Stefania Zanet

Piroplasmosis in wildlife: Babesia and Theileria affecting free-ranging ungulates and carnivores in the Italian Alps

BackgroundPiroplasmosis are among the most relevant diseases of domestic animals. Babesia is emerging as cause of tick-borne zoonosis worldwide and free-living animals are reservoir hosts of several zoonotic Babesia species. We investigated the epidemiology of Babesia spp. and Theileria spp. in wild ungulates and carnivores from Northern Italy to determine which of these apicomplexan species circulate in wildlife and their prevalence of infection.MethodsPCR amplification of the V4 hyper-variable region of the 18S rDNA of Babesia sp./Theileria sp was carried out on spleen samples of 1036 wild animals: Roe deer Capreolus capreolus (n = 462), Red deer Cervus elaphus (n = 52), Alpine Chamois Rupicapra rupicapra (n = 36), Fallow deer Dama dama (n = 17), Wild boar Sus scrofa (n = 257), Red fox Vulpes vulpes (n = 205) and Wolf Canis lupus (n = 7). Selected positive samples were sequenced to determine the species of amplified Babesia/Theileria DNA.ResultsBabesia/Theileria DNA was found with a mean prevalence of 9.94% (IC95% 8.27-11.91). The only piroplasms found in carnivores was Theileria annae, which was detected in two foxes (0.98%; IC95% 0.27-3.49). Red deer showed the highest prevalence of infection (44.23%; IC95% 31.6-57.66), followed by Alpine chamois (22.22%; IC95% 11.71-38.08), Roe deer (12.55%; IC95% 9.84-15.89), and Wild boar (4.67%; IC95% 2.69-7.98). Genetic analysis identified Babesia capreoli as the most prevalent piroplasmid found in Alpine chamois, Roe deer and Red deer, followed by Babesia bigemina (found in Roe deer, Red deer and Wild boar), and the zoonotic Babesia venatorum (formerly Babesia sp. EU1) isolated from 2 Roe deer. Piroplasmids of the genus Theileria were identified in Wild boar and Red deer.ConclusionsThe present study offers novel insights into the role of wildlife in Babesia/Theileria epidemiology, as well as relevant information on genetic variability of piroplasmids infecting wild ungulates and carnivores.

Epidemiology of Leishmania infantum, Toxoplasma gondii, and Neospora caninum in Rattus rattus in absence of domestic reservoir and definitive hosts

Isolated environments are privileged settings to study transmission of infection. Montecristo is a small island where no wild or domestic carnivores are present. Invasive Black rats Rattus rattus (n=78) were captured and tested by PCR for Leishmania infantum, Toxoplasma gondii and Neospora caninum. We wanted to test, for these parasites, the existence of a sylvatic cycle independent of reservoir or definitive hosts. None of the rats tested positive by PCR for either T. gondii or N. caninum. We recorded a 15.5% prevalence (CI95% 8-26%) of L. infantum in the rats and Phlebotomus mascittii was captured in Montecristo, leading us to identify it as possible vector of the parasite.

Encephalitozoon cuniculi, Toxoplasma gondii and Neospora caninum infection in invasive Eastern Cottontail Rabbits Sylvilagus floridanus in Northwestern Italy

Sylvilagus floridanus is a lagomorph introduced for hunting purposes from North America to Europe where, in certain areas like Northern Italy, its population reached high densities. Brain, kidney and skeletal muscle of 144 Eastern Cottontail Rabbits S. floridanus were examined by PCR for Encephalitozoon cuniculi, Toxoplasma gondii and Neospora caninum. DNA of E. cuniculi was found with a prevalence of 9.72% (CI 95% 0.058-0.156). T. gondii and N. caninum DNA was detected in 2.08% (CI 95% 0.0071-0.0595) and 2.78% (CI 95% 0.0109-0.0692) of the samples examined, respectively. This is the first report of E. cuniculi infection in a lagomorph species other than in its natural host Oryctolagus cuniculus, and this is also the first time N. caninum is found to naturally infect S. floridanus. E. cuniculi, T. gondii and N. caninum infect S. floridanus at low but relevant prevalences, considered the important role that these pathogens could play in both animal and human health.

Detection of rat hepatitis E virus in wild Norway rats (Rattus norvegicus) and Black rats (Rattus rattus) from 11 European countries

Rat hepatitis E virus (HEV) is genetically only distantly related to hepeviruses found in other mammalian reservoirs and in humans. It was initially detected in Norway rats (Rattus norvegicus) from Germany, and subsequently in rats from Vietnam, the USA, Indonesia, China, Denmark and France. Here, we report on a molecular survey of Norway rats and Black rats (Rattus rattus) from 12 European countries for ratHEV and human pathogenic hepeviruses. RatHEV-specific real-time and conventional RT-PCR investigations revealed the presence of ratHEV in 63 of 508 (12.4%) rats at the majority of sites in 11 of 12 countries. In contrast, a real-time RT-PCR specific for human pathogenic HEV genotypes 1-4 and a nested broad-spectrum (NBS) RT-PCR with subsequent sequence determination did not detect any infections with these genotypes. Only in a single Norway rat from Belgium a rabbit HEV-like genotype 3 sequence was detected. Phylogenetic analysis indicated a clustering of all other novel Norway and Black rat-derived sequences with ratHEV sequences from Europe, the USA and a Black rat-derived sequence from Indonesia within the proposed ratHEV genotype 1. No difference in infection status was detected related to age, sex, rat species or density of human settlements and zoological gardens. In conclusion, our investigation shows a broad geographical distribution of ratHEV in Norway and Black rats from Europe and its presence in all settlement types investigated.

Toxoplasma gondii in sympatric wild herbivores and carnivores: epidemiology of infection in the Western Alps

BackgroundToxoplasma gondii is an apicomplexan parasite that is able to infect almost all warm blooded animals. In Europe, the domestic cat is the main definitive host. Worldwide, 6 billion people are infected with this parasite. The goal of our research is to evaluate the prevalence of T. gondii infection in wild animals from a previously unsampled area in Northern Italy where 0.1% of women seroconvert during pregnancy each year.MethodsWe sampled and tested skeletal muscle and central nervous system tissue of 355 wild animals by PCR (n = 121 roe deer Capreolus capreolus, n = 105 wild boar Sus scrofa, n = 94 red fox Vulpes vulpes, n = 22 alpine chamois Rupicapra rupicapra, n = 13 red deer Cervus elaphus).ResultsThe overall prevalence of infection with T. gondii was 10.99% (confidence interval (CI) 95% 8.14%-14.67%). A higher rate of infection was recorded in carnivores and omnivores (red fox 20.21%, CI 95% 13.34%-29.43%; wild boar 16.19%, CI 95% 10.36%-24.41%) compared to ruminants (2.48%, CI 95% 0.85%-7.04% in roe deer; 0.00%, CI 95% 0.00%-22.81% in red deer, and 0.00% alpine chamois (CI 95% 0.00%-14.87%) confirming the importance of tissue cysts in transmitting infection.ConclusionsThe relatively high prevalence of T. gondii DNA in highly consumed game species (wild boar and roe deer) gives valuable insights into T. gondii epidemiology and may contribute to improve prevention and control of foodborne toxoplasmosis in humans.

Nosema ceranae has been infecting honey bees Apis mellifera in Italy since at least 1993

(2013). Nosema ceranae has been infecting honey bees Apis mellifera in Italy since at least 1993. Journal of Apicultural Research: Vol. 52, No. 2, pp. 60-61.

The use of loop-mediated isothermal amplification improves Toxoplasma gondii detection in wildlife

Toxoplasma gondii is among the most widespread parasites worldwide. Wildlife is recognized as an important reservoir and source of infection of T. gondii. The goal of the present work was to assess the performance of loop-mediated isothermal amplification (LAMP) as a diagnostic tool for T. gondii infection in the skeletal muscle and central nervous system (CNS) of free-ranging ungulates and carnivores. Fifty-seven wild animals were tested for the presence of T. gondii DNA by polymerase chain reaction (PCR) and LAMP. The use of LAMP amplification improved sensitivity in T. gondii molecular detection compared with conventional PCR on skeletal muscle (χ2 = 5.8, P < 0.05), having a lower minimum detection limit (0.1 tachyzoite) than PCR (1 tachyzoite). No significant differences existed between the detection capacities of both assays when performed on CNS. LAMP is a valid tool to improve the diagnosis of T. gondii infection in wild game meat. The technique provides a sensitive yet specific method that can be applicable to both field surveys and large-scale testing of wildlife samples.

Leishmania infantum (Trypanosomatida: Trypanosomatidae) phlebotomine sand fly vectors in continental Mediterranean Spain.

ABSTRACT Leishmania infantum—the causal agent of human and canine leishmaniasis in the Mediterranean basin-remains the most important of the phlebotomine sand fly-borne pathogens in the area. However, information on phlebotomine sand flies in certain European regions remains scarce and consequently epidemiological modeling, risk prediction, and disease control are difficult. Thus, we aimed to investigate the presence and distribution of phlebotomine sand fly vectors of L. infantum in an endemic region of continental Mediterranean Spain. Climatically stratified trapping of phlebotomine sand flies was performed over 39 points in south- central Spain. Later on, the effect of ecogeographical variables—geography trend, climate, habitat, and hosts—over the abundance of the predominant species—Phlebotomus perniciosus Newstead, 1911—was analyzed. Polymerase chain reaction was performed over pools of the captured species to search for L. infantum DNA. There were 152 phlebotomine sand flies (142 Ph. perniciosus and 10 Phlebotomus ariasi Tonnoir, 1921) captured. Model results showed that Ph. perniciosus abundance is expected to be higher in warm agricultural areas within the study region in agreement to previous findings in other climatic regions. Molecular analyses revealed the presence of L. infantum DNA in pools from locations in the study region displaying the highest abundance of phlebotomine sand flies. These findings suggest that along mainland Spain, warm agricultural landscapes are more prone to harbor higher abundances of Ph. perniciosus and account for a higher risk of exposure to L. infantum.

Horses infected by Piroplasms different from Babesia caballi and Theileria equi: species identification and risk factors analysis in Italy

Equine Piroplasmosis (EP) caused by Theileria equi and Babesia caballi is a disease affecting the health and the international movement of horses. In order to assess prevalence of Piroplasmid infection in the Northwestern part of Italy and to evaluate the associated risk factors, whole blood was collected from 135 horses from 7 different stables across the study area. PCR and sequencing were used to assess prevalence of infection and to identify detected Piroplasms to species level. A total of 23 horses (P=17.04%; CI95%: 10.70-23.38%) was found to be infected with Piroplasms and T. equi was the most prevalent species, found in 18 animals (P=13.33%; CI95%: 7.60%-19.07%). Although B. caballi was never detected, the presence of parasites belonging to the genus Babesia was confirmed by sequencing in 5 horses, 3 of which were infected with B. canis (P=2.22%; CI95% 0.76%-6.33%), and 2 with B. capreoli (P=1.48%; CI95% 0.41%-5.24%). The natural reservoir hosts of B. canis and B. capreoli are the domestic dog and roe deer Capreolus capreolus respectively. These findings pose attention to the need of considering in future epidemiological and clinical studies, other Apicomplexan species as able to infect horses.

Higher risk of gastrointestinal parasite infection at lower elevation suggests possible constraints in the distributional niche of Alpine marmots

Alpine marmots Marmota marmota occupy a narrow altitudinal niche within high elevation alpine environments. For animals living at such high elevations where resources are limited, parasitism represents a potential major cost in life history. Using occupancy models, we tested if marmots living at higher elevation have a reduced risk of being infected with gastro-intestinal helminths, possibly compensating the lower availability of resources (shorter feeding season, longer snow cover and lower temperature) than marmots inhabiting lower elevations. Detection probability of eggs and oncospheres of two gastro-intestinal helminthic parasites, Ascaris laevis and Ctenotaenia marmotae, sampled in marmot feces, was used as a proxy of parasite abundance. As predicted, the models showed a negative relationship between elevation and parasite detectability (i.e. abundance) for both species, while there appeared to be a negative effect of solar radiance only for C. marmotae. Site-occupancy models are used here for the first time to model the constrains of gastrointestinal parasitism on a wild species and the relationship existing between endoparasites and environmental factors in a population of free-living animals. The results of this study suggest the future use of site-occupancy models as a viable tool to account for parasite imperfect detection in eco-parasitological studies, and give useful insights to further investigate the hypothesis of the contribution of parasite infection in constraining the altitudinal niche of Alpine marmots.