Toxoplasma gondii infection in alpine red deer (Cervus elaphus): Its spread and effects on fertility

In contrast to the depth of knowledge on the pathological effects of parasitism in domestic animals, the impact of the vast majority of parasites on wildlife hosts is poorly understood and, besides, information from domestics is rarely usable to disclose the parasites' impact on free-ranging populations' dynamics. Here we use Toxoplasmosis as a study model since, until now, the infection process and the protozoan's effects in natural conditions has received little attention. We analysed 81 sera from red deer (Cervus elaphus) sampled in Italian Alps and through generalized linear models we evaluated (1) the epidemiological factors influencing T. gondii infection dynamics; (2) its impact on female fertility [corrected]. High seroprevalence of T. gondii infection was recorded in yearling (1 year-old; prevalence = 52.4%) and adult (>2 year-old; prevalence = 51.3%) red deer, while calves (<1 year-old) did not contract the infection suggesting horizontal transmission as the main route of infection. The stable prevalence between yearlings and adults and the higher serological titres of younger individuals lead to two alternative infection processes suggesting a difference between age classes or in acquiring the infection or in responding to the pathogen. No associations between T. gondii serological titres and pregnancy status was observed indicating no direct effect on the probability of being pregnant; nevertheless a relation between females' higher serological titres and lower foetal development emerged, suggesting potential effects of the parasite infection on deer reproduction. The results demonstrate high seroprevalence of T. gondii infection in free-ranging red deer and, furthermore, the effect on foetal development suggests the potential impact of the parasite on red deer fertility and thus on its population dynamics

Increased hormonal stress response of Apennine chamois induced by interspecific interactions and anthropogenic disturbance

Responses of animals to environmental changes and their interactions with other species play an important role in conservation. Sharing a common habitat may lead to interspecific competition for resources, but field assessment of these biological events is not always easily accomplished. By using a non-invasive method, we evaluated the physiological stress responses of Apennine chamois (Rupicapra pyrenaica ornata) to the presence of cattle, sheep and goat, red deer (Cervus elaphus), people (hikers), and predators to identify which factors may affect this endangered species. During September 2012, November 2012, and July 2013, a total of 318 faecal samples were collected in representative sites and analysed for faecal cortisol metabolites (FCM). FCM concentration was analysed through linear mixed-effect models. A significant increase in FCM values in Apennine chamois sharing their habitat with domestic animals was recorded during all study periods. On the contrary, stress responses to red deer and people were limited in time and emerged only during summer months, when hikers are more frequent and red deer extend their altitudinal range reaching chamois\u2019 habitat. The observed effects of domestic animals, red deer, and hikers should be considered in future Apennine chamois management plans, which should include the regulation of pastured domestic livestock, anthropogenic disturbances, and possible interferences with other wild species within parks

Molecular identification of cryptic cysticercosis : Taenia ovis krabbei in wild intermediate and domestic definitive hosts

The complex life cycle of taeniids represents an ideal model of a multi-host system. The complexity of these parasites can therefore cover the epidemiological issues of the interface between wild and domestic animals, especially once spatial overlap between wild and domestic definitive and intermediate hosts occurs. Here we use the occurrence of Taenia ovis krabbei in two model areas as an example of this epidemiological complexity. In two contiguous areas in the Italian northern Apennines, two hunted roe deer (Capreolus capreolus) showed numerous cysticerci in the muscles of their whole body and an adult tapeworm was recorded in a semi-stray dog (Canis lupus familiaris). Through molecular typing of the mitochondrial cytochrome c oxidase I (cox1) gene, cysticerci and the adult tapeworm of T. krabbei were identified. Taenia krabbei cysticercosis was recorded for the first time in Italy. Although the role of dogs in the parasite's life cycle emerges, the overlap between wild and domestic definitive hosts and the increase of wild population densities raise concerns about the temporal (old or new) introduction and the spread of this parasite by one of these canid species (wolf (Canis lupus) or dog). Although T. krabbei is not a public health issue, economic concerns emerged for hunters and meat producers, related to the damage of carcasses by cysticerci. Therefore, there is a need to evaluate the spread of T. krabbei in the intermediate and definitive host populations, and to ensure the relevant sanitary education for hunters in order to avoid practices that could favour the spread and maintenance of its life cycle

The cooler the better? Indirect effect of spring-summer temperature on fecundity in a capital breeder

Female reproductive performance is a central component of ungulate population dynamics, and it can be influenced by individual, social, and environmental factors. Researchers have often assumed direct effects of different predictors on reproduction, yet more complex relationships should be considered when investigating temporal variations in life\u2010history traits within a broader eco\u2010evolutionary context. In this study, we explored direct effects of individual, social, and environmental predictors on female reproductive performance and investigated potential causal chains among variables. We analyzed the variation in fecundity, measured as the probability of being pregnant, in 215 adult female deer Cervus elaphus culled on the Italian Alps, with respect to age, body mass, kidney fat, jaw length, lactation status, population size, temperature and precipitation in spring\u2013summer, temperature and snow depth in winter, and the delayed effect of spring\u2013summer temperature. We used random forest and logistic regression models to select variables whose direct effects best explained variation in fecundity. Path analysis was used to test for alternative hypotheses of direct/indirect effects between pre\u2010selected weather (spring\u2013summer temperature) and individual (age, kidney fat index [KFI]) predictors. The most important direct predictors of fecundity were age, kidney fat, and the interaction between kidney fat and spring\u2013summer temperature. Path analysis supported the hypothesis that higher spring\u2013summer temperature had negative, indirect effects on the probability of being pregnant, mediated by decreasing values of KFI. Our study revealed some complex, cause\u2013effect relationships between weather stochasticity, body condition, and reproduction, possibly suggesting a conditional trade\u2010off between opportunity for reproduction and survival, and emphasizing how environmental variations and individual characteristics may interact to shape life\u2010history traits in ungulate population

Torque Teno Sus Virus (TTSuV) Prevalence in Wild Fauna of Northern Italy

Torque teno sus virus (TTSuV) is a non-enveloped circular ssDNA virus which frequently infects swine and has been associated with hepatic, respiratory, and autoimmune disorders. TTSuV’s pathogenic role is still uncertain, and clear data in the literature on virus reservoirs are lacking. The aims of this study were to investigate the presence of potentially zoonotic TTSuV in wild animals in Northern Italy and to evaluate their role as reservoirs. Liver samples were collected between 2016 and 2020 during four hunting seasons from wild boars (Sus scrofa), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and chamois (Rupicapra rupicapra). Samples originated from areas in Northern Italy characterized by different traits, i.e., mountains and flatland with, respectively low and high farm density and anthropization. Viral identification was carried out by end-point PCR with specific primers for TTSuV1a and TTSuVk2a species. TTSuV prevalence in wild boars was higher in the mountains than in the flatland (prevalence of 6.2% and 2.3%, respectively). In wild ruminants only TTSuVk2a was detected (with a prevalence of 9.4%). Our findings shed light on the occurrence and distribution of TTSuV in some wild animal species, investigating their possible role as reservoirs

Molecular detection of Toxoplasma gondii from a naturally infected Alpine chamois (Rupicapra r. rupicapra) from Italian Alps

Background The protozoan Toxoplasma gondii affects many species of domestic (1; 2) and wild (3; 4; 5) warm-blooded animals,raising public health issues related to its zoonotic potential. In this sense wild ungulates may therefore be a source of T. gondii infection for consumers (raw, undercooked meat and fresh sausages) (6; 7; 8) and for hunters and slaughterers through manipulation, evisceration and handling of carcasses (9; 10; 11). Alpine chamois (Rupicapra r. rupicapra) is the most hunted wild ungulate in the Italian Alps with a significant increase of density in the last decades (12); as positiveresultsofserologicaltestingforT. gondii have been reported in the population from the Italian Alps(13; 14) and in southern chamois (Rupicapra pyrenaica) from Spanish Pyrenees (6), we investigated the presence of the protozoan DNA in brain tissues in order to define the receptivity of this species to T. gondii infection and its role in the protozoan lifecycle. Materials and methods During the hunting season 2011, 11 samples of chamois brain tissues were collected in the Lepontine Alps (VB). DNA extraction was performed with the QIAamp DNA Mini Kit (Qiagen, Italy). All the samples were assayed by targeting a 529 bp non-coding region (15), then the positive one was confirmed by a PCR-RFLP assay targeting the 18S small-subunit ribosomal gene of T. gondii, using primers that identify also Neospora caninum and Sarcocystis spp. (16). Results and Discussion T. gondii DNA was detectedin a six-year-old male chamois hunted at an altitudeof 1700 m.a.m.s.l..The subject was in a good body condition and its behaviour was normal;the post-mortem examination did not reveal any systemic macroscopic lesions. The protozoan DNA was detected by both PCR protocols.The PCR-RFLP restriction enzyme analysis of the amplified product confirmed the presence of Toxoplasma gondii, excluding eventual cross-reactions with N. caninum and Sarcocystis spp., closely related to T. gondii. As far as we know, this is the first detection of T. gondii DNA from Alpine chamois. This result confirms the Alpine chamois as intermediate host of T. gondii and demonstrates the protozoan presence in the Alpine ecosystem, even in remote areas. Considering the sporadic presence of linx in the Italian Alps, feral cats are the only definitive hosts of T. gondii, even if transplacental transmissioncan not be excluded. The impact on chamois population dynamics can not properly be evaluated without a better understanding of the epidemiology of infection. In addition, the consumption of raw or undercooked chamois meat could be a possible source of T. gondii infection in humans. In particular, the fact that T. gondii usually affects the host without producing clinical signs (17) could increasethe risk of human infection ascribed to theapparent healthiness of chamois meat. Perspectives and future research priorities Further analysis are needed to define the epidemiology of T.gondii,in particular performing serological study of antibodies against the parasite and the genotyping of the present and future PCR positives samples in order to define (a) prevalence of T. gondii infection in Alpine chamois populations, (b) which parasite strainsare circulating in this alpine ruminant, (c) its pathogenicity and the related zoonosis risk. 6) Acknowledgements We wish to thank all the hunters of the Alpine hunting district (VCO2) in the province of Verbania for their helpduring research in the field, Maria Chiara Cerutti for her useful technical contribution, Donatella Ghidotti and Marzia Marchionni for their helpfulness and collaboration in the lab activities, Ilaria Marangi for her invaluable suggestions that improved the drafting of this manuscript. References 1. Masala G., Porcu R., Madau L., Tanda A., Ibba B., Satta G., Tola S. 2003. Survey of ovine and caprine toxoplasmosis by IFAT and PCR assays in Sardinia, Italy. Veterinary Parasitology, 117: 15-21. 2. Dubey J.P. 1992. Isolation of Toxoplasma gondii from a Naturally Infected Beef Cow. The Journal of Parasitology, 78: 151-153. 3. Jokelainen P., Isomursu M., N\ue4reaho A., Oksanen A. 2011. Natural Toxoplasma gondii infections in european brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization. Journal of Wildlife Diseases, 47: 154-163. 4. Sobrino R., Cabez\uf3n O., Mill\ue1n J., Pab\uf3n M., Arnal M.C., Luco D.F., Gort\ue1zar C., Dubey J.P., Almeria S. 2007. Seroprevalence of Toxoplasma gondii antibodies in wild carnivores from Spain. Veterinary Parasitology, 148: 187-192. 5. B\ue1rtov\ue1 E., Sedl\ue1k K., Liter\ue1k I. 2006. Prevalence of Toxoplasma gondii and Neosporacaninumantibodies in wild boars in the CzechRepublic. Veterinary Parasitology, 142: 150-153. 6. Gauss C.B.L., Dubey J.P., Vidal D., Cabez\uf3n O., Ruiz-Fons F., Vicente J., Marco I., Lavin S., Gort\ue1zar C., Almer\ueda S. 2006. Prevalence of Toxoplasma gondiiantibodies in red deer (Cervuselaphus) and other wild ruminants from Spain. Veterinary parasitology,136: 193-200. 7. Ross R.D., Stec L.A., Werner J.C., Blumenkranz M.S., Glazer L., Williams G.A. 2001. Presumed acquired ocular Toxoplasmosis in deer hunters. Retina, The Journal of Retinal and Vitreous Diseases, 3: 226-229. 8. Dubey J.P. & Beattie C.P. 1988. Toxoplasmosis of Animals and Man.CRC Press, Boca Raton, Florida. 220 pp. 9. Kapperud G., JenumP.A.,Stray-Pedersen B.,Melby K.K., Eskild A., Eng J. 1996. Risk Factors for Toxoplasma gondii Infection in Pregnancy Results of a Prospective Case-Control Study in Norway. American Journal of Epidemiology, 144: 405-412. 10. Dubey J.P. 1994. Toxoplasmosis. Journal of the American Veterinary Medical Association, 205: 1593-1598. 11. McDonald J.C., Gyorkos T.W., Alberton B., MacLean J.D., Richer G., Juranek D. 1990. An Outbreak of Toxoplasmosis in Pregnant Women in Northern Qu\ue9bec. Journal of Infectious Disease, 161: 769-774. 12. Pedrotti L., Dupr\ue8 E., Preatoni D., Toso S. 2001. Banca Dati Ungulati. Status, distribuzione, consistenza, gestione, prelievo venatorio e potenzialit\ue0 delle popolazioni di Ungulati in Italia. Istituto Nazionale per la Fauna Selvatica \u201cAlessandro Ghigi\u201d, 115-129 pp. 13. Gaffuri A.,Giacometti M., Tranquillo V.M., Magnino S.,Cordioli P., Lanfranchi P. 2006. Serosurvey of Roe Deer, Chamois and Domestic Sheep in the CentralItalian Alps. Journal of Wildlife Diseases, 42:685-690. 14. Gennero M.S., Meneguz P.G., MandolaM., MasoeroL., De MeneghiD., RossiL.1993. Indagini sierologiche su ruminanti selvatici in Piemonte. Atti Societa` Italiana delle Scienze Veterinarie, 47: 979-983. 15. Homan W.L., Vercammen M., De Braekeleer J., Verschueren H. 2000. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. International Journal for Parasitology, 30: 69-75. 16. Magnino S., Vigo P.G., Bandi C., Colombo M., De Giuli L., Fabbi M., Genchi C. 1998. PCR diagnosis for Neosporacaninuminfection in aborted bovine foetuses and for Toxoplasma gondii infection in hares and goats in Italy. Pp. 1269-1272. In: Proceedings of the IX International Congress of Parasitology, MakuhariMesse, Chiba, Japan, 24-28 August 1998. 17. Marco I., Velarde R., L\uf3pez-Olvera J.R., Cabez\uf3n O., Pumarola M., Lav\uedn S. 2009. Systemic toxoplasmosis and Gram-negative sepsis in a southern chamois (Rupicaprapyrenaica) from the Pyrenees in northeast Spain. Journal of Veterinary Diagnostic Investigation, 21:244-247

GIARDIA, CRYPTOSPORIDIUM AND EIMERIA INFECTIONS IN ALPINE WILD UNGULATES: EPIDEMIOLOGICAL INVESTIGATION AND MANAGEMENT IMPLICATIONS

Monitoring and management of wildlife health are of increasing importance in the last decades, first of all considering that more of 70% of emergent zoonoses are host in wild animals. Moreover the public health issue must be seen also in accordance with the European food hygiene regulations (Reg. EC 852, 853, 854/2004), that make hunters primary producers intended to auto-consumption and/or commercialization. We have to consider wildlife health value also in relation to possible interactions with livestock and to population dynamics and biodiversity preservation. We focused our attention on protozoa, parasites with the highest success spreading, in particular on Giardia, Cryptosporidium and Eimeria in chamois (Rupicapra rupicapra rupicapra and Rupicapra pyrenaica ornata), red deer (Cervus elaphus) and roe deer (Capreolus capreolus). Faecal samples were collected, in the biennium 2013-2014, from Lepontine Alps and from central Apennine. Through immunofluorescence emerged Giardia prevalence of 5,82% in R. r. rupicapra, and of 1,85% in R. p. ornata. Positive samples were confirmed by q PCR and end-point PCR, with identification of zoonotic assemblage A and livestock assemblage E. A parallel survey was conducted through immunoenzimatic methodology. Giardia showed prevalences of 4,5%, 2,5% and 8,4% while Cryptosporidium of 1,2%, 0,5% and 3,4% respectively in chamois, red deer and roe deer. Positive samples were subjected to molecular investigations. Assemblage A of Giardia was detected in chamois and red deer, suggesting a potential zoonotic risk, although rather low. Assemblage E was detected in chamois, highlighting a trasmission of this parasite between livestock and wildlife. Concerning Cryptosporidium, immunoenzimatic positivities haven\u2019t been confirmed by PCR. Roe deer shows higher prevalences for both protozoa, statistical analyses about Giardia infection highlight a higher probability of calves being infected than adult, and higher significant probability to contract the infection in animals culled at lower altitudes, probably due to impact originated by human activities. Finally faeces were analysed by FLOTAC methodology in order to quantify emissions of coccidian oocysts and nematode eggs. Eimeria shows prevalence of 81,1% in chamois, 46,1% in red deer and of 43,4% in roe deer. Overall prevalences for gastro-intestinal nematodes are 76,4% in chamois, 57,4% in red deer and 67,1% in roe deer. Statistical analyses were carried out to define the epidemiological role of coccidian infections in wild ungulates in alpine context. Data collected about coccidia and gastro-intestinal nematodes don\u2019t allow a correct definition of simultaneous parasitosis, considering nematodes possibility to infect several hosts. About that Eimeria represents a useful model to better understand interaction among pathogen, host and environment at intraspecific level, because of its strict host-specifity At the moment comparison between data about all protozoa investigated and alpine data census don\u2019t suggest any impact on population dynamics. However it can not be completely excluded because of limits related to period of sampling that not always correspond to critical period for these infections in wild ungulates. We need to increase sampling season, compatibly with weather conditions, and to produce a data series about emissions of oocysts to better understand the eco-epidemiological role of protozoa in wildlife