Tuberculosis in wildlife: new diagnostic tests and host respons to vaccination in red deer

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(2) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Trabajo de investigación presentado por. Jobin Thomas. Para optar al grado de Doctor por la Universidad de Castilla-La Mancha. Ciudad Real, agosto 2019 Grupo de Sanidad y Biotecnología (SaBio) Instituto de Investigación en Recursos Cinegéticos (IREC; CSIC-UCLMJCCM) Programa de Doctorado en Ciencias Agrarias y Ambientales Universidad de Castilla-La Mancha (UCLM).

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(4) The undersigned, as directors of this doctoral thesis, note that the thesis entitled “Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer” and prepared by Jobin Thomas, licensed in veterinary medicine, meets the requirements to qualify for the Doctoral Degree.. Thesis Directors. Prof. Dr. Christian Gortázar. Prof.ª Dra. María Ángeles Risalde. Ciudad Real, septiembre 2019 Grupo de Sanidad y Biotecnología (SaBio) Instituto de Investigación en Recursos Cinegéticos (IREC; CSIC-UCLMJCCM) Programa de Doctorado en Ciencias Agrarias y Ambientales Universidad de Castilla - La Mancha.

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(6) To my wife Gissa and my entire family for their love, endless support, inspiration and sacrifices. "Success is not final; failure is not fatal: It is the courage to continue that counts" Winston S. Churchill.

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(8) My gratitude and thanks to:. Spanish Government MINECO Plan Nacional I+D+I grant AGL201456305 MINECO Plan Nacional I+D+I grant WildDriver CGL2017-89866 Fondo Europeo de Desarrollo Regional (FEDER) CDTI and Glenton EU FP7 grant WildTBvac #613779 Indian Council of Agricultural Research-International Fellowship 201415 (ICAR-IF 2014-15).

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(10) INDEX. RESUMEN ...................................................................................................... 1 SUMMARY ..................................................................................................... 7 INTRODUCTION ........................................................................................ 13 Prevalence of animal TB .......................................................................... 15 Etiology ...................................................................................................... 17 Host Range ................................................................................................. 18 Transmission of TB ................................................................................. 20 Pathogenesis of animal TB ....................................................................... 20 Immunity towards TB .............................................................................. 21 Innate immune response ......................................................................... 21 Acquired immune response ...................................................................... 21 Cellular immune response.................................................................. 22 Humoral immune response ................................................................ 22 Diagnosis .................................................................................................... 24 TB like lesions (TBL) .............................................................................. 24 Post-mortem examination ................................................................. 25 Histopathological examination .......................................................... 25 Identification of the microorganism ...................................................... 25 Microscopy ........................................................................................ 25 Culture and identification ................................................................... 26 Tests based on immune response .......................................................... 27.

(11) CMI based diagnostics .......................................................................28 Antibody based tests ...........................................................................31 Confounding factors ..............................................................................38 Host factors ........................................................................................38 Environmental factors ........................................................................39 Factors related to prior sensitization .................................................39 Sample and sampling related factors .................................................40 Diagnostic technique related factors..................................................40 Improved diagnosis ................................................................................41 Selection of the appropriate test .........................................................41 Proper implementation and interpretation of the test ........................42 Combination of diagnostic tests .........................................................42 Control of Tuberculosis ............................................................................43 Population control ..................................................................................43 Farm biosecurity ....................................................................................43 Vaccination .............................................................................................44 Types of vaccines ................................................................................44 Delivery of vaccine with emphasis on oral bait deployment ..............52 Dose and frequency of vaccination ....................................................53 Characteristics of a good vaccine ......................................................53 Efficacy of vaccination .......................................................................55 Conclusion ..................................................................................................57 JUSTIFICATION AND WORK PLAN .....................................................59 OBJECTIVES ...............................................................................................65.

(12) CHAPTERS .................................................................................................. 71. CHAPTER 1: Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis .................................................................................... 74 Abstract ...................................................................................................... 75 Abbreviations ............................................................................................ 76 Background ............................................................................................... 77 Methods...................................................................................................... 80 Animals and experimental design ............................................................ 80 Microbiology............................................................................................ 81 Serum antibody detection ........................................................................ 82 IFNγ test ................................................................................................... 82 M. bovis proteins selection ...................................................................... 83 Statistical analyses................................................................................... 85 Results ........................................................................................................ 85 Discussion................................................................................................... 91 Conclusions ................................................................................................ 96 References .................................................................................................. 96 Additional files ........................................................................................ 102 CHAPTER 2.1: A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum .................................................... 107 Abstract .................................................................................................... 109 Introduction ............................................................................................. 110 Materials and methods ........................................................................... 114.

(13) Collection of samples .............................................................................114 Ethics statement .....................................................................................114 Serum antibody detection techniques .....................................................115 Indirect bPPD ELISA .............................................................................115 Indirect P22 ELISA ................................................................................115 Data treatment .......................................................................................116 Results ......................................................................................................117 Discussion .................................................................................................120 Conclusion ................................................................................................122 References ................................................................................................122 CHAPTER 2.2: Validation of a new serological assay for the identification of Mycobacterium tuberculosis complex-specific antibodies in pigs and wild boar...................................................................................127 Abstract ....................................................................................................129 Introduction .............................................................................................130 Materials and methods............................................................................135 Animals ..................................................................................................135 Bacteriology ...........................................................................................136 Serum antibody detection ......................................................................137 Indirect ELISA with P22 ..................................................................137 Indirect ELISA with bPPD ...............................................................138 Data analysis .........................................................................................138 Results ......................................................................................................139 Discussion .................................................................................................144 Conclusion ................................................................................................146.

(14) References ................................................................................................ 147 Supplementary files ................................................................................ 152 CHAPTER 3: Red deer response to oral administration of heatinactivated Mycobacterium bovis and challenge with a field strain ........ 155 Abstract .................................................................................................... 157 Introduction ............................................................................................. 158 Materials and methods ........................................................................... 160 Ethics statement ..................................................................................... 160 Study animals and experimental design................................................. 160 Vaccines ................................................................................................. 161 Heat-inactivated M. bovis (IV) ......................................................... 161 BCG ................................................................................................. 161 Challenge ............................................................................................... 161 Pathology ............................................................................................... 162 Microbiology.......................................................................................... 163 Gamma interferon test (IFNγ) ............................................................... 163 bPPD ELISA ......................................................................................... 164 Complement C3 ..................................................................................... 164 Cytokines (IL-1β, IL-10, IL-12, IFNγ and TNFα) .................................. 164 Statistical analyses................................................................................. 164 Results ...................................................................................................... 166 Oral vaccination with IV reduces TB lesion score in deer ................... 166 M. bovis infection was confirmed by culture in all deer ....................... 168 IFNγ response to the challenge with M. bovis ...................................... 168.

(15) Oral vaccination with IV and BCG does not induce antibody responses prior to the challenge ...........................................................................170 M. bovis infection increases plasma C3 concentration in red deer ......171 Oral vaccination with IV activates the innate immune response and Th1 cytokines in deer after the challenge ....................................................172 Discussion .................................................................................................174 Conclusion ................................................................................................177 References ................................................................................................178 GENERAL DISCUSSION .........................................................................181 CONCLUSIONS .........................................................................................189 BIBLIOGRAPHY .......................................................................................194 ABBREVIATIONS .....................................................................................225 ACKNOWLEDGEMENTS .......................................................................231.

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(17) Resumen. Resumen. 1.

(18) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. 2.

(19) Resumen. La Tuberculosis (TB) animal es una enfermedad infecciosa de carácter crónico que representa un importante problema sanitario para animales domésticos y salvajes, así como para el ser humano. El control y manejo de esta enfermedad constituye un área de investigación continúa debido a su impacto sobre la economía, la sanidad animal y la salud pública en general. Además, la TB ha sido recientemente considerada como una amenaza emergente para la conservación de las especies. Las nuevas aproximaciones al diagnóstico de la TB, especialmente en la fauna silvestre, están adquiriendo relevancia en los últimos años, ya que éstas constituyen un paso primordial para la investigación epidemiológica, así como para asegurar el éxito de las estrategias de control de la enfermedad. Dicho control resulta más fácilmente alcanzable cuando se basa en la integración de muchas estrategias diferentes, incluyendo el diagnóstico y la vacunación. Nuestro estudio fue diseñado con el objetivo de validar nuevos test diagnósticos basados en respuestas celulares y mediadas por anticuerpos para TB en rumiantes silvestres y ganado porcino, así como evaluar el efecto de la vacunación oral con la vacuna de Mycobacterium bovis (M. bovis) inactivada por calor (VI) en ciervos (red deer) mediante un estudio de infección experimental con M. bovis. Esta tesis doctoral comprende una introducción con una visión general de la TB y tres capítulos basados en el control de esta enfermedad, los cuales están relacionados con nuevas estrategias de diagnóstico y un estudio. 3.

(20) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. experimental de vacunación en fauna silvestre, seguidos por una discusión general y un apartado de conclusiones. El capítulo 1 habla sobre el desarrollo y la validación de un ensayo de interferón gamma (IGRA) con diferentes antígenos micobacterianos en ciervo. A pesar de que la intradermotuberculinización es la prueba diagnóstica ante-mortem oficial, ésta presenta muchas limitaciones respecto a la dificultad de manipular animales durante un periodo de 72 horas y a la variabilidad técnica a la hora de la lectura de los resultados. Nuestro objetivo consistió en desarrollar un test complementaro o de soporte a la intradermotuberculinización para ciervo con el fin de mejorar la precisión diagnóstica de dicha prueba en cérvidos. Para poder evaluar el potencial del IGRA, se llevó a cabo una infección experimental con 15 ciervos inoculados con M. bovis, donde se midió el interferón gamma (IFNγ) en respuesta a la estimulación in vitro de las células sanguíneas con un derivado proteico purificado de M. bovis (PPDb), el complejo inmunoproteico p22 obtenido a partir de PPDb o las proteínas específicas de micobacterias tuberculosas ESAT-6/CFP-10, Rv3615c y Rv3020c. Los resultados indican que antes de la infección todos los animales fueron negativos al IGRA, mientras que en el periodo post-infección la respuesta del IFNγ incrementó siguiendo patrones variados. Así, se pudieron detectar respuestas de IFNγ desde los 15 días postinfección, aunque la respuesta de anticuerpos frente a PPDb medida por ELISA fue más prominente durante las fases tardías de la enfermedad. El estudio muestra que los antígenos y el cut-off óptimos para la prueba del IFNγ son PPDb, P22 y la combinación de ESAT-6/CFP-10 y Rv3020c con un valor de cut-off de 0.05 ΔDO (densidad óptica). Este IGRA alcanzó niveles de detección de hasta el 100% de los ciervos positivos y negativos con las condiciones experimentales ensayadas; no obstante, la sensibilidad (Se) de la prueba disminuyó hacia las etapas tardías de la enfermedad. Esta técnica ayudará en la detección de TB tanto en ciervos que habitan normalmente granjas como en aquellos que se desplazan desde otras localizaciones, aunque debe ser aún validada en condiciones de campo. 4.

(21) Resumen. El diagnóstico basado en anticuerpos es una herramienta de gran utilidad, económica y rápida para el diagnóstico ante-morten y post-mortem de la TB en animales domésticos y silvestres. El capítulo 2 evalúa el uso de la P22, un nuevo complejo proteico inmunopurificado derivado de la PPDb, en el diagnóstico de anticuerpos específicos del complejo M. tuberculosis para mejorar la especificidad (Sp) de los test serodiagnósticos de TB en la fauna silvestre. La validación de la P22 mediante un ELISA casero (“in-house”) fue llevada a cabo en ciervo (capítulo 2.1) y en suidos, jabalí (Sus scrofa) y cerdo (capítulo 2.2), en comparación con el comúnmente usado ELISA con PPDb. En ciervo, se observó una elevada Sp (99%) para el ELISA de P22 sin comprometer por ello la Se (70.1%) al compararlo con el ELISA de PPDb (Sp - 91.6%, Se - 70.1%). En suidos, ambos ELISAs mostraron un buen valor diagnóstico, aunque se alcanzó una mayor Se y Sp en el ELISA de P22 (Se 84.1%, Sp - 98.4%) con respecto al ELISA de PPDb (Se - 77.3%, Sp 97.3%). Ambos ELISAs para PPDb y P22 alcanzaron un valor óptimo del 100% de Sp cuando se utilizaron muestras de cerdos blancos (criados bajo sistemas de control intensivos). Estos hallazgos indican que los antígenos específicos o purificados, especialmente la P22, podrían contribuir a mejorar el diagnóstico de la TB en un amplio rango de hospedadores silvestres, tal como ocurre en cérvidos y suidos. Los cérvidos son hospedadores del complejo Mycobacterium tuberculosis, de modo que el control de la TB en estos animales a través de distintas estrategias, incluyendo la vacunación, es un área de investigación continua. El capítulo 3 se centra en la eficacia de la VI oral en ciervos en un modelo experimental que emplea desafío con M. bovis, en comparación con la vacuna oral convencional Bacillus Calmette-Guerin (BCG). Este estudio se apoyó en la base de los prometedores resultados obtenidos en otras especies domésticas y silvestres. En este capítulo pusimos en marcha un experimento con ciervos de 5 meses de edad vacunados con VI, BCG o sin vacunar (n = 5/grupo), los cuales fueron inoculados con una cepa virulenta de M. bovis a los 70 días después de la vacunación y sacrificados 60 días después de dicho 5.

(22) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. desafío.. Los. resultados. obtenidos. sugieren. que. la. VI. redujo. significativamente la tasa de infección con respecto al grupo control. Además, empleando la vía oral como ruta de administración de la vacuna no se detectó ninguna interferencia de la vacunación con los test diagnósticos utilizados. Los mecanismos de inmunidad conferidos por la VI no han llegado a ser completamente elucidados en este estudio, pero los resultados obtenidos sugieren que el factor del complemento C3, el ratio de IFNγ/IL-10, y el TNFα tienen un papel relevante en la inmunidad protectora desencadenada por la vacunación. No obstante, la eficacia y estabilidad de la vacuna en condiciones de campo deben ser aún evaluadas. Por lo tanto, se necesitarán futuros estudios para validar dicha vacuna en condiciones de campo, así como para describir en profundidad los mecanismos protectores asociados a esta vacunación.. Esta tesis doctoral proporciona nuevos hallazgos para luchar frente a la TB en la fauna silvestre, mejorando el diagnóstico de dicha enfermedad y contribuyendo con información novedosa acerca del uso de la VI como candidato potencial en cérvidos para contribuir a las estrategias de control de la TB existentes en estas especies.. Palabras clave: diagnóstico; ELISA; fauna silvestre; IGRA; inmunocomplejo P22; tuberculosis; vacuna M. bovis inactivada por calor.. 6.

(23) Summary. Summary. 7.


(25) Summary. Animal Tuberculosis (TB) is a chronic infectious disease, which represents a major health problem in domestic and wild animals as well as in humans. The control and management of TB is an actively ongoing research subject because of its impact on economy, sanitary standards and public health. Moreover, it is recently considered as an emerging threat for conservation. New approaches to diagnosis of TB, especially in wildlife, are gaining relevance in recent years because it is a paramount step for epidemiological investigation, as well as to ensure the success of control strategies. In addition, TB control is more likely to be achieved by an integration of many strategies, including vaccination. However, there is a paucity of information with respect to the efficacy of vaccines like M. bovis heat-inactivated vaccine (IV) in cervids, which has proven not to interfere with the available diagnostic tests. So, considering all these points, our study was designed with the objective to validate new cellular and antibodymediated diagnostic tests for TB in wildlife ruminants and swine, as well as to assess the effect of oral vaccination with IV in red deer in an experimental challenge study. The thesis comprises an introduction regarding the overview of TB and three chapters, dealing with new diagnostic approaches and an experimental vaccination study for the control of TB in wildlife, followed by a general discussion and conclusion.. 9.

(26) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Chapter 1 deals with development and validation of an interferon gamma assay (IGRA) with different mycobacterial antigens in red deer. While the skin test is an official antemortem test, it has many limitations with respect to difficulty in handling animals for a period 72 hours and technical variability in reading the results. Our aim was to develop an ancillary or supporting test to the skin test in red deer in order to improve the diagnostic accuracy in cervids. For it, an experimental infection with M. bovis was carried out in 15 red deer to evaluate the potential of IGRA in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The results indicate that before infection, all the animals were negative to IGRA, while in post-infection period, interferon-gamma (IFNγ) response increased but in varied patterns. We could detect the IFN-γ responses as early as 15 days post infection, but antibody response to bPPD by ELISA was prominent during the later stages of the disease. The study shows that optimal antigens and cut-off for IFN-γ test are bPPD, P22 and the combination of ESAT-6/CFP-10 and Rv3020c with a cut off value of 0.05 ΔOD. This IGRA yielded detection levels up to 100% of the positive and negative deer under experimental conditions; however, the Se of the assay got decreased towards the later stages of the disease. This technique will aid in TB testing of farmed and translocated deer, however, it has yet to be validated in field conditions. Antibody-based diagnosis is a powerful, cost effective and rapid tool for ante-mortem and post-mortem TB diagnosis and large-scale surveillance, especially with regard to swine. Chapter 2 addresses the use of P22, a new immunopurified protein complex derived from bPPD, in M. tuberculosis complex-specific antibodies diagnosis to improve the specificity (Sp) of TB serodiagnostic tests in wildlife. The validation of P22 in an in-house ELISA has been performed in red deer (chapter 2.1) and swine (chapter 2.2) in 10.

(27) Summary. comparison to the commonly used bPPD ELISA. In red deer, we observed a high Sp (99%) in the P22 ELISA without compromising the sensitivity (Se) (70.1%) compared to bPPD ELISA (Sp - 91.6%, Se - 70.1%) In swine, both ELISAs yielded a good diagnostic value, however, a higher Se and Sp was achieved with the P22 ELISA (Se - 84.1%, Sp - 98.4%) when compared to the bPPD ELISA (Se - 77.3%, Sp - 97.3%). An optimum Sp of 100% was attained with white pigs (reared under intensive management systems) for both the bPPD and P22 ELISA. These findings indicate that specific or purified antigens, especially P22, could contribute to improved performance of TB diagnosis in multiple wildlife hosts, as it occurs in cervids and swine. Deer species are often part of the Mycobacterium tuberculosis complex maintenance host community, and TB control in deer, including vaccination, is consequently an area of ongoing research. Chapter 3 focuses on the efficacy of the oral M. bovis heat-inactivated vaccine in red deer in an experimental challenge model in comparison with oral conventional BCG vaccine. This study was performed on the basis of promising results in other domestic and wildlife species. In this chapter, we ran an experiment with five month-old red deer vaccinated with M. bovis heat-inactivated, BCG or unvaccinated controls (n=5/group), which were challenged with a virulent M. bovis strain 70 days later and necropsied at 60 days post-challenge. The results suggest that oral M. bovis heat-inactivated vaccine produced a significant reduction in infection burden with respect to the control group. Moreover, by this vaccine delivery route there was no interference with diagnostic tests due to vaccination. The mechanisms of the immunity conferred by the M. bovis heat-inactivated vaccine are not fully revealed in our study, but there are indications that complement component C3, ratio of IFN-γ and IL-10, and TNF-α have prominent roles in protective immunity. Nevertheless, the efficacy and stability of the vaccine in field conditions has yet to be investigated. Also, further studies are needed to be validated in field. 11.

(28) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. conditions, as well as to describe the exact protective mechanism with respect to vaccination. This thesis provides new insights to fight against TB in wildlife, improving TB diagnosis and contributing with valuable information about the use of M. bovis heat-inactivated vaccine as a potential candidate vaccine to aid the existing TB control strategies.. Key words: diagnosis; ELISA; IGRA; M. bovis heat-inactivated vaccine; P22 immunocomplex; tuberculosis; wildlife.. 12.

(29) Introduction. Introduction. 13.


(31) Introduction. Animal tuberculosis (TB) is a worldwide zoonosis caused by members of Mycobacterium tuberculosis complex (MTC), which are able to infect a wide range of domestic and wild mammals (Bailey et al., 2013) and it is a major public health concern in developing countries (Dürr et al., 2013). The TB is a disease listed in the World Organization for Animal Health Terrestrial Animal Health Code (OIE, 2009). It is now the world’s most deadly infectious disease, with an estimated 9.6 million new cases and 1.5 million deaths annually (WHO, 2016). In industrialized countries, the main reason for TB control is economic, resulting in severe losses in animal industry due to trade restrictions and slaughter compensations for test-positive animals (Bailey et al., 2013).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., 2015). Hence, animal TB control is considered globally important for public health, economics, high sanitary standards and conservation. 1. Prevalence of animal TB The World Animal Health Information Database reports that TB in domestic animals was present in Africa, Asia, European Union (EU), America and Oceania (WAHIS, 2018). No disease data was available in most of northern, northwestern and central Africa countries, China or some countries of southern Latin America and in a few eastern EU countries. 15.

(32) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Recently, the scientific report of EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control) offered a valuable overview of the M. bovis infection in Europe (EFSA, 2017).Bovine TB (bTB) has been subjected to compulsory eradication programs in the EU for many years, and 18 EU member states have already achieved official bTB-free status(OTF). Nevertheless, in several other countries, the disease is still endemic and prevalence levels have remained constant or even increased in recent years (EFSA, 2017) (Figure 1).. Figure 1. Status of European countries regarding bTB in 2016 (EFSA, 2017).. Despite the fact that the Spanish eradication program has reduced the TB herd prevalence from 11.1% in 1986 to 2.3% in 2017 (MAPAMA, 2017), Spain is the third country with the highest animal TB herd prevalence in the EU, behind the United Kingdom and the Republic of Ireland (EFSA, 2015). In 2017, the prevalence of TB in herds remained high in the south-central regions of Spain (SCS) (MAPAMA, 2017), affecting mainly the Communities of Andalusia (12.3%), Castilla-La Mancha (10.3%) and. 16.

(33) Introduction. Extremadura (9.7%). Here, the animal TB is highly prevalent in some big game species such as Eurasian wild boar (Sus scrofa) and red deer (Cervus elaphus), which are natural MTC reservoirs (Gortázar et al., 2012).. 2. Etiology The mycobacteria come under the genus Mycobacteria, family Mycobacteriaceae, order Actinomycetales and phylum Actinobacteria (King et al., 2017). Those members isolated in culture have been classified as fast or slow growing. This last group includes the main mycobacteria of importance in veterinary medicine and public health, like Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium complex (MAC) (Table 1). Table 1. Species in Mycobacterium tuberculosis and avium complex. M. tuberculosis complex. M. avium complex. M. tuberculosis. M. avium. M. bovis. M. avium paratuberculosis. M. bovis BCG. M. aviumsilvaticum. M. africanum. M. aviumhominissuis. M. microti. M. colombiense. M. caprae. M. indicuspranii. M. pinnipedi. M. intracellulare. M. canetti M. mungi M. orygis M. suricattae. The main species involved in the pathogenesis of animal TB are M. tuberculosis, M. bovis, M. bovis BCG and M. caprae. These species are characterized by: ̵. M. tuberculosis was the first mycobacteria identified by Robert Koch in 1882 (Koch, 1882) and it mainly affects primates. Although it has also been isolated from domestic and wild animals (Fetene et al., 2011). 17.

(34) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. ̵. M. bovis is the main etiologic agent of TB in cattle (Pollock and Neill, 2002; Palmer et al., 2007), presenting a wide range of domestic and wild hosts (Chambers, 2013; Gortázar et al., 2015). It has also been isolated in human (Grange, 2001). ̵. M. bovis Bacillus Calmette-Guerin (BCG) is an attenuated M. bovis strain obtained by subculturing M. bovis to produce a genetic drift and it is mainly used as a vaccine. ̵. M. caprae is the main pathogen in goats, although it can also affect other animal species such as cattle, pigs, deer, wild boar and European bison (Krzysiak et al., 2018), or humans (Rodríguez et al., 2011).. 3. Host Range Animal TB affects all the mammals including many domestic and wild animals and human. With regard to the domestic animals, the cattle is commonly regarded as the primary or definite host (Donnelly et al., 2007), although it also affects buffalo, domestic pigs, goats (mainly M. caprae), llamas, alpacas, camel and rarely reported in sheep, cat, horse and donkey (Gortázar et al., 2015). Wild animals can act either as maintenance host or as spill over host depending on the status of infection. The main wildlife reservoir host of animal TB includes Eurasian wild boar (Sus scrofa), red deer (Cervus elaphus) and fallow deer (Dama dama) in the Iberian Peninsula (Gortázar et al., 2015), African buffalo (Syncerus caffer) and Lechwe antelope (Kobus leche) in southern Africa (Van der Heijden et al., 2016; Michel et al. 2010), brushtail possum (Trichosurus vulpecula) in New Zealand (Tait et al., 2017), Eurasian badger (Meles meles) in the British Isles (McCulloch and Reiss, 2017), white-tailed deer (Odocoileus virginianus)in Michigan and Minnesota USA (Lavelle et al., 2016; Palmer et al., 2007). Many species of cervidae and suidae families have a great epidemiological role in the maintenance and transmission of TB, the details of which are illustrated in figures 2 and 3, respectively.. 18.

(35) Introduction. Figure 2. Importance of cervidae in animal TB. 19.

(36) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Figure 3. Importance of suidae in animal TB.. 4. Transmission of TB Infected animals shed mycobacteria via oro-nasal mucus, sputum, urine, feces and wound discharges (Barasona et al., 2017).The transmission of MTC among animals varies between species, being mainly by aerosols during close contact (Lavelle et al., 2016) and by indirect transmission at shared watering and feeding sites in cross species infection (Cowie et al., 2016; Barasona et al., 2017). The zoonotic transmission is through the consumption of contaminated raw milk and dairy products, or raw or improperly cooked meat from diseased animals (Dean et al., 2018).. 5. Pathogenesis of animal TB Animal. TB. is. a. chronic. granulomatous. caseous-necrotizing. inflammatory process that mainly affects the lymph nodes (LN) and lung. After the initial infection, the viable mycobacteria are readily transported by phagocytic cells through lymphatic capillary vessels to the draining LN, where they establish a new infection focus. This dual infection is known as 20.

(37) Introduction. the primary complex. The typical gross lesion of TB is known as tubercle, which is a circumscribed yellowish granulomatous inflammatory nodule. Histologically, small tuberculous granulomas are formed by neutrophils, epitheloid macrophages (MΦs) and a few Langhans-type multinucleated giant cells, which can be encapsulated by connective tissue and often contains central caseous necrosis and mineralization (Vordermeier et al., 2002). If the initial immune response is ineffective, a primary infection may generalize which is known as early generalization. The most common form of generalization is military TB, characterized by a large number of small whiteyellowish caseous foci without clear-cut delimitations.. 6. Immunity towards TB Immunity in TB is a combination of many factors which includes innate and acquired immune parameters.. 6.1. Innate immune response The innate immune response consists of cells and primary protection barriers (mucous membranes, intact skin, pH, etc.) to defend non-specifically against infection. Neutrophils are attracted to and accumulate at the site of initial infection after the inflammatory process triggered by mycobacteria (Flynn & Chan, 2001). Another essential cellular component after infection are the natural killer (NK) cells, capable of directly destroying the mycobacterium and/or the infected MΦs to prevent the advance of the pathogen.. 6.2. Acquired immune response The acquired immune response requires a first contact with the antigen so that the immunological memory is developed, and in case of reinfection, the immune response is triggered in the host in a faster way (Waters et al., 2011b).. 21.

(38) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. 6.2a. Cellular immune response In TB, the most relevant immune response is the cell-mediated immunity (CMI), in which the main cell type involved are T lymphocytes (Baldwin et al., 2015). The T cells are differentiated into two populations: T helper (Th) (CD4+) and T cytotoxic (CD8+) cells that actively participate in the immune response (Lin & Flynn, 2015). In experimentally infected animals with M. bovis, it has been observed that CD4+ T lymphocytes are the main cellular population producing IFN-γ, while CD8+ T lymphocytes are responsible for lysis of infected cells to avoid the advance of the infection (Liébana et al., 1999).. 6.2b. Humoral immune response The humoral-based immune response is not relevant in the early stages after infection, but antibody response acquires greater intensity as the infection progresses (Pollock et al., 2001) and it is associated with the development of lesions (Pollock et al., 2005) (Figure 4). As the cellular based response decreases, a change in the domain of the lymphocytic population occurs, with the Th2 lymphocyte population predominating (Surcel et al., 1994).. Figure 4. Pathology associated to animal TB (adapted from Gortázar et al., 2019).. 22.

(39) Introduction. The cytokines regulate the inflammatory and acquired immune responses throughout the activation of different cell types. The most important cytokines involved in the immune response against TB are listed: a) Interferon-gamma (IFN-γ): a key cytokine in TB infection, produced by sensitized T lymphocytes. Its main role in the specific response is the activation of MΦs against infection (Gormley et al., 2006). b) Tumor necrosis factor alpha (TNF-α): cytokine produced by the antigen presenting cells (APCs), among others, which has a main role in MΦs activation and immunoregulation (Waters et al., 2003). c) Interleukin-1β (IL- β): a major cytokine produced by APCs in response to TNF-α. It has stimulatory and inhibitory actions on various cell types, and also induces cellular apoptosis. Its lack causes bacterial multiplication and alterations in the formation of granulomas, favoring the advance of infection (Coad et al., 2010). d) IL-6: cytokine produced by Th2 lymphocytes, MΦs, endothelial cells and fibroblasts. It has pro and anti-inflammatory properties (Gormley et al., 2006). e) IL-12: cytokine produced by T helper cells that serves as a growth factor for T cells, increasing the production of specific IFN-γ and acts as protection marker in TB vaccine studies (Chen et al., 2017). f) IL-17: cytokine produced by T lymphocytes whose regulation is induced by IL-23. It acts as a mediator in delayed inflammatory reactions. It has been shown that its basal levels are decreased in infected animals with active TB (Fan et al., 2015). g) IL-4: produced by Th2 lymphocytes, mast cells, basophils, stromal cells of the bone marrow and NK cells. It suppresses the production of specific IFN-γ and the activation of MΦs (Toossi and Ellner, 1998). h) IL-10: cytokine produced mainly by Th2 lymphocytes, MΦs and B lymphocytes. It antagonizes the inflammatory response by decreasing the production of IFN-γ, TNF-α, IL-2 and IL-12, and favors the progression of TB infection (Waters et al., 2003). 23.

(40) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. i) Transforming growth factor beta (TGF-β): cytokine produced mainly by T lymphocytes and platelets. It is related to tissue damage and fibrosis that originate in TB lesions (Gormley et al., 2006).. 7. Diagnosis Diagnosis is an essential part in the disease surveillance as well as a very crucial step in the successful disease control and eradication programmes. Ideally, a diagnostic test should be easy to perform, easy to interpret, relatively inexpensive and with high sensitivity (Se) and specificity (Sp). The methods/tests used for diagnosis of TB are represented in figure 5.. Figure 5. Main diagnostic methods in animal TB.. 7.1. TB like lesions (TBL) Presence of TBL is an indication of active TB and is observed either macroscopically during post-mortem examination or microscopically during histopathological examination. 24.

(41) Introduction. 7.1.1. Post-mortem examination Post-mortem examination can be performed on hunted/dead animals by macroscopic examination of LNs and abdominal and thoracic organs, especially lungs to assess the TBL (Mendoza et al., 2006; Vicente et al., 2007). In wild boar, generalized infection is not so common, gross lesions are most frequently observed in the mandibular LNs, but retropharyngeal, tracheobronchial and mediastinal LNs or abdominal organs are also frequently involved (García-Jiménez et al., 2013). Generalized lesions are more common in deer, where medial retropharyngeal LNs are critical points (Schmitt et al., 1997). In deer, more lesions are found in the thoracic region, but more abdominal lesions are observed in fallow deer (Schmitt et al., 1997; Martín-Hernando et al., 2010). However, a confirmatory diagnosis cannot be made following the macroscopic examination because pathogens other than mycobacteria may cause indistinguishable lesions (Cardoso-Toset et al., 2015). Moreover, the lesions are predominantly seen in advanced cases of TB, so that early stages of infection cannot be detected by necropsy. 7.1.2. Histopathological examination The histological diagnosis consists of pathological observation of tuberculous granulomas, which are usually formed by cells of inflammatory character such as MΦs, giant cells of Langhans and lymphocytes (Davis & Ramakrishnan, 2009). 7.2. Identification of the microorganism Identification of MTC organisms is a confirmatory method in diagnosis that can be performed by direct microscopy/culture and identification/nucleic acid recognition methods. 7.2.1. Microscopy Direct microscopy with the acid-fast staining (Ziehl-Neelsen staining) is a simple, rapid and economical method for the detection of MTC. Samples can be collected from a slaughtered/hunted animal or a live animal (samples 25.

(42) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. of tracheobronchial washes). But this method can give non-specific results due to other mycobacteria. Fluorescence staining includes the staining of acid-fast bacteria using fluorescent markers. Fluorescence microscopy gives faster and more accurate results than acid fast staining, but it is laborious and economically not viable and sometimes polyclonal antibodies can give non-specific results due to other mycobacteria (Hendry et al., 2009). 7.2.2. Culture and identification Culture and identification is considered as the gold standard method for the diagnosis of TB. Identification of the microorganism after cultural isolation can be done by cultural characteristics, biochemical tests or nucleic acid recognition methods. Identification of each member of MTC can be performed based on the differences in phenotypic characteristics and biochemical properties (Aranaz et al., 1999). The Se of culture and isolation varies depending on the stage of the disease, on the number and selection of tissues processed and on sample quality (Boadella et al., 2011). Nucleic acid recognition methods can be applied using DNA extracted directly from the tissue samples or, for more accuracy, it can be performed from the culture isolated samples. The diagnosis of MTC by simple polymerase chain reaction (PCR) is fast, highly sensitive and of great value in epidemiological studies (Cedeño et al., 2005). Another nucleic acid recognition method is genotyping, which is routinely applied during epidemiological investigation. The important methods used for MTC genotyping are the spoligotyping, Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) analysis (Andrievskaia et al., 2018). Genotyping can also be used to discriminate different members and strains of MTC species, to large-scale molecular epidemiology studies (Milian-Suazo et al., 2016) or to detect outbreaks and their sources by MIRU-VNTR (Carvalho et al., 2016). 26.

(43) Introduction. 7.3. Tests based on immune response Immunological methods for detection of animal TB are inevitable in the surveillance programmes, control and management of TB are concerned. The immunological TB diagnosis is based on cell mediated as well as antibody mediated diagnostic tests against different antigens, which are listed below: ̶ Bovine purified protein derivative (bPPD): also known as bovine tuberculin. It is a combination of proteins discovered by Robert Kochin 1890 and used as standard in the diagnosis of TB (Cousins et al., 2011). ̶ Early secretary antigenic target-6 kDa (ESAT-6): protein secreted in the initial phases after MTC infection (Brodin et al., 2006). It is absent in M. bovis BCG and 90% of the mycobacteria other than MTC or MAC (Chambers, 2013). It has been shown to be capable of inducing both cellular (Moradi et al., 2015) and humoral immune responses (Leng et al., 2014). The protein ESAT-6 is usually used for diagnosis in conjunction with the CFP-10 protein (Vordermeier et al., 2001). ̶ Culture filtrate protein-10 (CFP-10): protein whose expression is regulated by a gene of the RD1 region of the genome (van Pinxteren et al., 2000). This protein is absent in BCG and it can be used for cell (de Araujo et al., 2014) and humoral-based tests (Zhu et al., 2014). ̶ Rv3615c: protein secreted in MTC infected animals (Sidders et al., 2008) and absent in BCG (Millington et al., 2011), which induce cellular immune responses (Sidders et al., 2008; Jones et al., 2012). ̶ Rv3020c: cell wall protein secreted in high amounts in animals infected with MTC members (Jones et al., 2012). The protein Rv3020c has demonstrated specific immunological reactivity in cellular based diagnostic tests (Jones et al., 2010). ̶ Mobility protein of M. bovis 83 (MPB83): glycosylated lipoprotein from the surface of the mycobacterium membrane (Waters et al., 2006). It was identified in M. tuberculosis H37Rv (MPT83), M. bovis in high amounts (Lyashchenko et al., 2001) and M. bovis BCG (Wiker, 2009). Its 27.

(44) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. diagnostic potential in serological tests has been demonstrated in conjunction with the MPB70 protein (Chambers, 2013). ̶ Mobility protein of M. bovis 70 (MPB70): soluble protein secreted in high amounts in M. bovis infections (Lyashchenko et al., 2001). It was identified in M. tuberculosis H37Rv (MPT70) and M. bovis BCG (Wiker, 2009). It is an important antigen used in serological tests along with the MPB83 protein (Marassi et al., 2014). ̶ P22 complex: immunopurified complex obtained from bPPD which includes main proteins MPB70, MPB83, ESAT-6 and CFP-10. It is a highly specific antigen used in serodiagnostic tests in many animal species (Casal et al., 2017; Infantes-Lorenzo et al., 2017, 2019).. 7.3.1 CMI based diagnostics ➢ Skin test: It is an ante-mortem TB diagnostic test based on the principle of delayed hypersensitivity reaction (type IV). This method is performed as approved by OIE (2009), where a tuberculous antigen is usually inoculated in the mid neck region in deer (Jaroso et al., 2010a) and inguinal region in wild boar (Jaroso et al., 2010b) or caudal part of the ear in warthog (Roos et al., 2018a), although in swine, it is rarely practiced. According to the skin intradermal test (SIT), a standard reactor is defined if the reaction is ≥4 mm; inconclusive if the reaction is >2 mm and <4 mm; or negative in the rest of the cases. In order to avoid nonspecific reactions to MAC, a single comparative cervical intradermal test (SICCT) has been developed in which avian PPD (aPPD) is also used in addition to bPPD (Jaroso et al., 2010b). The interpretation of the SICCT test is that any deer with a skin fold increase > 2mm to bPPD and > 1mm larger than the increase to aPPD was considered as TB reactor (Griffin et al., 1991). The tuberculin skin test has some drawbacks including the need to handle the animals twice in a 72-hour period (Harrington et al., 2008), low Sp (Queirós et al., 2012) and technical variability (Fernández-de-Mera et al., 2009). 28.

(45) Introduction. ➢. Interferon Gamma Release Assay (IGRA): The IGRA is used as an alternative or supplementary assay to the skin. test, in which CMI response can be measured in vitro by an assay that detects the IFN-γ produced by peripheral blood mononuclear cells (PBMCs) exposed to M. bovis antigens. Although, bPPD is the most commonly used antigen, some others have also been described as potential diagnostic targets (e.g. ESAT-6/CFP-10, Rv3615c or Rv3020c) in livestock and wildlife. The IGRA based on ESAT-6/CFP-10 helps to discriminate between BCG vaccinated and infected animals (Kang et al., 2005). Cervigam assay was a commercial IGRA test in cervids (Palmer et al., 2004; Harrington et al., 2006), but it is not commercially available nowadays. A lack of adequate response to mycobacterial antigens has been reported for infected white-tailed deer, elk and fallow deer (Waters et al., 2008). In suidae, IGRA provided a tool with a high Sp for the surveillance and management (Pesciaroli et al., 2012). The test avoids the continuous stimulation of the animal with mycobacterial antigens, its excessive handling and the technical variability associated with skin tests (de la Rua-Domenech et al., 2006). However, the test involves logistical constraints as strict laboratory conditions and the need for fast processing of samples (Pesciaroli et al., 2012). Moreover, the possibility of acute stress in tested animals could induce high levels of cortisol, resulting in reduced IFN-γ response by the inhibition of NF-κB transcription factor (Waters et al., 2008). ➢. Lymphocyte stimulation test (LST): This test measures the reactivity of blood lymphocytes to mycobacterial. antigens. The test has been performed in deer (Griffin et al., 1991; Surujballi et al., 2009) and pig (Faldyna et al., 2007). This method is complicated to perform for large scale diagnosis, being more suitable its use in research. The details of cell mediated diagnostic tests in cervidae and suidae are listed in Tables 2 and 3, respectively. 29.

(46) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Table 2. Details of cell mediated diagnostic tests in deer Assay Species test Skin test. N/E. Fallow deer N. nSe + nSp Antigens. Se. 21. bPPD. 80.1. bPPD. 97. 81. 91.4. 98.7. Corrin et al., 1993. 46.9. Griffin et al., 1994 Waters et al., 2003. White tailed N+E 60+56 deer. Reference Jaroso et al., 2010a Palmer et al., 2001. Red deer. E. 60+1157 bPPD. Red deer. N. 218. bPPD. Elk. N. 7+3. bPPD. 100. 100. Reindeer. E. 13+4. bPPD. 92. 25. 91+44. bPPD. 91. 98. 51. bPPD. 90a, 78b. 106. ESAT6:CFP10 bPPD ESAT6:CFP10. 100c 96d 91a 83b 94c, 87.5d. bPPD ESAT6:CFP10 bPPD. 98a, 92b 97c, 95d. Waters et al., 2008. 70. 74. Hutchings and Wilson, 1995 Shury et al., 2014 Griffin et al., 1991. IGRA White tailed E deer Elk. Reindeer. LST. Sp. N. N. White tailed N deer. 95. Elk. N. 433. Elk. N. 33 + 450 bPPD. 83. 64. Red deer. N. 39+16. bPPD MPB70. 95 72. 44 50. Red deer elk E hybrid. 10+15. bPPD. 65.7. 92.5. Palmer et al., 2006 Palmer et al., 2004 Waters et al., 2008 Waters et al., 2008. Harrington et al., 2007. N/E -Natural or experimental infection; nSe- number of TB positive animals used for evaluation of Se; nSp-number of negative animals used for evaluation of Sp; Se: sensitivity; Sp: specificity; Se and Spare expressed as percentage values; LSTLymphocyte stimulation test; IGRA-Interferon gamma release assay;acut off bPPDaPPD and bPPD –PBS< 0.1; bcut off bPPD-aPPD and bPPD –PBS< 0.05; ccut off ESAT-6:CFP10–PBS< 0.1;dcut off ESAT-6:CFP10–PBS< 0.05. 30.

(47) Introduction. Table 3. Details of cell mediated diagnostic tests in swine Assay test Skin test. IGRA. Species. N/E. nSe +nSp. Antigen. Se. Sp. References. Eurasian wild boar. E. 4+21. bPPD. 77-100a. 48.477.4a. Jaroso et al., 2010b. Common warthog. N. 16+18. bPPD. 69b,81c. 100d. Roos et al.,2018a. Domestic pig. N. 19+31. bPPD. 78.9. 100. Pesciaroli et al., 2012. N/E - Natural or experimental infection; nSe- number of TB positive animals used for evaluation of Se; nSp-number of negative animals used for evaluation of Sp; Se: sensitivity; Sp: specificity; a range of values due to the difference in reading criteria; b single intradermal tuberculin test (SITT); c Comparative intradermal skin test (CITT);dSp for both SITT and CITT is the same; IGRA-Interferon gamma release assay.. 7.3.2 Antibody based tests Antibody assays are convenient to perform as they require only onetime handling of animals and do not need fast processing of samples. They ensure large scale screening of samples, ante-mortem as well as post-mortem, and are able to diagnose TB in animals with progressive disease (Welsh et al., 2005; McNair et al., 2007). ➢ Enzyme Linked Immunosorbent Assay (ELISA): ELISA is the most extensively used serodiagnostic technique which detects circulating antibodies against MTC. The most common antigen used in ELISAs is bPPD, but many other specific or purified antigens (MPB83, MPB70, ESAT-6 and CFP10) have been tested to improve the diagnostic accuracy both in swine and deer. The use of antigenic cocktails (MPB70, MPB83) in the assay, ethanol extract of M. bovis antigen (EVELISA) and prior pre-absorption of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies or antibodies to other environmental mycobacteria as M. phlei have also proven promising results (Wadhwa et al., 2013). 31.

(48) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. ➢. Fluorescence polarization assay (FPA): This test comprises the use of the target antigen MPB70 with a. fluorescent molecule bound to it, in order to detect antibodies in serum. This assay was first described by Surujballi et al. (2002) in cattle, and later it has been validated in red deer (Surujballi et al., 2009) and elk (Shury et al., 2014), but with comparatively low diagnostic value in cervids when the test is used alone (Shury et al., 2014). ➢. Multiantigen print immunoassay (MAPIA): It uses a panel of 12 mycobacterial antigens including 8 purified. recombinant proteins (ESAT-6, CFP10, MPB64, MPB59, MPB70, MPB83, Acr1, and the 38 kDa protein), two protein fusions (CFP10/ESAT-6 and Acr1/MPB83), and two native antigens, bPPD and M. bovis culture filtrate (Lyashchenko et al., 2000). The assay enables the identification of speciesspecific immunodominant proteins as well as the reactivity patterns over the course of the disease which, in turn, helps in the selection of antigens for other diagnostic tests (Lerche et al., 2008). The MPB83 alone or in combination with the protein Acr1 was found to be the most serodominant antigen in different wild species, followed by ESAT-6/CFP10 and MPB70 proteins in deer and wild boar, respectively (Lyashchenko et al., 2008). The MAPIA gave almost equal or even high Se and Sp in comparison to other rapid tests, ELISA or immunoblot, but there is a practical difficulty to implement this assay for screening large number of samples. ➢. Immunoblotting: Electrophoresis and immunoblot assays are usually performed using. whole cell sonicate antigen (Waters et al., 2005) or recombinant MPB83 (O’Brien et al., 2008), but it is not routinely used for diagnosis in any species. ➢. Lateral flow tests: These tests are based on the immunochromatography. The main lateral. flow diagnostic tests are listed below: 32.

(49) Introduction. ̶. Cervid TB STAT-PAK (Chembio Diagnostic Systems, Inc., Medford, NY). This diagnostic test is used in deer and employs a unique cocktail of MPB83, ESAT-6 and CFP10 antigens (Lyashchenko et al., 2008). Its advantages include easiness to perform in field with a small volume of blood, serum or plasma, and detection of immunoglobulin A (IgA), IgM, and IgG antibodies to MTC (Gowtage-Sequeira et al., 2009). ̶. DPP tests (Chembio Diagnostic Systems, Inc., Medford, NY). This assay involves the independent delivery of the tested sample and the antibody-detecting reagent (protein A/G hybrid conjugated to colloidal gold particles), in contrast to the single-strip format used in the Cervid TB STAT-PAK test (Greenwald et al., 2009). The assay has been validated in different species of cervidae and suidae. Another test based on DPP technology is DPP WTB which makes use of two antigens in separate (MPB83 and MPB70). This assay is mainly focused for diagnosis in suidae, since MPB70 antigen is more serodominant than CFP10/ESAT-6, unlike what happens in deer (Che’ Amat et al., 2015; Roos et al., 2016; Cardoso-Toset et al., 2017). ̶. INgezim TB-CROM (INGENASA S.A., Madrid, Spain). Recently developed lateral flow test which uses MPB83 antigen. In domestic pig, a similar or even high Se and a slightly less Sp was obtained when compared to other in-house (bPPD, tPPD) or commercial ELISAs (bPPD, MPB70 + MPB83) (Cardoso-Toset et al., 2017). The details of antibody mediated diagnostic tests in cervidae and suidae. are listed in Tables 4 and 5, respectively.. 33.

(50) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Table 4. Details of antibody mediated diagnostic tests in deer. Assay Test. Species. N/ E. ELISA. Red deer N. Red deer N. nSe + nSp Antigens. Se. Sp. Reference. 6 +15. bPPD. 72.7a. 100a. GarcíaBocanegra et al., 2012. MPB83. 100. 100. GarcíaBocanegra et al., 2012. 88. 52. Griffin et al., 1991. 80. 79. Griffin et al., 1991. 104 + 56 bPPD MPB70. FPA. Red deer N. 94+ 217 bPPD, MPB 70, aPPD. 45.7. 100. Griffin et al., 1994. Elk. 108 +48 MPB70. 51.9. 100. Kang et al., 2016. MPB83. 49.1. 97.9. Extract of M. bovis. 86.7. 93.3. Kang et al., 2016 Wadhwa et al., 2013. 51. 96. Boadella et al., 2012a. N. Red deer E. 15 +15. Fallow deer. N. 73 +157 bPPD. White tailed deer. N. 12+329. LAM 66.7b enriched antigen 58.3c. 95.1b 97.3c. O’Brien et al., 2008. Reindeer E. 11 +4. LAM. 100. 50. Waters et al., 2005. Red deer N and elk. 16. MPB70. 81. Elk. N. 33 + 450 MPB70. 40. Red deer-elk hybrid. E. 10. 33.33. MPB70. 34. Surujballi et al., 2009 81. Shury et al., 2014 Harrington et al.,2008.

(51) Introduction. Assay Test Cervid TB STATPAK. DPP Vet TB. Species Elk. N/ E N. nSe + nSp Antigens. Se. Sp. Reference. 34 +141 ESAT-6, CFP10, 82 MPB83. 93. Waters et al., 2011a. Fallow deer. N. 32 +107 ESAT-6, CFP10, 91 MPB83. 91. Waters et al., 2011a. Elk. N. 33 +450 ESAT-6, CFP10, 62 MPB83. 87. Shury et al., 2014. Elk. N. 98.3. Red deer-elk hybrid. E. 31 + 842 ESAT-6, CFP10, 87.1 MPB83 10 ESAT-6, CFP10, 72.5 MPB83. Nelson et al., 2012 Harrington et al.,2008. Red deer. N/E 52 + 105 ESAT-6, CFP10, 86.5 MPB83. 83.8. Buddle et al., 2010. Fallow deer. N. 21. ESAT-6, CFP10, 80.1 MPB83. White tailed deer. N. 22+724. ESAT-6, CFP10, 54.5 MPB83. 98.1. O’Brien et al., 2008. White tailed deer. N/E 26+435. ESAT-6, CFP10, 44.7 MPB83. 85.7. Lyashchen -ko et al., 2008. ESAT-6, CFP10, 85.7a MPB83. 94.8 a. GowtageSequeira et al., 2009 Waters et al., 2011a. Jaroso et al., 2010a. Mixed N Deer sp.. 7 + 425. Elk. N. 34 + 141 ESAT-6, CFP10, 79 MPB83. 98. Fallow deer. N. 32+107. 99. Waters et al., 2011a. Red deer N/E 52 + 105 ESAT-6, CFP10, 84.6 MPB83. 91.4. Buddle et al., 2010. Fallow deer. N. ESAT-6, CFP10, 71 MPB83. 88. Boadella et al., 2012a. White tailed deer. N/E 63+903. ESAT-6, CFP10, 65.1 MPB83. 97.8. Lyashchen -ko et al., 2013. 73+157. ESAT-6, CFP10, 91 MPB83. 35.

(52) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Assay N/ Species Test E MAPIA Elk N Fallow deer. IB. nSe + nSp Antigens. Se. Sp. Reference. 34. M. bovis Antigensd M. bovis Antigensd. 82. Waters et al., 2011a. 97. Waters et al., 2011a Harrington et al.,2008. N. 32. Red E deer-elk hybrid White N tailed deer Reindeer E. 10. M. bovis Antigensd. 76.7. 22+727. M. bovis Antigensd. 68.2. 97.1. O’Brien et al., 2008. 11+23. M. bovis Antigensd. 100. 85. Waters et al., 2005. White N tailed deer White N tailed deer Reindeer E. 13+333. Whole cell sonicate. 46.2. 92.5. O’Brien et al., 2008. 20+671. MPB83. 55. 93. O’Brien et al., 2008. 11+4. Whole cell sonicate. 90.9. 50. Waters et al., 2005. ELISA- Enzyme Linked Immunosorbent Assay; FPA- Fluorescent Polarization Assay; Cervid TB STAT-PAK and DPP Vet TB- Lateral flow tests; IB-Immunoblotting; Se: sensitivity; Sp: specificity (Se and Sp are expressed as percentage values); N/E -Natural or experimental infection, nSe- number of TB positive animals used for evaluation of Se, nSp-number of negative animals used for evaluation of Sp; aSe and Sp were evaluated based on mixed species of animals; b cut off OD≥0.25, c cut off OD≥0.3; d ESAT-6, CFP10,MPB59, MPB64, MPB70, MPB83, the16-kDa protein, the 38-kDa protein, two fusion proteins comprising CFP10/ESAT-6 and the 16kDa protein/MPB83, and two native antigens, bovine PPD and M. bovis culture filtrate.. 36.

(53) Introduction. Table 5. Details of antibody mediated diagnostic tests in swine. Assay test. Sample. N/ E. nSe +nSp. Antigen. Se. Sp. References. ELISAprotein G. Eurasian wild boar. N. 96+104. bPPD. 79.2. 100. Boadella et al., 2011. ELISAIgG. Eurasian wild boar. E. 17+17. bPPD. 94.4. 100. Beltrán-Beck et al., 2014. Domestic pig. N. 66+23. bPPD. 86.4. 65.2. Mohamed et al., 2012. Ag85-B. 87.9. 86.9. ELISAIgM. Eurasian wild boar. E. 17+17. bPPD. 77.7. 100. Beltrán-Beck et al., 2014c. ELISAprotein A&G. Eurasian wild boar. N. 16+15. bPPD. 72.7a. 100a. MPB83. 100. 100. GarcíaBocanegra et al., 2012. Eurasian wild boar. N. 22+43. MPB83. 86. 100. De Val et al., 2017. Common warthog. N. 16+19. bPPD. 88. 89. Roos et al.,2016. Common warthog. N. 25. bPPD. 92. Eurasian wild boar. N. 73+112. bPPD. 72.6. 96.4. Aurtenetxe et al., 2008. Common warthog. N. 16+19. bPPD. 88. 79. Roos et al.,2016. Domestic pig. N. 59+88. bPPD. 72.9. 100. Cardoso-Toset et al., 2017. Common warthog. N. 25. bPPD. 86. ELISAINgezim TB. Domestic pig. N. 59+88. MPB7, MPB83. 74.6. 98.9. Cardoso-Toset et al., 2017. DPP WTB. Eurasian Wild boar. E. 16+16. MPB83, MPB70. 94.11. 100. Beltrán-Beck et al., 2014c. ELISA TB -VK. 37. Roos et al., 2018b. Roos et al., 2018b.

(54) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. Assay test DPP VetTB. Sample Eurasian wild boar. INgezim TBCROM. N/ E N. nSe +nSp. Antigen. Se. Sp. References. 64+113. 76.6. 97.3. Lyashchenko et al., 2008. 89.6. 90.4. Boadella et al., 2011. 77.8. 100. Beltrán-Beck et al., 2014c. 75. 89. Roos et al., 2016. 74.6. 98.9. Cardoso-Toset et al., 2017. 90.2. 100. FrescoTaboada al., 2019. Eurasian wild boar. N. 96+104. Eurasian wild boar. E. 17+17. Common warthog. N. 16+19. Domestic pig. N. 59+88. MPB83, ESAT-6, CFP10 MPB83, ESAT-6, CFP10 MPB83, ESAT-6, CFP10 MPB83, ESAT-6, CFP10 MPB83. Eurasian Wild boar. E. 51+9. MPB83. Eurasian Wild boar. N. 30+25. MPB83. 93.3. 96. et. FrescoTaboada et al., 2019. N/E -Natural or experimental infection, Se: sensitivity; Sp: specificity; n Se- number of TB positive animals used for evaluation of Se, nSp-number of negative animals used for evaluation of Sp;aSe and Sp were evaluated based on mixed species of animals; ELISA. TB –VK- ELISA test kit (Vacunek, Spain); ELISA- INgezim TB- ELISA test kit (INGENASA, Spain); DPP WTB and DPP VetTB- Rapid lateral flow test kits (Chembio Diagnostic Systems, Inc., USA), INgezim TB-CROM- Rapid lateral flow test kit (INGENASA S.L., Spain).. 7.4. Confounding factors There are many factors related to host, environmental, sampling and diagnosis technique which can affect the performance of the test: 7.4.1. Host factors There are no age-related differences (21 months age to5-year-old) in the responsiveness to skin test in red deer. However, age related differences are more pronounced in red deer and fallow deer (after 5-year-old) (Fernández-de-Mera et al., 2008; Jaroso et al., 2010a). The gender also provokes differences in the cellular immune responses in red deer and fallow 38.

(55) Introduction. deer, which could be due to differences in reproductive effort and energy expenditure (Clutton-Brock et al., 1982; Vicente et al., 2007). Moreover, males tend to have a thicker skin than females, so skinfold increases are relative to the thickness of the skin in red deer (Fernández-de-Mera et al., 2008, 2011) and fallow deer (Jaroso et al., 2010a). In wild boar, an increase in skin responsiveness with age was noticed, but there was no sex by age interaction (Jaroso et al., 2010b). Serodiagnostic techniques in white-tailed deer showed no age or sex related differences (Immunoblot, MAPIA, ELISA, CervidTB STAT-PAK) (O’Brien et al., 2008). In wild boar, infected piglets had lower Seat ELISA and DPP tests, as compared to yearling, juvenile or adult wild boar (Che’ Amat et al., 2015; Fresco-Taboada et al., 2019). Gender based variation is not seen in DPP test (Boadella et al., 2011). 7.4.2. Environmental factors Skin responsiveness to mitogen in winter was found to be significantly high compared to the response in summer and the difference was more prominent in adult red deer (Fernández-de-Mera et al., 2011). This may be due to seasonal presence of non-tuberculous mycobacteria which is a main confounding factor in TB diagnosis (Quieros et al., 2012). 7.4.3. Factors related to prior sensitization The previous skin testing for TB could also lead to the diagnosis of false positive animals. Griffin et al. (1994) reported that Se of ELISA and LST was considerably increased in red deer 10 days after skin test compared to the Se before skin test (45.3% to 85.3% in ELISA and 85.9% to 90.8% in LST, respectively). In experimental infection in white-tailed deer, reindeer and red deer, an elevated antibody response (in ELISA, immunoblot analysis, MAPIA and rapid tests) could be detected shortly afterskin testing (Waters et al., 2004, 2005; Harrington et al., 2008; Buddle et al., 2010). However, assays of repeated comparative skin testing in red deer at 6 months interval confirmed that it did not affect serological results (Che’ Amat et al., 2016).. 39.

(56) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. 7.4.4. Sample and sampling related factors The in vitro production of IFN-γ is influenced by the blood storage temperature and duration of storage until processing (recommended storage temperature is 4°C and maximum time for processing is 24 hours), as well as the type of anticoagulant (optimal response with heparin) (Bosward et al., 2010; Gerace et al., 2018). Source of the sample can also cause variation in diagnostic test results; being observed that serological test results for hunter or veterinarian harvested samples had higher level of agreement with culture results than samples from carcasses with TBL (O’brien et al., 2008). Both, repeated freeze thawing and haemolysis, can affect the diagnostic technique (Boadella and Gortázar, 2011). High levels of haemolysis decreased antibody test Se and this effect was more evident for the bPPD ELISA in red deer, however rapid tests are neither get affected by hemolysis in red deer (Boadella and Gortázar, 2011) or white-tailed deer (O’brien et al., 2009), which are designed for use even with whole blood. 7.4.5. Diagnostic technique related factors The Se and Sp of a test predominantly depends on the type of antigen used. Use of specific or purified antigens improves diagnostic accuracy (please, see Tables 2-5). There can be technical variation in skin test results depending on the person who performs the test (Fernández-de-Mera et al., 2009) and with the use of McLintock syringe that can cause non-specific reaction (Roy et al., 2019).In IGRA, there is variation in Se depending on the type of assay used to measure IFN-γ (ELISA/ELISPOT) (Lalvani and Pareek, 2010) and the use of maintenance media for samples can be compromised with the delay in processing the samples (Gerace et al., 2018). In the humoral diagnostic tests, species-specific conjugate (anti-IgG pig) gave better performance in ELISA than protein G conjugate in wild boar (Che’Amat et al., 2015). The strong IgG responses in wild boar appear in agreement with the reported observation that Ig heavy and light chains were up-regulated during M. bovis infection (Naranjo et al., 2006). 40.

(57) Introduction. On the other hand, reference tests used have a major influence on the diagnostic value of the tests. The gold standard test, mycobacterial culture has been widely used in validation; however, it has low Se and heavy reliance on the number and quality of tissues examined at necropsy (Chambers, 2013). In some studies, skin test or presence of TBL was used as reference standards in deer and swine (Griffin et al., 1991; Boadella et al., 2012a; Kang et al., 2016) or Bayesian analytical technique is proved to be a good alternative (Shury et al., 2014). 7.5. Improved diagnosis 7.5.1. Selection of the appropriate test The selection of an appropriate test is based on many factors like species being tested, stage of the disease, diagnostic accuracy of the test, economic feasibility and ease of performing the test, etc. Skin test has limited application in suidae (Jaroso et al., 2010b), whereas it is the standard ante mortem screening test in deer (OIE, 2009). Stage of the disease is a major factor involved in the selection of a test, being CMI based tests more accurate in identifying the early stages of infection in contrast to antibody based tests which are useful in later stages (Chambers, 2013). The antigen MPB83 is detected early in the course of experimental MTC infections (Waters et al., 2006, 2010), unlike MPB70, that elicits a humoral response to MTC in the later stages of the disease (Fifis et al., 1992; Bezos et al., 2014). On the other hand, CMI tests can only be applied in diagnosis of live animals, while antibody based ones can be applied in both live and dead animals. Among the latter, the ELISA techniques are useful for evaluating a large number of samples (Lyashchenko et al., 2008) whereas rapid tests are easy to perform and give rapid results, but these are not economically viable for screening large number of field samples. All these factors must be taken into consideration while choosing a diagnostic test for the detection of TB.. 41.

(58) Tuberculosis in wildlife: new diagnostic tests and host response to vaccination in red deer. 7.5.2. Proper implementation and interpretation of the test Proper implementation of the test procedure is very important to minimize the diagnostic errors as well as for better results. The Se and Spare the major factors evaluated for assessing the test result, being a highly Se test related to a low Sp and vice versa (Aurtenetxe et al., 2008).In addition, predictive values, likelihood ratio and diagnostic odds ratio have importance in interpreting the results. A higher prevalence tends to lead to an increased positive predictive value (PPV) and a decreased negative predictive value (NPV), whilst a lower prevalence tends to lead to an increased NPV and a decreased PPV (Okeh and Okoro, 2012). 7.5.3. Combination of diagnostic tests Combination of two diagnostic test results can enhance the diagnostic accuracy. Two test results can be combined in parallel (Shury et al., 2013) and in series (Jaroso et al., 2010a). Parellel testing is a method in which two screening tests performed at the same time and the results are subsequently combined, resulting in higher Se, but lower Sp. Usually, CMI based tests are interpreted in parallel to antibody based tests resulting in very high Se which aids to detect a maximum number of animals in different stages of disease and thereby facilitating test and removal strategies for the disease control in livestock (Casal et al., 2017; Bezos et al., 2018). In the same way, combination of SICCT and Cervid TB STAT-PAK allowed the detection of all M. bovis confirmed by culture in fallow deer (Jaroso et al., 2010a), and the combination of LST and Cervid TB STAT-PAK improved the results in elk deer (Shury et al., 2014). The use of multiple antigens in the same diagnostic test (IGRA, ELISA or rapid tests) and its further interpretation improves the diagnostic accuracy. On the other hand, two diagnostic tests can be performed sequentially in which second screening test is performed only if the result of the first screening test is positive (serial testing). Pathological lesions and culture results are usually considered in series to determine true infection status which minimizes the number of false positives (Jaroso et al., 2010a). 42.