Accepted Manuscript Title: Animal tuberculosis maintenance at low abundance of suitable wildlife reservoir hosts: a case study in northern Spain Authors: C. Gort´azar, L.M. Fern´andez-Calle, J.A. Collazos-Mart´ınez, O. M´ınguez-Gonz´alez, P. Acevedo PII: DOI: Reference:

S0167-5877(17)30176-9 http://dx.doi.org/10.1016/j.prevetmed.2017.08.009 PREVET 4303

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Received date: Revised date: Accepted date:

2-3-2017 27-7-2017 11-8-2017

Please cite this article as: Gort´azar, C., Fern´andez-Calle, L.M., Collazos-Mart´ınez, J.A., M´ınguez-Gonz´alez, O., Acevedo, P., Animal tuberculosis maintenance at low abundance of suitable wildlife reservoir hosts: a case study in northern Spain.Preventive Veterinary Medicine http://dx.doi.org/10.1016/j.prevetmed.2017.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Animal tuberculosis maintenance at low abundance of suitable wildlife reservoir hosts: a case study in northern Spain

C. Gortázara*, L.M. Fernández-Calleb, J.A. Collazos-Martínezb, O. Mínguez-Gonzálezb, P. Acevedoa

a

SaBio Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM). Ronda de Toledo

12, 13005 Ciudad Real, Spain. b

Servicio de Sanidad Animal, Dirección General de Producción Agropecuaria e Infraestructuras

Agrarias, Consejería de Agricultura y Ganadería. Junta de Castilla y León, Valladolid, Spain. * Corresponding author [email protected]

Abstract Animal tuberculosis (TB), which is caused by infection with members of the Mycobacterium tuberculosis complex (MTC), is a typical multi-host infection that flourishes at the livestockwildlife interface. TB epidemiology is well characterized in the Mediterranean woodland habitats and Atlantic regions of southwestern Europe. However, much less is known about huge regions that do not form part of the two abovementioned settings, which have a low abundance of wild reservoirs. We hypothesized that MTC would be maintained in multi- rather than singlehost communities in which wildlife would make a relatively low contribution to the maintenance of TB. Between 2011 and 2015, 7729 Eurasian wild boar (Sus scrofa) and 1729 wild ruminants were sampled for culture during hunting events on unfenced sites. In addition, 1058 wild ungulates were sampled on 23 fenced hunting estates. Infection prevalence data were modeled along with official data on cattle and goat TB, on livestock distribution and management, and on wild boar abundance. The mean individual MTC infection prevalence was 4.28% in wild boar, while the cattle skin test reactor percent was 0.17%. The prevalence of MTC infection in wild ungulates (mostly wild boar) from the fenced hunting estates was 11.6%. Modeling revealed 1

that the main driver of TB in cattle was their management (beef; communal pastures). However, wild boar abundance, the prevalence of MTC infection in wild boar and the presence of fenced hunting estates also contributed to explaining cattle TB. The model used for goat TB identified communal pastures as a risk factor. The model for the prevalence of MTC infection in wild boar included wild boar abundance and communal pastures. We conclude that the MTC maintenance host community is most likely of a multi-host nature. While cattle and communal pastures pose the main risk regarding TB, it is also necessary to consider increasing wild boar densities and specific risks owing to fenced wildlife. We infer several management implications regarding wildlife management, the wildlife sampling strategy and laboratory testing, the peculiarities of fenced hunting estates, and the wildlife-livestock interface.

Keywords: Castilla y León; Communal pastures; Epidemiology; Low-prevalence region; Shared infections; Wildlife-livestock interface.

Introduction Animal tuberculosis (TB) is caused by the infection with Mycobacterium bovis and other closely related members of the M. tuberculosis complex (MTC). M. bovis is a typical multi-host pathogen that flourishes at the livestock-wildlife interface in a diversity of situations, worldwide (Gortázar et al. 2015). In some settings, such as the British Isles, New Zealand and Michigan (USA), MTC is maintained in relatively simple cattle-wildlife networks (O'Brien et al. 2006, Gormley and Corner 2013, Nugent et al. 2015). In others, for instance in Sub Saharan Africa, several wildlife species contribute to multi-host MTC maintenance communities (De Garine-Wichatitsky et al. 2013). Two main settings have been described in continental Europe. First, one associated with wild ungulates in Mediterranean woodland habitats of the southwestern quarter of the Iberian Peninsula (e.g. Gortázar et al. 2008, 2012, Vicente et al. 2013, Cowie et al. 2016). Second, one linked with the Eurasian badger (Meles meles) and the Eurasian wild boar (Sus scrofa) on the 2

northern Atlantic coast of Spain and in parts of France (Muñoz-Mendoza et al. 2013, Boschiroli and Bénet 2014). The former, in the southwestern quarter of the Iberian Peninsula, is characterized by dense and often overabundant red deer (Cervus elaphus) populations (e.g. an average of 21 per km2 of suitable habitat; Acevedo et al. 2008) and equally dense wild boar populations (>10 per km2; Acevedo et al. 2007). These wildlife densities are often a consequence of human intervention through fencing, feeding and insufficient harvesting (Gortázar et al. 2008). Badgers are also present, but at relatively low densities (0.05). Wildlife populations and their TB status Figure 3 presents the CyL wild boar hunting harvest in the season 1980/81, and from 2000/01 to 2012/13. The 2013 annual wild boar harvest per square km was 0.33. The total increase in the wild boar harvest during these 33 years was 763% (an annual increase of 5.86%), and the increase between 2000/01 and 2012/13 was 248% (an annual increase of 6.02%). Historical data for red deer were unavailable. However, recent trends (2010/11 to 2012/13) have been stable (min 6915 in 2011/12, max 8722 in 2010/11), thus indicating no significant expected population growth under the current environmental conditions. The 2013 annual red deer harvest per square km was 0.09. Table 1 presents the prevalence of MTC infection in the wildlife in CyL sampled throughout the study period. The prevalence of mean individual wild boar MTC infection was almost double that recorded for red deer (4.28 vs. 2.24, respectively). This difference was significant on a regional scale (Chi square 10682, 1 d.f., p30 in all LVUs and particularly in all 43 fenced estates, since sample sizes >30 are currently only available for 1/4. Communal pastures were identified as risk factors in all models, signaling their relevance in TB epidemiology owing to the facilitation of intra- and inter-species contact (Guta et al. 2014). As stated in previous research (e.g. Garcia-Saenz et al. 2014, Guta et al. 2014), the main risk for cattle as regards TB are other cattle: cattle-related factors such as type of farming (beef) and the 10

surface of communal pastures were identified as drivers for the prevalence of TB in cattle in CyL. Goats, by contrast, were not identified as significant drivers of cattle TB in this study, possibly because the prevalence of TB in goats is higher in central CyL, where beef cattle are less abundant (Appendix S1). The LVUs with proportionally more beef cattle and more communal pastures - and hence those with a higher prevalence of TB in cattle- are mainly situated along the borders of the study area. These LVUs are also characterized by more suitable wildlife habitats when compared to the cereal lowlands of central CyL, and coincide in the north and south with the areas of highest predicted wild boar abundance (Appendix S1). This implies that cattle are more likely to come into contact with wild boar on sites on which the prevalence of TB is highest in both host species, and vice-versa. Using observational data impedes identifying the directionality of MTC transmission between cattle and wild boar. However, it is likely that both the wild and the domestic host species have some effect on TB in their counterparts. The cattle model suggests that wild boar abundance, the prevalence of TB in wild boar, and the presence of fenced hunting estates all contribute to the risk of detecting skin-test reactors cattle (Table 3). The prevalence of TB in goats was apparently unaffected by that in cattle and wild boar. However, communal pastures shared by goats, cattle and sheep were included in the model, suggesting some epidemiological link. The apparent lack of interrelation between cattle TB and goat TB could be owing to the more local presence of goats, as compared to the more widespread one of cattle. Moreover, TB testing in goats originally began in the peripheral provinces with the highest prevalence of TB, thus leading to the current situation in which most of the remaining goat TB is found in the interior lowlands, where both beef cattle and wild boar are less abundant. Only wild boar abundance and communal pastures were included in the model for TB prevalence in wild boar, suggesting that the infection maintained in this host species is more independent. Communal pastures with cattle, goats and sheep were, however, identified as a risk factor in the 11

wild boar model, perhaps an indication that there is a multi-host network in this kind of settings (Gortázar et al. 2008). Once infected, wild boar populations might be able to maintain MTC circulation without a steady spill-over from infected livestock (Naranjo et al. 2008), even in scenarios where the risk is supposed to be low (Mentaberre et al. 2014). The shape of the wild boar population trend graph and the large annual growth rate (6%) indicate that the CyL wild boar population has not yet reached an asymptote, i.e., that the carrying capacity has not been reached. Moreover, as occurs in other areas in Europe (Massei et al. 2015), the number of hunting licenses in CyL declined by 20% between 2011 and 2014 (data not shown), indicating a progressively lower hunting pressure. These data evidence that the CyL hunting harvest is insufficient if wild boar population growth is to be avoided. The prevalence of individual wild boar MTC infection is one order of magnitude higher than the prevalence of individual cattle skin-test reactors (4.28% vs 0.17%, respectively). Given that the 2013 annual wild boar harvest in CyL was 24,994, and that the wild boar population is steadily growing, as in other European areas (e.g. Massei et al. 2015), a four-fold of this figure could be regarded as a reasonable estimate of the current wild boar population size (i.e., 100,000 individuals). At a prevalence of 4%, this gives an estimated 4000 MTC-infected wild boar in CyL. This figure has to be compared with 1781 individual cattle TB reactors detected in 2014. In other words, the number of TB-positive wild boar in CyL might be roughly double that of cattle, and while cattle are tested annually and eventually slaughtered and their numbers are at best stable, the hunting harvest is insufficient to avoid the growth of the wild boar population (e.g. QuirósFernández et al. 2017). This implies that, while the current situation is not worrisome, MTC maintenance in growing wild boar populations might create conflicts in the future, particularly on sites with an abundant suitable habitat and frequent inter-species contacts (Barasona et al. 2014). Another important aspect is the role of fenced hunting estates. Our data reveal that the prevalence of MTC infection in wild ungulates from fenced hunting estates triples that of the 12

mean wild boar infection recorded for open areas. This observation fits with previous comparisons between fenced and open ungulate populations (Vicente et al. 2013). It is also in concordance with the fact that the presence of fenced hunting estates was selected by the models as a risk factor for cattle TB. Proximity to fenced hunting estates had already been identified as a risk factor for cattle TB in neighboring Castilla – La Mancha, a region with a higher prevalence of TB (LaHue et al. 2016). Management implications We infer management implications regarding regional wildlife management, the wildlife sampling strategy and laboratory testing, the peculiarities of fenced hunting estates, and the wildlife-livestock interface in CyL. One of the weaknesses of this study was the lack of data on wildlife densities. Such data would be of great value in the framework of a regional TB control scheme, particularly for wild boar, badger and red and fallow deer. It is evident from the hunting harvest data that the hunting and predation of wild boar are currently unable to control its population growth (see e.g. Nores et al. 2008, Quirós-Fernández et al. 2017). In order to avoid future conflicts, it is advisable to work with the appropriate stakeholders and discuss options for a more efficient wild boar population control. The current wildlife sampling strategy is ambitious and achieves its goals, providing sound data on the main wildlife MTC host, the wild boar, based on the gold standard technique, culture. However some other species, notably badgers, are insufficiently surveyed. Setting up a targeted surveillance of all fenced estates is also desirable. Considering the expense and the logistic constraints of using culture as the main wildlife TB surveillance tool, we suggest switching to antibody detection tests in the case of the wild boar (e.g. Boadella et al. 2011a,b, Richomme et al. 2013, Pérez de Val et al. 2017). Blood tests simplify sampling and would allow the sample size to be increased in remote areas and under-sampled LVUs. Selected individuals can still be sampled for culture in order to generate isolates suitable for molecular epidemiology - hopefully based on whole genome sequencing rather than only spoligotyping, in order to allow a better 13

insight into transmission directionality (Trewby et al. 2016). The reduction in costs achieved by substituting most wild boar sample culturing for a simple ELISA could eventually make it possible to invest more resources in the sampling and culturing of other potentially relevant wildlife, including badgers. Game from fenced hunting estates has a higher prevalence of TB than free-ranging wildlife (Vicente et al. 2013 and this study), and can affect surrounding wildlife and livestock (LaHue et al. 2016 and this study). Fenced big game is usually managed through supplementary feeding and watering, thus increasing the risk of TB (Vicente et al. 2013). Our recommendation is that the managers of those fenced estates be informed about their TB situation, and about the consequences of TB for farming and wildlife management and game production (Barasona et al. 2016). Specific (tailor-made) action plans carried out by means of, for example, changes in feeding and watering, could eventually be designed in collaboration with each fenced estate, with the objective of stabilizing or hopefully improving the current situation. Finally, two hotspots of wildlife TB were identified in this study, one in the northern LVUs of León and Palencia, and one in Ávila in the south. While wildlife is not the only risk (cattle models show that cattle management and communal pastures are relevant), it is advisable to take specific action to help local cattle farmers improve their farm biosafety. A case-control study comparing TB-positive and TB-free farms in those risk areas would allow further insights to be attained.

Acknowledgements PA is supported by the Spanish Ministerio de Economía y Competitividad (MINECO) and Universidad de Castilla-La Mancha (UCLM) through a ‘Ramón y Cajal’ contract (RYC-2012-11970) and partially by the AGL2016-76358-R grant (MINECO-FEDER, UE). CG acknowledges a shortstay grant from CYTEMA and the study benefitted from the logistics of MINECO and FEDER grant AGL2014-56305. 14

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Table 1.- Prevalence of Mycobacterium tuberculosis complex infection in wildlife from Castilla y León (Spain) for the period 2011-2015. Based on microbiological culture.

Species

Prevalence (95% CI) 4.28% (3.67-4.55)

Remarks

Wild boar (Sus scrofa)

Sample size (positive) 7676 (329)

Red deer (Cervus elaphus)

1515 (34)

2.24% (1.8-3.28)

Local distribution

Fallow deer (Dama dama)

135 (11)

8.14% (4.28-13.98)

Very local distribution

Roe deer (Capreolus capreolus)

62 (0)

0% (0-6.05)

Widespread distribution

Mouflon (Ovis aries)

22 (0)

0% (0-15.17)

Very local distribution

Spanish ibex (Capra pyrenaica)

6 (0)

n.d.

Very low sample size

Chamois (Rupicapra pyrenaica)

1 (0)

n.d.

Very low sample size

European badger (Meles meles)

1 (0)

n.d.

Very low sample size

Red fox (Vulpes vulpes)

2 (0)

n.d.

Very low sample size

Widespread distribution

21

Table 2.- Number of animals tested, number positive and prevalence of TB (in %) in cattle (2014; skin testing), goats (2014; skin testing) and wild ungulates (total 2011-2014, culture) in Castilla y León, Spain. Livestock tests refer to single intradermal tuberculin tests, while prevalence signifies reactor prevalence. In the case of wildlife, testing refers to the sampling of hunterharvested individuals for culture regarding the prevalence of Mycobacterium tuberculosis complex infection in wild boar and wild ruminants sampled on non-fenced sites. Cattle (2014)

Goats (2014)

Province

Sample size (positive)

Prv.

Sample size (positive)

Prv.

Wild boar (2011-2014) Sample Prv. size (positive)

Wild ruminants (2011-2014) Sample Prv. size (positive)

Ávila

189,482 (310)

0.16

55,245 (184)

0.33

1110 (72)

6.48

249 (15)

6.02

Burgos

62,686 (57)

0.09

6906 (1)

0.01

797 (19)

2.38

76 (0)

0

León

98,322 (189)

0.19

22,691 (81)

0.36

953 (63)

6.61

234 (6)

2.56

Palencia

52,081 (50)

0.09

2939 (30)

1.02

709 (54)

7.61

238 (1)

0.42

Salamanca

457,845 (730)

0.15

5927 (24)

0.40

959 (31)

3.23

245 (7)

2.85

Segovia

69,188 (242)

0.34

2557 (17)

0.66

1183 (47)

3.97

114 (4)

3.5

Soria

18,605 (102)

0.54

2332 (35)

1.50

495 (15)

3.03

478 (8)

1.67

Valladolid

27,082 (40)

0.14

3831 (157)

4.10

547 (10)

1.82

25 (0)

0

Zamora

78,968 (61)

0.07

11,765 (3)

0.03

923 (18)

1.95

70 (1)

1.42

Total CyL

1,054,259 (1781)

0.17

114,193 (532)

0.46

7676 (329)

4.28

1729 (42)

2.42

22

Table 3.- Fixed and random effects included in the final models, statistical coefficients and 95% credible intervals (CR). S is the structured spatial random effect. Model Variable Cattle Extensive cattle farms (%) individual TB prevalence Fenced estates (presence) Wild boar abundance Goat TB prevalence Communal pastures cattle + small ruminants Communal pastures cattle Wild boar TB prevalence S Intercept Goat individual Communal pastures cattle TB prevalence + goats S Intercept Wild boar MTC Wild boar abundance infection prevalence Communal pastures cattle + small ruminants S Intercept

Coefficient 0.1403

CR 2.5% 0.1392

CR 97.5% 0.1413

15.9715 2.7622 -30.3160 0.0003

15.8466 2.7360 -30.8023 0.0003

17.1471 0.2734 0.452 34.2942 0.0005

17.1023 0.2597 -2.862 34.2047