Preliminary Seroepidemiological Investigation Regarding Mycobacterium Avium Infection in Wild Boars and Foxes

1 1 2 1 Ioana Alexandra RĂȚOI1 *, Luciana Alexandra CRIVEI1 , Daniela POREA , Anca Mihaela1 DASCĂLU1 , Andreea Paula COZMA1 , Luanda Elena OȘLOBANU , Dragoș Constantin ANIȚĂ , Adriana ANIȚĂ , Gheorghe1 Department SAVUȚA of Public Health. The University of Agricultural Sciences and Veterinary Medicine Iasi, . 2 Centre for the Study of Transborder and Emergent Diseases and Zoonoses Department, Danube Delta National Institute for Research and Development, Tulcea, Romania *Corresponding author: [email protected]

Bulletin UASVM Veterinary Medicine 77(2)/2020 Print ISSN 1843-5270; Electronic ISSN 1843-5378 doi:10.15835/buasvmcn-vm:2020.0010

Abstract Mycobacterium avium Sus scrofa Vulpes vulpes The present study aimed at identifying seroprevalence in wild boars ( ) and red foxes ( ) samples, from the Eastern region of Romania, by using an indirect ELISA assay. A total of 367 wild animal samples were collected from eight counties: 275 wild boars sera samples from Iasi (n=209), Botosani (n=11), Bacau (n=17), Galati (n=27) and Covasna (n=11) and 92 fox samples of thoracic fluid from Iasi (n=30), Suceava (n=20), Neamt (n=19), Vaslui (n=11) and GalatiMycobacterium (n=12). Specific avium antibodies were not detected in wild boar serum samples tested. From the fox samples, one was positive (1.08%), originating from Galati County. Our results may be correlated with a reduced distribution of subspecies in the environment and a low prevalence of infections caused by these bacteria in wild animals. The current assessment shows that foxesKeywords: have a limited Mycobacterium role in the epidemiology avium, of nontuberculous mycobacterial infections.

seroprevalence, wildlife

Introduction et al et al Mycobacterium avium conductedet inal various countries as Scotland (Beardet al Infections caused by nontuberculous myco­ ., 2001), Spain (BoadellaSus scrofa., 2011), Portugal bac­teria (NTM) of the Com- (Matos ., 2015) or Norway (Tryland ., plex (MAC) were reported in variousMycobacterium domestic or 2004). The wild boars ( ) are amongst aviumwild species, ruminant,paratuberculosis omnivorous or carnivoMyco- the species with a large territorial distribution in bacteriumrous. MAC aviummembers, mostavium notably Europe. NTM infection and dissemination can be subsp. (MAP) and possible in wild boars, considering the species subsp. (MAA), are the most adaptability to environmental changes and the commonly reported species, causinget inal some wild- ability to cross natural barriers, the omnivorous life species similar lesion aspects to those encoun- etdiet, al the contact with many other wild and domestic tered in domestic animals (Thorel ., 2001). animal species and the wide distribution (Boadella Evidence of natural infection with MAC ., 2011). Numerous studies have highlighted members in wild animals is provided by studies the role of feral pigs in maintaining and spreading 166 et al.

RĂȚOI Mycobacterium bovis et al et al etmycobacteria al from MAC and The purpose of this study was to conduct a (Trcka ., 2008; Naranjo ., 2008, Richomme seroprevalence study regarding the presence of ., 2019). The lesions are rarely observed, anti-MAC antibodies in wild boars and wild foxes, more frequently in adult animals and are usually from seven counties located in Eastern Romania present in the lymph nodes. MAC infections in and Materialsone in the centraland methods region of the country. carnivores are most often localized at the digestive tract and occur as a result of ingestion of the bacilli during feeding, environmental contamination In this study, 275 randomly collected wild being unlikely. Small rodents, birds and rabbits boar sera samples from the following counties included in fox diet are the most likely the source in Eastern Romania were tested: Iasi (n=209), of infection. Botosani (n=11), Bacau (n=17), Galati (n=27), Determining the prevalence of nontuberculous along with eleven samples from Covasna County. mycobacterial infections among wild animal Sampling was performed during the 2017-2018o populations provides significant additional hunting seasons, sera being stored at -20 C, until information on the distribution and transmission the time of testing. Age, gender and the origin of these infections. In the context of a common hunting fund of wild boars from Iasi County are habitat, where there may be interactions between presented in Table 1. These data were not available cattle and wild animals and taking into account the for the wild boars Vulpesin the othervulpes counties. opportunistic infectious capacity of the NTM, their In addition, 92 samples of thoracic fluids resistance in the environment and the possibility of from wild foxes ( ) were tested. Fox developing a sensitisation to tuberculin condition samples were collected during 2015, after rabies in cattle, information regarding the epidemiology vaccination campaign and previously tested for of NTM infection in wildlife is of valuable use in effectiveness of oral rabies vaccinations. The study assessment of the tuberculosis control program included fox samples originated fromMycobacterium five counties efficiency. Furthermore, the evaluation of the aviumin N-E area of Romania (Figure 1). health status of wild animal populations should For the identification of anti- not be neglected. IgG antibodiesMycobacterium in serum avium samples and thoracic To our knowledge, no serological investigations fluids, an indirect ELISA commercial kit was used have been carried out so far regarding infections (ID Screen® Indirect Multi- with MAC members in wild animals, in Romania. species, ID.vet, Innovate Diagnostics, France).

Figure 1.

Bulletin UASVM Veterinary Medicine 77 (2) / 2020 Geographic origin of the fox samples Mycobacterium Avium 167

Preliminary Seroepidemiological Investigation Regarding Infection in Wild Boars and Foxes Table 1

. Data regarding the distribution and origin of the samples from Iași County Gender Age category Number of tested Hunting fund (HF) samples /funds M F Yearling Juvenile Adult

1 Tatarusi 1 1 1 2 Motca 2 2 2 3 Miroslovesti 2 2 2 4 Halaucesti 1 1 1 5 Stolniceni Prajescu 4 4 4 6 Stroesti 1 1 1 7 Harmanesti 1 1 1 8 Siretel 2 2 2 9 Bahlui 24 8 16 8 16 10 Catalina 3 2 1 2 1 17 Gropnita 1 1 1 22 Turia Perieni 23 11 12 6 17 23 Larga Jijia 2 1 1 2 24 3 2 1 3 25 Letcani 2 2 2 27 Victoria 8 4 4 1 7 28 Golaesti 3 2 1 1 2 29 Tutora 2 2 2 30 Prisacani 4 4 4 31 3 3 3 32 Crivesti 5 3 2 4 1 33 Braiesti 6 2 4 6 35 Sinesti 4 4 4 37 Gheorghitoaia 8 8 3 5 38 Horlesti 4 2 2 2 2 40 Mogosesti 7 2 5 1 2 4 41 Barnova 1 1 1 42 Poieni 14 4 10 1 3 10 43 4 2 2 2 2 44 Pietrosu 7 1 6 3 4 45 Pocreaca 2 1 1 2 46 Bradicesti 3 3 3 47 Bunesti 2 1 1 1 1 48 Crasnita 14 11 3 1 2 11 49 4 3 1 1 3 50 Raducaneni 7 5 2 7 51 16 11 5 3 13 52 4 1 3 2 2 53 Tibana 1 1 1 54 Tibănesti 4 2 2 2 2 Bulletin UASVM Veterinary Medicine 77 (2) / 2020 168 et al.

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et al Sample testing was performed according to the hosts or super-shedders for bovine tuberculosis manufacturer’sMycobacterium specifications. avium The. testhominissuis presents a (Richomme ., 2019). wide applicability, not only to detect the circulation Studies on MAC infection in wild boar of subsp at populations are quite a few. The wild boar is an herd level, in domestic pigs, but can also be used omnivoreLepus europeus which, in the wild, occasionallyIthaginis feeds on serum and plasma samples from a variety of on small animalsPerdix perdixand birds, such as rabbits domestic (sheep, goats, cattle and pigs) and wild ( ), pheasants ( spp.), animals. According to the values etprovided al by the partridges ( ) or young ruminants. manufacturer and preliminary studies (Lesceu The wild boar also feeds on remains etof alcarcasses and Pourquier, 2011; Eisenberg ., 2012), the from dead wild animals or domestic animals used ELISA method presents a sensitivity ranging improperly disposed of (Machackova ., 2003). from 34.48% to 44% and a specificity of 100%. Thus, mycobacterial infection may be transmitted The plates were read at 450nm wavelength, through both respiratory (during digging) and using the ELISA Sunrise™ Absorbance Reader digestive (ingestionet al of debris from contaminated (Tecan Group Ltd., Männedorf, Switzerland). The pastures, offal from infected dead animals) routes evaluation of the obtained results was performed (Machackova ., 2003). according to the protocol formulas, samples In general, a low frequency of MAP is expected being considered negative if the Cut-off was in wild boars, up to 5% according to the resultset lower than 40% and positive if higher than 50%. alreported by Alvarez inet Spainal or Machackova in Confidence intervals were calculated using the on- various countries in central Europe (Alvarez line platform VassarStats: Website for Statistical ., 2005, Machackova ., 2003). Moreover, it is ComputationResults and(http://vassarstats.net/). discussions considered that in areas where other mycobacterial etinfections al are prevalent,et wild al boars are not the most relevant marker for paratuberculosis (Carta Following the serologicalMycobacterium testing of the avium 275 ., 2013). Boadella . (2011) evaluating wild boar sera, with the use of a commercial the presence for MAP antibodies (PPA 3) in wild indirect ELISA kit, no anti- boars’ sera, using an ELISA method, suggested antibodies were detected in evaluated serum that wild boars cannot be consideredet alsentinels samples. As such, no correlation could had been for MAC infections. MAA infection in wild boars is made with gender and age.Mycobacterium Only one fox sample avium very difficult to detect, however Lara . (2011) from Galati County was detected positive (1.08%, have succeeded in isolating the mycobacteria from CI 95%: 0,1% - 5.9%) for , mesenteric and submaxillary lymph nodes. antibodies. The lack of seroconversion revealed by this Amongst MAC members MAP is the nontuber­ study could beet explained al by a real low level of culous mycobacteria with the greatest negative exposure to MAC mycobacteria. Using the same impact on animal welfare.et al The importance of method, Matos . (2015) identified a prevalence wildlife in paratuberculosis transmission is so far of 23.9% for the wild boar tested in Portugal. recognizedSuidae (Stevenson ., 2009), theCanidae aetiological Moreover, our results can be correlated with a lower agent being isolated from different wild species, number of paratuberculosis outbreaks (n=27) including -wild boars and -foxes identified between 2014-2018 in our country and (EFSAMycobacterium AHAW Panel, bovis 2017). reported in the World Animal Health Information Wild boarset al are known to be relevantet al reservoirs System (WAHIS) portal of World Organisation for in the Iberian Peninsula for Animal Health (WOAH-OIE). Moreover, in the (Aranaz ., 2004, Boadella et al., 2011), N-E region of the country, only one outbreak was especially in Spain, where game-management reported in Iasiet alCounty (1 outbreak in 2015) and practices are common (Naranjo ., 2008). two in Botosani (two outbreaks in 2016). Moreover, tuberculous mycobacteria were isolated Garrido . (2010) observed that in case from this species in other European countries also of MAA experimental infection in wild boars, and depending on the epidemiological context, clinical signs are absent, and lesions occur in theet wild boars may act as spill-over hosts, maintenance head and mesenteric6 lymph nodes at an infective Bulletin UASVM Veterinary Medicine 77 (2) / 2020 dose of 2.5x10 mycobacterial cells (Garrido Mycobacterium Avium 169

Preliminary Seroepidemiological Investigation Regarding Infection in Wild Boars and Foxes al

., 2010). These observationset al are reinforced by the research in the context of nontuberculous the lesions observed by other authors in natural mycobacterial infections in wildlife, and further infections (Machackova ., 2003). In addition, analysis is considered appropriate in order to it was suggested that depending on the path observe whether in our country the mycobacterial of infection, respiratory (in close contact with infection shifted in wildlife direction and even if shedders) or digestive tract (consumption of thereAcknowledgments. is an underestimation in livestock. infected tissues), the development of the immune response etis aldifferent and may be correlated with This work was undertaken the mycobacterial infection manifesting potential by the project team involved in the POC grant (Garrido ., 2010). entitled: “Regional Center of Advanced. Research The use of serological methods for MAC for Emerging Diseases, Zoonoses and Food Safety infections evaluation in wild mammals is not – ROVETEMERG”,References Smis 107563 widely etperformed, al making a difficult analysis of our results. Using the same testing procedure, 1. MycobacteriumAlvarez J, de Juanavium L, Brionesparatuberculosis V, Romero B, Aranaz Matos . (2015) identified a single positive foxet A, Fernandez-Garayzabak JF, Mateos A (2005). alsample out of 42et tested al (2.4%). MAP was isolated subsp. in fallow or identified by PCR in foxes in Europe (Beard deer and wild boar in Spain. Veterinary Record, 156: 212– ., 2001,et Floroual ., 2008). In Portugal, infection 213. with MAP is common among wild carnivores 2. Aranaz A, de Juan L, Montero N, Sánchez C, Galka M, (Matos ., 2014). DelsoMycobacterium C, Alvarez bovisJ, Romero B, Bezos J, Vela AI, Briones V, Mateos A, Domínguez L (2004). Bovine tuberculosis Furthermore, in countries where the paratu­ ( ) in wildlife in Spain. Journal of ber­culosis prevalence is higher, evidence of clinical microbiology, 42(6): 2602–2608. infection is encountered in wild animal hosts. 3. Beard PM, Daniels MJ, Henderson D, Pirie A, Rudge K, Our results may be correlated with a reduced Buxton D, Rhind S, Greig A, Hutchings MR, McKendrick I, distribution of MAC members in the environment Stevenson K, Sharp JM (2001). Paratuberculosis infection of nonruminant wildlife in Scotland. Journal of clinical and a low prevalence of infections caused by microbiology, 39: 1517–1521. these bacterial agents in animals, either wild or 4. Boadella M, Lyashchenko K, Greenwald R, Esfandiari domestic. A lack of involvement or a limited role J, JarosoMycobacterium R, Carta T, Garrido bovis JM, VicenteMycobacterium J, de la Fuente avium J, of foxes in the epidemiology of nontuberculous Gortázar C (2011).paratuberculosis Serologic tests for detecting antibodies mycobacterial infections is revealed from the againstSus scrofa scrofa and current assessment. As in many grazing areas subspecies in Eurasian wild boar ( ). Journal of Veterinary Diagnostic from the assessed counties cattle, sheep and goats Investigation, 23(1): 77–83. are raised in extensive systems, the common 5. Carta T, Álvarez J, Pérez de la Lastra JM, Gortázar C (2013). interface between domestic and wildlife is Wildlife and paratuberculosis: a review. Research in created. Furthermore, as wild lagomorphs are Veterinary Science, 94(2): 191–197. linked to foxes and considered to pose the highest 6. EFSA AHAW Panel (EFSA Panel on Animal Health and MAP transmission risk for livestock, of the non- Welfare): More S, Bøtner A, Butterworth A, Calistri P, ruminant hosts, a more accurate epidemiologic Depner K, Edwards S, Garin-Bastuji B, Good M, Gortazar Schmidt C, Michel V, Miranda MA, Nielsen SS, Raj M, situation may be revealed by extending the number Sihvonen L, Spoolder H, Stegeman JA, Thulke H-H, of fox samples and continue the evaluation in other Velarde A, Willeberg P, Winckler C, Baldinelli F, Broglia species,Conclusions such as rabbits. A, Zancanaro G, Beltran-Beck B, Kohnle L, Morgado J, Bicout D (2017). Scientific Opinion on the assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No To the extent of our knowledge, this is the 2016/429): paratuberculosis. EFSA Journal, 15(7):4960, first MAC seroprevalence study conducted in our p. 47. country on wild animal species. Considering the 7. Eisenberg T, Volmer R, Eskens U, Moser I, Nesseler A, fact that serosurveys on MAC infection in wildlife Sauerwald C, Seeger H, Klewer-Fromentin K, Möbius are not widely performed, and only few published MycobacteriumP (2012). Outbreak avium of reproductivehominissuis disorders and mycobacteriosis in swine associated with a single strain of results are available, the comparison of our subspecies . Veterinary results proved to be difficult. Though negative, the microbiology, 159(1-2): 69–76. serologic response identified in this study opens Bulletin UASVM Veterinary Medicine 77 (2) / 2020 170 et al.

RĂȚOI Mycobacterium tuberculosis

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Bulletin UASVM Veterinary Medicine 77 (2) / 2020