Intestinal parasites in Rupicapra spp. populations: study in the framework of the relevant italian project (PRIN)

Intestinal parasites can seriously threaten the performances and well-being of wild ungulates. In this study, we investigated the occurrence and parasitic burden of protozoans and gastro-intestinal helminths (GIH) in Rupicapra spp. From September 2013 to January 2016, 352 fresh fecal samples were collected from Rupicapra rupicapra rupicapra in the Alps (N=262) and from Rupicapra pyrenaica ornata in the Apennines (N=90). Samples were examined using standard copro-parasitological methods for Eimeria and GIH and an immunofluorescence test for Cryptosporidium and Giardia duodenalis. Parts of gp60 and ssRNA/gdh/βgiardin genes were used to identify these protozoa species/genotypes. In R.r.rupicapra and in R.p.ornata, 7 and 6 parasite taxa were identified, respectively, with a mean number of 1.7 species/host (min-max 0-5) and 2.05 (min-max: 0-4), respectively. Overall, 85.3% (95%, C.I.=81.5-89.1) of the animals investigated scored microscopically positive to Eimeria spp. with a mean intensity of emission (m.i.e.) of up to 776 o.p.g.; 5.4% (95%, C.I.=3.07.7) were positive to G. duodenalis and 82% (95%, C.I. 77.91-86.15) to GIH with a m.i.e. of up to 147 e.p.g. Prevalence in R.r.rupicapra was 81.2% with a m.i.e. of 380 o.p.g. for Eimeria, 6.87% for Giardia, and 77.45% for GIH with a m.i.e. of 142 e.p.g. Prevalence in R.p.ornata was 94.4% with m.i.e. of 1,093 o.p.g. for Eimeria, 1.1% for G.duodenalis, and 94.4% for GIH with a m.i.e. of 151 e.p.g. Assemblages A/AI and E were identified in R.r.rupicapra and assemblage A/AIII in R.p.ornata. None of the animals tested positive for Cryptosporidium. The results show that the prevalence of Eimeria, G.duodenalis and GIH in both host species is nonnegligible, with a significantly higher parasitic burden in R.p.ornata. The detection of G. duodenalis in Rupicapra spp. is noteworthy. This study updates the data on parasitic fauna of these wild bovids. The impact of these parasites on chamois population dynamics will be inferred from the results/variables obtained throughout the entire interdisciplinary project

High prevalence and genetic diversity of Treponema paraluisleporidarum isolates in European lagomorphs

The bacterium Treponema paraluisleporidarum causes syphilis in lagomorphs. In a set of 1,095 samples from four species—European brown hare, mountain hare, Corsican hare, and European rabbit—we tested for infection and genotyped the strains that infect wild lagomorphs. Samples originate from Sweden, the Netherlands, the United Kingdom, Germany, the Czech Republic, and Italy. The phylogenetic analyses of two informative gene targets (tp0488 and tp0548) showed high genetic diversity among the lagomorph-infecting treponemes. More specifically, we found a high number of nucleotide variants and various short repeat units in the tp0548 locus that have not been described for human syphilis and primate yaws causing Treponema pallidum. While the functional aspect of these short repeat units remains subject to ongoing investigations, it likely enables the pathogen to better survive in its lagomorph host. Our data did not support any geographic clustering, which is equally reflected in the host population genetics as shown by mitochondrial genome data corresponding to the sampled lagomorph populations. This is unexpected and in contrast with what has been shown for nonhuman primate infection with T. pallidum. In the future, the combination of multi-locus sequence typing and whole genome data from modern and ancient samples from a wide geographic range and multiple lagomorph species will contribute to a better understanding of the epidemiology and evolutionary path of lagomorph-infecting treponemes. In conclusion, our current study demonstrates widespread infection and a high genetic variation of the syphilis-causing pathogen in a higher number of positively PCR-tested European lagomorphs (n = 302/1,095)

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed

Pathology and distribution of trombiculosis in northen chamois (rupicapra rupicapra rupicapra) in the Italian Alps

Trombiculosis has been reported in some wild ruminant species. We investigated the occurrence of trombiculosis in the northern chamois (Rupicapra rupicapra rupicapra) in the Western Italian Alps, and we describe the related histopathologic changes. Superficial scrapes and skin samples were taken from 191 chamois from the Lombardy and Piedmont regions during the hunting season in September–December 2015. Numbers of eosinophils, mast cells, cluster of differentiation (CD)3-, CD79a-, CD68-positive cells were determined on immunohistochemically stained skin sections using a semiautomatic analysis system. Forty (20.9, 40/191) chamois were positive for trombiculid larvae on skin scrapings. Of the positive animals, 15 were from Lombardy and 25 from Piedmont, with similar prevalences. Macroscopic lesions were light with involvement of body regions that had contact with the ground, especially head (pinnae and areas around eyes and mouth) and limbs, where stylostome was easily formed due to thin skin. Histologically, trombiculosis caused a focal moderate dermatitis with epidermal necrosis, thin crusts, and hyperkeratosis. Inflammatory infiltrates were suggestive of a granulomatous reaction centered on a stylostome, formed by mite saliva and necrotic host tissue debris. However, we detected some difference in cutaneous immune response with some chamois showing a prevalent T-cell response and others having an increased Bcell count associated with a higher number of eosinophils, mast cells and a lower number of T cells

Detection of Zoonotic Cryptosporidium ubiquitum in Alpine Wild Ruminants

Cryptosporidium is a widespread apicomplexan protozoan of major zoonotic importance, characterized by a wide host range, and with relevant economic implications and potential negative effects on livestock and wildlife population dynamics. Considering the recent strong demographic increase of alpine ungulates, in this study, carried out in the Italian Northwestern Alps, we investigated the occurrence of Cryptosporidium spp. in these species and their potential involvement in environmental contamination with Cryptosporidium spp. oocysts. The immune-enzymatic approach revealed a Cryptosporidium prevalence of 1.7% (5/293), 0.5% (1/196) and 3.4% (4/119) in alpine chamois (Rupicapra rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), respectively. Positive samples were subjected to polymerase chain reaction (PCR) amplification for the COWP and gp60 genes. The presence of Cryptosporidium was confirmed in one chamois and four roe deer. Sequences obtained clustered within Cryptosporidium ubiquitum, currently recognized as an emerging zoonotic species. This finding represents the first detection of zoonotic C. ubiquitum in chamois and in the Alpine environment. Despite the low observed prevalences, environmental contamination by oocysts could play a role as a potential source of infections for humans and livestock

Detection of Zoonotic Cryptosporidium ubiquitum in Alpine Wild Ruminants

Cryptosporidium is a widespread apicomplexan protozoan of major zoonotic importance, characterized by a wide host range, and with relevant economic implications and potential negative effects on livestock and wildlife population dynamics. Considering the recent strong demographic increase of alpine ungulates, in this study, carried out in the Italian Northwestern Alps, we investigated the occurrence of Cryptosporidium spp. in these species and their potential involvement in environmental contamination with Cryptosporidium spp. oocysts. The immune-enzymatic approach revealed a Cryptosporidium prevalence of 1.7% (5/293), 0.5% (1/196) and 3.4% (4/119) in alpine chamois (Rupicapra rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), respectively. Positive samples were subjected to polymerase chain reaction (PCR) amplification for the COWP and gp60 genes. The presence of Cryptosporidium was confirmed in one chamois and four roe deer. Sequences obtained clustered within Cryptosporidium ubiquitum, currently recognized as an emerging zoonotic species. This finding represents the first detection of zoonotic C. ubiquitum in chamois and in the Alpine environment. Despite the low observed prevalences, environmental contamination by oocysts could play a role as a potential source of infections for humans and livestock