Tuberculosis in Wild Boar and Red Deer: Diagnosis and Control in Intensive Management Systems

Tuberculosis in wild boar and red deer: Diagnosis and control in intensive management systems

Azlan Bin Che’ Amat

Tesis Doctoral

Tuberculosis in wild boar and red deer: Diagnosis and control in intensive management systems

Trabajo de investigación presentado por

Azlan Bin Che’ Amat

Para optar al grado de Doctor por la

Universidad de Castilla-La Mancha

Ciudad Real, diciembre 2017

Grupo de Sanidad y Biotecnología (SaBio)

Instituto de Investigación en Recursos Cinegéticos (IREC; CSIC-UCLM-JCCM)

Programa de Doctorado en Ciencias Agrarias y Ambientales

Universidad de Castilla-La Mancha (UCLM)

The undersigned, as directors of this doctoral thesis, note that the thesis entitled

“Tuberculosis in wild boar and red deer: Diagnosis and control in intensive management systems” and prepared by Azlan Bin Che’ Amat, licensed in veterinary medicine, meets the requirements to qualify for the Doctoral Degree.

Thesis Directors

Prof. Dr. Christian Gortázar Dr. Maria Ángeles Risalde

Ciudad Real, diciembre 2017

Grupo de Sanidad y Biotecnología (SaBio)

Instituto de Investigacíon en Recursos Cinegéticos (IREC; CSIC-UCLM-JCCM)

Programa de Doctorado en Ciencias Agrarias y Ambientales

Universidad de Castilla - La Mancha

This work of thesis has been realized with gratitude and thanks to:

Spanish Government MINECO Plan Nacional I+D+I grant AGL2014-56305

Fondo Europeo de Desarrollo Regional (FEDER)

CDTI and Glenton

EU FP7 grant WildTBvac #613779

Scholarship Department of the Higher Education Ministry, Government of Malaysia

Faculty of Veterinary Medicine, Universiti Putra Malaysia

ACKNOWLEDGEMENT

In the name of God, the Entirely Merciful, the Especially Merciful.

All praises be to God, with His guidance and permission,

I was able to complete this thesis as required.

Alhamdulillah

Here, I would like to acknowledge Prof. Dr. Christian Gortázar, my doctoral programme supervisor. Thank you for always motivated, helped and guidance throughout the period since day one. I really appreciate your kindness, especially when you were accepting my application to pursue my doctoral study in IREC under your supervision. “Beware of Attack Boss” sign on your door always scared me out most of the times, however you are truly kind, humble and always give a hand whenever needed.

Like me, sometime (even always…) good to give an attack to wake me up. Without your ideas, constructive comments and opinions, definitely it would not be easy and smooth for me to carry out the studies. I must say here that you are the vital organs of all these works. I would like to extend my gratitude to Prof Christian because he directed me to my co-supervisor, Dr. Maria Ángeles Risalde, to whom I am extremely grateful for her important role in these last 4 years. You have been exceptionally supportive and helped me keeping my motivation, either from far away or during my stay in Ciudad Real.

The back-up of a good both of you makes all the difference in the hard times, and in that respect I feel that I was very fortunate.

También quiero agradecer a todo el personal de SaBio-IREC, especialmente a Encarni, que fueron muy amables al ayudarme por todo lo relacionado con toda la documentación oficial. A Paqui, gracias por su ayuda en asuntos de laboratorio, que hizo que mi trabajo fuera fluido. A todos mis compañeros, Iratxe, José (Barasona, Armenteros, José-Fran), David G. Barrio, María Cruz, Yolanda,

Jobin, Jordi y Roxana, muchas gracias por una gran amistad, apoyo ayudado y amabilidad. Nunca

olvidaré su cálida hospitalidad. Mi disculpa por cualquier mal comportamiento de la mía.... No olvidé a

Joaquín, Fran y Cristina, Isabel, Ursula y Pelayo, todos ustedes son gente verdaderamente agradable y me hacen reconfortar a lo largo de los años de mi estancia en IREC. Tal vez muchos otros no mencionados aquí, pero créanme, todos los equipo de SaBio-IREC (presentes y pasados) son parte de mi familia y el recuerdo permaneció para siempre, debo muchas cosas a todos ustedes.

To all research partners and collaborators, Mariana Boadella (SaBiotec), Beatriz Romero

(VISAVET), Javier Bezos (VISAVET, MAEVA SERVET), Jose Antonio Ortiz (Cadiz), Paloma Rueda,

Angel Venteo (INGENASA), Konstantin Lyashchenko (Chembio, USA), Daniel O’Brien (Michigan,

USA) and others not mentioned here, thank you for the extraordinary assistance and scientific opinions. I always keep the words by Christian, ‘working as a team is always the winner’ and no doubt it is very true.

Also, I would like to thank the Government of Malaysia through the Ministry of Higher

Education for permitting and granted a scholarship for my doctoral programme in Spain. At my university, Universiti Putra Malaysia (UPM), thank you for accepting my proposal application to further my doctoral programme in IREC-UCLM, especially Prof. Dr. Aini Ideris (current Vice Chancellor of

UPM). To current dean of Faculty of Veterinary Medicine, Prof. Dr. Hair Bejo and former deans, current head of Department of Veterinary Clinical Studies, Assoc. Prof. Dr. Jalila Abu and former heads of department, thank you for keep supporting and patient with me. With a very fruitful experience here, hope

I will bring back those for the betterment to the faculty and department.

I leave the most important to the end now and thank those who are very dear to me, especially my lovely mother, Ainun Anisah Basharuddin, and all our big family; Azhan and Zarina, Azmin and Atikah,

Azlinda and Raslan, Azwan and Aida and to my lovely younger brother and sister, Azral and Azliana.

Thank you for believing and supporting for whatever things and decision I have made, even sometime all of you are concerned for certain things. I know everybody of you keep praying for me. May God bless all of you…Special thanks to my beloved wife Haslinda Abu Hassim, for making our home a warm and loving place to return to at the end of each working day, for taking care of our princess, Nur Alya

Humaira and for cherishing the differences between us. My apology because we have to be a thousand

miles away from each other for a while, but believe me, all these are for our better future. Thank you for always supporting and adapting the situation. You are my inspiration and happiness in my quest for knowledge.

Muchas gracias todos!

Danke euch allen!

Thank You everyone!

Terima Kasih semua!

.….This is also for you Abah (Che’ Amat Senawi)..Al-Fatihah.

Table of Contents

Page RESUMEN………………………………………………………………………………………… . 1

SUMMARY………………………………………………………………………………………... . 5

ORGANIZATION OF THE THESIS…………………………………………………………….. 9

CHAPTER 1: Overview 1.1 General Introduction…………………………………………………………………………. . 11 Tuberculosis and the Mycobacterium tuberculosis Complex……………………………… 12 Epidemiology of Mycobacterium tuberculosis complex…………………………………... 12 Transmission of M. tuberculosis among humans and to animals……………………… 13 Transmission of zoonotic TB (to humans and among humans)……………………..... 13 Transmission of animal TB among animals (intra- and inter-species)………………... 14 Pathogen and host factors in M. bovis infection…………………………………………… 17 Pathogenesis and development of TB-like lesions……………………………………...... 17 Global view of bovine TB…………………………………………………………………. 18 Importance of animal TB………………………………………………………………….. 20 Zoonotic and public health significance………………………………………………. 20 Impact on economy…………………………………………………………………… 20 Conservation related issues…………………………………………………………… 21 Potential cross border animal TB transmission………………………………………. 22 The burden of TB in wild animals………………………………………………………… 22 General background of wildlife TB…………………………………………………… 22 Wild boar and cervids: the Mediterranean wildlife reservoir………………………… 23 Ante-mortem diagnostic tests for MTC in wild boar and red deer……………………….. 25 CMI-based tests………………………………………………………………………. 26 Antibody detection tests……………………………………………………………… 27 Effects of co-infections on the diagnostic response………………………………………. 30 Domestic animals…………………………………………………………………….. 30 Wild animals…………………………………………………………………………. 31 Justification…………………………………………………………………… …………. 32

1.2 Open questions and recent advances in the control of a multi-host infectious disease: animal tuberculosis…………………………………………………………………………………….. 33 Abstract………………………………………………………………………………………… . 34 Introduction……………………………………………………………………………………... 35 MTC Reservoirs………………………………………………………………………………… 36 Case study: Southeast Asia…………………………………………………………………. 40 Research needs: MTC reservoirs…………………………………………………………… 41 Host Pathology, Ecology and TB Epidemiology……………………………………………….. 41 Research needs: host pathology, ecology and TB epidemiology…………………………... 44 Time Trends in Wildlife TB…………………………………………………………………..... 44 Research needs: time trends………………………………………………………………... 46 Attempts to Control TB………………………………………………………………………… 46 Population control…………………………………………………………………………. 46 Farm biosafety……………………………………………………………………………... 47 Research needs: controlling TB……………………………………………………………. 47 Vaccination……………………………………………………………………………………... 48 Case study: planning for vaccination in Michigan, USA…………………………………... 48 Case study: Montes de Toledo, Spain……………………………………………………… 54 Research needs: vaccination……………………………………………………………….. 55 Discussion……………………………………………………………………………………… . 56 Acknowledgements……………………………………………………………………………... 57 References……………………………………………………………………………………… . 57

1.3 STUDY HYPOTHESIS AND OBJECTIVES………………………………………………. . 67

RESULTS - CHAPTER 2: Testing wild boar piglets for serum antibodies against Mycobacterium bovis………………………………………………………………………………………………..... 69 Abstract……………………………………………………………………………………………… 70 Introduction…………………………………………………………………………………………. 71 Material and Methods………………………………………………………………………………. 73 Animals and sampling…………………………………………………………………………. 73 Bacterial culture and characterization………………………………………………………… . 73 Antibody detection tests……………………………………………………………………….. 74

ELISA bPPD IgG and IgM………………………………………………………………… . 74 ELISA bPPD Protein-G (IgG detection)…………………………………………………… 75 ELISA-DR…………………………………………………………………………………. 75 DPP tests…………………………………………………………………………………… . 76 Data treatment…………………………………………………………………………………. 76 Results………………………………………………………………………………………………. 77 Discussion…………………………………………………………………………………………... 81 Acknowledgements………………………………………………………………………………… 82 References………………………………………………………………………………………….. 83

RESULTS - CHAPTER 3: Effects of repeated comparative intradermal tuberculin testing on test results: a longitudinal study in TB-free red deer…………………………………… …... 85 Abstract…………………………………………………………………………………………….. 86 Background………………………………………………………………………………………… 87 Methods…………………………………………………………………………………………….. 89 Animals………………………………………………………………………………………... 89 Diagnostic tests………………………………………………………………………………... 90 Intradermal skin test………………………………………………………………………... 90 Serological tests……………………………………………………………………………. 90 Data analysis…………………………………………………………………………………... 92 Results………………………………………………………………………………………………. 92 Discussions…………………………………………………………………………………………. 97 Conclusion………………………………………………………………………………………….. 100 Abbreviations………………………………………………………………………………………. 100 Acknowledgements………………………………………………………………………………… 100 Funding…………………………………………………………………………………………….. 100 Availability of data and material…………………………………………………………………... 101 Author’s contributions……………………………………………………………………………... 101 Competing interests………………………………………………………………………………… 101 Consent for publication…………………………………………………………………………….. 101 Ethics and consent to participate…………………………………………………………………... 101 References…………………………………………………………………………………………... 101 Additional file………………………………………………………………………………………. 105

RESULTS - CHAPTER 4: Is targeted culling a suitable means for tuberculosis control in wild boar?...... 111 Abstract…………………………………………………………………………………………….. 112 Introduction………………………………………………………………………………………… 113 Material and Methods……………………………………………………………………………… 114 Study area……………………………………………………………………………………... 114 Data collection and analysis………………………………………………………………….. 116 Results………………………………………………………………………………………………. 119 Discussion…………………………………………………………………………………………... 122 Acknowledgements………………………………………………………………………………… 124 References………………………………………………………………………………………….. 125

RESULTS - CHAPTER 5: The porcine respiratory disease complex causes significant mortality among wild boar piglets………………………………………………………………………….. 127 Abstract…………………………………………………………………………………………….. 128 Introduction………………………………………………………………………………………… 129 Material and Methods……………………………………………………………………………… 130 Results………………………………………………………………………………………………. 132 Discussion…………………………………………………………………………………………... 135 Acknowledgements………………………………………………………………………………… 137 References………………………………………………………………………………………….. 137 Suppelementary file………………………………………………………………………………… 139

GENERAL DISCUSSION AND SYNTHESIS………………………………………………….. 141 Future research needs……………………………………………………………………….. 145

CONCLUSION……………………………………………………………………………………. 147

REFERENCES ……………………………………………………………………………………. 149

RESUMEN

La tuberculosis animal (TB) es una de las enfermedades infecciosas de la ganadería y la fauna silvestre de mayor preocupación en todo el mundo. Esto se debe a su enorme impacto en aspectos como la salud pública, la economía, la gestión de la vida silvestre y su conservación. El propósito de esta tesis es aportar información sobre la implementación de pruebas de diagnóstico y prácticas de manejo en las dos especies de ungulados silvestres más prevalentes en la Península ibérica, jabalíes y ciervos, utilizando estas herramientas como estrategias de control para la TB. Además, estudiamos el papel desempeñado por otras infecciones en la mortalidad de los rayones de jabalí.

Esta tesis se compone de una introducción general y de cuatro capítulos de investigación, donde se incluyen estudios específicos sobre diversas pruebas de diagnóstico ante-mortem, un ensayo de campo para el control de la TB basado en el diagnóstico y sacrificio selectivo, y un estudio de las patologías en jabalí sometido a un manejo intensivo.

El primer capítulo es una revisión general de la enfermedad, dividida en dos partes. La primera parte describe el estado actual del diagnóstico ante-mortem de la TB en animales de vida libre, los problemas relacionados con la TB y estos animales, la economía y la conservación. Además, en este capítulo se describen exhaustivamente las pruebas de diagnóstico actuales para micobacterias del Complejo

Mycobacterium tuberculosis (CMT) utilizadas en distintas especies de fauna silvestre a nivel mundial. La segunda parte trata sobre la distribución mundial de la TB y la importancia de los reservorios de vida libre; reconoce aspectos insuficientemente conocidos de la patología, la ecología y la epidemiología de la enfermedad en estas especies; presenta estudidos temporales de la TB en animales silvestres; y resume la investigación actual sobre el control de la TB, proporcionando información adicional sobre la vacunación frente a esta enfermedad.

En el segundo capítulo se evalúan los resultados de varias pruebas de diagnóstico de la TB mediante la detección de anticuerpos frente al CMT en rayones de jabalí de 2 a 6 meses de edad, expuestos naturalmente al patógeno. Este estudio transversal incluyó necropsias con la identificación macroscópica de lesiones compatibles con TB, la identificación de micobacterias del CMT por cultivo y la comparación de diversas pruebas serológicas para la detección de anticuerpos frente al CMT. Un total de 126 muestras de jabalí que murieron en verano en 2012 y 2013 fueron incorporadas al estudio. El cultivo bacteriano confirmó una prevalencia del 33,9%, mientras que las seroprevalencias estimadas por anticuerpos variaron entre el 19,0 y el 38,0%, consiguiendo la mejor sensibilidad (entre el 61,5% y el 69,2%) la prueba de inmunocromatografía rápida basada en la tecnología de doble vía en comparación con distintos protocolos de ELISA. Este capítulo pone de manifiesto que cerca del 33% de los rayones de jabalí pueden infectarse a una edad temprana en áreas con una elevada prevalencia de TB. La serología fue capaz de detectar anticuerpos frente al CMT en rayones de jabalí; sin embargo, esta técnica es menos sensible que en el jabalí adulto. Por todo ello, concluimos que las pruebas rápidas para el diagnótico de la TB en animales pueden contribuir al control de la enfermedad mediante programas sanitarios de diagnóstico y sacrificio.

El tercer capítulo está enfocado en determinar si las pruebas en piel repetidas en ciervos no tuberculosos con derivados de la proteína purificada aviar y bovina (PPDa, PPDb) para el diagnóstico de la TB, por ejemplo cada 6 meses, podrían afectar a la capacidad de respuesta de la prueba piel o a los niveles de anticuerpos contra los antígenos de micobacterias. Esta pregunta se nos planteó debido a que la intradermotuberculinización es la prueba rutinariamente usada para el diagnóstico de la TB en rumiantes, incluyendo los animales silvestres. En este estudio, hemos observado que las repetidas tuberculinizaciones comparadas no ocasionan una pérdida de la sensibilidad contra PPDb en piel o en el

ELISA para la detección de anticuerpos frente a PPDb y MPB70. Sin embargo, observamos que los anticuerpos frente PPDa y PPA3 aumentan en determinadas épocas, probablemente debido a la exposición a Mycobacterium avium paratuberculosis (MAP) o a las reacciones cruzadas con

micobacterias ambientales. Así, este método de diagnóstico se puede aplicar en el programa de control de la TB sin miedo a una sensibilización progresiva o a la desensibilización.

El cuarto capítulo se concentra en una estrategia específica de control de la TB para reducir la prevalencia de micobacterias del CMT mediante la implementación de un programa de eliminación selectiva, es decir, eliminando los rayones de jabalí seropositivos a pruebas inmunocromatográficas rápidas en una determinada área de caza. Esta captura-prueba-liberación fue llevada a cabo en los veranos de 2012-2014. En rayones capturados anualmente, la seroprevalencia a anticuerpos frente al CMT disminuyó significativamente en un 39% desde el incio del programa, de 2012 a 2013, sin reducción adicional durante la tercera temporada de captura. Adversamente, la prevalencia de CMT en el sitio de liberación aumentó significativamente en un 60% de la población abatida por los cazadores. La eliminación selectiva de animales positivos como estrategia de control de la TB dentro de un área de alta presión de infección no logró reducir la prevalencia, sin embargo ha generando valiosos conocimientos sobre esta herramienta de control y sobre la epidemiología de la TB en áeras de alta presión de infección.

Finalmente, en esta área de alta presión de infección de TB, llevamos a cabo un estudio para elucidar las causas que provocaban una marcada mortalidad de los rayones en verano. Nuestros resultados evidenciaron una mortalidad esperada de rayones extremadamente alta (71%) durante el verano de 2015, medida por la disminución en la proporción rayones/adultos observada en los comederos mediante cámaras trampa y entrevistas a los guardas del coto. Los rayones menores de 4 meses hayados muertos mostraron una deficiente condición corporal, presentando el 88,9% de ellos lesiones respiratorias compatibles con el Complejo respiratorio porcino (CRP), y siendo el circovirus porcino tipo 2 (PCV2) el patógeno más frecuente identificado (en el 39% de los casos). Sin embargo, no se observó ninguna clara relación entre las lesiones pulmonares y la prevalencia de los agentes infecciosos asociados (PCV2,

Mycoplasma hyopneumoniae), ni con la infección del CMT. Los hallazgos pulmonares caracterizados por

las lesiones neumónicas, pero no por la infección per se del CMT, fue la razón más plausible de la alta mortalidad registrada en los rayones de jabalí.

Palabras clave: Pruebas diagnósticas, ensayo inmunoenzimático (ELISA), mortalidad, Complejo

Mycobacterium tuberculosis, ciervos rojos, enfermedades respiratorias, eliminación selectiva, control de la tuberculosis, prueba de la tuberculina en piel, rayones de jabalí.

SUMMARY

Animal tuberculosis (TB) is one of the infectious diseases of livestock and wildlife of more concern, worldwide. This is due to its huge impact on aspects such as public health, economy, wildlife management and conservation. The purpose of the thesis is to provide information on the implementation of practical and accessible TB diagnostic tests in two wild ungulates, wild boar and red deer, using these tools as control strategies for TB in wild animals. In addition, we aimed to study the role played by other infections in wild boar piglet mortality.

The thesis is composed of an introductory main overview section and four research chapters, each of them dealing with specific studies on different ante-mortem tests, a field TB control trial based on targeted culling, and a disease investigation in wild boar under intensive management.

Chapter one is a general overview of TB and defines basic terminology. It also details the information and knowledge related to the research of the thesis. Chapter one is subdivided into two parts.

Part one describes the state of the art regarding TB diagnosis in wildlife, TB hazard related issues among animals and humans, economy and conservation. It also comprehensively describes the current diagnostic tests for MTC that have been studied or used in wildlife species globally. Part two deals with specific reviews on the current knowledge on global TB distribution and the significance of wildlife hosts; recognizes insufficiently known aspects of host pathology, ecology and epidemiology; presents selected time series in wildlife TB; and summarizes ongoing research on TB control, providing additional insight on vaccination.

Chapter two assesses the performance of several antibody detection tests for M. bovis infection in free-living, naturally-exposed 2–6 month-old piglets during summer. This cross sectional study included necropsies with macroscopic identification of TB-like lesions, Mycobacterium tuberculosis complex

(MTC) bacterial culture and identification, and six different serological antibody tests. A total of 126 samples of wild boar piglets which died within summer in 2012 and 2013 were incorporated in the analysis. Bacterial culture yielded a prevalence of 33.9%, while prevalences estimated by serology ranged from 19.0 to 38.0%, achieving the best sensitivities between 61.5% and 69.2% for rapid immunochromatographic tests based on the dual path platform (DPP) technology as compared the other four plate ELISAs. This chapter evidences that about 33% of the wild boar piglets can become infected at early age in high TB prevalence sites. Serology able to detect antibodies against MTC in piglets however is rather less sensitive than in adult wild boar. Rapid animal-side tests can contribute to TB control using test and cull schemes.

Chapter three addresses the question whether repeated skin testing with avian and bovine purified protein derivative (aPPD, bPPD), for instance every 6 months,could affect the skin test responsiveness or the antibody levels against mycobacterial antigens in TB-free red deer. This question arises since the tuberculin skin test is the routinely used one for TB diagnosis in ruminant species, including wild animals. We observed that repeated comparative skin testing did not cause progressive changes or specifically cause a loss in skin test responsiveness against bPPD tuberculin or in ELISA antibody responses against bPPD and MPB70 antigens. However, the results showed ELISA antibody increases through times against avian PPD and PPA3 antigens, which possibly were due to exposure to

Mycobacterium avium paratuberculosis (MAP) or to cross reactions with environmental mycobacteria.

Thus, this diagnostic method can be applied for the TB control programme without the fear for progressive sensitization or de-sensitization.

Chapter four focuses on one specific control strategy to reduce TB transmission between wildlife and livestock, namely targeted culling of infected wildlife. The main aim of the research reported in this chapter was to reduce the TB prevalence in the control site by implementing a targeted removal strategy, i.e. by selectively removing seropositive wild boar (using immunochromatographic DPP rapid tests) from

one hunting estate and harvesting them by hunting on the release site. This capture-test-release trial was conducted in summer 2012-2014. The annual summer seroprevalence of antibodies to the MTC declined significantly by 39% in live-captured wild boar piglets from the treatment site, from 2012 to 2013 with no further reduction during the third capture season. Adversely, the MTC prevalence in the release site increased significantly by 60% in the hunter-harvested population. Targeted removal attempted in this TB control strategy within a high infection pressure area failed to reduce the prevalence, nevertheless generating valuable knowledge on this specific TB control tool and on TB epidemiology at very high force of infection.

Finally, in the site with high force of infection, we led a cross sectional investigation to elucidate the causes for a marked mortality among wild boar piglets during summer. The findings were, that the expected piglet mortality during summer 2015 was extremely high (71%) as measured by the decrease of the piglet-to-adult ratio at the feeders through video-trapping analysis, as well as by interviewing the game rangers. In addition to that, dead piglets aged less than 4-month-old have indicated poor body condition, 88.9% had PRDC-compatible respiratory lesions predominantly characterized by pneumonia, and Porcine Circovirus Type 2 (PCV2) was the most prevalent single pathogen at 39%. However, no clear relationship between prevalence and lung lesions was observed for the PRDC associated pathogens

(PCV2, Mycoplasma hyopneumoniae), nor for MTC infection. Respiratory infection characterized by pneumonia, but not MTC infection per se, was the most plausible reason of the high mortality recorded among early age wild boar piglets.

Keywords: Diagnostic tests, enzyme-linked immunosorbent assay (ELISA), mortality, Mycobacterium tuberculosis complex (MTC), red deer, respiratory disease, targeted removal, tuberculosis control, tuberculin skin test, wild boar piglets.

ORGANIZATION OF THE THESIS

This thesis is organized according to the usual order of a scientific work. Starting with an introduction, in which there are two parts: a general review on the epidemiology of Mycobacterium tuberculosis complex (MTBC) in different hosts, in which particular attention was given to wild boar and red deer; and the second part discusses on the current knowledge and control strategy for wildlife tuberculosis (TB) as well as future research needs.

This thesis deals with the study of MTBC in wild boar and red deer by focusing on the usage of selected humoral and cell mediated immunity tests. Features of this work are that: (i) the work is carried out with wild boar and red deer infected under natural conditions, (ii) the evaluation of different humoral antibody tests by means of ELISAs and rapid immunochromatographic tests is done using sera samples that are collected during hunting season in autumn-winter and capture-recapture management in summer,

(iii) the comparative tuberculosis skin testing in red deer is done repeatedly in different seasons and studied its effect on serological tests (iv) and (v) the rapid immunochromatographic tests is applied in the field for TB control programme in an attempt to control TB in wild boar.

The thesis is structured such that, once introduced the relevance of serological tests in a specific age group of animals, this is followed by the application of the tests with other diagnostic tools to characterize disease status in high TB pressure farms. Then, the TB ELISA tests were used in concurrent events with

TB skin tests in red deer. In last place, the rapid tests that have been used in the previous sections are now used in selected removal of wild boar for TB control programme.

The whole thesis is written in English and some chapters correspond to scientific articles in different publication status, so that each of the chapters has the typical structure of an article beginning with summary, introduction, material and methods, results, discussion and references. Synthesis of the thesis is

performed by a brief discussion of the general results obtained in the main chapters and finally listed the general conclusions obtained in this work.

Chapter I

INTRODUCTION

1.1. General Introduction

Tuberculosis and the Mycobacterium tuberculosis Complex

Tuberculosis (TB) is an ancient, worldwide distributed, disease of humans and animals caused by infection with the Mycobacterium tuberculosis complex (MTC). This complex of slow-growing mycobacteria includes M. tuberculosis, M. cannettii, M. africanum, M. bovis, M. pinnipedii, M. caprae,

M. microti and M. mungi (LoBue et al., 2010; Thoen et al., 2014). Tuberculosis is one of the most fatal diseases in the world with 95% human cases occurring in developing nations (WHO, 2015). Human TB is caused predominantly by M. tuberculosis infection causing pulmonary and systemic disease. Only a small proportion of human TB cases are attributed to zoonotic TB, caused by M. bovis, M. caprae and others.

However, the true incidence of zoonotic TB in many countries is probably not well monitored due to unsystematic surveillance for MTC members other than M. tuberculosis, and lack of appropriate laboratory procedures to diagnose and differentiate mycobacteria (Olea-Popelka et al., 2016). M. bovis and M. caprae are globally important mycobacteria causing TB in domestic and wild animals (Thoen et al., 2014). From the public health point of view, M. bovis is an important species because of being the second most pathogenic MTC member after M. tuberculosis, and the one with the widest host range (Une and Mori, 2007). Mycobacterium bovis is often associated with non-pulmonary TB, and higher case- fatality, in humans (Palacios et al., 2016).

Epidemiology of Mycobacterium tuberculosis complex

The primary mode of intra-species transmission for M. bovis is through aerosol, however the infection may influenced by animal age, behavior, environment and climate, and prevailing farm practices

(Pollock and Neil, 2002). The transmission of MTC is dependent on several factors that include the number of infected and susceptible animals, routes of infection, the anatomical site of disease invasion, structure of tuberculous lesions, routes of pathogen excretion, and the minimum infective dose by each infection route (Corner, 2006). A schematic diagram of MTC transmission in humans and animals is presented in Figure 1.

Transmission of M. tuberculosis among humans and to animals

Humans are the reservoir for M. tuberculosis, with transmission occurring person-to-person via the respiratory route by direct aerosol transmission. Yet the close personal contact with airborne transmission is hard to occur (Punjabi et al., 2016; Shiloh, 2016). Pulmonary TB is more likely to produce high levels of bioaerosols and continuous tidal breathing could transport M. tuberculosis within a size range that could be inhaled by a susceptible contact (Wurie et al., 2016). The likelihood of transmission of M. tuberculosis is proportional to the duration of exposure to an infectious person and the concentration of infectious droplet nuclei in the air (Pienaar et al., 2010). However, the findings of viable M tuberculosis in 10% of water samples and 1% of soil samples in Tehran City

(Iran) suggest environmental routes of TB transmission among human beings (Velayati et al., 2015).

Certain risk groups are more susceptible to get infected and develop active TB including those immunocompromised (e.g. HIV) where high mortality occurs (Fogel, 2015).

Transmission of zoonotic TB (to humans and among humans)

The transmission of zoonotic TB caused by M. bovis and closely related members of the MTC can be a direct contact (aerogenous route or contaminated secretion) with the animal reservoir host

(primarily cattle); consumption of unpasteurized dairy products; and more exceptionally improperly handled raw meat (Thoen et al., 2016). Exposure to M. bovis is highly linked to work risks mainly including crop and livestock farmers as well as possible exposure in patients born in countries with a high prevalence of bovine TB (Rodríguez et al., 2009). Transmission of M. bovis among humans is usually very limited, however close contact airborne transmission from person to person has been suggested in (e.g. in UK and France) where evidence showed that the patients visited the same area with no links of consuming unpasteurized dairy products (Evans et al., 2007; Sunder et al. 2009).

Recent evidence also suggested transmission by the respiratory route and close contact between humans where a person came from Mexico to US (with a history of working in a dairy farm) and the other person possibly became infected when they visited the same church (Buss et al., 2016).

Transmission of animal TB among animals (intra- and inter-species)

Cattle is the primary host for bovine TB due to M. bovis with a complex epidemiological pattern, which includes the transmission of infection to animals and occasionally to humans. Animal tuberculosis affects a wide host range of domestic and wild animals and widely distributed (Pesciaroli et al., 2014). Other than cattle, high infection rates also occur in other domestic species mainly swine, sheep and goats and camelids, which possibly play a role in the inter-species disease (Pesciaroli et al.,

2014, details in Table 1).

In cattle, the main route of infection is through aerosol exposure, facilitated by close contact between animals (Neill et al., 1991). Less commonly, infection occurs by ingestion (oral route) of contaminated products such as pasture and water (Menzies and Neill, 2000). In calves, transmission during milk feeding would be an important route since the M. bovis has been isolated from the milk from cattle and buffalo (Jha et al., 2007).

Generally, it was accepted that the most effective route of infection between and within wildlife species and from wildlife to domestic animals is the direct contact through airborne and oral route

(Corner, 2006). Nevertheless, a study on the TB host community (cattle, domestic pigs, wild boar and red deer) in southwest Spain showed that indirect intra-species contacts were frequent compared to extremely rare direct inter-species interactions. Thus, disease transmission within species is unlikely to be of a direct nature (Cowie et al., 2016).

In fact, ‘indirect’ interspecies transmission is gaining relevance, particularly where several domestic and wild host species interact at focal risk sites such as water points, in which the M. bovis seems to survive, particularly in the mud. This poses a great risk for infection transmission (Kukielka et al 2013; Barasona et al., 2016; Carrasco-Garcia et al., 2016).

Figure 1. Origin and transmission of human and animal TB among human, domestic and wildlife species.

Table 1. Domestic animal species and their roles in MTC maintenance

Primary Animals/Hosts Modes of MTC infection and transmission Susceptible MTC

Cattle M. bovis Primary transmission by inhalation (direct contact). Mainly shed

through respiratory tissues (tonsils, mucosa and turbinates) and

nasal mucus with less significant transmission through milk, urine

and feces. Secondary by ingestion (e.g. contaminated pastures,

water, fomites). Congenital infections and vertical transmission to

calves are uncommon.

Water buffalo M. bovis Shedding similar as in cattle. Mainly by respiratory route (close

contact transmission due to being a social species forming dense

groups on available dry land during the wet season.

Goat M. bovis and M. Similar as in cattle. Close contact transmission intra- and

caprae; interspecies (cattle and small ruminants) transmission,

particularly when they share resources.

Sheep M. bovis and M. Nasal discharge (TB lesions mostly confined to the respiratory

caprae; tract). Aerosol transmission (direct contact). Sheep should be

considered as potential sources of MTC especially when sharing

farms and pastures with TB-infected cattle and/or goats.

Camelids M. bovis or M. Close contact (high susceptibility when reared in intensive

microti and NTM conditions and stress-associated). Lymph nodes, whose afferents

drain the nasal cavities, the nasopharynx, the auditory tubes and

the lung (sputum, mucus, or stool).

Domestic pig Mainly M. bovis Respiratory and oral route (ingestion of milk, milk products or

offal from infected cows). Prevalence in pig population was

usually correlated to that in cattle. Increasing spread of outdoor

pig farming systems has led to the reemergence of M. bovis

infection in domestic pigs.

Sources: Pollock and Neill, 2002; Corner 2006; Pesciaroli et al., 2014; Muñoz-Mendoza et al., 2016.

Pathogen and host factors in M. bovis infection

Viability (small vs high infective dose), size (fine aerosol suspensions of low viscosity are most effective) and consistency of mycobacterial bacilli are pathogen-related factors playing important roles for M. bovis establishing infection (Pollock and Neill, 2002). Complex properties of the outer and inner layers of the mycobacterial cell wall (lipids and proteins) mediate the virulence through the intracellular resistance, maintaining growth within the tuberculous granuloma and favoring or inhibiting macrophage activation (Hett and Rubin, 2008). Other virulence factors may be involved in the interaction of MTC and the host macrophage as well as playing a role in modulation of the host immune response (Forrellad et al.,

2013). Host influences such as age (older waning protective capability) and external influences such as nutritional deficiencies (e.g. lymphocyte subpopulation), stress-associated events, immunosuppressive effects (e.g. co-infection) might reduce resistance to TB (Pollock and Neill, 2002). Recent exposures to infectious organisms or environmental mycobacteria could provide a certain degree of protection (Pollock and Neill, 2002).

Pathogenesis and development of TB-like lesions

In general, the tuberculous mycobacteria invade the macrophage by cell surface binding of the phagocyte. Ingestion by phagocytes protects the bacilli from the natural defenses in the serum and lysosomes-phagosome fused leading phagocytes to destroy the bacillus. Due to mycobacterial virulence factors and the host immune response, the bacilli have the ability to escape killing, reactivate and release new bacilli causing an infection (Olsen et al., 2010). Using the human TB model, inhalation droplet nuclei containing tubercle bacilli reach the alveoli of the lungs leading to ingestion by alveolar macrophages.

Most bacilli are destroyed or inhibited with a few ones remaining. These will multiply intracellularly.

When macrophages die, the bacilli continue to spread through the lymph or bloodstream to tissues or organs in which TB disease is most likely to develop. This primed immune system attracts macrophages

and other immune cells to respond against extracellular bacilli, confining them in the formation of granulomas (LoBue et al., 2010).

In cattle, infection with droplet nuclei containing M. bovis through the respiratory system begins with the formation of lesions in the lungs, primarily in the tracheobronchial lymph nodes (LN). Lung tissues and LN of the head are the second most frequent site for tuberculous lesions to appear, usually in the form of nodules with very small size, white to yellowish. Later, lesions develop in the lungs unilaterally and/or bilaterally and most of them occur in caudal lobe. Epithelioid and giant cells (macrophage-fusion) form the center of young tubercles subsequently surrounded by a zone of lymphocytes, plasma cells and monocytes. The peripheral tubercle ultimately forms fibroplasia, necrotized in the center and mineralization can be observed in the caseous necrotic area (Neill et al., 2001).

In wild boar within high MTC prevalence scenarios in Mediterranean Spain, lesions are most often observed in the mandibular LN (retropharyngeal and parotid LN were only detected if lesions present in mandibular LN). Generalization to other anatomic regions was frequent, including lesions in thoracic LNs and lungs, and less frequently abdominal organ lesions including liver and spleen (Martin-Hernando et al., 2007). Tonsil and ileo-cecal valve lesions were mostly detected by histopathology, whereas mammary gland and renal or renal LN lesions were rare. However, in low prevalence settings such as the Atlantic region of Spain, only a low proportion of MTC infected wild boar displayed generalized TB lesions

(Muñoz-Mendoza et al., 2013).

Global view of bovine TB

Bovine TB in domestic animals is prevalent in many parts in the world. The World Animal Health

Information Database (WAHIS Interface, 2017) in 2015 reported that bovine TB in domestic animals was present (including suspected) in many members of Africa, Asia, EU, Americas and Oceania. No disease

information data was available in most of northern, northwestern and central Africa, part of central Asia including China, southern Latin America and in a few eastern EU countries (Figure 1).

A markedly different scenario was observed in the same period regarding data for wildlife TB.

Reported cases came mainly from northern America and the EU, while information from other regions is scarce and scattered (Figure 2). Two recent reviews assessing the relevance of wildlife diseases showed that bovine TB ranked among the top four infectious diseases reported at the wildlife-livestock interface, along with avian influenza, rabies and salmonellosis (Wiethoelter et al., 2015) or with Coxiella burnetii, pathogenic Escherichia coli and again (highly pathogenic) avian influenza (Gortázar et al., 2016), respectively.

Figure 2. Global domestic animals bovine TB distribution maps (July-December 2015 report).

Figure 3. Global wild animal bovine TB distribution maps (July-December 2015 report).

Importance of animal TB

Zoonotic and public health significance

The consumption of unpasteurized milk or milk products still remains a risk for infection in

countries (notably low-income or developing nations) where bovine TB has not been eradicated,

infection in cattle is enzootic and where HIV is prevalent causing both pulmonary and significant

extra-pulmonary TB and marked pediatric cases (de Kantor et al., 2010, Thoen et al., 2016).

Impact on economy

Animal TB causes significant economic burden principally for the cattle industry with estimated

USD 3 billion annually (Waters et al., 2012). If left uncontrolled, the disease might cause high

morbidity, a decrease in livestock productivity and death in cases of advanced TB (Verma et al.,

2014). National or international economic impact includes effects on animal production, impact on trade due to movement restriction of animals within or between countries, and the real cost of implementing surveillance and control programs including a well-defined testing and culling program.

Such programs use the intradermal tuberculin test where positive reactors are culled and compensation payments for these animals are often in place. Sustainment of such TB control programs and ongoing research to improve control strategies all add to the impact of TB on economy (El Idrissi and Parker,

2012; Waters et al., 2012; Torgerson 2013; Caminiti et al., 2016).

Conservation related issues

Consuming MTC-contaminated meat by zoo animals (e.g. M. bovis transmission to African lion

(Panthera leo) due to feeding contaminated raw meat, see Adeogun et al., 2016) or MTC-infected prey by endangered predators may implicate conservation issues. Known examples of transmission of TB influencing conservation of endangered species were the Spanish Iberian lynxes (Lynx pardinus), which were found infected with MTC belonging to the molecular types common in wild ungulates

(Gortázar et al., 2008) and African lions contracting TB from African buffalo (Syncerus caffer) or possibly other wildlife (Viljoen et al., 2015). Close contact between enclosures and exposure through the poor hygienic habits by the visitors or zookeepers favor the maintenance and survival of human- origin TB in zoo animals due to M. tuberculosis. This pathogen was detected in captive wild ungulates, non-human primate species and large felines (e.g. lion) in zoological gardens (Michel et al.,

2003; Adeogun et al., 2016). Another conservation issue are the conflicts that TB may cause between livestock farmers (e.g. spillback from wildlife, limited land for grazing cattle as to prevent wildlife- livestock interface), hunters (e.g. hunting is not permitted or prohibition in supplemental feeding and baiting for purposes of hunting) and conservationists (e.g. opposition to culling or depopulation of wildlife reservoirs), in different parts of the world (Gortázar et al., 2008; Shury and Bergeson 2011;

Shury et al., 2015).

Potential cross border animal TB transmission

Complex socio-ecological dynamics affect livestock-wildlife disease control with the potential of

cross-border transmission. For instance foot and mouth disease (FMD) control in southern Africa is

hampered by multi factorial events including damage of ‘cordon fences’ (Mogotsi et al., 2016), and

movement of livestock and wild animals between different areas (Cameron et al., 1999).

Another risk factor identified with the emergence of disease from wildlife has been the significant

increment in trade and consumption of bushmeat in many parts of the world. The current critical

increasing estimation of poaching and animals seized for illegal market for exportation either for

animal trade or meat consumption may carry a potential risk of spreading pathogens (and subsequent

emergence of zoonotic diseases) to other regions with serious implications for humans and domestic

animals (Bouslikhane, 2015; TRAFFIC, 2016).

The burden of TB in wild animals

General background of wildlife TB

Across the world, several evidences have shown severe problems associated with wildlife

reservoirs ofMTC, involving different host species in different geographical conditions:

 New Zealand, the brushtail possum (Trichosurus vulpecula) causing spread to cattle, deer and

ferrets (Caley et al. 1999; Caley et al., 2001; Nugent et al., 2015);

 British Isles (Republic of Ireland and UK), European badger (Meles meles) causing spillover to

domestic cattle (Corner et al., 2011);

 Iberian Peninsula (Spain and Portugal), red deer (Cervus elaphus) and the Eurasian wild boar (Sus

scrofa), causing threat to livestock and wild animals including the endangered Iberian lynx

(Aranaz et al., 2004; Vicente et al., 2006; Gortázar et al., 2008; Naranjo et al., 2008; Madeira et

al., 2015)

 North America (Michigan, Mexico), white-tailed deer (Odocoileus virginianus) (O’Brien et al.,

2011); Canada, wood bison (Bison bison athabascae), elk (Cervus elaphus) and white-tailed deer

(Nishi et al., 2006; Shury and Bergeson, 2011);

 South Africa, African buffalo (Syncerus caffer) with spillover infection to other wild animals

(Renwick et al., 2007);

 Zimbabwe (southern Africa), lechwe antelope (Kobus leche) causing lechwe population decline

(Bengis et al., 2004; Munyeme et al., 2010).

Wild boar and cervids: the Mediterranean wildlife reservoir

The most common host communities for TB hazard in Europe are ‘cattle-deer-wild boar’ in

which the wild boar poses the greatest hazard of all the wildlife species and wild boar-deer have

the greatest susceptibility and ability to transmit disease to cattle and other animals (Hardstaff et

al., 2014). In Spain, Eurasian wild boar and red deer are known to be wildlife MTC reservoirs

(Gortazar et al., 2011). The persistence of bovine TB in beef cattle (in the south-central region of

Spain) is associated with the presence of wildlife reservoirs especially farms with large pasture

areas which eventually increase the probability of interaction (Guta et al., 2014; LaHue et al.

2016). In addition, the use of extensive farm resources by wild ungulates (particularly wild boar

and red deer) is frequent and widespread (Carrasco-Garcia et al., 2016). As evidenced by García-

Jiménez and colleagues (2016), the similar spoligotypes shared by wild boar and cattle (93.5%)

support the role of wild boar as main maintenance host. Although many deer species have been

recorded to be susceptible to MTC infection, a large portions of them seems incapable for frequent

intra-species transmission and hence to maintain infection within their respective populations.

White-tailed deer, red deer and fallow deer are the most known cervids to serve as true MTC

reservoir. To fit the purpose of this thesis, this review focused on red deer (and wild boar).

In Ciudad Real province (south-central Spain), the wildlife hunting estate surveillance (from year 2000 to 2012) showed a high TB lesion prevalence of 63% in wild boar (Vicente et al., 2013).

This high prevalence was due to an increase in wild boar numbers, dry summers and increasing wildlife management through fencing, feeding and watering. Down to southern regions a high wild boar MTC infection prevalence was reported in Doñana National Park (NP) by culture

(52.4%; Gortázar et al., 2008), and a 52.3% seroprevalence was recorded using a combination of

ELISA tests throughout Andalusia region (García-Bocanegra et al., 2012). A high prevalence of wild boar TB has been noted in one fenced estate in central Spain (near Madrid) in which the prevalence increased from 45.9% to 83.3% in 2007 to 2012 respectively (García-Jiménez et al.,

2013). Epidemiological trends suggest that TB is still increasing in south-central and southern

Spain (Vicente et al. 2013).

The TB trends in wild boar show a different pattern in another region in Spain. Within 2008-

2012, low prevalence was noted in the northwest of Spain (Asturias and Galicia). In this Iberian

Atlantic coastline area, the mycobacterial culture prevalence was 2.5% with majority of lesions

(84.4%) restricted to localized TB-like lesions in mandibular LN (Munóz-Mendoza et al., 2013).

In northeastern Spain (Catalonia region) during 2004-2012, a 24.7% TB prevalence was recorded based on the combination of TB-like lesions and MTC culture, with 70% infected in adult wild boar (Mentaberre et al., 2014). Recent findings showed a 22% cumulative prevalence based on

TB-like lesions (Pérez de Val et al., 2017). Thus, wild boar in these regions are less likely to play an important reservoir role for MTC taking into account factors such as the low prevalence among wild boar and lower frequency of generalized TB lesions. However, their presence and the wild boar population growth may pose a future hazard for TB in livestock and other animals.

Wild deer have been implicated in establishing new foci of wildlife infection due to these animals having large home ranges and a long life span (Buddle et al., 2015). Transmission of

infection from wild deer to domestic livestock such as cattle has been reported in white-tailed

deer, the main wildlife reservoir in Michigan (O’Brien et al., 2011). However in New Zealand,

feral deer are believed to act only as spillover hosts, less likely important in the epidemiology of

TB considering the low population density and low animal aggregation as a result of no artificial

feeding among other factors (Buddle et al., 2015). In Spain, red deer are able to maintain the MTC

without the presence of domestic livestock and show an increasing trend of TB lesion prevalence

particularly in the southwest (Gortazar et al., 2011). In the south, MTC infection prevalence in red

deer increased by 50% from 1998–2003 to 2006-07 in Doñana NP (Gortázar et al., 2008). Up to

50% herd prevalence is found around this region from 1999-2004 (Vicente et al., 2006). However,

temporal trends for TB in red deer are more stable and at a rather low prevalence as compared to a

wild boar. This is possibly because of the lower demographic turnover and mortality, different

contact rates, exposure, susceptibility, course of TB and excretion rates in red deer (Vicente et al.,

2013). Red deer also emerged playing an important role of maintaining for M. caprae-TB in the

Alpine region mainly in Austria as a results of increased red deer aggregation at winter feeding

sites (Fink et al., 2015).

Ante-mortem diagnostic tests for MTC in wild boar and red deer

A crucial issue of managing wildlife TB is the availability of diagnostic tools, which are often limited to those developed for domestic animals and humans (Maas et al., 2013). Many reviews emerged in recent years on understanding the diagnosis of animal TB, including a range of ante-mortem techniques

(Schiller et al., 2010; Chambers, 2013; Maas et al., 2013; Bezos et al., 2014). In wildlife disease surveillance, an extreme care should be taken to ensure the validity of diagnostic tools used to identify pathogens applied to a particular wild animal species and the sensitivity and specificity of the tests used should be included in the analysis and interpretation (OIE, 2009). Mycobacterial culture is still known as the gold standard diagnosis for animal TB and used for comparison for validating diagnostic assays.

Studies aiming at the humoral and the cell-mediated immune (CMI) response have been conducted in

wild boar and red deer (Tables 2 and 3). The performance of ante-mortem tests in general had a moderate sensitivity and specificity, although the variability is huge and is not only test- but also species-dependent

(Buddle et al., 2015).

CMI-based tests

Delayed type hypersensitivity and the interferon gamma (IFN-γ) assay are the most common

tools for diagnosis of bovine TB in ruminant species. The in-vivo tuberculin skin test is officially used

for diagnosis of TB based on the measurement of the skinfold increase after intradermal inoculation of

a purified protein derivative (PPD) (Good and Duignan, 2011). However, testing for CMI response has

some limitations. Using bovine/avian purified protein derivatives (bPPD/aPPD) as antigens may

influence the specificity of the test since PPDs comprise a complex mixture of proteins (de Lisle and

Havill, 1985). Repeated skin testing in cattle may cause a reduction in skin test responsiveness and

have a consequence of inability to detect reactors (Thom et al., 2004; Coad et al., 2010).

Desensitization occurred in experimentally infected red deer after repeated comparative skin

testing (CST) within 3-7 days and the sensitivity increased with the interval of more than 60 days

(Corrin et al., 1993). In this species, high non-specific sensitization to the single skin test (SST) was

due to contact with non-tuberculous mycobacteria causing false positive reactors (Queiros et al.,

2012). In addition, age, sex, season, body condition and type of management can affect the skin test

responsiveness in deer (Fernández-de-Mera et al., 2011; Queiros et al., 2012). In wild boar, CMI-

based tests have been studied using mycobacterial antigens bPPD, aPPD and non-mycobacterial

antigen utilizing phytohaemagglutinin, PHA (Jaroso et al., 2010). The CST sensitivity was relatively

good (75-100%) in wild boar, although with low specificity (48-77%). However, application of skin

testing in wild boar is questionable when taking into account that handling these animals is dangerous,

needs repeated measurement and still yields a suboptimal test performance (Jaroso et al. 2010).

In vitro CMI-based diagnostic tests to detect IFN-γ produced by blood leukocytes in response to mycobacterial antigens such as Cervigam assay have been used in cervids such as in white-tailed deer and reindeer (Palmer et al., 2004; Waters et al., 2006a). The IFN- γ in response to bPPD and bPPD,

ESAT-6-CFP-10 was significantly greater in experimentally M. bovis-infected white-tailed deer and reindeer respectively as compared with uninfected control. In a red deer, the detection of mycobacterial antigen-specific responses, IFN-γ mRNA was evaluated in experimentally TB-infected animals and quantified using real-time qRT-PCR assay (Harrington et al., 2007). The qRT-PCR assay had the highest sensitivity (79%) as compared to commercial Cervigam ELISA (70%), and both had a specificity of more than 90%. Ongoing research on IFN-γ testing in deer is taking place at IREC

(Jobin Thomas, personal communication).

Antibody detection tests

Antigen recognition patterns for MTC infection in different animal hosts varies, however protein

MPB83 is the most serodominant one in cattle and many wildlife species (Waters et al., 2006;

Lyashchenko et al., 2008). In wild boar, MPB83 protein antigen is serodominant with M. bovis infection, followed by MPB70, ESAT-6 and CFP10 (Lyashchenko et al., 2008). In experimentally

MTC-infected white-tailed deer, ESAT-6 and CFP10 were the second common antigens after dominant MPB83 and MBCF antigens and less frequent MPB70 (Waters et al., 2004; Lyashchenko et al., 2008).

Serology has advantages in wild animals due to being a single test. Besides, it allows testing many samples, simply, rapidly, and inexpensively and the protocol can be standardized in different laboratories. It also has advantages in terms of logistics due to stability of antibodies during transport, storage and handling (Maas et al., 2013). In wild boar, MTC antibody detection by ELISA and rapid

(lateral flow) tests had a good sensitivity and specificity. Hence, these diagnostic tools are regularly used as a method to determine TB status in this species (Lyashchenko et al., 2008; Boadella et al.,

2011 details in Table 2). The limitations of serological tests are that the humoral responses are

generally developed at advanced stages of disease. For instance, there is a tendency to have a

correlation between higher TB lesion scores and antibody levels in natural and experimentally infected

wild boar (Boadella et al., 2011; Garrido et al., 2011). Enhanced diagnostics are a critical need to

detect all infected individual animals, especially in occasions where individual animals are infected

and show subclinical disease or no visible lesions (Fitzgerald and Kaneene, 2012).

Table 2 Performance of ante-mortem diagnostic tests used in wild boar (Sus scrofa).

Wildlife Test Sensitivity Specificity Antigens/ Reference species details Wild boar ELISA 79.2% 100% bPPD, protein-G HRP Boadella et (Sus conjugate. al., 2011 scrofa) ELISA 72.6% 96.4% bPPD, protein-G HRP Aurtenetxe et conjugate. al., 2008

DPP VetTB 89.6% 90.4% MPB83 and Boadella et CFP10/ESAT-6. al., 2011

DPP VetTB 76.6% 97.3% MPB83 and Lyashchenko CFP10/ESAT-6. et al., 2008

Intradermal 75-100% 48.4-77.4% bPPD, aPPD Jaroso et al., skin test intradermally at 2010 inguinal region (different skin measurement criterion).

Bovine purified protein derivative (bPPD); Avian purified protein derivative (aPPD); DPP (Dual path platform); HRP (horseradish peroxidase); Interferon-gamma enzyme linked immunoassay (IFN-γ ELISA); Multiantigen print immunoassay (MAPIA); Comparative skin test (CST).

Table 3 Performance of ante-mortem diagnostic tests used in red deer (Cervus elaphus).

Wildlife Test Sensitivity Specificity Antigens/ Reference species details

DPP VetTB 75% 91.4% MPB83 and Buddle et al., CFP10/ESAT-6. 2010

Red deer DPP VetTB 79% 98% MPB83 and Waters et al., (Cervus CFP10/ESAT-6. 2011 elaphus) CervidTB 86.5% 83.8% MPB83 and Buddle et al., STAT-PAK CFP10/ESAT-6. 2010

CervidTB 82% 93% MPB83 and Waters et al., STAT-PAK CFP10/ESAT-6. 2011

Cervigam 70% 100% bPPD Harrington et (IFN-γ ELISA) al., 2007

Real time RT- 79% 97.5% bPPD / detection of Harrington et PCR IFN-γ mRNA using al., 2007 whole blood

ELISA 85.3-95% 100% bPPD, aPPD, MPB70 Griffin et al., 1994

ELISA 72.7% bPPD García- Bocanegra et al., 2012

ELISA 86.7% Extracted M. bovis Wadhwa et surface antigen. al., 2013

MAPIA 82% ESAT-6, CFP10, Buddle et al., MPB59,MPB64, 2010; Waters MPB70, MPB83, 16- et al., 2011 kDa protein, 38-kDa protein, 2- fusion proteins: CFP10/ESAT- 6 and the 16-kDa /MPB83, 2 native antigens, bPPD and M. bovis culture filtrate.

bPPD and aPPD

Intradermal 91.4% 98.7% Lateral side of neck Corrin et al., tuberculin skin 1993 test (CST) 30-80% Griffin et al., 1994

Lymphocyte 95.7- 98% Griffin et al., transformation 95.9% 1994 assay (IFN-γ

Effects of co-infections on the diagnostic response

Susceptibility to other pathogens showed that co-infection is an important factor for consideration in epidemiological surveys, particularly in wild animals where multiple infections are generally present.

Thus, the potential risk of susceptible hosts or reservoirs of infectious diseases other than TB should be taken into account in epidemiological studies. Realizing that the gold standard for bovine TB diagnosis is still depending on bacterial isolation (dead animals) and delayed type hypersensitivity (for live animals), advancing towards improved humoral tests is a need. This is in spite of their variable outcome concerning sensitivity, specificity, and side-factors that could affect their performance. Generating more knowledge on in vivo TB diagnostic tests is needed to aid in disease control.

Domestic animals

Cattle with liver fluke infestation had reduced skin test responsiveness to bPPD (Flynn et al.,

2009). Experimentally fluke- and MTC-infected dairy calves showed that co-infected calves (with

liver flukes) reacted less strongly to the CST and IFN-γ test than those infected with M. bovis alone

(Claridge et al., 2012). Co-infection between Mycobacterium avium paratuberculosis (MAP) and M.

bovis in cattle has been showen to have an effect on CMI IFN-γ assay with an apparent 28% reduction in sensitivity in animals with a mixed infection with MAP compared to all animals suffering TB

(Álvarez et al., 2009). However, recent data showed a higher number of IFN-γ positive animals with no visible TB lesion; this lead to speculating about some protection conferred by prior infection with

MAP. Nevertheless, the combined use of SIT and IFN-γ was able to detect all confirmed cases of TB as compared to SIT alone (Seva et al., 2014).

Antibody responses in natural bovine MTC infection influences PTB diagnosis, and vice-versa

(Lilenbaum et al., 2009). In addition, the proteins MPB70 and MPB83 respond to bovine TB and PTB in cattle, thus M. bovis-specific antibodies detection should be interpreted with care (Marasi et al.,

2010). Refinements in antigens used for MTC diagnosis, including cattle, are ongoing (e.g. the P22 antigen developed by IS Carlos III in collaboration with VISAVET and IREC; Lucas Domínguez, personal communication).

Wild animals

Contact with MAC or other environmental mycobacteria may have been more likely among farmed wild boar although avian TB is less frequent. This could explain the high skin test response against aPPD and bPPD in wild boar (Jaroso et al., 2010). In farmed red deer, natural infection with

MAP increased the risk of detection of cross-reactors upon the tuberculin skin test (Stringer et al.,

2011). Antibody detection from M. bovis–positive wild boar had a tendency to react with the (avian)

PPA3 antigen, however not in the case of M. bovis–negative wild boar - suggesting that cross reactions exist between members of the M. avium complex (MAC) rather than a genuine antibody response to

MAP (Boadella et al., 2011). The MAP could also generate false positive results in TB tests by using humoral based detection tests such as the STAT-PAK lateral flow test in red deer (Buddle et al.,

2010).

Justification

The MTC causing TB is a hazard to the livestock and wildlife communities and their zoonotic implications are a major concern in public health all over the world, mostly in developing nations. The role of the wildlife reservoir and evidence on indirect means of interspecies MTC transmission

(environmental reservoir) is expanding our understanding of TB epidemiology. Expansion in research on diagnostic tests for MTC infection in wildlife (as well as continuing in domestic species) has up surged and improves our capacity for TB detection in multiple animal hosts. However, substantial setbacks of diagnostic test performance (e.g. less sensitive and/or specific, practicality and cost) limit their acceptance for routine use, notably in wildlife. Humoral-based antibody detection provides a simple and non- technically-demanding procedure providing rapid results. Yet the antibody staging, release and detection may be different particularly with age and disease exposure. Furthermore, certain tests may also influence other tests, thus making the interpretation problematic. In such a way, enhancing knowledge and understanding ante-mortem diagnostic tests in detecting TB in wildlife species is a great advantage and is of paramount importance in animal health.

Dealing with TB control requires the development of strategies that reduce pathogen transmission between wildlife and domestic animals and the need to explore single intervention tools such as targeted culling because there is no replicated information available on the effect on TB of using single control measures such as farm biosafety, feeding ban or population control. Targeted culling strategy is more likely to succeed in isolated populations such as in fenced wildlife hunting estate and will probably result in certain reduction in disease prevalence in the wildlife host.

Chapter I

INTRODUCTION

1.2. Open questions and recent advances in the control of a multi-host

infectious disease: animal tuberculosis

Christian Gortázar*, Azlan Che Amat, Daniel J. O’Brien

Mammal Review. 2015, 45: 160-175.

This thesis chapter is prepared in the style format of Mammal Review

ABSTRACT

1. Animal tuberculosis (TB) control is globally important for public health, economics and conservation.

Wildlife species are often part of the Mycobacterium tuberculosis complex (MTC) maintenance community, complicating TB control attempts.

2. We describe the current knowledge on global TB distribution and the significance of wildlife hosts; identify insufficiently known aspects of host pathology, ecology and epidemiology; present selected time series in wildlife TB; and summarize ongoing research on TB control, providing additional insight on vaccination.

3. Six specific research needs are identified and discussed, namely: 1) complete the world map of wildlife

MTC reservoirs and describe the structure of each local MTC host community; 2) identify the origin and behaviour of generalized diseased individuals within populations, and study the role of factors such as co- infections, re-infections and individual condition on TB pathogenesis; 3) quantify indirect MTC transmission within and between species; 4) define and harmonize wildlife disease monitoring protocols, and apply them in a way that allows proper population and prevalence trend comparisons in both space and time; 5) carry out controlled and replicated wildlife TB control experiments using single intervention tools; 6) analyse cost-efficiency and consider knowledge transfer aspects in promising intervention strategies.

4. We believe that addressing these six points would push ahead our capacities for TB control. A remaining question is whether or not interventions on wildlife TB are at all justified. The answer depends on the local circumstances of each TB hotspot, and is likely to evolve during our collective progress towards TB control in livestock and in wildlife.

Keywords: Disease ecology, monitoring, Mycobacterium bovis, shared infection, wildlife reservoir

INTRODUCTION

The Mycobacterium tuberculosis complex (MTC) is a group of multi-host pathogens thriving at the wildlife–livestock interface. Animal tuberculosis (TB) is due to infection with Mycobacterium bovis and other closely related members of the MTC, such as Mycobacterium caprae. Zoonotic TB is the disease caused by non-Mycobacterium tuberculosis members of the MTC transmitted from animals to humans. It often causes extra-pulmonary disease in humans and is still a major public health concern in developing countries, often linked with the consumption of raw dairy products or close contact with infected livestock (Dürr et al. 2013).

In industrialized countries, the main reason for TB control is economic: cattle TB results in severe losses due to trade restrictions and slaughter compensations for test-positive animals (Amanfu 2006).

Recently, TB has become of concern in other livestock sectors such as the pig industry (Bailey et al.

2013). TB acquired by consuming MTC-infected prey affects lion Panthera leo conservation in South

Africa (Renwick et al. 2007) and Iberian lynx Lynx pardinus conservation in the Iberian Peninsula (López et al. 2014). Moreover, TB-mediated conflicts between farmers, hunters and conservationists can affect animal health and conservation strategies in and around protected natural areas (Gortázar et al. 2010).

Hence, animal TB control is considered globally important for public health, economics and conservation.

In most industrialized countries, successful cattle TB control is based on intensive cattle test and slaughter, and on movement control policies, with sporadic whole herd culling (Brooks-Pollock et al.

2014). However, the eradication of cattle TB remains unlikely if the role of all hosts is not clear enough for relevant reservoirs to be targeted at the same time (O’Reilly & Daborn 1995, Gortázar & Cowan

2013). TB control in wildlife is extremely difficult (Fitzgerald & Kaneene 2013). Several reviews have addressed wildlife TB, underlining the need to understand epidemiological complexity and to use integrated approaches for TB control at the wildlife–livestock interface (Gortázar et al. 2012, Palmer et al.

2012, Fitzgerald & Kaneene 2013, Palmer 2013). In this non-systematic review, we describe the significance of wildlife as part of complex multi-host MTC maintenance communities; identify

insufficiently known aspects of host pathology, ecology and TB epidemiology; present selected time series in wildlife TB; and summarize ongoing research on TB control. Specific research needs are identified and discussed.

MTC RESERVOIRS

TB control is difficult in large areas with insufficient livestock movement control and complex host communities (Acevedo et al. 2013). MTC host communities are often composed of both domestic and wild hosts. The domestic portion of this host community often includes goats, pigs or other domesticated species in addition to cattle (Gortázar et al. 2011b), while wild hosts often go beyond the local ‘key’ reservoir species to include additional hosts with larger or smaller contributions to MTC maintenance

(Palmer 2013). In addition, the environment itself might contribute to maintaining viable MTC in water or soil, further complicating TB epidemiology (Ghodbane et al. 2014, Walter et al. 2014). More complex scenarios provide better opportunities for MTC survival, and greater challenges for TB control. In this context, the term reservoir means the whole system (domestic hosts, wild hosts and environment) rather than a single-host species (sensu Haydon et al. 2002). In our view, cattle are an active part of the MTC maintenance community, while also being the main target of disease control.

Worldwide, the best-known wildlife TB reservoir situations occur in the British Isles and the Iberian

Peninsula in Europe (Gortázar et al. 2012), in sub-Saharan Africa (Michel et al. 2006), in parts of North

America (Wobeser 2009, Carstensen & DonCarlos 2011, O’Brien et al. 2011a, Shury & Bergeson 2011,

Miller & Sweeney 2013) and in New Zealand (Hutchings et al. 2013). These areas represent global wildlife TB hotspots. Details on host ecology, pathology, surveillance and control attempts have recently been reviewed for each of these hotspots by Fitzgerald and Kaneene (2013). Pathology and epidemiology were also recently reviewed by Palmer (2013). Three hotspots are currently regarded as two-host settings, with one target species (cattle) and one single wildlife reservoir: the Eurasian badger Meles meles in the

British Isles; the introduced Australian brushtail possum Trichosurus vulpecula in New Zealand; and

white-tailed deer Odocoileus virginianus in Michigan, USA. However, the epidemiological complexity might actually be larger in some of these systems.

By contrast, multi-host situations are known to occur in Mediterranean habitats of the Iberian

Peninsula and in sub- Saharan Africa. In the Iberian Peninsula, several wild ungulates, namely the

Eurasian wild boar Sus scrofa, red deer Cervus elaphus and fallow deer Dama dama, are regarded as members of the wildlife MTC maintenance host community, and TB epidemiology is further complicated by the co-existence of several suitable domestic hosts, including cattle, goats and pigs (Gortázar et al.

2011b). In southern Africa, MTC is probably mainly maintained by the African buffalo Syncerus caffer in several settings, and by the lechwe antelope Kobus leche in Zambia’s Kafue National Park (Michel et al.

2006). But MTC infects a long list of other wild African mammals, some of which might contribute to complexity (Michel et al. 2006).

In addition, MTC infection is endemic in feral pigs on the island of Molokai in Hawaii (Miller &

Sweeney 2013), in American bison Bison bison in Wood Buffalo National Park, Alberta, Canada (Joly &

Messier 2004, Wobeser 2009), and in red deer in the Tyrolean Alps of Austria and neighbouring countries

(Schoepf et al. 2012). Figure 1 represents the geographical distribution of scientific literature on wildlife

TB, evidencing the existing bias towards a few regions. By contrast, information on wildlife TB from

Asia, South America and northern Africa is extremely scarce. Table 1 lists examples of underreported wildlife TB.

Fig. 1. Numbers of literature references in the Scopus database relating to different geographical areas found using the search terms ‘wildlife’ and

‘tuberculosis’ from the period 1994 to 2013. It is evident that scientific activity is biased towards a few hotspots in Europe, North America, sub-

Saharan Africa and New Zealand, while very little information is available for Asia and Latin America. Light grey areas are those in which almost no studies of bovine TB were found

Table 1.- Examples of situations where the role of wildlife in Mycobacterium tuberculosis complex (MTC) maintenance is unknown, underreported or still under debate.

Host species Country or region MTC prevalence and remarks on host status References Low seroprevalence, sporadic confirmation by PCR, Barrios-Garcia et al. (2012); White-tailed deer Mexico reservoir status unknown Medrano et al. (2012)

Locally high Mycobacterium caprae prevalence in Alpine countries, central red deer, linked to winter feeding, with spillover to Red deer Schoepf et al. (2012) Europe cattle

Sporadic reports on Mycobacterium bovis isolation, Feral pig or South America reservoir status unknown Meikle et al. (2011) introduced wild boar

Generally low prevalence, links with cattle TB Atlantic Spain, France, Muñoz-Mendoza et al. (2013); Eurasian wild boar breakdowns, reservoir status unknown other regions in Europe Richomme et al. (2013)

Confirmed infection with spill over to Eurasian lynx European bison Poland Lynx lynx and wild boar, reservoir status unknown Krajewska et al. (2014)

Balseiro et al. (2011); Atlantic Spain, France, Links with cattle TB breakdowns, reservoir status Richomme et al. (2013); Eurasian Badger other regions in Europe unknown except for the British Isles Payne et al. (2013)

Kruger National Park, Lion South Africa Confirmed infection, reservoir status under debate Renwick et al. 2007

Ward et al. (2009); Deer species and Confirmed infection, not regarded as reservoirs, but England Foyle et al. (2010); wild boar expanding Ward & Smith (2012) PCR, Polymerase Chain Reaction; TB, tuberculosis.

Case study: Southeast Asia

One example of a region where the epidemiology of animal TB and the role of wildlife hosts is poorly understood is Southeast Asia. Between 2005 and 2013, the Office International des Epizooties

(OIE) World Animal Health Information Database (Anonymous 2013a) recorded TB reports in four countries of the region: Malaysia (75 cases in goats and cattle), Thailand (36 cases in cattle and buffaloes), Myanmar (9 cases in unknown wildlife species) and suspected cases in wildlife in Vietnam

(Fig. 2).

Fig. 2. Map of Southeast Asia, with animal shapes indicating the distribution of potential wildlife hosts for the Mycobacterium tuberculosis complex, and stars indicating countries in which animal tuberculosis (TB) was reported between 2005 and 2013 (Myanmar, Thailand, Vietnam, Malaysia and Indonesia). The box shows total estimated livestock numbers (source: Anonymous 2013b).

In addition to these OIE data, a literature review revealed the possible implication of other animal species in MTC epidemiology, including Asian elephants Elephas maximus (Angkawanish et al. 2010, but see Ong et al. 2013) and nonhuman primates (Payne et al. 2011). Hence, TB is present in the region both in livestock and in wildlife. However, the extent of the problem and the role of wildlife in MTC epidemiology are largely unknown. Wild suids Sus scrofa for instance are regarded as suitable MTC maintenance hosts in some parts of Europe (Gortázar et al. 2012) and America (Miller & Sweeney 2013).

In Southeast Asia, wild suids (several species) are widely distributed (Fig. 2), and occur at high densities

(e.g. up to 78 per square km in Malaysia, Ickes 2001). Similarly, deer of the subfamily cervinae (genera

Axis and Cervus) are present in Southeast Asia (Fig. 2). Thus, after improving the knowledge on TB in livestock, it would be interesting to investigate the presence and prevalence of TB in wild suids and deer from different sites throughout the region, including sites with and without contact with livestock, and representing the range of wild ungulate densities. Since contact between domestic and wild suids is likely in Southeast Asia, there is an increasing trend in deer farming, and trans-border movement of potential wildlife reservoirs is likely, wildlife hosts need to be studied along with the domestic MTC hosts.

Research needs: MTC reservoirs

Two aspects require further research: first, completing the map of wildlife MTC reservoirs (as noted by Palmer 2013) and second, understanding the structure of each regional MTC host community: host species and their roles.

HOST PATHOLOGY, ECOLOGY AND TB EPIDEMIOLOGY

The distribution and characteristics of TB lesions in infected hosts of different species is of paramount importance for understanding MTC transmission. It is assumed that TB lesion characteristics and their distribution are related to mycobacterial excretion and may be related to transmission risk. In ungulates (bovids, cervids and suids), lung and thoracic lymph node involvement is frequent (about 20–

60% of infected individuals; O’Brien et al. 2001, Martín-Hernando et al. 2007, 2010, Shury & Bergeson

2011, Fitzgerald & Kaneene 2013,Muñoz-Mendoza et al. 2013). In white-tailed deer, the most common site for lesion development is the medial retropharyngeal lymph node (Palmer 2013). This is consistent with MTC shedding mainly by oral and nasal excretion and secondarily also by faecal excretion. In badgers, the prevalence of lung involvement is also about 50%. However, kidneys are often affected, too, and infected bite-wounds or draining subcutaneous lymph nodes might also produce additional shedding routes (Gallagher & Clifton-Hadley 2000). Badgers are thought to transmit MTC to cattle mainly by indirect contact, for instance by contaminating cattle feed (Wilson et al. 2011). Infected possums rarely present lung lesions, but do develop draining lesions in subcutaneous inguinal and axillary lymph nodes, which are likely to contribute to inter-species transmission (Paterson & Morris 1995). These different potential MTC shedding routes might have significant consequences for TB epidemiology, including environmental contamination. Knowing the shedding routes may contribute to improved control.

Host sex and age are well-known predictors of risk of MTC infection. Other less often evidenced individual risk factors include belonging to infected family groups or social groups (e.g. Blanchong et al.

2007, Gortázar et al. 2011a), and certain individual genetic backgrounds (Acevedo- Whitehouse et al.

2005). Known risk factors at the population level include having direct and indirect contact networks (risk varies with inter-specific contact rate) to other infected host species, existing at high densities and forming large spatial aggregations at feeding sites or waterholes (O’Brien et al. 2006, Vicente et al.

2007b). Co-infections have been suggested to drive TB epidemiology. In Mediterranean Spain, correlations were found (at the population level) between wild boar TB prevalence, generalized TB and the prevalence of porcine circovirus type 2, a widespread immunosuppressive virus (Risco et al. 2013).

However, such correlations were less evident at the individual animal scale (Díez-Delgado et al. 2014b).

Human influences (via activities such as fencing, feeding, translocating) have often contributed to the introduction and maintenance of TB in wildlife (Palmer 2007, Fitzgerald & Kaneene 2013, Vicente et al.

2013). Thus, while co-infections could be relevant, confounders such as management impede our understanding of them in non-experimental settings.

Not all infected individuals contribute equally to MTC maintenance within a certain host category and in a given epidemiological setting. Animals with generalized lesions are more likely to shed MTC

(Palmer 2013 and the authors, unpublished data), and those that do not shed may play a limited epidemiological role or not play a role at all (Corner 2006). Unfortunately, the drivers of TB generalization are less well-known than the drivers of infection, since lesion scoring is not carried out as frequently as is the simple detection of infection. In fact, proper quantitative or semi-quantitative lesion scoring is usually only done in experimental infections (e.g. Ballesteros et al. 2009b), as opposed to observational studies in the wild (Vicente et al. 2013). For instance, the practical necessity of maintaining large-scale surveillance programmes over long periods often restricts routine sampling to those anatomical sites (e.g. head region) that are most frequently infected.

It is interesting to note that, while in TB-endemic Mediterranean regions, 57% of MTC-infected wild boar develop thoracic lesions (Martín-Hernando et al. 2007), less than 20% do so in non-endemic areas of

Atlantic climate such as Asturias in northern Spain (Muñoz-Mendoza et al. 2013). This suggests that in non-endemic areas, some factors, maybe lower infection pressure, less frequent re-infection events, or differences in the rate of co-infections and in general host condition, contribute to limiting the likelihood of animals developing severe disease. In Spain, dry seasons have been identified as one of those drivers of

TB generalization in wild boar (Vicente et al. 2013).

TB is largely regarded as a respiratory disease, in which airborne transmission by aerosols plays a major epidemiological role (Hopewell 1994). However, there is increasing scientific evidence suggesting that direct contacts (those facilitating aerosol transmission) between species are scarce (Kukielka et al.

2013), and direct intra-species transmission is not easily replicated in experiments (e.g. lack of transmission among fenced feral pigs in New Zealand, Nugent 2011). Moreover, observational (Vicente et al. 2007b) and experimental studies (Palmer et al. 2004, Barasona et al. 2013) have shown that indirect

transmission can occur at feeding sites or waterholes. Considering this information together, we hypothesize that MTC transmission between wild and domestic hosts is mostly indirect.

Research needs: host pathology, ecology and TB epidemiology

Research should focus on delivering better knowledge of the origin and behaviour of generalized tuberculous individuals within populations, and of the role of co-infections, re-infections and individual condition (among other factors) in TB pathogenesis. Research quantifying indirect MTC transmission within and between species is duly needed to inform decisions on investment in control options that tackle different potential transmission routes.

TIME TRENDS IN WILDLIFE TB

Unfortunately, few long-term studies in which serial monitoring is conducted are available for wildlife TB. A selection of the longer ones is shown in Fig. 3. Generally speaking, stable or declining trends below 10% prevalence occur in cervids (e.g. elk Cervus elaphus in Riding Mountain National Park,

Canada) and in badgers in Woodchester Park, England, while increasing trends occur in African buffalo in the Kruger National Park, South Africa and in wild boar in Ciudad Real, Spain. The available information represents changes over time in prevalence, as opposed to true time trends. Comparisons between species are difficult due to variability in data type.

Fig. 3. Selected time trends of tuberculosis (TB) prevalence in wildlife hosts. Source: authors’ estimates based on figures in Cross et al. 2009, O’Brien D.J. (personal communications), Shury and Bergeson 2011, Vicente et al. 2007a, 2013. The UK badger data refer to prevalence of animals found to be excreting bacilli, not the prevalence of infected animals.

Analysing the significance of these trends and inferring their drivers is beyond the scope of this review. However, one could argue that wildlife TB control has been relatively successful in (almost single-host) North American cervid populations, as shown by the progressive declining trend of white- tailed deer TB in Michigan, and the success of interventions in Minnesota (Carstensen & DonCarlos

2011) and Manitoba (Shury & Bergeson 2011); while in high prevalence populations in multi-host settings with limited (if any) control intervention, TB keeps getting worse (e.g. Vicente et al. 2013). We hypothesize that two factors determine the observed trends: the capability to intervene with costly or potentially contentious measures such as population control, feeding bans and biosafety improvements; and intervention in single-host vs. multi-host settings.

Research needs: time trends

There is a clear lack of constant, harmonized information derived from large-scale disease and population monitoring Even the best available time series data refer to local areas, and information on wildlife abundance and relevant metadata is not as readily available as information on TB prevalence in livestock or even in wildlife. Hence, there is an urgent need to define and harmonize wildlife disease monitoring protocols, and to apply them in a way that allows proper trend comparisons in both space and time. This information is of paramount importance for assessing the effect of any future intervention

(Boadella et al. 2011, Gortázar et al. 2015).

ATTEMPTS TO CONTROL TB

The goal of cattle TB eradication requires the development of strategies that reduce pathogen transmission between wildlife and domestic animals (O’Reilly & Daborn 1995). This is a cross- disciplinary task where inter-agency teams of vets and ecologists are needed (Fitzgerald & Kaneene

2013). Currently available tools for disease control at the wildlife–livestock interface were recently reviewed (Delahay et al. 2009, O’Brien et al. 2011b, Gortázar et al. 2015) and range from interaction management (farm biosafety improvement), through random (culling) or targeted (test and cull) population control, to vaccination. Examples of successful MTC eradication in wildlife are scarce, although there are cases consistent with wildlife culling impacting on cattle TB (see Table 2). One aspect to consider is reporting bias: failed experiments are less likely to be reported than successful ones.

Population control

The UK randomized badger culling trial (Donnelly et al. 2006, 2007), and the Irish four areas experiments (Corner et al. 2008), along with data on wild boar culling in Spain (Boadella et al. 2012), are among the few studies where random culling was tested as a single TB control tool. In our view, random culling of wildlife reservoir hosts might contribute to cattle TB control through reduction of densities, contact rates or specific high-risk individuals. However, it provokes a strong debate among stakeholders

and is not sustainable as a long-term tool in Europe. Possible exceptions might include culling invasive

(pest) species such as possums in New Zealand (O’Brien et al. 2011b) or water buffalo Bubalus bubalis in

Australia (Radunz 2006), and short-term culling in response to new outbreaks (Carstensen & DonCarlos

2011, Gortázar et al. 2015).

Targeted culling (selective culling) failed to yield convincing results in attempts targeting white- tailed deer in Michigan (Cosgrove et al. 2012b) or wild boar in Spain (the authors, unpublished data).

This tool depends on capturing a large proportion of an animal population, testing it, and selectively removing test-positive individuals. This kind of intervention is probably more suitable for captive wild animals (Gortázar et al. 2015).

Farm biosafety

Farm biosecurity practices are known preventative tools in livestock farming. The rationale consists of using fencing and other physical barriers to reduce contact between livestock and potentially infected wildlife (Gortázar et al. 2015). For instance, sheet metal gates and fencing, feed bins and electric fencing were shown to prevent badgers from entering cattle farm facilities in the UK (Judge et al. 2011). In Spain, segregating wild ungulates and livestock from common resources such as waterholes reduced cattle TB incidence (Barasona et al. 2013). Nonetheless, fences are vulnerable to certain animal species that can destroy them, and are expensive to set up and maintain (Jori et al. 2011). As in the case of culling, there are few situations where biosafety improvements have been the only approach to TB control at the wildlife–livestock interface (Renwick et al. 2007, Barasona et al. 2013).

Research needs: controlling TB

There is a need for experiments enabling the evaluation of the benefits of each single intervention tool. Although valuable examples of on-farm prevention do exist, there is no replicated information available on the effect on cattle TB of using preventive farm biosafety measures alone, in the absence of other interventions such as feeding bans or population control.

Table 2. Selected examples of tuberculosis (TB) control attempts at the wildlife–livestock interface and their reported outcomes

Host and setting TB control tools Outcome (in wildlife and cattle) References Variable outcomes, including reduction in Boadella et al. 2012; Wild boar in multi-host Wild boar population control, (also in wild boar TB prevalence, and in TB García-Jiménez et al. 2013; settings, Spain cattle depopulation and re-stocking) prevalence in sympatric deer and cattle Mentaberre et al. 2014 Variable, depending on study site, Badger in the British Isles, Badger population control treatment surface and type of control. Corner et al. 2011 UK Positive effects on cattle TB in Ireland. White-tailed deer population control, White-tailed deer in feeding bans, cattle biosafety TB no longer detectable in wildlife, cattle Carstensen and DonCarlos Minnesota, USA improvements, voluntary cattle buyout herds cleared 2011 program White-tailed deer in White-tailed deer population control, Significant reduction, but TB still endemic O’Brien et al. 2011a; Michigan, USA feeding bans, cattle biosafety in deer, sporadic in cattle, other wildlife Ramsey et al. 2014b Buffalo and other mammals Electrified fencing to avoid escapes Some species are able to cross fences Renwick et al. 2007 in Kruger NP, South Africa and contact with cattle Wild boar and red deer on Changes to waterholes making them Reduction in cattle TB prevalence below Barasona et al. 2013 beef cattle farm, Spain cattle or wildlife-proof previous records and below control areas Brushtail possum, New Oral BCG vaccination Lower incidence in vaccinated possums Tompkins et al. 2009 Zealand Lower test positivity in vaccinated Chambers et al. 2011; Badger, UK Parenteral BCG vaccination badgers, unvaccinated cubs. Lower Wilson et al. 2011; infection likelihood in vaccinated setts Carter et al. 2012 Lower infection prevalence in vaccinated Wild boar, Spain Oral IV vaccination Díez-Delgado et al. 2014a wild boar Brushtail possum, New Nationwide population control Lower possum TB prevalence and less Hutchings et al. 2011 Zealand (poison) spill-back to cattle. Cattle TB declining. White-tailed deer, Impractical due to low prevalence; Local test & cull attempt Cosgrove et al. 2012a,b Michigan, USA expensive, eradication unlikely American bison, Elk Island Local test & cull attempt Success (small isolated population) Wobeser 2009 NP, Canada BCG, Bacille Calmette-Guérin; RoI, Republic of Ireland.

VACCINATION

Of the three main intervention tools, namely biosafety measures, culling and vaccination, vaccination is most likely to provide hope in the decade ahead. This is because biosafety measures alone can help to reduce cattle TB prevalence, but are unlikely to eradicate MTC (Barasona et al. 2013), and because culling is generally regarded as a non-sustainable disease control tool in the long term (Chambers et al.

2011, Corner et al. 2011, Boadella et al. 2012).

Recent reviews have addressed wildlife TB vaccination (O’Brien et al. 2011b, Beltrán-Beck et al.

2012, Buddle et al. 2013, Gormley & Corner 2013). Controlled and replicated field experiments are available for possums (Tompkins et al. 2009), badgers (Chambers et al. 2011) and for wild boar (Díez-

Delgado et al. 2014a). Here we use two case studies to discuss some practical aspects of wildlife vaccination in single-host and multi-host contexts.

Case study: planning for vaccination in Michigan, USA

Vaccination of wildlife to eliminate pathogens at landscape scales has proven nearly impossible where multiple maintenance hosts exist (Plumb et al. 2007). Because only one maintenance host other than cattle, i.e. white-tailed deer, has thus far been identified in Michigan (O’Brien et al. 2006, 2011a), vaccination is a viable option, and has been included in modelling of likely outcomes of control strategies

(Ramsey et al. 2014a).

In Michigan livestock, 67 TB-positive herds have been identified (49 beef, 13 dairy cattle, 4 farmed deer and 1 bison). Forty-five outbreaks (67%) have been officially attributed to wildlife exposure. Eight cattle farms have experienced multiple breakdowns (7 twice, 1 three times), accounting for 27% (17 out of 62) of the cattle farm total. Elimination of TB from Michigan cattle appears to be achievable: the number of herds that break down each year is low (mean: 3.7, range: 1–8, variance: 2.2 in 1998–2014), most herds had two or fewer culture-positive cattle at post mortem, electronic identification has theoretically made all Michigan cattle traceable, and improvements in farm biosecurity have been

partially implemented (O’Brien et al. 2011a, Walter et al. 2012). Who would pay for vaccination has not yet been seriously discussed.

One argument justifying vaccination of white-tailed deer is stewardship, the responsibility to leave a healthy deer herd for sustained use by future generations. Notably, vaccination is not justified by negative population-level effects of bovine TB on white-tailed deer. White-tailed deer are abundant: the estimated population in Michigan is 1.7 million (range 1.69–1.97 million, 2011–2013; Michigan Department of

Natural Resources, unpublished data; estimation via sex–age kill; Mattson & Moritz 2008). Population- level TB mortality is minimal, and white-tailed deer are of economic and cultural, rather than conservation, significance (O’Brien et al. 2006). Vaccinating deer is arguably less justifiable than vaccinating cattle, but vaccination of white-tailed deer is partially driven by the current lack of a viable bovine vaccine, and by social concerns that make cattle an unpalatable target for vaccination (Plumb et al.

2007). Cattle vaccination is likely to be far simpler logistically and more cost effective than deer vaccination. Ultimately, however, vaccination of white-tailed deer is justified to eliminate the self- sustaining wildlife reservoir from the landscape.

Prevention of TB in white-tailed deer, while desirable, is not necessary. Vaccination needs only to reduce or prevent transmission among deer and to other species. As the vaccine, Danish Bacille Calmette-

Guérin (BCG) is the likely candidate (Waters et al. 2012). In 19- to 20-week studies, BCG has proven safe and effective (Palmer et al. 2007, 2009, 2014b, Nol et al. 2008). Oral administration has been most effective (Nol et al. 2008). Given the greater tissue persistence of parenteral BCG (Palmer et al. 2010b,

2014c), oral administration seems likely to be the preferred delivery route. Research into bait uptake is underway (Palmer et al. 2014a). Limited secondary transmission of BCG has occurred from vaccinates to unvaccinated in-contact deer, but not to indirectly exposed cattle (Palmer et al. 2010a, Nol et al. 2013).

Studies of duration of immunity, reduction of transmission, and the effects of BCG overdose in white- tailed deer are underway (M. V. Palmer, personal communications). The geographical scale of a potential

vaccination programme is relatively well-defined: both white-tailed deer and cattle in the core outbreak area, Deer Management Unit (DMU) 452, have the highest risk of infection based on prevalence (Table

3). Within DMU 452, cattle farms occupy less than one-third of the area (Fig. 4). Modelling suggests that vaccinating deer in the vicinity of those farms could quickly reduce herd breakdowns (Ramsey et al.

2014b).

Table 3. Apparent prevalence of Mycobacterium bovis infection in free-ranging white-tailed deer and percentage of cattle farms with tuberculosis (TB+) by geographical area, Michigan, USA, 1975–2014

Area TB+ free-ranging white-tailed deer TB+ cattle farms

DMU 452 579/27476 2.1% 16/88 18%

Rest of endemic area 143/53010 0.27% 24/585 4.1%

Rest of Michigan 22/120759 0.018% 13/~12953* 0.1%

DMU, Deer Management Unit; *(Anonymous 2014).

Fig. 4. Locations of Mycobacterium bovis infected free-ranging white-tailed deer and tuberculosis (TB)- positive cattle farms in Deer Management Unit 452, Michigan, USA, 1975–2012.

Female white-tailed deer and their female offspring exhibit remarkable fidelity to natal range (Van

Deelen et al. 1998, Nelson & Mech 1999). In the TB-endemic area, most females first breed as yearlings; about half produce twins. Females typically give birth to twins annually from age 2 onward. If infected, matriarchial groups are likely to maintain bovine TB locally, and males act as between-group spreaders

(O’Brien et al. 2002, Palmer et al. in press). Annual mean white-tailed deer harvest in DMU 452 is ∼5400 of the population of ∼25000–30000, comprising 40% of the adult male and 16% of the female and fawn population per year. Between 2001 and 2011, 40% (range 31–49%) of the white-tailed deer harvested were ≤1.5 years old (Michigan Department of Natural Resources, unpublished data). If, as in other species (Ballesteros et al. 2009a, Waters et al. 2012), young white-tailed deer are optimal targets for vaccination, increased hunting pressure on juveniles resulting in high juvenile mortality may prolong the time to bovine TB eradication. Annual vaccination of 90% of susceptible deer with a 90% efficacious vaccine would take about three decades to achieve eradication (Ramsey et al. 2014a). Models predict that the shortest time to eradication will occur by vaccinating deer in midsummer (Palmer et al. in press).

However, the desire to minimize human exposure to persistent BCG in harvested venison (Palmer et al.

2010b, 2014c) may dictate vaccination in winter.

Designing a field trial presents many challenges. A combined field feasibility and modelling study of a live-trap/ test/cull or vaccinate approach suggested that 30 years of application in DMU 452 would cost

∼US$50 million and carry only a 34% probability of bovine TB eradication from white-tailed deer

(Cosgrove et al. 2012a, b). Consequently, mass distribution of oral baits is likely to be necessary. Costs notwithstanding, sacrificing large numbers of deer in order to measure the effectiveness of vaccination may not be feasible because of opposition by hunters. Potential alternatives may include greatly expanded testing of hunter-harvested deer for BCG, and a before–after control–impact study to measure the rate of

TB extinction (MacKenzie et al. 2006).

Delivery of oral BCG could occur either via bait stations (Ballesteros et al. 2009a) or via aerial bait drops (Rosatte et al. 2009, Muller et al. 2012). Accomplishing uniform spatial coverage with bait stations would necessitate unfettered access to privately owned lands, which comprise 93% of DMU 452

(Carstensen et al. 2011). Increased aggregation of deer at bait stations (Thompson et al. 2008) and the consequent increased MTC transmission (Becker & Hall 2014, Ramsey et al. 2014a) might attenuate vaccination’s positive effects. Agency use of bait where baiting by hunters is banned could prove problematic (Rudolph et al. 2006). Aerial bait distribution would be likely to lead to better spatial coverage of white-tailed deer, but there would be less control of BCG exposures to non-target species, notably cattle.

Given current modelling results, it seems that no strategy, including vaccination, will be successful with only short term commitment to its application. It is not yet clear whether Michigan’s policymakers, hunters, farmers and public are sufficiently committed to TB eradication to warrant embarking on a long- term vaccination programme in earnest (Ramsey et al. 2014a).

Case study: Montes de Toledo, Spain

Starting in 2007, the first controlled laboratory wild boar vaccination trials employed oral and parenteral BCG and resulted in the characterization of the wild boar immune response to vaccination and infection (Ballesteros et al. 2009b, Pérez de la Lastra et al. 2009). Later, a heat inactivated field strain of

Mycobacterium bovis (inactivated vaccine, IV) was successfully used in new controlled vaccination and challenge trials. The IV yielded protection levels similar to BCG (Garrido et al. 2011). In additional experiments in captive animals, protection levels of above 80% were achieved through re-vaccination, both for BCG (Gortázar et al. 2014) and for IV (Beltrán-Beck et al. 2014a). In parallel, suitable oral baits

(Ballesteros et al. 2009c), piglet-selective deployment cages (Ballesteros et al. 2009a) and efficient bait deployment strategies were designed and field-tested (Ballesteros et al. 2011). Since the summer of 2012, the first field trials are ongoing in the high-prevalence region of Montes de Toledo in Spain using BCG and IV in different sites (Díez-Delgado et al., unpublished data).

Safe and specific vaccine deployment is a key concern in oral vaccination strategies. In addition, confirmation of bait uptake (i.e. the use of marked baits) is needed to generate sound scientific data. In the wild boar field vaccination experiments, safety and specificity were confirmed through camera trap surveys and analyses of target and non-target host tissues for BCG (Beltrán-Beck et al. 2014b).

Furthermore, daily bait deployment at dusk and collection of non-consumed baits immediately after dawn improved bait specificity and limited BCG inactivation due to high environmental temperatures.

However, this procedure is labour-intensive and could be avoided by deploying only the IV.

Assessing vaccination efficacy under field conditions is challenging. In the ongoing vaccination field trials, sites were selected away from cattle farms, since risks of cattle contamination with live BCG could not be excluded a priori. No results will become available in terms of reductions in outbreaks of cattle

TB. So far, this has also been the case in other field vaccination trials in possums and badgers (Tompkins et al. 2009, Chambers et al. 2011). Further studies on and around TB-positive cattle farms are required.

However, the effects of wild boar vaccination are measured in the wild boar target host. Efficacy can be tested at the individual level (in piglets with and without the biomarker) and at the population scale

(treatment sites before and after treatment; treatment sites vs. controls). Preliminary information regarding lesion and culture scores in piglets is encouraging (Díez-Delgado et al., unpublished data).

No cost estimations are currently available for this field experiment development during 4 years.

Modelling suggests that clear results at population scale will become evident after 5 years of vaccination

(Anderson et al. 2013). The newly developed vaccine and selective baiting tools require knowledge transfer from the laboratory to the market, and attainment of regulatory approval for distribution in the field. Here, the challenges faced are beyond those of traditional science, but are equally important.

Research needs: vaccination

As shown by the case studies, field vaccination trials can be challenging in terms of deployment logistics and accurate efficacy assessment, and often need to take place over periods longer than those

covered by the usual research grant funds. Some critical aspects to be considered are not scientific, but include cost-efficiency analyses and knowledge transfer aspects.

DISCUSSION

In a review on wildlife disease monitoring using mycobacterial diseases as a case study, Boadella et al. (2011) identified several conceptual steps, from disease discovery and descriptive epidemiology, through risk factor identification and monitoring, to disease control. In this review, we identify six specific research needs, broadly corresponding to these conceptual steps, as follows:

1. Complete the world map of wildlife MTC reservoirs and describe the structure of each local MTC host community, and the role of the environment (disease discovery and descriptive epidemiology).

2. Identify the origin and behaviour of generalized diseased individuals within populations, and study the role of factors such as co-infections, re-infections and individual condition on TB pathogenesis (risk factor identification).

3. Quantify indirect MTC transmission within and between species (risk factor identification).

4. Define and harmonize wildlife disease monitoring protocols, and apply them in a way that allows proper population and prevalence trend comparisons in both space and time (monitoring).

5. Carry out properly designed wildlife TB control experiments enabling the evaluation of the benefits of each single intervention tool.

6. Analyse cost-efficiency and consider knowledge transfer aspects in promising intervention strategies

(control). We believe that addressing these points would push forward our capacity for TB control, through improving our ability to identify measure and control TB in multihost systems. However, many other open questions remain. Vaccination, for instance, usually targets a single-host species. Where MTC is maintained in a host community, the benefits of targeting just one host species might be limited (Plumb et al. 2007).

Another question derives from the fact that human influences have often contributed to the introduction and maintenance of TB in wildlife (Carstensen et al. 2011, Fitzgerald & Kaneene 2013,

Vicente et al. 2013). Efforts to deal with social aspects and management-related public conflicts deserve more support in order to achieve better stakeholder and society buy-in (O’Brien et al. 2011a). For instance, farmers’ opinions about TB can be influenced by their experience of the disease and their interactions with wildlife (e.g. hunters vs. non-hunters; Cowie et al. 2013).

Since TB is more difficult to control in wildlife than in cattle (Fitzgerald & Kaneene 2013), and since interventions in natural systems are prone to conflicts (Artois et al. 2001), a further remaining question is whether or not interventions on wildlife TB are at all justified. The answer varies depending on the local circumstances in each TB hotspot, and is likely to evolve during our collective progress towards TB control in livestock and in wildlife.

ACKNOWLEDGEMENTS

TB research funding was provided via a Plan Nacional grant AGL2011-30041 (MINECO, Spain and

FEDER) and an EU FP7 grant WildTBvac #613779. The corresponding author acknowledges additional support from Campus de Excelencia Internacional (CYTEMA). Azlan Che Amat acknowledges a grant from the Malaysian Government.

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1.3. STUDY HYPOTHESIS AND OBJECTIVES

Key Issues

The development of diagnostic tools for the in vivo detection of Mycobacterium tuberculosis complex (MTC) infection in wild animals (wild boar and red deer in particular) has been widely explored.

However, the performance of these tests is still limited, particularly in certain groups such as ruminants, and specific age-classes, notably young ones. These groups generally lack in test performance, especially with regards to ante-mortem tests. Certain tests, particularly rapid antibody detection tests, are needed in targeted TB control trials. This thesis provides new information on the performance of in vivo tests and the suitability of targeted culling as a TB-control tool.

Hypothesis

Infection by the MTC causes a variable humoral and cell mediated immune response (CMI). We hypothesize that these responses vary depending on the host species and the specific age-class under study. We also hypothesize that a better understanding of the performance of antibody-mediated and

CMI-mediated ante-mortem diagnostic tests in wildlife will improve our capacity to control animal TB.

These diagnostic tools will find their application in epidemiology and surveillance, as well as in disease control.

General Objective

The CMI responses are predominant during early infection in bovine TB while serum antibodies are generally evident at a more advanced stage and increasing bacilli burden. The overall objective of the thesis is to generate additional information on implementation of practical and accessible diagnostic tests in wild boar and red deer and using the tool in control strategies for tuberculosis in wild animals.

Improving knowledge on TB pathogenesis such as understanding the role played by other infection is also

needed. Gaining understanding wild boar and red deer immune responses is beneficial for improving diagnostic methods to facilitate control of animal TB under all conditions.

Specific Objectives

Objective 1: To assess the performance of several antibody detection tests (in-house and developing commercial ELISAs and rapid immunochromatographic tests) for Mycobacterium bovis infection in 2–6 month-old piglets during summer.

Objective 2: To evaluate the effect of repeated skin testing for avian and bovine tuberculosis and phytohaemagglutinin on serological tests results in TB-free herd in red deer and their temporal variations in skin and serological tests.

Objective 3: To assess the suitability of targeted removal as a means for TB control in intensely managed

Eurasian wild boar (Sus scrofa) hunting estates.

Objective 4: To investigate infection status of wild boar piglets less than 4-month old in fenced intensive game management system and to determine the interaction of certain pathogens connected with marked mortality among wild boar piglets.

Result - Chapter II

Testing Eurasian wild boar piglets for serum antibodies against

Mycobacterium bovis

Che’ Amat, A., González-Barrio, D., Ortiz, J.A., Díez-Delgado, I., Boadella, M., Barasona, J.A.,

Bezos, J., Romero, B., Armenteros, J.A., Lyashchenko, K.P., Venteo, A., Rueda, P., Gortázar, C.*

Preventive Veterinary Medicine, 2015. 121: 93–98

This chapter is prepared in the style format of Preventive Veterinary Medicine

ABSTRACT

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (Sus scrofa).

Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2 to 6 month-old piglets.

In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (n=53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19-38%, reaching sensitivities between

15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between

DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2 to 6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account.

Keywords: ELISA, Mycobacterium tuberculosis complex, sensitivity, Sus scrofa

1. INTRODUCTION

Animal tuberculosis (TB) is caused by infection with Mycobacterium bovis (M. bovis) and closely related members of the M. tuberculosis complex (MTC). The infection is often reported in wildlife, even in developed countries (Gortázar et al., 2012). The native Eurasian wild boar (Sus scrofa) is considered the greatest TB hazard of all wildlife species in Europe (Hardstaff et al., 2014) and particularly is the single most important MTC maintenance host species in Mediterranean ecosystems of the Iberian

Peninsula (Gortázar et al., 2008; Naranjo et al., 2008). High TB prevalence of above 60% is sometimes reported in wild boar from sites with a high infection pressure in central and southern Spain (Vicente et al., 2013).

The prevalence of M. bovis infection increases with age in wild boar. In 7-12 month old yearlings

(those harvested by hunters in their first autumn-winter season), prevalences reported ranged from 20 –

52% (Vicente et al., 2006, 2013; Díez-Delgado et al., 2014). Infection of yearling wild boar was also noted in a survey on the MTC member M. microti in Italy (11.11%; Chiari et al., 2015). However, published information refers almost exclusively to wild boar over six months of age, since it is generally based on hunter-harvested individuals sampled in autumn and winter. Data on MTC prevalence are almost lacking in piglets (less than 7 months old). Exceptionally, a study based on culled (not hunted) wild boar in the TB endemic Doñana National Park in southern Spain found 3 out of 3 piglets sampled in summer to be M. bovis infected (Gortázar et al., 2008). In addition, a recent survey in eastern Spain also based on culled wild boar showed a summer prevalence of 6.3% in wild boar piglets less than 6 months

(Mentaberre et al., 2014). Field observations show that lesion scores are lower in piglets than in yearling or subadult wild boar (Díez-Delgado, pers. comm.). Considering the correlation between lesion scores and serum antibody levels against M. bovis reported in wild boar (Garrido et al., 2011) and the predominance of early infections in piglets with still immature immune system, serology interpretation may be particularly challenging in this age category.

The gold standard confirmation for MTC infection is microbiological culture and identification (OIE,

2009). However, serological methods are often used in wildlife because of their relatively low cost, simplicity, speed and the advantage of easily conducted retrospective studies (Chambers, 2009). The serology can be performed in live animals and is a suitable test towards the harmonization of wild boar testing for large scale surveys eventually involving several countries (Beerli et al., 2015). A conventional enzyme-linked immunosorbent assay (ELISA) using bovine purified protein derivative (bPPD) has shown a moderate sensitivity (73%) and good specificity (96%) for detection of infected wild boar (Aurtenetxe et al., 2008). This finding led to subsequent improvements of sensitivity (79%) and specificity (100%) with no significant difference between yearling, subadult and adult wild boar (Boadella et al., 2011). In

10-month old domestic pigs experimentally infected with M. bovis, the sensitivity of a modified bPPD

ELISA using anti-pig immunoglobulin G (IgG) was 94% (Beltrán-Beck et al., 2014).

Rapid immunochromatographic tests, such as the DPP (Dual Path Platform) VetTB and DPP WTB

(Wildlife TB) assays (Chembio, New York, USA), were developed for animal-side applications. The DPP assays use MTC antigens, including recombinant MPB70, MPB83, and CFP10/ESAT-6 fusion protein.

High diagnostic accuracy was reported for wild boar (77-90% sensitivity and 90-97% specificity;

Boadella et al., 2011; Lyashchenko et al., 2008). Recent findings in domestic pigs vaccinated with heat inactivated M. bovis and challenged with a field strain showed a range of DPP assay sensitivity from 78 to

94% and 100% specificity (Beltran-Beck et al., 2014). The antibody detection rate in wild boar with macroscopic lesions was five times higher (83%) than that in animals without visible lesions (17%;

Lyashchenko et al., 2008).

Since only limited knowledge exists on TB prevalence and serology utility in wild boar piglets, the goal of this study was to assess the performance of several antibody detection tests for M. bovis infection in 2 to 6 month-old piglets during summer. We hypothesized that serology test sensitivity might be lower

in this age category largely representing relatively recent infections with less frequent and/or early seroconversions.

2. MATERIALS AND METHODS

2.1. Animals and Sampling

This study took place in an intensively managed private hunting estate in Sevilla province, southern

Spain. As in other intensively managed estates with hunting purposes, wild boar piglets are captured in early summer and maintained in enclosures for about 3 months until the opening of the hunting season.

Piglets are released back into the wild after the first hunting event. This procedure is expected to reduce piglet mortality during the dry summer months and during the first hunting event. A total of 126 wild boar piglets (52 males and 74 females) captured during June and July 2012 and 2013 died during captivity and were submitted to the laboratory. The range of body weights was 3.24 to 15.24 kg (mean = 7.53 ± 2.17

S.D.). Blood was taken and serum samples stored at -20°C until testing. All piglets were necropsied and pooled lymphoid tissues from a subsample of 53 piglets were submitted for microbiology. A TB lesion score ranging from 0 – 26 was calculated based on observation of 13 sites. These included left and right mandibular, left and right bronchial and mediastinal lymph nodes, lungs (7 lobes) and mesenteric lymph nodes (see Díez-Delgado et al., 2014 for further details).

2.2. Bacterial culture and characterization

Bacterial culture was used as gold standard and readers of this test were masked to the serology results. Pooled tissues were cultured for Mycobacterium bovis as previously described (Corner and

Trajstman, 1988). Briefly, the samples were thoroughly homogenized in sterile distilled water (2 g in 10 ml or equivalent) and decontaminated with hexadecylpyridinium chloride at a final concentration of

0.35% (wt/vol) for 1 h. The samples were centrifuged at 1,500 g for 30 min and the pellets cultured onto

Coletsos and 0.20% (wt/vol) pyruvate-enriched Lowenstein-Jensen media (Difco FSM, Madrid, Spain) at

37°C. The culture media were checked weekly over 12 weeks for growth. All isolates were spoligotyped in order to confirm the strain identification (Kamerbeek et al., 1997).

2.3. Antibody detection tests

Table 1 present the tests used for detection of antibodies against MTC antigens. All tests were run and interpreted in presence of the same expert (first author).

2.3.1. ELISA bPPD IgG and IgM

The ELISA tests for IgG and immunoglobulin M (IgM) were carried out with a readjusted procedure used by Hu et al. (2008) and Chen et al. (2005) respectively. Briefly, the testing plates were coated with bovine purified protein derivative (bPPD) tuberculin (CZ Veterinaria S.A., Pontevedra, Spain) at 5 µg/ml and incubated overnight at 4°C. Skim milk powder solution (5% concentration) was added to each well and left 60 minutes at room temperature. Following three washes with phosphate buffered saline (PBS) solution containing 0.05% Tween 20 (PBST), piglet sera were added at 1:100 dilution and incubated for

30 min at 37°C. For the detection of IgG antibodies, 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-pig IgG antibodies (Bethyl Laboratories Inc., Texas, USA) at a 1:5000 dilution were added, whereas for the detection of IgM antibodies, 100 μl of HRP-conjugated goat anti-pig IgM antibodies at a

1:10000 dilution were added. After the addition of the respective conjugate, the plates were incubated for

30 min at 37°C and subsequently washed with PBST three times. Color was developed by adding 50 μl of

3,3′,5,5′-Tetramethylbenzidine (TMB) (Promega Corp., Madison, USA). Protected for 15 minutes from the light at room temperature, the reaction was stopped with 50 μl of H2SO4 (2M). The optical densities

(OD) were measured at 450 nm with an ELISA reader. For positive control ELISA-IgG, pooled anti-

PPD–positive serum was obtained from wild boar previously described as M. bovis culture positive and negative controls from TB- free wild boar previously described as M. bovis culture negative from bTB- free areas. ELISA results for IgG were expressed by using the formula: [sample E% = (mean sample OD /

2 x mean negative control OD) x 100]. Values higher than 100 were considered positive. For ELISA-IgM,

since no controls were available, cut-off was determined by plotting a vertical graph of the net-OD. The most evident jump in OD was chosen as positive value (OD > 0.4).

2.3.2. ELISA bPPD Protein-G (IgG detection)

Serum samples were tested for anti-PPD antibodies by means of an ELISA using bPPD as antigen and protein-G horseradish peroxidase (Bethyl Laboratories Inc., Texas, USA) as a conjugate applying the previously described protocol (Boadella et al., 2011). Briefly, after coating the plates (at 5 µg/ml) for 18 hours at 4°C, wells were washed with PBST and blocked for 1 hour at room temperature (RT) with 140 µl to each well of 5% skim milk in PBST. Sera were added directly on plate (100 µl/well) at a dilution of

1:200 in PBS and incubated for 1 hour at 37°C. Samples, blanks, and positive and negative controls were tested in duplicate in each plate. Protein G was added (100 µl/well) at a dilution of 0.5 µg/ml in PBST and incubated at 37°C for 1 hour and 20 minutes. After revealing with FastOPD (SigmaFast™ OPD, Sigma,

St. Louis, USA), the reaction was stopped with 50 µl/well of sulfuric acid (H2SO4; 3N), and OD was measured in an ELISA reader at 450 nm. Sample results were expressed as an ELISA percentage (E%) and E% values greater than 100 were considered positive.

2.3.3. ELISA-DR

The methodology was developed by INGENASA (Madrid, Spain). The principles of double recognition ELISA (DR-ELISA) was developed with recombinant protein (MPB83) serving both as coating antigen and conjugated form (horseradish peroxidase (HRP) labelled), the signal capture. The results were read by ELISA spectrophotometry and 0.3 was chosen for cut-off OD. This assay is able to recognize not only IgGs but also other immunoglobulins, such as IgMs. Briefly, the recombinant MPB83 expressed in baculovirus system was used as antigen to coat 96-well microtitre plates (25ng/well). After an incubation at 4oC, the wells were blocked and a 1/200 dilution of serum in 0.05% Tween, 0.35M NaCl in Phosphate-Buffered Saline (PBS) was incubated for one hour at RT. Bound antibodies were detected

by incubation with MPB83-HRP one hour at RT and subsequent addition of the substrate (TMB-MAX,

Neogen Corporation, KY, USA).

2.3.4. DPP Tests

We used two immunochromatographic antibody assays, the DPP VetTB and DPP WTB tests, developed by Chembio Diagnostic Systems, New York, USA. DPP VetTB is USDA-licensed (for cervids and elephants) and commercially available for use in any animal species, while DPP WTB is not licenced but available for investigational assays. Sera were tested for the presence of specific antibodies as previously described (Boadella et al., 2011). Any visible band in the test area 20 min after adding sample buffer, in addition to the control line, was considered an antibody positive result, whereas no test band in presence of control line was considered a negative result.

2.4. Data treatment

Sensitivity and area under curve (AUC; based on ROC curve analysis; Fawcett 2006) of serology were analyzed using MTC culture and tuberculosis-like lesions as reference standards, separately. The accuracy was derived from the AUC value. It represents the probability that the test result for a randomly chosen positive case will exceed the result for a randomly chosen negative case. Agreement between test systems was calculated by using the Cohen kappa (Cohen, 1960) coefficient significance value <0.05.

The General Linear Models (one for each serological technique) used serological response (binomial variables, coded as 0 = negative and 1 = positive) as a dependent variable, while culture or lesions where included as categorical factors in separate models. We categorized the tuberculosis-like lesions into 3 categories (Score 0; score less than 5; score more than 5). Parameter estimates (β) were calculated using a reference value of 0 for the ‘3’ level (highest TB score) in the variable ‘score group’. We used a binomial error and a logit link. All analyses were computed using SPSS Statistical Package (ver.20.0, IBM Corp.,

New York, USA).

3. RESULTS

Bacterial culture yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19 to 38% (Table 1). Table 2 shows the sensitivity and AUC values of the six serological tests using TB lesion presence and culture as reference, respectively. The

Cohen kappa coefficient of agreement between TB lesion presence and MTC culture was 0.82 (Sig.

0.000; p<0.05), indicating an almost perfect agreement. Regarding the serum antibody tests, agreement between DPP WTB and culture was moderate (0.45, p>0.05) and all other serological tests used had poor to fair agreements (Table 2). Figure 1 displays the performance of each serological test in detecting antibodies against MTC antigens in wild boar piglets as compared to the culture results. The diagnostic sensitivities varied between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for DPP tests. Specificity was not calculated since it required true negative animals from zero prevalence areas, which were not available in this study. Figure 2 shows the relationships between TB lesion scores and seropositivity rates. Antibody prevalence increased significantly with score lesion group in the DPP WTB assay (p=0.004; β score group 1 = -2.2±0.08; β score group 2 = -1.0±0.007), DPP VetTB assay (p=0.023;

β score group 1 = -2.1±0.08; β score group 2 = -1.5±0.009) and ELISA-DR test (p=0.015; β score group 1

= -1.7±1.0; β score group 2 = -0.6±0.01). The patterns of ELISA-IgG and ELISA Protein-G were different although not significant (p>0.05), which groups with highest TB lesion scores showing lower proportions of seropositive results.

Table 1: Prevalence of serum antibodies against the Mycobacterium tuberculosis complex (MTC) according to six different tests; prevalence of M. bovis infection assessed by culture; and prevalence of tuberculosis-compatible lesions in two to six month-old wild boar piglets.

Test system Details No. of MTC antibody positive prevalence (95% CI) samples DPP WTB Immunochromatographic single band 48/126 38.0% test, MPB70 and MPB83 antigens, (29.5-46.4) Chembio Diagnostic Systems, Inc. DPP VetTB Immunochromatographic 48/126 38.0% two-band test, MPB83 and (29.5-46.4) CFP10/ESAT-6 antigens, Chembio Diagnostic Systems, Inc. ELISA-IgG Plate test, PPDb antigen, HRP 37/126 29.3% conjugated goat anti-pigs IgG, in- (21.3-37.2) house ELISA ELISA-Protein-G Plate test, PPDb antigen, Protein-G 26/126 20.6% HRP conjugate, in-house ELISA (13.5-27.6) ELISA-DR Plate test, MPB83 antigen, 26/126 20.6% INGENASA® Madrid, Prototype (13.5-27.6) ELISA-IgM Plate test, PPDb antigen, HRP 24/126 19.0% conjugated goat anti-pigs IgM, in- (12.1-25.8) house ELISA M. bovis culture VISAVET, Madrid 18/53 33.9% (21.1-46.6) TB-compatible lesion IREC-UCLM-JCCM 27/126 21.4% (14.2-28.5)

Table 2: Sensitivity and area under curve (AUC) of six tests for the detection of serum antibodies against the Mycobacterium tuberculosis complex (MTC), using tuberculosis (TB)-compatible lesions and MTC antigens culture as references, respectively.

Test system TB lesion MTC culture K K Sensitivity AUC Sensitivity AUC (sig <0.05) (sig <0.05) 0.283 0.452 DPP WTB 66.7% 0.682 69.2% 0.735 (0.001) (0.004) 0.210 0.342 DPP VetTB 59.3% 0.635 61.5% 0.678 (0.011) (0.029) 0.211 0.288 ELISA-IgG 48.1% 0.620 46.2% 0.638 (0.016) (0.067) 0.259 0.186 ELISA-DR 40.7% 0.628 23.1% 0.578 (0.004) (0.160) 0.212 0.051 ELISA-ProteinG 37.0% 0.604 23.1% 0.523 (0.018) (0.736) 0.091 -0.076 ELISA-IgM 25.9% 0.544 15.4% 0.466 (0.305) (0.613)

K: Kappa agreement expressed as value and significant <0.05

Figure 1: Performance of six serological assays in two to six month-old Eurasian wild boar (Sus scrofa) piglets as compared to culture: two immunochromatographic tests, DPP WTB and DPP VetTB assays (Chembio, NY, USA); three in-house enzyme-linked immunosorbent assays (ELISA) for immunoglobulin G (IgG), Protein G and immunoglobulin M (IgM); and the ELISA-DR prototype (Ingenasa, Madrid, Spain).

Figure 2: Seropositivity in relation to three tuberculosis (TB) lesion score categories (0, <5, ≥5) in two to six month-old Eurasian wild boar (Sus scrofa) piglets using two immunochromatographic tests, DPP WTB and VetTB assays (Chembio, NY, USA); three in-house enzyme-linked immunosorbent assays (ELISA) for immunoglobulin G (IgG), Protein G and immunoglobulin M (IgG); and the ELISA-DR prototype (INGENASA, Madrid, Spain). Bars represent the standard error (S.E.).

4. DISCUSSION

This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2 to 6 month-old wild boar piglets naturally infected with M. bovis. Both traditional plate

ELISA tests and rapid animal side tests can be used, although the best performance was demonstrated for the latter. The results supported our hypothesis of a lower sensitivity of serology for detecting MTC- infected piglets, as compared to yearling, juvenile or adult wild boar.

Based on the results of microbiological culture, one third of the annual cohort of wild boar was infected very early in life, at least under the high infection pressure existing in intensely managed hunting estates within TB endemic areas. In general, ELISA test sensitivity in piglets was lower than those reported in previous studies on wild boar of all ages (>70%; Boadella et al., 2011, Lyashchenko et al.,

2008) and in pigs (94%; Beltrán-Beck et al., 2014). In human TB, diagnosis in pediatric patients is also difficult by any available method and serology is not an exception. The antibody responses to all mycobacterial antigens in pediatric patients were much lower than in adult patients. This may be due to reduced serological responses to MTC infection in young or early infected individuals (Achkar and

Ziegenbalg, 2012).

The poor performance of antibody detection tests in piglets means that pre-movement testing during wild boar piglet translocations can easily miss detecting infected individuals. Also, piglets will yield lower prevalences than other age classes in cross-sectional serosurveys. This sub-optimal sensitivity must also be taken into account in longitudinal studies, where tagged piglets are later re-tested at more advanced age: some delayed seroconversions could be due to the insufficient test sensitivity in piglets.

The test based on IgM aimed at detecting early infections. However, this test had the poorest sensitivity. Two explanations can be found. First, the lack of a proper positive control hampered establishing a proper cut-off. Second, IgG-based tests consistently yielded higher sensitivities, suggesting that the detected contacts of the piglets with MTC were not necessarily recent.

In this study, the antibody levels detected by the bPPD ELISAs for IgG did not correlate with the lesion score. This is in contrast to previous findings where the bPPD ELISA results correlated with the

lesion score in wild boar, suggesting that ELISA may be used for classifying infected animals as showing a more or less advanced disease (Garrido et al., 2011). Nevertheless, the findings of this particular study on wild boar piglets should not be compared with studies including older subjects with a more developed humoral immune response. On the other hand, lesion scores did correlate with DPP assay results also detecting serum IgG antibodies. Therefore, the difference observed do not only stem from age or early infection, but may represent an example of inter-assay differences. Our sample also lacked of culture and lesion negative piglets from non-endemic sites, thus precluding a specificity analysis.

All plate tests, ELISA IgG, ELISA protein-G and ELISA-DR had a good agreement among them except the ELISA IgM. Both DPP assays had a better sensitivity than the plate ELISAs. Sera went only through one freeze-thawing cycle, making it unlikely that freeze-thawing negatively affected test performance (Boadella and Gortazar, 2011). A possible explanation for the higher sensitivity of the DPP

WTB assay as compared to DPP VetTB assay is that the former has a double amount of the target antigen

(MPB83+MPB70) immobilized in the test line, as compared to DPP VetTB assay (MPB83 only). This finding is in agreement with prior studies suggesting that ESAT-6 and CFP10 (antigen in DPP VetTB assay) are not essential for antibody detection in wild boar (Lyashchenko et al., 2008).

The results described that one third of the wild boar piglets can become infected at early age in a high TB prevalence sites. While we show that serology is less sensitive in piglets than generally observed in adult wild boar, performance varies among tests. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre- movement testing. However, sub-optimal test performance in piglets as compared to older wild boar should be taken into account.

Aknowledgements

Authors would like to acknowledge many students and collaborators who contributed to field work over the study period, and wish to express their gratitude to the managers and gamekeepers of the hunting estate. This is a contribution to Spanish Government MINECO Plan Nacional I+D+I grant AGL2014-

56305 and FEDER, to a contract between CDTI and Glenton, and to the EU FP7 grant WildTBvac

#613779. Azlan Che Amat has a PhD grant from the Malaysian Government, and José Angel Barasona and Iratxe Diéz-Delgado acknowledge PhD grants from the Spanish Government.

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Result - Chapter III

Effects of repeated comparative intradermal tuberculin testing on

test results: a longitudinal study in TB-free red deer

Azlan Che-Amat, Maria Ángeles Risalde, David González-Barrio,

Jose Antonio Ortíz, Christian Gortázar*

BMC Veterinary Research, 2016, 12:184

This chapter is prepared in the style format of BMC Veterinary Research

Abstract

Background: Diagnosing tuberculosis (TB) in farmed red deer (Cervus elaphus) is challenging and might require combining cellular and humoral diagnostic tests. Repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might sensitize or desensitize the subjects to both kinds of diagnostic tools. We evaluated the effect of repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA responsiveness in farmed red deer hinds from a TB-free herd. Eighteen 8-month old hinds were inoculated with bovine and avian PPDs and the mitogen phytohaemagglutinin (PHA), as positive control and concurrently tested by ELISA for antibodies against avian (avian PPD, aPPD and protoplasmatic antigen 3, PPA3) and bovine antigens (bPPD and MPB70). Blood serum was also sampled three weeks after each skin testing round and tested for antibodies against aPPD and bPPD, in order to detect eventual antibody level boosts. Testing took place every six months from winter 2012 until winter

2015.

Results: The skin test response to both PPDs peaked during the second and third test round, returning to standard values thereafter. Individual variability was particularly high at the first year and early second year testing rounds (first intradermal test and blood sampling; first winter). The antibody response to avian antigens increased through time, while no such increase was recorded for bovine antigens. The antibody boost three weeks after skin testing was more marked for avian PPD. However, there was no consistent trend in the boosting response through time.

Conclusion: Repeated comparative skin testing at six month intervals did not cause progressive increments in skin test responsiveness or antibody production. Specifically, we observed no loss of the skin test response to bPPD and also no progressive loss of the boosting effect in the ELISA responses.

However, we recorded increases through time in the antibody levels against avian mycobacterial antigens, possibly due to the progressive exposure to MAP or to other cross-reacting environmental mycobacteria.

These findings should be taken into account in designing and interpreting TB testing schemes in farmed deer.

Keywords: Cervus elaphus; farmed deer; immune response; mycobacterial diseases; temporal variability.

Background

The red deer (Cervus elaphus) is an important game species with a broad geographical range worldwide and a developed farming industry [1,2]. Red deer are susceptible to tuberculosis (TB) due to infection with Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex (MTC), often becoming true reservoir hosts [3]. In addition, red deer (and particularly farmed red deer) are also susceptible to Mycobacterium avium paratuberculosis (MAP), the causative agent of paratuberculosis (PTB) or Johne’s disease [4]. Finally, infection due to other mycobacteria can occur and sensitization to these non-tuberculous mycobacteria can cause diagnostic cross-reactions [5]. Thus, it is crucial to test deer in farms and prior to movement in order to avoid sanitary risks [6].

The tuberculin skin-test based on the intradermal inoculation of M. bovis–derived purified protein derivative (bovinePPD, bPPD), is regarded as the standard in-vivo diagnostic method for TB control by the World Organization for Animal Health [7]. The intradermal skin test is based on the detection of the delayed hypersensitivity reaction that appears 72 h after the inoculation of bPPD and, in the case of the comparative intradermal test, its comparison with the reaction elicited by the inoculation of aPPD.

Currently there is no formal requirement of TB testing for farmed deer in Spain. However, pre-movement tests are compulsory for translocations of live deer. For such cases, the single intradermal comparative cervical tuberculin (SICCT) test is the only approved diagnostic method. The bPPD skin response is expected to be greater in deer infected with MTC members. Likewise, if the animal is infected with mycobacteria other than the MTC, greater responses to aPPD than those observed to bPPD are expected

[5,8-10]. However as PPDs comprise a complex mixture of proteins in certain situations the specificity of the test may be compromised inducing false positive reactors [11]. Moreover, in cattle, it has been documented that repeated skin testing may cause a reduction in skin test response in natural and experimental infections with M. bovis. This has negative consequences on the ability to detect reactors

[12,13]. Many other factors such as age, sex, season, body condition and type of management can affect the skin test responsiveness in deer compromising the sensitivity and specificity [5,14]. In cases of

advanced TB, some animals become unable to respond to any diagnostic technique that detects cell mediated immune response. Such cases can be filtered-out using the mitogen phytohaemagglutinin (PHA) on a third injection site [15,16]. The PHA skin fold increase is independent of the mycobacterial infection status and hence not affected by the PPD skin test results [17]. The use of PHA also allows for control in the variation in the general responsiveness to intradermal injected antigens [14]. The gamma interferon test has also been used for in vivo TB diagnosis in deer [18].

The detection of humoral responses by means of serology represents an alternative for screening herds of livestock and wild animals for mycobacterial infections [19,20]. Serological tests for humoral response detection may be used as an alternative or more often as a complementary tool for screening mycobacterial infections in deer [21]. Unfortunately, tests aiming at MTC or MAP-specific antibody detection in red deer are not always very sensitive (72.7% to 86.7% for MTC, references: [22-26]; 50% to

91% for MAP, references: [22,27]) and not too specific (83.8% to 98.0% for MTC; [23,26]; 88% to

99.5% for MAP; [22,27]). Antigens used in these tests are summarized in Supplementary Table S1. One additional issue measuring humoral responses is that the titer of antibodies changes significantly during the infection and they are mainly produced in advanced stages of infection [28].

It is known that the inoculation of bPPD for skin testing for TB boosts the antibody responses to the

MTC in M. bovis-infected cattle [29-32]. Hence, it has been suggested that serological tests for TB in cattle should be performed after skin tests. Recently, Casal et al. [32] demonstrated that the use of serological testing performed after skin testing, in combination with traditional skin test procedures, increased the detection likelihood of tuberculous animals within TB-infected cattle herds, as compared to skin tests alone. However, the effects of serial injections of PPDs for skin testing on the responsiveness to skin tests and on serum antibody responses, as well as the duration and quality of the antibody boosts, have not been fully evaluated in red deer.

We hypothesized that repeated (every 6 months) skin testing with aPPD and bPPD could have an effect on the skin test responsiveness or on the antibody levels against mycobacterial antigens, causing progressive changes in these responses. This study of red deer hinds from a TB-free farm aimed to, 1)

evaluate the effect of multiple skin testing with aPPD and bPPD on the skin test responsiveness; and 2) to evaluate the effect of multiple skin testing with aPPD and bPPD on the antibody levels measured by

ELISA (aPPD, bPPD, MPB70 and PPA3) immediately before and (for the PPDs) three weeks after each skin testing round.

Methods

Animals

The present study was carried out in a TB-free (no positive cases since 2003) red deer farm in southern Spain. It is a farm with a semi-intensive management scheme, with pasture-rotation and year- round food supplementation. The farm is surrounded by a double fence that limits with a red deer hunting estate of the same ownership. TB-positive deer are sporadically detected during meat inspection of hunter-harvested deer from this estate. Moreover, MTC infection in feral pigs (Sus scrofa) and in badgers

(Meles meles), both present at low density in the hunting estate, cannot be excluded.

Farmed deer are handled two times per year (summer and winter) for skin-testing, measurement, blood sampling and administration of antiparasitic drugs. Study animals included 18 deer hinds born in spring 2011 (8-month old at first testing). No initial assessment of the TB status was performed before the study started. These 18 hinds were individually identified with an ear tag and remained in the farm during the study period (2011–2015). We used hinds because stags usually are sold for release at the age of 1.5 years. Handling procedures and sampling frequency were part of the farm routine and not influenced by the study. These procedures were designed to reduce stress and health risks for subjects, according to

European (86/609) and Spanish laws (RD 223/1988; RD 1021/2005), and current guidelines for ethical use of animals in research [33].

Diagnostic tests

Intradermal skin test

All hinds were skin-tested and sampled seven times during the study period (in winter 2012, summer

2012, winter 2013, summer 2013, winter 2014, summer 2014 and winter 2015). Animals were handled twice per skin test, at times 0 and 72 hours. Deer were moved from the paddocks to the farm enclosures and then immobilized by physical restraint using a hydraulic crush. At time 0, each animal was identified, weighed and blood samples were collected. Three areas of 3 cm × 3 cm were shaved at the left side of the mid-neck with an electric shaver and skin fold thickness was measured employing a digital cutimeter

(Hauptner Instrumente GmbH, Zurich, Switzerland) three different times in each area, to the closest 0.1 mm, by the same operator. Then (from cranial to caudal) 0.1 ml of avian and bovine purified protein derivative (PPD; 25,000 IU/ml; CZ Veterinaria SL, Porriño, Spain) and the plant derived mitogen phytohaemagglutinin (250 g of PHA; Sigma, Barcelona, Spain) diluted in phosphate buffered saline as positive control, were inoculated using 1-ml syringes fitted with a 25-G ½-in. needle. At time 72 h, each animal was immobilized again by physical restraint, identified, and the skin fold thickness was measured again at each injection site three different times by the same operator. Animals were considered TB reactors in the SICCT if the skin fold increase to bPPD was greater than 2 mm and more than 1 mm larger than the skin fold increase to aPPD [34,35]. Avian reactors were defined as those with a skin fold increase to aPPD >3 mm [36]. Deer with skin fold thickness increases of less than 0.5 mm to all 3 antigens were considered unresponsive animals [17]. Clinical signs such as evident pain, necrosis or exudation were also considered as positive reactions.

Serological tests

Blood samples were collected from the jugular vein before inoculation with the antigens for skin tests in different seasons (winter 2012, summer 2012, winter 2013, summer 2013, winter 2014, summer

2014 and winter 2015). In order to evaluate antibody boosts in animals after antigen stimulation with aPPD and bPPD, blood samples were also obtained three weeks after selected skin testing rounds

(summer 2012, winter 2013, winter 2014 and winter 2015). Sera obtained by centrifugation (3000g for 10 minutes) from blood samples were tested for antibodies against bovine PPD and avian PPD, paratuberculosis protoplasmatic antigen 3 (PPA3; Allied Monitor, Fayette, MO, USA) and M. bovis antigen MPB70 (Lionex Diagnostics & Therapeutics GmbH, Braunschweig, Germany) using an in-house

ELISA as previously described [37,38]. Briefly, after coating the plates overnight at 4°C with 50 µl/well of antigen solution in carbonate–bicarbonate buffer (Sigma, Barcelona, Spain), wells were washed with phosphate buffered saline (PBS) solution containing 0.05% Tween-20 (PBST) and blocked for 1h at room temperature with 140 µl of blocking solution (5% skim milk in PBST). The adsorbed sera were diluted

(1:10, v/v) in blocking solution and 100 µl/well was added into duplicate wells of the antigen-coated plate. After a 1h and 30 min incubation period at 37ºC, the plates were washed three times with PBST and

100 µl/well was added (0.002 mg/ml in PBS) of protein G horseradish peroxidase conjugate (Sigma,

Barcelona, Spain) and incubated at room temperature for 1h. After three washes, 100 µl/well of substrate solution (Fast OPD, Sigma, Barcelona, Spain) was added. The reaction was stopped with 50 µl/well of

H2SO4 3N and the optical density (OD) was measured in a spectrophotometer at 450 nm.

Deer negative and positive control sera were included in every plate in duplicate . Pooled anti-PPD– positive serum was obtained from deer previously described as M. bovis culture positive and negative sera obtained from an experimental facility belonging to the University of Castilla-La Mancha with no clinical history of TB and PTB and repeated negative culture results. OD values of the animals between different plates were normalized according to the values of the negative controls included in each plate.

All ELISAs were performed at the same time by two experienced researchers with no previous knowledge of which sample was being analyzed. Sample results were expressed as an ELISA percentage

(E%) that was calculated using the following formula: [sample E% = (mean sample OD/2 mean of negative control OD) x 100 ]. Cut-off values were defined as the ratio of the mean sample OD to the double of mean OD of the negative control. Serum samples with E% values greater than 100 were considered positive [38]. Boosting was only investigated for avian and bovine PPD.

Data analysis

Apparent prevalence rates were calculated based on frequencies of cases over the total number of cases sampled. The Spearman correlation test was used to assess the relationship among skin and serological test results. Pairwise comparisons were used to compare the seasonal effect on skin and serological tests results. Differences between group means and correlation coefficients among skin and serological test results were considered significant at p<0.05. We used the Kolmogorov–Smirnov test to assess that the data was normally distributed. All analyses were performed using IBM SPSS Statistic

Processor (Version 20.0).

Results

Figure 1 shows the mean skin test responses of 18 red deer hinds for seven consecutive (six- monthly) testing rounds from winter 2012 to winter 2015. No animal was unresponsive. The mean skin test response (in mm; ±SD) was 2.42 (±2.27) to aPPD, 1.36 (±1.25) to bPPD, and 1.83 (±1.16) to PHA, respectively. The shape of the graphs for aPPD and bPPD was similar (rs=0.96; p<0.05), while there was no correlation with the independent PHA (rs<0.51; n.s.). The mean skin test response to both PPDs peaked during the second and third testing round, returning to standard values thereafter, while the response to PHA had an initial peak and later a second one at the fifth testing round. Two individuals matched the definition of a reactor to the comparative skin test (responding >2mm to bPPD and displaying a bPPD response at least 1 mm larger than aPPD). These individuals were positive in only one of seven testing rounds (round 2 and 5, respectively). However, 13 of 18 individuals (72.22%) tested positive for the single aPPD skin test (response to aPPD >3mm), and 7 of them tested positive in several

(2 to 5) rounds. Regarding the single bPPD skin test (response to bPPD >2mm), 13 of 18 individuals

(72.22%) tested positive for the single bPPD skin test, and 6 of them tested positive in several (2 to 4) rounds. Individual variability was high, particularly at the first year and early second year testing rounds

(Figure 2).

Fig 1. Red deer mean (±SD) seasonal (winter 2012 to winter 2015) skin test response in mm to the intradermal injection of (a) avian purified protein derivative (aPPD), (b) bovine PPD (bPPD) and (c) the mitogen phytohaemagglutinin (PHA). The dashed lines indicate the cut-off value for each PPD.

Fig 2. Individual seasonal (winter 2012 to winter 2015) skin test response in mm of 18 red deer hinds to the intradermal injection of (a) avian purified protein derivative (aPPD) and (b) bovine PPD (bPPD). Dashed lines indicated for cut-off value.

Figure 3 displays the mean response (in E%) to two avian and two bovine antigens, measured on serum samples taken before PPD injection for skin testing. The response to avian antigens (aPPD and

PPA3; panels a and b of Figure 3) tended to increase through time. This increase was significant for aPPD

(rs=0.78, p<0.05) but not for PPA3 (rs=0.64, n.s.). A total of 15 of 18 individuals (83.33%; 14 for PPA3,

12 for aPPD) yielded a positive ELISA for antibodies against avian antigens in at least one testing round.

Regarding the bovine antigens (bPPD and MPB70; panels c and d of Figure 3), these did not increase through time (rs<0.47; n.s.). Rather, there was an increase in the ELISA response to both bovine antigens at the 6th round of testing. A total of 12 of 18 individuals (66.66%; 12 for MPB70, 11 for bPPD) yielded a positive ELISA for antibodies against bovine antigens in at least one testing round. Animals responding to avian antigens tended to repeat and even increase their positive response through time, while responses to bovine antigens tended to be weak and sporadic, occurring more often in the 6th and 7th testing round

(Supplementary Table S2). Only in one case, and only in one testing round, a deer hind tested positive to bovine antigens (a weak 102% to bPPD, cut-off set at 100%) without testing positive to avian antigens, too.

The ELISA responses to different antigens were correlated with the shape of the graphs for aPPD and bPPD was similar (rs=0.96; p<0.05), while there was no correlation with the independent PHA

(rs<0.51; n.s.), reflecting likely cross-reactions. In both the avian and bovine ELISAs, a marked drop was recorded at testing round 5. This drop coincided in time with a marked peak in the skin test response to

PHA (Figures 1c and 3).

Blood sera taken three weeks after selected skin testing rounds (in summer 2012, winter 2013, winter

2014 and winter 2015), allowed measuring eventual boosts in antibody levels against the antigens used for skin stimulation. Figure 4 presents the mean (±SD) response (in E%) in the production of antibodies against aPPD and bPPD before and three weeks after inoculation for each testing round (boosting effect).

Antibodies against both antigens increased after skin-testing at all four times studied, i.e. the boosting effect was stable through time. This boosting effect was more evident for avian PPD than for bovine PPD

(Figure 4).

Fig 3. Red deer mean (±SD) seasonal (winter 2012 to winter 2015) antibody levels (in ELISA percentage, E%) against avian (a and b) and bovine (c and d) mycobacterial antigens

Fig 4. Red deer mean (±SD) antibody levels (in ELISA percentage, E%) against avian PPD (a) and bovine PPD (b) measured by ELISA immediately before and three weeks after selected skin testing rounds (*p≤0.05).

Discussion

Based on the observed results, we rejected our initial hypothesis that repeated (six monthly) skin testing with aPPD and bPPD would cause progressive changes in skin test or ELISA responsiveness in farmed red deer. Specifically, we observed no progressive loss of the skin test response to bPPD, in contrast to previous findings in repeatedly tested cattle [12,13]. The desensitization phenomenon described in cattle was observed in animals in which the intradermal skin test was repeated in a period of

time lower than six months, which is the interval respected in this study. In addition, some studies in cattle were performed in infected animals [12, 13]. These differences may contribute to explain the results of this study.

There was also no progressive change of the boosting effect on the ELISA responses. In our study, consistent increases in response through time were only recorded for single ELISAs using avian antigens, particularly aPPD. This increase did not correlate with the skin test data, suggesting they were independent. Moreover, this increase was not recorded for the bovine antigens, suggesting that it might have been due to exposure to MAP or to other cross-reacting environmental mycobacteria in the farm. In the same way, the boosted response seen to bPPD, smaller than the one to aPPD, may represent cross reactive antibodies to shared antigens between aPPD and bPPD, or cross-reactions with environmental mycobacteria. The presence of avian and environmental mycobacteria and their potential interference in

TB/PTB diagnosis in this specific deer farm had been reported earlier [5].

However, we recorded a possible temporal effect of the first skin testing round on skin-test and

ELISA responsiveness in the 2nd and 3rd testing rounds. Apparently, stimulation with avian and bovine

PPD during the first skin test caused an increase in the mean response to these antigens (but not with the control mitogen, PHA) in the 2nd and 3rd skin tests, and also a consistent increase in the antibodies against avian and bovine antigens in the 2nd testing round. Since our study site was a deer farm where all deer are first skin tested at the age of 8 months (1st testing round), we could not differentiate if this increased responsiveness recorded at the age of 14 months (2nd testing round) was actually an effect of the 1st skin test or due to developmental factors. However, the response to the independent mitogen PHA declined from the 1st to the 2nd round, suggesting that there was no age-related increase in the cellular responsiveness to intra-dermal antigens. An alternative or complementary explanation for this transitory increase in the skin test response to PPDs is that, as seen in cattle, the infection or exposure to M. avium or MAP could cause a transient increased cellular immune response [39].

At the 3 mm cut-off for aPPD and the 2 mm cut-off for bPPD, the intradermal skin test detected a high percentage of deer (72%) as avian and as bovine positives, in at least one of the seven testing rounds.

This occurred more often in the first three testing rounds (Figure 2). The fact that we recorded a clear increase in the response to PPDs in the 2nd and 3rd testing rounds, and in some individuals even already in the first round, can lead to false-positive reactors and needs to be accounted for when interpreting skin test results in calf and yearling hinds (i.e. until their second winter or 4th testing round).

When MTC infection happens, the decrease of the cell mediated immune response may correlate with higher levels of antibodies and the development of extended TB lesions [28,40]. In this study, the opposite situation was recorded at testing round 5, with higher skin test responses, particularly to PHA.

However, as this farm was TB-free, our interpretation is that this unexpected peak was due to environmental factors. The combination of methods based on the cellular response against M. bovis along with serological tests may increase the chances of detection of the infectious agent and facilitate to manage TB outbreaks [32, 41]. This study showed that in TB-free red deer, there was no permanent boosting effect on serological test results after repeated tuberculin tests. As expected, the ELISA responses to different mycobacterial antigens were correlated, reflecting the likelihood of cross-reactions.

Animals responding to avian antigens tended to repeat and even increase their positive response through time, suggesting true contact with avian or environmental mycobacteria. In contrast, responses to bovine antigens tended to be weak and sporadic, occurring more often in the last (6th and 7th) testing rounds, possibly as a consequence of increasing cross-reaction with avian or environmental mycobacteria.

However, re-testing for antibodies responses three weeks after each skin test helped in discarding possible cross-reactions, since the boosts were much more evident for the avian antigen (aPPD) than for the bovine antigen (bPPD) (Figure 4). This boosting effect has a benefit by maximizing the detection of reactors through serological tests. In studies on M. bovis infected cattle, the injection of PPDs for skin testing boosted the responses to certain antigens (i.e. MPB83 and MPB70; [42]). In human tuberculosis, the boosting effect is maximal if the interval between the initial and second test is between 1 to 5 week

[43] and is much less frequent if the interval is only 48 hours [44] or more than 60 days [43], although boosting has been detected one or more years after a first negative tuberculin test [44,45]. In experimental

goat tuberculosis (M. caprae), it showed a similar trend as described in cattle and human tuberculosis.

The sensitivity of ELISA against MPB83 increased dramatically 2 weeks after boosting with SICCT [46].

Conclusion

In summary, repeated (every six months) administration of avian and bovine PPDs for skin testing did not result in continued sensitizing or desensitizing effects on the skin test response in red deer from a

TB-free herd. Repeated skin testing had also no continued effect on serum antibody responses against avian and bovine mycobacterial antigens. The boosting effect of skin testing on antibody levels recorded three weeks later was stable. However, transitory increases in skin test responsiveness during one year

(two testing rounds) and in ELISA results during six months (one testing round) cannot be discarded. The findings observed in the present work may been taken into account when designing and interpreting TB diagnosis schemes in TB-free farmed red deer and similar wildlife species.

Abbreviations

TB: Tuberculosis PTB: Paratuberculosis MTC: Mycobacterium tuberculosis complex MAP:

Mycobacterium avium paratuberculosis aPPD/bPPD: avian/bovine purified protein derivatives PPA3:

Paratuberculosis protoplasmatic antigen-3 ELISA: Enzyme-linked immunosorbent assay PHA:

Phytohaemagglutinin SICCT: Single intradermal comparative cervical tuberculin test PBST: Phosphate buffer saline with 0.05% Tween-20 OD: Optical density E%: ELISA percentage

Acknowledgements

The authors thank F. Talavera for technical assistance.

Funding

Research funding is acknowledged to Plan Nacional grant AGL2014-56305 (MINECO, Spain and

FEDER). ACA was supported by a grant from University Putra Malaysia, Malaysia, and MAR had a research contract from Junta de Comunidades de Castilla-La Mancha (JCCM), Spain.

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Availability of data and material

The dataset supporting the conclusions of this article is included within the article (and its additional file).

Authors’ contributions

CG, ACA, MAR participated in the conceptual aspect of the work. DGB, JAO, ACA, MAR participated in sample collection and analysis. All authors provided consultation and coordination. CG, ACA & MAR wrote the first draft of the manuscript, with all authors involved in reviewing. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interest.

Consent for publication

Not applicable.

Ethics and consent to participate

Handling procedures and sampling frequency were part of the farm routine and not influenced by the study. Therefore, no formal ethics approval was required in this particular study. The procedures carried out during this work were designed to reduce stress and health risks for subjects, with written informed consent of the owner and according to European (86/609) and Spanish laws (RD 223/1988; RD

1021/2005), and current guidelines for ethical.

References

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Additional file

Table S1. ELISAs for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium paratuberculosis (MAP)-specific antibody detection in red deer (Cervus elaphus)

Sensitivity Specificity Assay test Antigens Reference (%) (%)

CervidTB ESAT-6/CFP-10, MPB83 75.0, 82.0 83.8, 93.0 Buddle et al 2010, Waters et al 2011 STAT-PAK

Buddle et al 2010, DPP VetTB ESAT-6/CFP-10, MPB83 75.0, 79.0 91.4, 98.0 Waters et al 2011

MAPIA ESAT-6, CFP10, MPB59, 82.0 Buddle et al 2010, MPB64, MPB70, MPB83, Waters et al 2011 the 16-kDa protein, the 38- kDa protein, two fusion

proteins comprising CFP10/ESAT-6 and the 16-

105

kDa protein/MPB83,

two native antigens, bovine PPD and M. bovis culture filtrate.

bPPD

MPB83

Extracted M. bovis surface García-Bocanegra et al 2012 ELISA antigen 72.7

Johnin PPD (jPPD) Wadhwa et al 2013

86.7

Crawford et al PpAg 50.0, 84.0 2006, Griffin et al 2005 93.0, 99.5

Griffin et al 2005

91.0 88.0

Table S2. Individual results for red deer hinds under repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA response (in E%). In the skin test the animals were considered TB reactors if the skin fold increase to bPPD was greater than 2 mm and more than 1 mm larger than the skin fold increase to aPPD; and avian reactors were defined as those with a skin fold increase to aPPD >3 mm (all single reactors are in yellow). In the ELISA tests for antibodies against avian antigens (aPPD and PPA3) and bovine antigens (bPPD and MPB70), sera with E% values greater than 100 were considered positive (in red).

106

Animal Time Season Date Skin aPPD Skin bPPD Skin PHA PPA3 (E%)PPA3pn aPPD (E%) aPPDpn bPPD (E%) bPPDpn MPB70 (E%)MPB70pn 62 1 winter 2012 1 W 12,33 9,00 52,00 77,22 Neg 73,25 Neg 59,91 Neg 91,44 Neg 62 2 summer 2012 2 S 117,00 73,00 10,00 122,66 Pos 59,09 Neg 45,37 Neg 111,71 Pos 62 2a summer 2013 2a S 85,18 Neg 62,01 Neg 62 3 winter 2013 3 W 68,30 22,30 9,30 113,56 Pos 67,35 Neg 61,94 Neg 92,34 Neg 62 3a winter 2013 3a W 125,18 Pos 82,53 Neg 62 4 summer 2013 4 S 25,00 16,33 5,33 88,25 Neg 73,05 Neg 53,33 Neg 65,39 Neg 62 5 winter 2014 5 W 20,33 11,33 29,33 128,27 Pos 79,02 Neg 48,05 Neg 56,78 Neg 62 5a winter 2014 5 W 116,71 Pos 85,36 Neg 62 6 summer 2014 6 S 21,00 3,33 9,67 155,05 Pos 114,20 Pos 120,12 Pos 102,96 Pos 62 7 winter 2015 7 W 32,00 12,67 16,00 152,62 Pos 110,33 Pos 72,29 Neg 63,63 Neg 62 7a winter 2015 7 W 125,24 Pos 112,04 Pos 63 1 winter 2012 1 W 4,33 1,67 12,00 98,11 Neg 71,11 Neg 123,10 Pos 96,66 Neg 63 2 summer 2012 2 S 82,67 60,00 17,67 108,43 Pos 66,52 Neg 69,79 Neg 85,71 Neg 63 2a summer 2013 2a S 66,46 Neg 80,59 Neg 63 3 winter 2013 3 W 54,30 21,00 34,70 91,23 Neg 87,43 Neg 57,32 Neg 76,34 Neg 63 3a winter 2013 3a W 126,07 Pos 50,92 Neg 63 4 summer 2013 4 S 45,33 23,33 16,33 89,64 Neg 90,58 Neg 52,95 Neg 64,07 Neg 63 5 winter 2014 5 W 29,33 16,33 62,67 99,81 Neg 55,90 Neg 45,10 Neg 61,75 Neg 63 5a winter 2014 5 W 129,47 Pos 79,37 Neg 63 6 summer 2014 6 S 18,33 0,00 12,00 132,65 Pos 100,76 Pos 123,77 Pos 129,54 Pos 63 7 winter 2015 7 W 48,33 36,33 16,33 110,71 Pos 86,03 Neg 84,28 Neg 79,88 Neg 63 7a winter 2015 7 W 130,81 Pos 114,70 Pos 75 1 winter 2012 1 W 3,00 4,33 27,00 89,55 Neg 60,42 Neg 44,56 Neg 74,32 Neg 75 2 summer 2012 2 S 40,00 20,33 8,00 141,72 Pos 69,60 Neg 64,51 Neg 86,47 Neg 75 2a summer 2013 2a S 101,17 Pos 62,06 Neg 75 3 winter 2013 3 W 39,00 19,30 19,70 151,34 Pos 89,45 Neg 71,32 Neg 81,78 Neg 75 3a winter 2013 3a W 73,37 Neg 38,05 Neg 75 4 summer 2013 4 S 12,00 5,33 11,00 158,73 Pos 103,58 Pos 78,71 Neg 68,28 Neg 75 5 winter 2014 5 W 28,33 6,33 16,33 76,51 Neg 58,42 Neg 58,92 Neg 28,27 Neg 75 5a winter 2014 5 W 88,13 Neg 47,03 Neg 75 6 summer 2014 6 S 13,33 3,00 14,33 166,29 Pos 124,15 Pos 121,87 Pos 109,11 Pos 75 7 winter 2015 7 W 25,33 10,67 17,00 136,45 Pos 89,52 Neg 80,56 Neg 60,21 Neg 75 7a winter 2015 7 W 73,76 Neg 49,31 Neg 79 1 winter 2012 1 W 126,33 51,33 21,33 71,18 Neg 49,91 Neg 42,42 Neg 80,11 Neg 79 2 summer 2012 2 S 61,67 23,33 13,33 112,78 Pos 79,67 Neg 56,39 Neg 108,68 Pos 79 2a summer 2013 2a S 121,89 Pos 46,64 Neg 79 3 winter 2013 3 W 55,30 29,00 13,70 97,23 Neg 72,56 Neg 49,45 Neg 87,12 Neg 79 3a winter 2013 3a W 53,98 Neg 39,10 Neg 79 4 summer 2013 4 S 30,33 10,33 10,00 89,68 Neg 73,32 Neg 38,77 Neg 46,44 Neg 79 5 winter 2014 5 W 27,67 17,33 45,00 102,95 Pos 54,46 Neg 42,02 Neg 36,24 Neg 79 5a winter 2014 5 W 80,39 Neg 45,87 Neg 79 6 summer 2014 6 S 20,00 5,00 14,67 138,27 Pos 100,68 Pos 107,67 Pos 88,08 Neg 79 7 winter 2015 7 W 18,00 8,00 11,00 114,96 Pos 82,00 Neg 51,63 Neg 45,25 Neg 79 7a winter 2015 7 W 69,69 Neg 51,47 Neg

107

Animal Time Season Date Skin aPPD Skin bPPD Skin PHA PPA3 (E%)PPA3pn aPPD (E%) aPPDpn bPPD (E%) bPPDpn MPB70 (E%)MPB70pn 89 1 winter 2012 1 W 5,33 2,00 39,33 59,91 Neg 32,28 Neg 37,57 Neg 57,27 Neg 89 2 summer 2012 2 S 27,00 16,67 14,67 71,87 Neg 73,38 Neg 65,07 Neg 66,58 Neg 89 2a summer 2013 2a S 73,370 Neg 38,05 Neg 89 3 winter 2013 3 W 33,30 4,70 25,70 70,34 Neg 58,67 Neg 43,98 Neg 54,39 Neg 89 3a winter 2013 3a W 62,23 Neg 44,65 Neg 89 4 summer 2013 4 S 10,67 4,00 8,67 73,82 Neg 42,98 Neg 27,50 Neg 34,93 Neg 89 5 winter 2014 5 W 7,67 11,67 24,33 70,85 Neg 38,51 Neg 29,52 Neg 30,84 Neg 89 5a winter 2014 5 W 95,32 Neg 50,80 Neg 89 6 summer 2014 6 S 1,33 0,67 10,67 44,60 Neg 42,71 Neg 37,06 Neg 58,48 Neg 89 7 winter 2015 7 W 18,33 15,33 7,00 84,21 Neg 73,12 Neg 53,23 Neg 61,66 Neg 89 7a winter 2015 7 W 65,01 Neg 52,47 Neg 100 1 winter 2012 1 W 18,67 4,67 28,33 43,23 Neg 43,23 0 32,91 Neg 48,84 Neg 100 2 summer 2012 2 S 60,33 47,00 8,33 55,76 Neg 68,47 Neg 75,08 Neg 74,70 Neg 100 2a summer 2013 2a S 63,98 Neg 89,10 Neg 100 3 winter 2013 3 W 53,70 26,70 16,70 73,45 Neg 84,54 Neg 73,25 Neg 76,54 Neg 100 3a winter 2013 3a W 70,25 Neg 65,45 Neg 100 4 summer 2013 4 S 24,67 3,67 0,00 85,93 Neg 103,08 Pos 72,86 Neg 79,46 Neg 100 5 winter 2014 5 W 13,00 7,67 27,33 69,72 Neg 63,00 Neg 50,06 Neg 50,19 Neg 100 5a winter 2014 5 W 51,64 Neg 45,70 Neg 100 6 summer 2014 6 S 9,00 10,33 16,00 74,37 Neg 69,03 Neg 49,56 Neg 58,79 Neg 100 7 winter 2015 7 W 10,33 4,33 18,67 71,15 Neg 61,73 Neg 37,97 Neg 48,82 Neg 100 7a winter 2015 7 W 103,12 Pos 50,92 Neg 111 1 winter 2012 1 W 0,00 2,67 31,00 33,73 Neg 30,71 Neg 27,25 Neg 45,56 Neg 111 2 summer 2012 2 S 10,00 22,33 14,33 69,92 Neg 46,88 Neg 27,12 Neg 87,10 Neg 111 2a summer 2013 2a S 62,23 Neg 44,65 Neg 111 3 winter 2013 3 W 42,70 44,00 20,00 63,25 Neg 58,34 Neg 39,25 Neg 72,67 Neg 111 3a winter 2013 3a W 66,57 Neg 47,92 Neg 111 4 summer 2013 4 S 16,67 16,00 6,00 60,30 Neg 66,96 Neg 44,60 Neg 61,62 Neg 111 5 winter 2014 5 W 15,67 14,67 9,00 53,83 Neg 44,41 Neg 26,38 Neg 39,45 Neg 111 5a winter 2014 5 W 69,03 Neg 51,80 Neg 111 6 summer 2014 6 S 11,00 11,33 18,67 120,35 Pos 93,24 Neg 107,37 Pos 105,62 Pos 111 7 winter 2015 7 W 21,33 17,33 15,33 86,03 Neg 63,78 Neg 43,05 Neg 52,09 Neg 111 7a winter 2015 7 W 91,39 Neg 45,48 Neg 115 1 winter 2012 1 W 18,67 9,33 12,00 65,95 Neg 66,08 Neg 50,60 Neg 65,01 Neg 115 2 summer 2012 2 S 22,67 11,67 14,00 103,27 Pos 61,36 Neg 63,44 Neg 68,41 Neg 115 2a summer 2013 2a S 70,25 Neg 65,45 Neg 115 3 winter 2013 3 W 41,00 19,30 10,00 87,34 Neg 57,39 Neg 49,37 Neg 52,18 Neg 115 3a winter 2013 3a W 61,62 Neg 42,32 Neg 115 4 summer 2013 4 S 9,00 9,00 10,00 30,28 Neg 46,36 Neg 38,88 Neg 40,64 Neg 115 5 winter 2014 5 W 16,33 15,00 16,67 37,63 Neg 45,41 Neg 23,99 Neg 30,28 Neg 115 5a winter 2014 5 W 63,51 Neg 45,70 Neg 115 6 summer 2014 6 S 7,33 6,00 12,00 13,38 Neg 59,17 Neg 36,81 Neg 55,59 Neg 115 7 winter 2015 7 W 6,67 9,67 13,33 50,80 Neg 33,71 Neg 26,27 Neg 27,56 Neg 115 7a winter 2015 7 W 98,66 Neg 46,98 Neg 136 1 winter 2012 1 W 7,67 12,67 17,00 34,55 Neg 23,29 Neg 29,77 Neg 44,93 Neg 136 2 summer 2012 2 S 86,33 42,33 16,67 57,84 Neg 33,61 Neg 27,19 Neg 42,79 Neg 136 2a summer 2013 2a S 66,57 Neg 47,92 Neg 136 3 winter 2013 3 W 5,70 15,00 16,00 61,23 Neg 57,69 Neg 56,39 Neg 45,89 Neg 136 3a winter 2013 3a W 92,14 Neg 79,09 Neg 136 4 summer 2013 4 S 19,67 3,00 8,67 65,58 Neg 67,78 Neg 79,96 Neg 48,24 Neg 136 5 winter 2014 5 W 9,67 14,67 8,00 35,49 Neg 25,88 Neg 18,09 Neg 20,73 Neg 136 5a winter 2014 5 W 43,68 Neg 97,23 Neg 136 6 summer 2014 6 S 9,67 2,33 5,33 53,64 Neg 59,92 Neg 52,26 Neg 58,04 Neg 136 7 winter 2015 7 W 1,00 0,67 10,67 69,93 Neg 37,59 Neg 48,90 Neg 54,90 Neg 108 136 7a winter 2015 7 W 82,40 Neg 79,15 Neg

Animal Time Season Date Skin aPPD Skin bPPD Skin PHA PPA3 (E%)PPA3pn aPPD (E%) aPPDpn bPPD (E%) bPPDpn MPB70 (E%)MPB70pn 148 1 winter 2012 1 W 14,67 10,33 54,67 52,80 Neg 41,10 Neg 53,62 Neg 59,28 Neg 148 2 summer 2012 2 S 24,67 14,33 3,00 70,55 Neg 52,93 Neg 42,16 Neg 74,01 Neg 148 2a summer 2013 2a S 61,62 Neg 42,32 Neg 148 3 winter 2013 3 W 17,70 10,70 20,30 113,24 Pos 95,23 Neg 57,87 Neg 81,24 Neg 148 3a winter 2013 3a W 197,21 Pos 51,69 Neg 148 4 summer 2013 4 S 19,00 6,67 14,33 133,35 Pos 106,34 Pos 66,08 Neg 88,76 Neg 148 5 winter 2014 5 W 10,00 7,00 15,33 79,59 Neg 45,79 Neg 24,12 Neg 39,70 Neg 148 5a winter 2014 5 W 103,90 Pos 57,18 Neg 148 6 summer 2014 6 S 19,67 6,67 11,33 60,24 Neg 86,93 Neg 82,73 Neg 79,40 Neg 148 7 winter 2015 7 W 7,33 14,33 16,33 137,36 Pos 76,54 Neg 70,99 Neg 72,74 Neg 148 7a winter 2015 7 W 87,08 Neg 54,30 Neg 176 1 winter 2012 1 W 5,33 9,33 23,00 60,98 Neg 41,22 Neg 44,12 Neg 52,05 Neg 176 2 summer 2012 2 S 12,33 0,00 9,00 152,86 Pos 106,73 Pos 86,85 Neg 81,94 Neg 176 2a summer 2013 2a S 192,14 Pos 79,09 Neg 176 3 winter 2013 3 W 56,00 48,30 16,70 163,21 Pos 121,34 Pos 82,34 Neg 4,29 Neg 176 3a winter 2013 3a W 164,23 Pos 92,54 Neg 176 4 summer 2013 4 S 15,33 17,00 11,33 170,79 Pos 130,15 Pos 87,06 Neg 82,79 Neg 176 5 winter 2014 5 W 13,33 1,33 62,67 110,68 Pos 88,51 Neg 62,81 Neg 51,57 Neg 176 5a winter 2014 5 W 86,85 Neg 46,81 Neg 176 6 summer 2014 6 S 26,33 18,67 18,00 192,10 Pos 157,86 Pos 121,26 Pos 104,63 Pos 176 7 winter 2015 7 W 18,67 10,00 10,67 148,67 Pos 135,91 Pos 67,65 Neg 62,26 Neg 176 7a winter 2015 7 W 187,02 Pos 83,26 Neg 204 1 winter 2012 1 W 63,33 25,67 17,33 68,53 Neg 62,24 Neg 55,82 Neg 89,55 Neg 204 2 summer 2012 2 S 17,33 15,33 40,00 78,73 Neg 46,19 Neg 58,28 Neg 59,28 Neg 204 2a summer 2013 2a S 119,83 Pos 67,89 Neg 204 3 winter 2013 3 W 20,00 5,70 10,30 82,45 Neg 51,76 Neg 52,39 Neg 60,39 Neg 204 3a winter 2013 3a W 101,84 Pos 173,99 Pos 204 4 summer 2013 4 S 12,33 4,67 18,33 86,97 Neg 56,51 Neg 47,07 Neg 62,05 Neg 204 5 winter 2014 5 W 34,33 5,67 33,00 48,87 Neg 50,50 Neg 32,47 Neg 38,19 Neg 204 5a winter 2014 5 W 107,97 Pos 79,59 Neg 204 6 summer 2014 6 S 6,67 5,33 14,33 73,56 Neg 98,18 Neg 86,12 Neg 79,15 Neg 204 7 winter 2015 7 W 23,33 6,33 34,00 124,83 Pos 98,25 Neg 86,26 Neg 76,69 Neg 204 7a winter 2015 7 W 88,30 Neg 86,80 Neg 225 1 winter 2012 1 W 12,67 16,33 12,00 61,17 Neg 59,16 Neg 49,02 Neg 79,23 Neg 225 2 summer 2012 2 S 104,00 30,67 25,00 69,23 Neg 40,97 Neg 37,51 Neg 55,44 Neg 225 2a summer 2013 2a S 64,23 Neg 42,54 Neg 225 3 winter 2013 3 W 76,30 11,00 17,30 62,56 Neg 51,29 Neg 45,76 Neg 53,91 Neg 225 3a winter 2013 3a W 200,78 Pos 94,02 Neg 225 4 summer 2013 4 S 25,00 8,67 12,33 57,54 Neg 55,78 Neg 42,09 Neg 50,13 Neg 225 5 winter 2014 5 W 37,00 5,67 14,00 45,29 Neg 46,98 Neg 25,94 Neg 30,84 Neg 225 5a winter 2014 5 W 291,59 Pos 127,68 Pos 225 6 summer 2014 6 S 51,33 20,67 23,67 95,37 Neg 69,32 Neg 102,51 Pos 90,36 Neg 225 7 winter 2015 7 W 32,67 13,67 11,33 82,99 Neg 73,50 Neg 44,19 Neg 48,22 Neg 225 7a winter 2015 7 W 239,11 Pos 86,99 Neg 251 1 winter 2012 1 W 0,00 11,00 27,00 66,65 Neg 42,92 Neg 45,63 Neg 60,35 Neg 251 2 summer 2012 2 S 12,67 1,00 11,00 108,81 Pos 113,15 Pos 109,19 Pos 97,48 Neg 251 2a summer 2013 2a S 141,84 Pos 103,06 Pos 251 3 winter 2013 3 W 4,70 6,00 21,00 107,43 Pos 98,71 Neg 74,32 Neg 67,83 Neg 251 3a winter 2013 3a W 137,16 Pos 94,56 Neg 251 4 summer 2013 4 S 5,33 6,33 10,33 109,00 Pos 72,94 Neg 53,74 Neg 52,67 Neg 251 5 winter 2014 5 W 21,67 11,67 17,00 104,71 Pos 77,58 Neg 57,79 Neg 55,03 Neg 251 5a winter 2014 5 W 117,94 Pos 88,30 Neg 251 6 summer 2014 6 S 1,00 6,33 14,00 116,90 Pos 121,67 Pos 97,80 Neg 103,08 Pos 109 251 7 winter 2015 7 W 20,33 16,33 23,67 169,70 Pos 133,64 Pos 106,07 Pos 88,99 Neg

251 7a winter 2015 7 W 141,06 Pos 51,64 Neg

Animal Time Season Date Skin aPPD Skin bPPD Skin PHA PPA3 (E%)PPA3pn aPPD (E%) aPPDpn bPPD (E%) bPPDpn MPB70 (E%)MPB70pn 257 1 winter 2012 1 W 1,33 5,67 44,33 97,61 Neg 69,67 Neg 80,99 Neg 79,86 Neg 257 2 summer 2012 2 S 0,33 13,00 9,00 137,95 Pos 109,63 Pos 76,53 Neg 84,58 Neg 257 2a summer 2013 2a S 200,78 Pos 124,02 Pos 257 3 winter 2013 3 W 23,70 10,70 21,00 112,23 Pos 107,45 Pos 68,21 Neg 65,78 Neg 257 3a winter 2013 3a W 197,05 Pos 123,79 Pos 257 4 summer 2013 4 S 15,33 8,33 15,00 104,72 Pos 106,61 Pos 51,10 Neg 49,59 Neg 257 5 winter 2014 5 W 27,00 14,67 61,33 159,42 Pos 98,37 Neg 46,23 Neg 41,90 Neg 257 5a winter 2014 5 W 153,48 Pos 122,13 Pos 257 6 summer 2014 6 S 12,33 9,33 19,33 187,21 Pos 102,34 Pos 124,30 Pos 101,00 Pos 257 7 winter 2015 7 W 8,33 5,67 21,33 225,44 Pos 212,07 Pos 155,50 Pos 203,87 Pos 257 7a winter 2015 7 W 254,60 Pos 177,56 Pos 260 1 winter 2012 1 W 6,00 13,33 22,00 63,94 Neg 66,14 Neg 46,26 Neg 61,30 Neg 260 2 summer 2012 2 S 6,67 5,33 25,00 132,35 Pos 87,67 Neg 93,46 Neg 78,16 Neg 260 2a summer 2013 2a S 137,16 Pos 94,56 Neg 260 3 winter 2013 3 W 25,70 21,30 11,70 129,34 Pos 89,56 Neg 76,93 Neg 75,23 Neg 260 3a winter 2013 3a W 83,29 Neg 80,98 Neg 260 4 summer 2013 4 S 24,33 4,67 9,67 126,07 Pos 92,65 Neg 66,71 Neg 72,93 Neg 260 5 winter 2014 5 W 18,67 9,33 22,67 128,02 Pos 68,22 Neg 102,58 Pos 48,12 Neg 260 5a winter 2014 5 W 217,94 Pos 93,29 Neg 260 6 summer 2014 6 S 6,00 5,33 12,00 203,49 Pos 141,61 Pos 127,33 Pos 107,74 Pos 260 7 winter 2015 7 W 22,33 17,33 23,33 164,39 Pos 154,21 Pos 116,63 Pos 86,10 Neg 260 7a winter 2015 7 W 172,479 Pos 134,28 Pos 266 1 winter 2012 1 W 24,33 15,00 10,00 110,30 Pos 79,77 Neg 44,60 Neg 59,92 Neg 266 2 summer 2012 2 S 15,67 10,00 21,00 177,60 Pos 146,70 Pos 111,20 Pos 111,33 Pos 266 2a summer 2013 2a S 197,05 Pos 123,79 Pos 266 3 winter 2013 3 W 58,30 50,30 27,70 168,23 Pos 132,43 Pos 87,00 Neg 85,24 Neg 266 3a winter 2013 3a W 125,18 Pos 82,53 Neg 266 4 summer 2013 4 S 22,00 5,67 11,67 142,21 Pos 112,00 Pos 81,41 Neg 86,12 Neg 266 5 winter 2014 5 W 7,67 31,00 15,67 163,19 Pos 108,35 Pos 61,62 Neg 62,06 Neg 266 5a winter 2014 5 W 155,38 Pos 88,19 Neg 266 6 summer 2014 6 S 23,67 10,00 13,33 138,50 Pos 115,41 Pos 117,01 Pos 79,88 Neg 266 7 winter 2015 7 W 11,33 2,67 8,33 114,50 Pos 133,03 Pos 94,99 Neg 77,68 Neg 266 7a winter 2015 7 W 161,39 Pos 70,16 Neg 303 1 winter 2012 1 W 19,00 24,67 38,67 144,05 Pos 106,10 Pos 38,83 Neg 93,08 Neg 303 2 summer 2012 2 S 17,00 9,00 9,67 96,35 Neg 102,52 Pos 89,55 Neg 95,47 Neg 303 2a summer 2013 2a S 133,29 Pos 80,98 Neg 303 3 winter 2013 3 W 24,00 7,30 12,00 98,23 Neg 100,34 Pos 79,23 Neg 89,21 Neg 303 3a winter 2013 3a W 197,21 Pos 63,01 Neg 303 4 summer 2013 4 S 4,33 0,67 9,33 131,97 Pos 103,71 Pos 74,56 Neg 84,36 Neg 303 5 winter 2014 5 W 16,33 7,00 17,00 44,97 Neg 49,56 Neg 49,43 Neg 44,03 Neg 303 5a winter 2014 5 W 101,50 Pos 63,17 Neg 303 6 summer 2014 6 S 6,67 5,67 18,33 142,98 Pos 91,04 Neg 79,50 Neg 113,14 Pos 303 7 winter 2015 7 W 15,67 5,33 10,00 126,04 Pos 121,79 Pos 74,26 Neg 58,01 Neg 303 7a winter 2015 7 W 169,25 Pos 87,63 Neg

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Result - Chapter IV

Is targeted removal a suitable means for

tuberculosis control in wild boar?

Che’Amat, A., Armenteros, J.A., González-Barrio, D., Lima, J.F., Díez-Delgado, I.,

Barasona, J.A., Romero, B., Lyashchenko, K.P., Ortiz, J.A., Gortázar, C.*

Preventve Veterinary Medicine, 2016. 135:132-135

This chapter is prepared in the style format of Preventive Veterinary Medicine

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ABSTRACT

We assessed the suitability of targeted removal as a means for tuberculosis (TB) control on an intensely managed Eurasian wild boar (Sus scrofa) hunting estate. The 60 km2 large study area included one capture (treatment) site, one control site, and one release site. Each site was fenced. In the summers of

2012, 2013 and 2014, 929 wild boar were live‐captured on the treatment site. All wild boar were micro‐chipped and tested using an animal side lateral flow test immediately after capture in order to detect antibodies to the Mycobacterium tuberculosis complex (MTC). The wild boar were released according to their TB status: Seropositive individuals onto the release site (hunted after summer), and seronegative individuals back onto the treatment site. The annual summer seroprevalence of antibodies to the MTC declined significantly in live‐captured wild boar piglets from the treatment site, from 44% in

2012 to 27% in 2013 (a reduction of 39%). However, no significant further reduction was recorded in

2014, during the third capture season. Fall‐winter MTC infection prevalence was calculated on the basis of the culture results obtained for hunter‐harvested wild boar. No significant changes between hunting seasons were recorded on either the treatment site or the control site, and prevalence trends over time were similar on both sites. The fall‐winter MTC infection prevalence on the release site increased significantly from 40% in 2011‐2012 to 64% in 2012‐2013 and 2013‐2014 (60% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted removal, failed to reduce TB prevalence when compared to the control site.

However, it generated valuable knowledge on infection pressure and on the consequences of translocating

TB‐infected wild boar.

Keywords: Animal side test; Mycobacterium bovis; Selective culling; Sus scrofa; Wildlife disease control

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INTRODUCTION

The primary means to control non‐vector borne infections shared with wildlife include preventive actions, host population control and vaccination (Gortázar et al., 2015). However, wildlife disease control often consists of an intervention in natural ecosystems and is, as such, controversial (Artois et al., 2001).

One potential socially acceptable wildlife disease control strategy that is employed as an alternative to random culling (also called population control) is selective culling. Selective culling is similar to the test and cull schemes used with domestic animals. These schemes are, however, expensive, and their feasibility depends on access to the animals, the availability of convenient sensitive and specific diagnostic tests, the prevalence of the infection and the spatial distribution of the target population

(Gortázar et al., 2015). Random and selective culling strategies are more likely to succeed in isolated populations than on large geographical scales, and the results will probably consist of a certain reduction in disease prevalence in the wildlife host and in the domestic host targeted, rather than in the total eradication of the infectious agent. The success of a culling scheme will also depend on the attributes of the specific infectious agent targeted (Boadella et al., 2012).

In the Iberian Peninsula, intense Eurasian wild boar (Sus scrofa) management is characterized by supplementary feeding, fencing and translocation, often including piglet weaning in an attempt to reduce piglet mortality. Increased densities and aggregation cause a high prevalence of infection by members of the Mycobacterium tuberculosis complex (MTC), which are the causative agents of animal tuberculosis

(TB) (Acevedo et al., 2007; Vicente et al., 2013). In this context, it has been shown that random wild boar culling may contribute to reducing wild boar TB prevalence, with positive effects on sympatric ruminants

(Boadella et al., 2012). However, given that wild boar have an economic and cultural value for the hunting community in Spain, an intensive random culling program is unlikely to be supported (Boadella et al., 2012; Cowie et al., 2015). In addition, the recent development of an animal side lateral flow test

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that detects MTC antibodies in wild boar serum (Boadella et al., 2011) provided the opportunity to selectively cull infected wild boar, thereby enabling a more targeted approach to control, which would also be more acceptable to the hunting community (Cowie et al., 2015).

Rather than culling, we assessed the suitability of targeted removal as a means to carry out TB control on intensely managed wild boar hunting estates. We hypothesized that selectively removing seropositive wild boar from one hunting estate and harvesting them by hunting on the release site would result in a progressive decrease in TB prevalence as compared to a control site. We also expected that culling by hunting would counteract the adverse consequences of releasing seropositive animals onto the release site.

MATERIAL AND METHODS

Study area

The study area, a 60 km2 large private hunting estate, is divided into three sites. Each one is surrounded with game species‐proof fencing, signifying that it is possible to differentiate the populations.

There are no badgers (Meles meles) on the study site, and red deer (Cervus elaphus) TB prevalence is low

(<5%). Wild boar MTC infection prevalence (based on culture) had been recorded since 2011 during the annual hunting events, resulting in a mean MTC infection prevalence of 57% (80 of 141 samples tested).

In 2011, prevalence was similar on two sites (66‐68%), but lower on the third one (37%). The details of hunting data per season from 2011‐2014 are presented in Table S‐1. We used this data as a basis to define one treatment site (30km2, initial prevalence 68%, captures and release of seronegative wild boar), one control site (17km2, initial prevalence 66%, no capture and testing), and one release site (13km2, initial prevalence 37%, release of seropositive wild boar; Figure S‐1).

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Table S-1. Wild boar hunting data per season (from 2011-2012 to 2013-2014) tabulated according to age class and study site. For each age and season, numerators indicate the number of Mycobacterium tuberculosis complex positive (by culture) wild boar and denominators indicate the number of individuals hunted. Apparent prevalence is shown with its 95% confidence interval (C.I.).

2011-2012 2012-2013 2013-2014

Yearling Subadult Adult Total Yearling Subadult Adult Total Yearling Subadult Adult Total

Capture site 3/8 11/13 17/24 31/45 1/4 2/3 14/20 17/27 2/6 0/2 2/3 4/11

Prevalence 37.5% 84.6% 70.8% 68.8% 25.0% 66.6% 70.0% 62.9% 33.3% 0.0% 66.6% 36.3%

(95% C.I.) (±33.5) (±19.6) (±18.2) (±13.5) (±42.4) (±53.3) (±20.0) (±18.2) (±37.7) (0) (±53.3) (±28.4)

Release site 3/4 3/17 12/28 18/49a 1/4 7/11 28/47 36/62a 1/4 11/15 17/25 29/44

Prevalence 75.0% 17.6% 42.8% 36.7% 25.0% 63.6% 59.5% 58.0% 25.0% 73.3% 68.0% 65.9%

(95% C.I.) (±42.4) (±18.1) (±18.3) (±13.5) (±42.4) (±28.4) (±14.0) (±12.3) (±42.4) (±22.3) (±18.3) (±14.0)

Control site 6/12 13/16 12/19 31/47 4/7 12/17 16/24 2/9 5/8 11/19 18/36

Prevalence 50.0% 81.2% 63.1% 65.9% 57.1% 70.5% 66.6% 22.2% 62.5% 57.8% 50.0%

(95% C.I.) (±28.3) (±19.1) (±21.7) (±13.5) (±36.6) (±21.6) (±18.8) (±27.1) (±33.5) (±22.2) (±16.3)

aSignificant at p<0.05 Note: Sampling included the mandibular lymph nodes (LN) and the oropharyngeal tonsils in the head region; the left bronchial LN and the lung in the thorax; and the mesenteric and hepatic LNs and the spleen in the abdomen. submitted to the EU reference laboratory for Mycobacterium bovis (VISAVET, Madrid)

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Figure S-1. Schematic representation of the study sites and their location in south-western Spain (the black shape represents the Sierra Norte de Sevilla Natural Park). Captures took place in the treatment site, and only seronegative captured individuals were released back into the treatment site. Seropositive wild boar from the treatment site were translocated to the release site. No intervention took place in the control site.

Data collection and analysis

All animal handling was carried out by personnel from the hunting estate. In summer

(May‐September) 2012, 2013 and 2014, gamekeepers live‐captured‐recaptured a total of 929 wild boar (604 piglets, 325 older animals) by means of cage traps followed by safe physical restraining devices. All live‐captured wild boar were micro‐chipped and tested for antibodies to MTC using an animal side lateral flow ELISA (Chembio DPP Vet test, New York, USA). This test has: a running time of 20 min; a sensitivity close to 90% in adult wild boar, and a high specificity (90.4%)

(Boadella et al., 2011). However, it has a lower sensitivity (61.5%) in the case of wild boar piglets

(Che’Amat et al., 2015). Juvenile and adult wild boar were immediately released according to their

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TB status: Seropositive individuals onto the release site and seronegative individuals back into the capture area, with both sites being subjected to routine hunting in fall‐winter.

Seronegative and seropositive piglets were separated and kept in captivity (weaning) until their release in November. Those individuals that re‐tested as seropositives were again moved to the release sites, and seronegative individuals went back into the capture area (Table 1; Time‐space schematic flow as in Figure S‐2). The aim of releasing seropositive animals onto the release sites was to keep them enclosed until the routine hunting season. This was expected to increase the chances of eliminating the infected animals by hunting, thereby producing a profit rather than the loss caused by culling.

Background data on wild boar abundance and spatial aggregation were obtained yearly in

September, i.e., after the annual summer captures, by applying the method based on recording fecal dropping abundance and distribution on linear transects, as described in Acevedo et al. (2007)

(Table S‐2).

All hunter‐harvested wild boar were sampled in fall and winter. Sex and age‐class were recorded (age based on tooth eruption: yearling if borne that season; subadult if between 12 months and 2 years of age; adult if over 2 years of age). Pooled tissues were cultured for MTC as in

Che'Amat et al. (2015).

Sterne’s exact method was used calculate 95% confidence intervals(CIs) of apparent prevalence, while we used Fisher’s exact tests (STATISTICA 9.0 software, version 7.1., StatSoft,

Inc, www.statsoft.com) in order to compare prevalence figures for a given age class or site before and after management measures were implemented.

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Figure S-2. Diagram summarizing the time-space of selected removal and targeted culling. Testing and segregation were conducted during summer (in capture site) and hunting during autumn-winter in all sites (including release-site).

Table 1. Number of wild boar captured per season and age, and number of wild boar release into the capture site and the release site, by season.

Removal rate (%) = Number of captured [removal Wild boar wild boar and tested Wild boar returned (translocated) translocated with rapid back to capture site number / total to release site serological test number in captured site] x 100

Older Older Older All ages Year Piglets* Piglets Piglets ages ages ages

2012 228 182 37 54 42 128 41.5%

2013 187 73 65 12 28 62 34.6%

No No 65.7% 2014 189 70 24 46 data data

Total 604 325 102 62 70 242 33.6%

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*Piglets were captured and maintained in captivity for weaning and tested piglets were separated according to their initial serological status, re-tested and released to their serological status sites in the next autumn. Number of piglets returned to capture site and translocated to release site not necessarily reflected the initial number of captured piglets Table S-2. Wild boar relative abundance (Fecal-based indirect index, FBII) and spatial aggregation (Z value of the runs test, Z) by site and year. Shadowed rows indicate years with capture and release taking place.

Capture site Control site Release sites Season FBII Z FBII Z FBII Z 2010 2.15 1.56 2.95 1.64 No data No data 2011 2.52 1.00 2.19 0.88 2.71 1.41 2012 1.66 1.83 1.74 1.11 2.37 1.28 2013 1.83 0.74 2.25 0.98 2.09 1.47 2014 1.55 1.04 1.20 1.01 2.11 2.12

RESULTS

Capture and release data for all years are described in Table 1. A total of 228 piglets and 182 older wild boar were captured on the capture site in summer 2012. While it was not possible to know the real number of wild boar present, this capture figure represents a minimum summer density of 13.67 wild boar per square kilometer in summer 2012. The annual summer seroprevalence of antibodies to MTC declined significantly in live‐captured wild boar piglets from the capture site, from 43.86% (±6.44) in 2012 to 26.74% (±6.34) in 2013 (a 39% reduction in seroprevalence). However, no significant further reduction was recorded in 2014 (33.33% ±7.12) during the third capture season. In older wild boar, the summer 2012 seroprevalence (70.3%) increased significantly to 85% in 2013, with no changes in 2014 (Figure S‐3).

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Fall‐winter MTC infection prevalence was calculated on the basis of culture results obtained from 345 hunter‐harvested wild boar from all study sites (Figure 1 and Table S‐1). No significant changes between hunting seasons were recorded on either the capture site or the control site, and prevalence trends over time were similar on both sites. However, on the release site, the seasonal fall‐winter MTC infection prevalence increased significantly in (subadult and adult) hunter‐harvested wild boar, from 33.3% in 2011‐2012 to 60.3% and 70.0% in 2012‐2013 and

2013‐2014, respectively (Table 1; Figure 1).

Figure S-3. Annual summer seroprevalence of antibodies against the MTC in live-captured wild boar from the treatment (capture) site. Inter-annual differences are statistically significant between 2012 and 2013 for piglets, and between 2012 and 2013 as well as between 2013 and 2014 for older ages (Fisher’s test). Error bars indicate 95% CI.

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Figure 1. Annual fall-winter prevalence of culture-confirmed Mycobacterium tuberculosis complex infection in non-yearling hunter-harvested wild boar. Inter-seasonal differences are statistically significant between 2011-2012 and 2012-2013 and between 2011-2012 and 2013-2014 only for the release site (Fisher’s test). Error bars indicate 95% CI.

In total, 14 recaptures of originally seronegative wild boar were recorded on the capture site during the study period. Of these, 10 had tested negative at piglet age, and were re‐tested one year later. Eight of these ten (80%) had already seroconverted. One piglet was recaptured two years later, and it had also seroconverted. Finally, three adults were negative at the first capture in summer

2012, and two had seroconverted two years later. A total of 17 additional recaptures (7 in 2013 and

10 in 2014) were of animals that had tested positive at the first capture. These had supposedly been translocated to the release sites, but were actually hunted or live‐recaptured on the capture site.

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Wild boar abundance and spatial aggregation are summarized in Table S‐2. Wild boar abundancedeclined on the capture site after the onset of summer wild boar capturing. However, spatial aggregation only declined in the second year. No evident changes in abundance or spatial aggregation were recorded on either the control site or the release sites, although inter‐annual variability was high on the latter.

DISCUSSION

In a previous single‐tool study, the random culling of wild boar resulted in a significant reduction in TB prevalence (Boadella et al., 2012). In contrast, this first attempt to control TB in wild boar through targeted removal failed to reduce TB prevalence as compared to the control site, at least under the circumstances of our study. However, this experiment provided a valuable insight into TB epidemiology in settings of high infection pressure.

The design of the present study was not ideal. We had to balance our goals with the management (for profit) of a private hunting estate. Thus, our results should be interpreted from the perspective of a 'practical test' of an intervention, within the confines of what could be realistically undertaken in the real‐world. This experiment was not replicated. There were several drawbacks contributing to the (apparent) failure of the control procedure. First, the actual population size of the capture site was unknown and the proportion of tested and removed wild boar was, therefore, also unknown, although our best guess is that at least two thirds of the population were captured.

Hunting, although limited, took place on the capture site during 2013 and 2014. This activity was not likely to interfere significantly with the tested targeted removal. Second, the lateral flow test used in this trial was less sensitive in the case of piglets (61.5%; Che’Amat et al., 2015), signifying that some false negative individuals were released back onto the capture site. Finally, we recorded the unintended movement of wild boar between the sites, owing to either human error or the wild

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boar crossing fences. There was no “cordon sanitaire” between sites, and fences are never completely wild boar‐proof.

Other explanations for the failure of the targeted removal experiment include the extremely high infection pressure in this wild boar population, possibly including a significant environmental reservoir (e.g. in the waterholes; Barasona et al., 2016). This was evidenced by the high (44%) antibody prevalence in piglets at around 4 months of age and the 80% seroconversion rate from piglets to subadults. Another explanation to be considered is a possible temporal/secular trend (e.g.

Vicente et al., 2013) driving the local TB dynamics, despite our intervention. Finally, the sample size of hunter‐harvested wild boar was relatively low on the capture site in 2013 and 2014, thus limiting the statistical power of our analysis.

However, our results yielded two interesting observations regarding TB epidemiology and TB control in wild boar. First, we managed to obtain valuable data on MTC infection pressure in wild boar, including a very high (44%) prevalence of seropositive piglets in July, an 80% seroconversion rate between their first and their second summer, and a still high 33% seroconversion rate per year in adults. After initiating the captures in summer 2012, piglet seroprevalence dropped by 40% in the summers of 2013 and 2014, suggesting that the intervention had some effect on reducing piglet infection pressure. However, most seronegative piglets seroconverted during the following year, indicating a very high likelihood of coming into contact with MTC on that site.

A second interesting result was the consequences of introducing MTC‐infected wild boar onto

(already infected) sites. In the summer and fall of 2012, a total of 180 seropositive wild boar were released into 13 km2 (thus increasing the density by almost 14 individuals per km2). While this release caused no consistent change in wild boar abundance and spatial aggregation in the following years (Table S‐2), infection prevalence in non‐yearling hunter‐harvested wild boar increased by

60%. The increasing prevalence of MTC on the release site could be explained by imbalances of lower hunting and high new infection rates in the susceptible population on that site. This evidences

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the potential negative effects of translocating and releasing MTC infected wild boar, something that might be occurring in other, uncontrolled, contexts.

Attempts to control wildlife diseases through targeted culling have also failed on other occasions in the case of, for instance, the Tasmanian devil (Sarcophilus harrisii) facial tumor

(Lachish et al., 2010) or, more recently, TB control in a fenced white‐tailed deer (Odocoileus virginianus) population in Michigan, USA (Cosgrove et al., 2012). However, culling through hunting would be almost the only realistic means of actively engaging hunters in targeted culling strategies, at least in the Iberian context. In Iberia, wild boar management through fencing and feeding is a risk in itself, since individuals aggregate at feeders or waterholes, thus facilitating the spread of infection (Vicente et al., 2013). One option for intervention would consist of habitat management by acting on feeder and waterhole characteristics. In farm‐like settings, where targeting a large proportion of wild boar is feasible, vaccination achieved a progressive reduction in

TB‐like lesion prevalence (Diez‐Delgado et al., 2016). Hence, although this specific trial failed, we suggest exploring integrated strategies, such as combinations of culling with vaccination (Abdou et al., 2016) or changes in water and feed distribution.

Acknowledgements

We acknowledge the many colleagues who contributed to field work over the study period, and the managers of the hunting estates. This is a contribution to Spanish Government MINECO Plan

Nacional grant AGL2014‐56305 and FEDER, to CDTI, and to the EU FP7 WIldTBvac#613779 grant. Azlan Che’Amat has a PhD grant from the Malaysian Government. Sally Newton revised the

English.

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Vicente, J., Barasona, J.A., Acevedo, P., Ruiz‐Fons, J.F., Boadella, M., Díez‐Delgado, I., et al., 2013. Temporal trend of tuberculosis in wild ungulates from Mediterranean Spain. Trans. Emerg. Dis. 60(S1), 92‐103.

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Result - Chapter V

The porcine respiratory disease complex causes significant mortality

among wild boar piglets

Che’Amat, A., Risalde, M.A., Díez-Delgado, I., Barasona, J.A., Xeidakis, A., González-Barrio, D.,

Vargas-Castillo, L., Vela, A.I., Pérez-Sancho, M., Ortíz, J.A., *Gortázar, C. (in preparation)

This chapter is prepared in the style format of European Journal of Wildlife Research

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ABSTRACT

Eurasian wild boar (Sus scrofa) mortality still remains relatively unexplored, particularly regarding piglet mortality rates and causes. In fenced hunting estates from a tuberculosis (TB) endemic area, we combined fieldwork (sampling and camera-trap monitoring) with laboratory analyses (pathology, microbiology and serology) to assess wild boar piglet mortality rates and causes focusing on <4 month old piglets during summer. Camera-trapping estimated a total summer piglet mortality rate of 71% based on the reduction of the piglet-to-adult ratio at the feeders. Dead piglets (n=54) were in poor body condition and respiratory lesions compatible with the Porcine

Respiratory Disease Complex (PRDC) were observed in 48 (88.9%). Porcine Circovirus type 2

(PCV2) was the most prevalent pathogen (54%) and its prevalence showed an increasing trend depending on the lung lesion score. The prevalence of other pathogens was not correlated with the lung lesion score and no association between pathogens was found. While TB was found to be irrelevant as a cause of death in this age class, PRDC explained or contributed to most of the recorded mortality. Disease-mediated piglet mortality is an important driver of wild boar population dynamics, at least in semi-intensive management regimes.

Keywords: Early mortality; Pneumonia; Porcine Circovirus type 2; Respiratory syndrome

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INTRODUCTION

Eurasian wild boar (Sus scrofa) mortality still remains relatively unexplored, despite being a key element for understanding population dynamics and for adjusting hunting harvest and population management. Existing information refers mostly to yearling and adult wild boar, where diseases are not generally the main cause of death (e.g. Barasona et al. 2016). In free-living, hunter- harvested wild boar populations, piglets i.e. under-one-year-old wild boar, make up 43-59% of the total population (Merta et al. 2015). However, only a few studies have assessed wild boar piglet mortality. For instance, Toïgo et al. (2008) reported a 55% mortality of ear-tagged wild boar piglets in France during their first year of life. Of this figure, 14% (1/4 of the total mortality) was defined as natural mortality, presumably disease-mediated. The study recruited 1 to 6 month old piglets and the study period included the first autumn/winter hunting season. Example in northern Spain showed the wild boar mortality rate was estimated at 38%, 12% from that caused by wolf predation and 75% were piglets (Nores et al. 2008). In a meta-analysis of wild boar mortality in central

Europe, Keuling et al. (2013) found that piglet mortality in half a year was 54%, again mostly due to hunting. In Mediterranean woodland habitats, piglets make up 58% of the total summer wild boar population (Maselli et al. 2014). However, piglet mortality during this season, at ages between one and six months, is probably high (Vicente et al. 2004) and unrelated to hunting as Spanish regulations restriction on piglet harvesting. In addition, reproductive performance among wild boar

(e.g. in Portugal) during autumn-winter constituted 6.3% post-natal mortality which might also count to the non-harvested mortality (Fonseca et al. 2011).

Even less is known about the pathogens contributing to wild boar piglet mortality. Porcine

Circovirus type 2 (PCV2) has repeatedly been implicated as a causal or at least predisposing factor of wild boar piglet mortality (Vicente et al. 2004; Díez-Delgado et al. 2014; Risco et al. 2015). It is the causal agent of PCV2–systemic disease characterized by a progressive loss of weight and respiratory distress in pigs with severe effects on domestic piglets at 2-4 months of age (Segalés et

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al., 2005; Segalés, 2012; Resendes and Segalés et al. 2015). Moreover, co-infections with this virus are frequent amongst natural populations, taking place early in life with complex effects on the infections and the hosts (Ellis et al. 2004; Díez-Delgado et al. 2014). One frequent co-infection- mediated condition is the Porcine Respiratory Disease Complex (PRDC), which has been reported in Mediterranean wild boar populations (Risco et al. 2015). This syndrome, characterized by its severe pulmonary lesions, is related to infection with various pathogens including viral agents such as PCV2, Swine Influenza virus (SIV), and Porcine Reproductive and Respiratory Syndrome virus

(PRRSV), as well as bacterial agents such as Mycoplasma hyopneumoniae, Haemophilus parasuis or Pasteurella multocida, among others. PRDC-compatible lesions were found in 83% of 210 hunter-harvested wild boar, with piglets showing a higher lesion severity (Risco et al. 2015).

However, to date no study has linked wild boar piglet mortality to PRDC.

We studied an intensely-managed (i.e. fenced and year-round fed) wild boar population in southern Spain. This population is known for a high Mycobacterium bovis (M. bovis) infection pressure, the causal agent of animal tuberculosis (TB), with 30% of the piglets already infected from/during the first summer of life (Che’Amat et al. 2015, 2016). High rates of piglet mortality are suspected during summer, and wild boar piglets are therefore captured at the feeders in July and kept in captivity until their release in autumn (Che’Amat et al. 2016). This created the opportunity to estimate mortality and study the causes of mortality in 54 wild boar piglets aged under 4 months.

We hypothesized that PRDC would be a frequent condition, often causing the death, and that TB would cause additional mortality or interact with PRDC as a relevant co-infection.

MATERIAL AND METHODS

The study was conducted during the summers of 2012-2015 in a group of fenced private hunting estates in Sierra Norte de Sevilla, southern Spain. These estates apply an intensive management system including year-round supplementary feeding and early piglet weaning. Wild boar piglets are captured during summer, mostly in July, as part of a farm management scheme

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aimed to reduce piglet mortality. Captured piglets remain in captivity for 3-4 months before being released back to the field in autumn. Dead piglets estimated at age less than 6-month old which spent less than 10 days in captivity (n=43) and piglets found dead in the field (n=11) were included in this survival analysis (n=54) to represent rapid mortality among piglets and this small subset was selected based on the sufficient necropsy and laboratory test analysis. Additionally, we set up camera traps at all (n=17) wild boar feeders for three consecutive nights during June, July, August and September 2015, respectively. Short (1 min) video footage was carefully analyzed in order to generate indicators of the piglet-to-adult ratio (Maselli et al. 2014) and piglet mortality was evaluated from the decrease of the piglet-to-adult ratio throughout that period. The health status of this population is routinely screened based on sampling hunted juvenile-adult wild boar during fall- winter. While the antibody prevalence against PCV2 was high (>60%), no SIV and PRRSV circulation was detected (data not shown). The fall-winter M. bovis infection prevalence among yearling and adult wild boar is high (>50%; Che’Amat et al. 2016).

For each piglet we took biometric data (Table S-1), recorded the presence of diarrhea and other relevant conditions, and collected blood for serum as well as selected tissues during post mortem- examination. These tissues included the tonsils, the mandibular lymph nodes and pieces of the cranial and caudal lung lobes, which were preserved for routine histopathological studies while small samples were stored frozen (-20°C) for further processing. The lungs were inspected, palpated and scored per lobe for pneumonia lesions. The percentage of pneumonia was categorized into 3 groups; mean lung lobes lesions under 10%, 10-30% and over 30%. Macroscopic TB-compatible lesions were scored as reported elsewhere (Díez-Delgado et al. 2014). Laboratory methods used for antibody or pathogen detection are summarized in Table 1. Pooled samples of mandibular lymph nodes, tonsils and lung were used for DNA extraction and microbial culture. We used descriptive statistics and non-parametric tests to assess relations between biometric data, lesion scores and contact with pathogens.

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RESULTS

Camera-trapping estimated a total summer 2015 (June to September) piglet mortality rate of

71.15% based on the reduction of the piglet-to-adult ratio (from 0.52 to 0.15) at the feeders. The 54 studied wild boar piglets were estimated to be less than 4-month old based on tooth eruption and body size. Their average body condition was poor (1.76 on a scale from 1 -emaciated- to 5 -fat-; mean KFI 14.31%; Table S-1).

Only six piglets (11.1%) were found to lack respiratory lesions. One of the aforementioned individuals presented a large hematoma on the right rib wall, and another one had an intense tick infestation. No particular feature was recorded for the remaining four individuals. PRDC- compatible respiratory lesions were observed in 48 (88.9%) of the dead wild boar piglets. The mean pulmonary consolidation status was 21.4% (95% C.I. 15.7-26.9%). The most affected lobes were the right and left cranial and the right medial ones, with a mean lung lesion score of 37.4%, 27.5% and 26.1%, respectively. Gross lesions recorded included enlargement, restricted lung expansion, congestion and marked areas of consolidation with adjacent emphysematous patches, in multiple lobes. Focal hemorrhages and fibrin adhesions were also noted. The ventral areas were the most affected ones, with presence of a diffuse congestion and moderate-to-severe multifocal dark purple- to-tan consolidations and pulmonary edema corresponding to pneumonia. Histopathology showed inflammatory changes in the lung tissue, confirming the pneumonia. The pulmonary parenchyma had evidences of interstitial pneumonia with alveolar septal thickening as a result of moderate-to- severe interstitial aggregations composed mainly of lymphocytes and macrophages and to a lesser extent neutrophils associated with the presence of bacterial colonies in some animals (Figure S-1).

Table 1 presents the proportion of piglets in contact with selected pathogens. TB-compatible lesions were detected in 11/54 piglets (20.4%), and the mean TB lesions score was low (0.75 on a scale from 0 to 26 (95% C.I. 0.27-1.24; n=54). The pathogen yielding the highest prevalence of contact (i.e. combining serology and PCR) was PCV2 at 53.7% (95% C.I. 40.4-67.0). PCV2 prevalence showed an increasing trend depending on the lung lesion score (Chi2= 3.04, 2 d.f.,

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p>0.05; Figure 1). No relation between prevalence and lung lesion score was observed for the other

pathogens tested as well asno positive or negative association between pairs of pathogens was

evidenced using Fisher’s tests (p>0.05 in all cases).

Table 1. Tests used for antibody and antigen detection for selected pathogen with the apparent prevalence (95% confidence interval) recorded in 54 dead- found less than 4-month old wild boar piglets sampled during the summers 2012-2014. Apparent Test used/ Tested Pathogen Reference prevalence (%) methods positive (n) (95% C.I.) d29.6% Serology: bPPD ELISA- MTC d16 (54) (17.5-41.8) IgG (in-house); aDPP Che’ Amat WTB test. Macroscopic TB et al. 2015

lesion 11 (54) 20.4% IREC-UCLM-JCCM. (9.6-31.1)

Serology: PCV-2 Risco et al. indirect ELISA 2014 e53.7% e29 (54) (bINGEZIM CIRCO (40.4-67.0) Porcine IgG. Cságola et al. Circovirus type DNA extraction 2012 2 (PCV2) (cNucleoSpin®Tissue). Jiang et al. 38.9% 21 (54) Single PCR for PCV2. 2010 (25.9-51.9) Risco et al. Serology: blocking 2014 ELISA (bINGEZIM

Mycoplasma HYO Compac). Cságola et al. hyopneumoniae DNA extraction e11.7% 2012 e6 (51) (M. hyo) (cNucleoSpin®Tissue). (2.9-20.6) Calsamiglia et Nested PCR for M. hyo. al. 1999 Cultured onto Columbia Lactobacillus sp.: 27.8% agar + 5% sheep blood Vela et al. 15 (54) (15.8-39.7) in aerobical or 2015 Enterococcus sp:12 (54) 22.2% Bacterial anaerobical conditions. (11.1-33.3) isolation Incubation at 37°C PantoeaAgglomerans: 9 16.7% and during 24-48h. (54) (6.7-26.6) identification Bacterial identification Randall et al. Aerococcus 12.9 % by MALDI-ToF Mass 2015 (4.0-21.9) Spectrometry (Bruker Viridans: 7 (54) 12.9% UltrafleXtreme (4.0-21.9)

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platform, Bruker Leoconostoc 9.3% Daltonics). mesenteroides: 7 (54) (1.5-17.0) Streptococcus sp. 5 (54) 7.4% (0.4-14.4) Carnobacterium 3.7% sp.: 4 (54) (0.0-8.7) Staphylococcus 3.7% Aureus: 2 (54) (0.0-8.7) a Chembio Diagnostic Systems, New York, USA; b INGENASA, Madrid, Spain; c Macherey-Nagel, Düren, Germany; d The result is based on the combined positivity to either serological test; eCombination of PCR and serology.

Figure 1.- Severity of the pulmonary lesions compatible with the porcine respiratory disease complex and its association with the detection of Porcine Circovirus type 2 antigen by PCR, in 54 dead-found wild boar piglets. The bottom boxes indicate the number of piglets in each lung lesion group.

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DISCUSSION

We only found partial support to our hypotheses only in part: wild boar piglets died due to

PRDC, or at least PRDC contributed a lot to the recorded deaths. However, TB was not a cause of death in this age class (in contrast with adults, Barasona et al. 2016), and had no apparent relation with PRDC either.

The recorded wild boar piglet mortality during summer 2015 was extremely high (71%) by the decrease of the piglet-to-adult ratio at the feeders and this estimation was similar to the mortality monitored by the gamekeepers through habitual direct observations at the feeders. Although the true estimates cannot be drawn using this method, thus capture-mark-recapture-recovery design should have been required to provide an acceptable natural mortality estimate (Toïgo et al. 2008). Almost

90% of this mortality was associated with respiratory problems. The PCV2 prevalence was relatively high: 54% (39% by PCR) in our sample of summer-mortality piglets as compared to 14% by PCR in (apparently healthy) hunter-harvested piglets sampled in similar habitats in autumn

(Risco et al. 2015). Subjective lesion scoring, presence of subclinical PCV2 infections, undetected histopathological lesions and a low detection rate of PCV2 in lung tissue samples (Segalés 2012) could contribute to explain the lack of statistical association among PCV2 and PRDC-compatible lesion scores. However, Figure 1 suggests a link between PCV2 and PRDC, possibly as a predisposing factor to secondary infections (Ellis et al. 2004). Moreover, the role of artificial management (high density, spatial aggregation) and adverse environment (dust, high temperature) in triggering respiratory disease cannot be excluded (Brockmeier et al. 2002).

The 30% contact prevalence with M. bovis is similar to the one found in previous studies within the same estates, thus confirming once more the endemicity of TB in this area, where wild boar piglets are exposed to this pathogen at a very early age (Che’Amat et al. 2015, 2016). Our data do not allow inferring whether PCV2 or PRDC may contribute in predisposing wild boar to M. bovis infection or to increased disease severity.

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In domestic pigs, enzootic pneumonia caused by M. hyopneumoniae limits the development and weight gain in piglets (Maes et al. 1999). Generally, body condition for these piglets are poor, however, our survey detects a rather low prevalence (12%) of this pathogen, as compared to studies focusing on older ages (e.g. Chiari et al. 2014; Risco et al. 2015).

A number of bacteria were isolated from samples of lungs, mandibular lymph nodes and tonsils of these piglets (Table 1) which all may be likely associated with bacterial flora or environmental contamination. However, opportunistic infection execution due to immunosuppression cannot be ruled out. Our isolates Enterococcus sp. or Lactobacillus sp., could be normally inhabit the gastrointestinal tract of various mammals including swine (Tannock et al. 1999). However, their pathogenic nature cannot be completely ruled out since infection by E. faecium causing domestic pig mortality, and infections with E. faecalis including large bacterial loads in the lung have occasionally been reported (Lu et al. 2002; Jiang et al. 2011). We found almost 17% prevalence of

Pantoea agglomerans which be able produces an endotoxin causing numerous pathologic effects including respiratory lesions, however the finding is inconclusive due to the ubiquity of this bacteria in the environment such as soil and dust, (Dutkiewicz et al. 2015). Aerococcus viridans has been isolated from 24% of lungs of domestic pigs with pneumonia, with none of other important pathogens causing pneumonia being isolated (Martín et al. 2007). Similar in our findings, 13% A. viridans prevalence was significant, however their cause for the respiratory lesions is uncertained.

Swine respiratory-associated disease is often the result of co-infection by main and opportunistic infectious agents (Brockmeier et al. 2002). Thus, these bacteria and others could be opportunistic pathogens to be considered in the list of possible etiological agents of wild boar PRDC in our study.

In conclusion, respiratory disease characterized by pneumonia was the potential cause of the high mortality among early age wild boar piglets during summer, and PCV2 was the most prevalent pathogen recorded in found-dead piglets. Future research should focus on gathering experimental evidence on the involvement the aforementioned and other pathogens, and should consider the importance of disease-mediated mortality in wild boar piglets, at least of those reared under

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intensive management conditions. Therefore in this type of management, preventive medicine such as vaccination against PCV2 and/or antimicrobial prescription for other respiratory-associated bacteria might be considered especially in known endemic area.

Acknowledgements

The corresponding author acknowledges VISAVET and many colleagues at SaBio-IREC for the provision of both technical laboratory and field assistance. Animal side TB tests were kindly provided by K. Lyashchenko, Chembio Diagnostic Systems, New York, USA. ACA is a grant recipient from the Malaysian Government and UPM. This is a contribution to Spanish Government

MINECO Plan Nacional grant AGL2014-56305 and FEDER, and to CDTI.

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Supplementary material

Table S-1. Biometric data of (n=54) under >6 month old summer-mortality piglets in southern

Spain.

Biometric data n Range Mean (95% C.I.)

Body weight (kg) 32/54 3.08-8.56 5.23 (4.65-5.82)

Body score index (1-5) 53/54 1-3 1.76 (1.55-1.96)

Kidney fat index (%) 44/54 3.7-38.09 14.31 (11.80-16.81)

Head and body length (cm) 44/54 42.0-76.5 59.16 (57.13-61.19)

Thoracic perimeter (cm) 44/54 24.0-48.0 37.71 (36.24-38.18)

Hind foot length (cm) 44/54 9.0-15.0 13.29 (12.89-13.68)

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Figure S-1. Wild boar piglet lungs with lesions compatible with the PRDC. Macroscopically, the lungs appear enlarged and fail to collapse. Diffuse and multifocal pneumonia and congestion ranges from moderate (A) to severe (B). Moreover some lungs present fibrin adhesions between the pleura and the thoracic wall (B). In the pulmonary parenchyma, there is evidence of interstitial pneumonia with alveolar septal thickening and hemorrhages (C), with severe thickening in various cases and associated with vascular findings and bacterial colonies (arrowhead, D). In panels C and D, H&E staining; bar = 50 μm.

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GENERAL DISCUSSION AND SYNTHESIS

Tuberculosis (TB) has a complex epidemiology, which includes multiple animal hosts and is influenced by climate and habitat. The role of wild and domestic hosts in animal TB varies amongst regions. Cattle are the most important domestic animal reservoir for Mycobacterium bovis (M. bovis) in relation to bovine TB and zoonotic TB in humans. However, as bovine TB prevalence has been reduced in livestock driven by the compulsory test and slaughter campaigns in cattle, the relative epidemiological and socio-economic significance of wildlife reservoirs and the need for disease management in these species has grown.

Wild ungulates, for example wild boar and feral pig; red deer, fallow deer and white-tailed deer;

African buffalo and other mammals such as badgers and possums are among the paramount important maintenance hosts for animal TB occurring worldwide. Suitable diagnostic tests, particularly for in vivo

TB diagnosis, and efficient control tools are duly needed in this context.

Animal TB due to Mycobacterium tuberculosis complex (MTC) infection is of great relevance within the Spanish framework of livestock farming, commercial game farming, wildlife conservation and the interface with public health. Although TB has been documented throughout the country, there are fundamental inter-regional differences in bovine TB epidemiology. The largest impact occurs in central- southern Spain. Drivers of TB in this region are well disclosed and include a high density of wildlife reservoirs, mainly wild boar and red deer (Vicente et al. 2013, LaHue et al. 2016). Other important risk factors include host genetic susceptibility, spatial aggregation at feeders and waterholes, scavenging and social behavior. In industrialized countries including the European Union (EU) members, the main reason for TB control is economic as a result of severe losses due to trade restrictions of cattle TB and slaughter compensations for test-positive animals.

In this context, we hypothesized that that the humoral and cell mediated immune responses would vary depending on the host species and the specific age-class under study. This is indeed the case as shown in the studies in chapters 2, 3 and 4. We also hypothesized that a better understanding of the performance of antibody-mediated and CMI-mediated ante-mortem diagnostic tests in wildlife would

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improve our capacity to control animal TB. In this case, we failed to confirm the hypothesis, since even novel and improved diagnostic tools were not sufficient to successfully implement a targeted culling program in wild boar from a high-prevalence site. However, we are sure these new insights into the studied diagnostic tools will find their application in epidemiology, surveillance and in disease control.

In Chapter 1, six specific research needs are identified: 1) complete the world map of wildlife MTC reservoirs and describe the structure of each local MTC host community; 2) identify the origin and behaviour of generalized diseased individuals within populations, and study the role of factors such as co- infections, re-infections and individual condition on TB pathogenesis; 3) quantify indirect MTC transmission within and between species; 4) define and harmonize wildlife disease monitoring protocols, and apply them in a way that allows proper population and prevalence trend comparisons in both space and time; 5) carry out controlled and replicated wildlife TB control experiments using single intervention tools; 6) analyse cost-efficiency and consider knowledge transfer aspects in promising intervention strategies. Based on some of these points, the aim of this work was contributing to improved TB monitoring and control by further investigating and understanding the ante-mortem techniques for diagnosis in wild boar and red deer. Furthermore, to apply such tests in a TB control field trial in one these maintenance hosts, the wild boar. In addition, we investigated the impact of other infections on the maintenance host in TB endemic areas. For this direction, four studies were conducted that are summarized below.

Chapter 2 describes a cross-sectional seroprevalence survey against MTC antigens on 2-6 month- old wild boar piglets within a TB endemic area using 6 different serological tests. A highlighted result showed that the dual-path platform (DPP) tests are the most sensitive ones (Sn: 61.5-69.2%) among other serological tests. This is despite being lower than sensitivities reported for adult age classes, and was not unexpected considering that piglets have an immature immune system development. This study confirms that the specific M. bovis antigens, such as MPB83 (as immobilized in one DPP test line) are common

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and serodominant in wildlife, including in wild boar piglets. The results also suggest that wild boar piglets were highly exposed to MTC at the early stage of life. This is an indication of already very high infection pressure. In field situations, a requirement for a practical test is performing fast and reliably.

Thus, rapid tests such as the DPP test could suite the purpose and this is the basis for the targeted culling study we conducted in Chapter 4. Although IgG is predominant against MTC in these wild boar piglets, the antibody development dynamics in piglets could not be established. Antibody development should be measured in an experimental study on a cohort of individuals with no maternal antibodies.

Serological tests have the advantage to overcome issues for bovine TB diagnosis in wildlife species, particularly the need of immobilizing the animals twice for skin-testing. However, the sensitivity and specificity of antibody-based tests, generally good in suids, is only moderate in wild ruminants, such as in red deer. In addition, the fear exists that repeated skin testing with both bovine and avian PPD for ante- mortem TB testing might influence the skin test or/and serological tests. Therefore, Chapter 3 studies the effect of repeated comparative skin testing in MTC-free red deer. This study confirmed that no sensitization or desensitization on skin response or ELISA reads resulted despite 6-monthly skin testing.

Nonetheless, a progressive skin sensitization against aPPD and serological ELISA aPPD boosting suggested either infection or exposure to avian TB or MAP and this also explained few transient cross- reactions in skin test and ELISA against bovine antigens. We suggest that the combination of comparative intradermal skin testing with ELISA is beneficial for accurate TB diagnosis in farmed red deer. Although other proteins (e.g. ESAT-6, CFP-10, etc) can be tested to enhance the sensitivity, understanding the response of red deer to bPPD and aPPD is important since this test system is the SaBio IREC testing routine, and possibly the only one currently applied to this species in Spain.

Animal TB needs to be controlled in intensely managed hunting estates in order to reduce mortality and trophy losses among game and to prevent the spread of infection to domestic livestock, with economic implications and ultimately consequences on human health. In Chapter 4, we explore targeted

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removal of seropositive wild boar of all ages utilizing the DPP rapid test system in naturally MTC infected wild boar. Seropositive (‘targeted’) wild boar were culled by routine hunting, thus getting hunters involved in this selective control tool. Clearly, this strategy resulted unable to sustainably decrease infection prevalence in the treatment site compared to the control site (with no intervention).

Indeed the TB prevalence was markedly increased only in the release site (where seropositives were translocated). We believe this tool can be further explored by testing using more sensitive tests, since test sensitivity was low particularly for the piglets, as shown in chapter 2) and strict movement control of animals between study sites. One important insight is that intensive and well managed hunting estates provide an ideal environment for intervention. This could support further investigation efforts, particularly assessing the use of integrated control actions, combining several of the available tools.

Findings in the last Chapter 5 showed high mortality (>70%) among wild boar piglets during the summer season (June-September). We expected TB to significantly contribute to this mortality.

Pathologically, almost 90% of the dead piglets had respiratory lesions compatible with the Porcine

Respiratory Disease Complex (PRDC). A number of pathogens were isolated but none of them had a correlation with respiratory lesions (including TB-compatible lesions). The porcine circovirus type 2

(PCV2) was the most prevalent one (54%). An increasing trend for PCV2 detection was found with higher lung lesion scores. In this context at least, we believe high mortality among wild boar piglets could be due to disease, particularly PRDC. However, it is not caused by TB. Rather, early exposure to other pathogens might facilitate MTC infection and later, TB progression. This supports recent findings by

Risco and colleagues (2015), which recorded a high prevalence PRDC-like lesions in hunter harvested wild boar piglets. Experimental studies are proposed in order to further explain the roles played by the pathogens in causing mortality among wild boar piglets either as a single infection or due to multiple interactions. Our findings may also encourage preventive measures such as vaccination (e.g. PCV2, pasteurella, etc) and even targeted and limited prophylactic antibiotic treatments, especially during specific times such as the summer season for the piglets.

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Future research needs

One of the research needs in the field of animal TB epidemiology and control, as mentioned in the introduction chapter, is to complete the world map of wildlife MTC reservoirs and describe the structure of each relevant local MTC host community. The prospect would be the developing nations such as many of the Asian region, where the wide range of wild boar, other suid, and feral pig populations exist and should be assessed to study the epidemiological differences as compared to known reported data from other regions. In addition, it would be interesting to perform screenings on other wildlife species that might be potential hosts for MTC.

In chapter 2, a suboptimal sensitivity of serology tests was evidenced in wild boar piglets. This might have occurred due to under- developed IgG antibodies at early age. Future studies should, for example, look for more sensitive technical procedures such as using an IMAPlate 5RC96, a disposable multi-utility lab device is used as a self-uptaking microcuvette array to read out the result of the ELISA that is performed in the normal 96-well plate with reduced substrate solution, stop solution and ability to measure low concentrations of analytes such as the low-abundant serum protein (Spies et al., 2010).

Further analyzing and improvement of MTC ELISA anti-IgM protocols is demanded since antibody response during early age (e.g. in non-human primates, see Holbrook et al., 2015) are predominantly IgM and the antibodies are less efficiently activated following infection.

In chapter 3, repetitive comparative tuberculin skin testing did not affect the cell mediated immune response as well as the antibody production in deer. However, this has only been tested in non MTC infected deer. Thus, a comparative study should evaluate MTC infected deer in order to further improve the TB testing protocol in farmed deer.

In chapter 4, indeed major issues has been raised in the study design notably uncontrolled movements of animals between sites and lower sensitivity of the test in piglets which could have had a

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serious effect on the results. Nevertheless, from the perspective of a practical test in a real field scenario, these intervention and study design might be improved and further evaluated such as optimizing control movement of animal across fences and increasing the volume of capture and test since the serology rapid tests are the only promising, applicable and practical ones in field work. Also, combinations of targeted culling with other intervention tools such as random culling, vaccination and others, should be tried.

In chapter 5, our findings evidences a marked mortality among piglets in fenced hunting estates during the summer period. However, the precise factors causing the problem remain elusive. Thus, for a future study, an extensive and thorough investigation including comprehensive pathological evaluation, microbiological, management and environmental studies should be applied to elucidate the probable causative factors of this mortality.

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CONCLUSIONS

1. Wild boar and red deer are important wildlife host species for the Mycobacterium tuberculosis

complex (MTC). Infection of these and related host species may occur worldwide. While their role is

well established in Mediterranean Iberia, it needs to be investigated for understanding animal TB

epidemiology in many other regions.

2. In high MTC prevalence sites, over one third of the wild boar piglets can become infected at early

age. This finding has implications for game management and disease control in endemic regions.

3. Serology for tuberculosis (TB) diagnosis is less sensitive in piglets than generally observed in adult

wild boar, and performance varies among tests.

4. However, certain antibody-detection tests, notably the rapid animal-side ones, can contribute to TB

control strategies by enabling the setup of test and cull schemes or improving pre-movement testing.

5. Diagnosing TB in farmed red deer is challenging and might require combining cellular and humoral

diagnostic tests.

6. We expected that repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might

sensitize or desensitize the subjects to both kinds of diagnostic tools. However, repeated comparative

skin testing at six month intervals did not cause progressive increments in red deer skin test

responsiveness or antibody production.

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7. The first attempt to control TB in wild boar through targeted removal as a single tool failed to reduce

TB prevalence. Although this specific trial failed, we suggest exploring integrated strategies, such as

combinations of culling with vaccination.

8. The targeted culling experiment provided valuable insights into the MTC infection pressure in high-

prevalence settings: we recorded an 80% seroconversion rate between the first and second summer in

piglets, and a still high 33% seroconversion rate per year in adults.

9. The experiment also revealed the consequences of introducing MTC-infected wild boar into already

infected sites, where infection prevalence in non-yearling hunter-harvested wild boar increased by

60%. This evidences the potential negative effects of translocating and releasing MTC infected wild

boar.

10. The Porcine Respiratory Disease Complex, and not TB, was the primary disease condition linked

with the high mortality among early age wild boar piglets during summer. Porcine Circovirus type 2

was the most prevalent pathogen recorded in found-dead piglets. Future research should focus on

gathering experimental evidence on the involvement of the aforementioned and other pathogens, and

should consider the importance of disease-mediated mortality in wild boar piglets, at least of those

reared under intensive management conditions.

11. Since TB is more difficult to control in wildlife than in cattle, and since interventions in natural

systems are complex and prone to conflicts, a remaining question is whether or not interventions on

wildlife TB are at all justified. We suggest that the answer varies depending on the local

circumstances in each TB hotspot, and is likely to evolve during our collective progress towards TB

control in livestock and in wildlife.

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