Boitani, L., Mattei, L., Nonis, D., Corsi, F., 1994. Spatial and activity patterns of wild boars in Tuscany, Italy. J. Mammal. 75, 600–612. Bollo, E., Ferroglio, E., Dini, V., Mignone, W., Biolatti, B., Rossi, L., 2000. Detection of Mycobacterium tuberculosis complex in lymph nodes of wild boar (Sus scrofa) by a target-amplified test system. J. Vet. Med. B 47, 337–342. Bosch, J., Peris, S., Fonseca, C., Martinez, M., de La Torre, A., Iglesias, I., ˜ M.J., 2012. Distribution, abundance and density of the wild Munoz, boar on the Iberian Peninsula, based on the CORINE program and hunting statistics. Folia Zool. 61, 138–151. Burnham, K.P., Anderson, D.R., 2002. Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach. SpringerVerlag, New York, USA, pp. 1–488. Calenge, C., Maillard, D., Vassant, J., Brandt, S., 2002. Summer and hunting season home ranges of wild boar (Sus scrofa) in two habitats in France. Game Wildl. Sci. 19, 281–301. Carter, S.P., Delahay, R.J., Smith, G.C., Macdonald, D.W., Riordan, P., Etherington, T.R., Pimley, E.R., Walker, N.J., Cheeseman, C.L., 2007. Culling-induced social perturbation in Eurasian badgers Meles meles and the management of TB in cattle: an analysis of a critical problem in applied ecology. Proc. Biol. Sci. 274, 2769–2777. Carstensen, M., O’Brien, D.J., Schmitt, S.M., 2011. Public acceptance as a determinant of management strategies for bovine tuberculosis in freeranging U.S. wildlife. Vet. Microbiol. 151, 200–204.

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Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017

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Please cite this article in press as: García-Jiménez, W.L., et al., Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: Effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain. PREVET (2013), http://dx.doi.org/10.1016/j.prevetmed.2013.02.017