Meteorological, demographic, and environmental risk factors for Leishmania infantum in wildlife.

Leishmania infantum is an important and neglected vector-borne zoonotic protozoa endemic in the Mediterranean basin. Several wild and domestic mammals can contribute to maintaining its circulation but their importance as effective reservoirs is still under discussion and varies depending on local ecological communities. By combining environmental, climatic, and individual information, this study assessed the presence of L. infantum DNA in a set of wild species from Northwestern Italy and the potential ecological factors related to the risk of infection. From 2020 to 2022, 304 free-ranging wild animals were analyzed for the detection of L. infantum DNA in the spleen and popliteal lymph node (when available). The prevalence obtained in wild boar (Sus scrofa) and roe deer (Capreolus capreolus) was higher than those previously reported (% ± confidence interval 95%; 42.9 ± 18.4% and 27 ± 6.6% in wild boar and roe deer, respectively), and this is the first report of this parasite infecting the coypu Myocastor coypus (60 ± 34.7%). L. infantum DNA was detected in all the seasons including those free of adult sandflies and seasonal differences were minimal, suggesting a long course of infection. The models revealed that animals from rainy areas with higher greenness during the summer, highly populated by humans and predominantly covered by water surfaces had a higher risk of L. infantum. This study contributes to confirming previous findings on the existence of a sylvatic cycle for L. infantum in certain regions of Italy, as well as on the potential epidemiological role of roe deer for this parasite given the elevated prevalence found.

Integrating environmental, entomological, animal, and human data to model the Leishmania infantum transmission risk in a newly endemic area in Northern Italy.

INTRODUCTION: Historically, leishmaniasis in Italy was constrained to areas with Mediterranean climate. In the last 20 years, sand fly vectors (Phlebotomus perniciosus), cases of canine leishmaniasis (CanL) and cases of human visceral leishmaniasis (VL) have been observed in Northern Italian regions, traditionally classified as cold areas unsuitable for sand fly survival. AIM: We aim to evaluate through a One-Health approach the risk of endemic transmission of Leishmania infantum in the Piedmont Region, Northern Italy. METHODS: We collected environmental, entomological, animal, and human data. We applied a geostatistical binomial model to map the probability of P. perniciosus presence in the study area, using selected environmental parameters as predictors. We evaluated the spatial relationship between the probability of P. perniciosus presence and the geographical distribution of CanL and VL cases observed between 1999 and 2013. RESULTS: Between 1999 and 2003, 142 sampling sets (17%) out of 839 resulted positive for P. perniciosus. Elevation, degree of slope, normalized difference vegetation index (NDVI) and summer temperatures were associated with positive sampling sets. During the study period, 164 (13.6%) of Piedmont municipalities reported at least one autochthonous case of CanL, while 89 VL cases were observed in 54 municipalities (4.5%). We observed an association between municipalities affected by autochthonous CanL cases and the estimated probability of P. perniciosus presence (Odds Ratio for 10% increase of probability: 2.66; 95% confidence intervals (CI): 2.16-3.37). We found that human VL incident cases were positively associated with the probability of the municipality of residence of being endemic for CanL (Incidence Rate Ratio for 10% increase of probability: 1.49; 95% CI 1.02-2.16). CONCLUSIONS: Using a One-Health approach, we quantified the spatial association between the distribution of P. perniciosus, municipalities endemic for CanL and incident cases of human VL, suggesting that the disease has become endemic in the Piedmont region.

Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples.

Sarcocystis spp. are protozoan parasites which can infect a wide range of vertebrates, including humans; the latter can act as definitive hosts for two cattle Sarcocystis spp.: Sarcocystis hominis and Sarcocystis heydorni. Reports of intestinal sarcocystosis are well documented in the literature, but PCR-based methods have been scarcely used to identify Sarcocystis species in human stools, and have been limited to the molecular analysis of 18S ribosomal RNA (18S rRNA) gene sequences. Since the mitochondrial cytochrome c oxidase subunit I (COI) gene is one of the most promising tools for distinguishing between closely related Sarcocystis spp., and taking into account the lack of publicly available S. hominis COI sequences, in the present study we obtained the first partial COI sequence of S. hominis from human stool samples of patient with gastrointestinal symptoms. We designed specific COI primers to develop a multiplex PCR method for the identification of Sarcocystis spp. in cattle. The submission of the COI sequence described herein and the unambiguous identification of S. hominis through the application of the new multiplex PCR is important for determining the prevalence of this zoonotic Sarcocystis spp. in meat and the risk for consumers.

Gastrointestinal helminths of wolves (Canis lupus Linnaeus, 1758) in Piedmont, north-western Italy.

Free-ranging grey wolves (Canis lupus), which are presently recolonizing Italy, can be parasitized by a diversity of helminths, but have rarely been subject to studies of their parasites. Therefore, this study aims to determine the prevalence of gastrointestinal helminths of road-killed grey wolves from the Piedmont region of Italy. Forty-two wolves were collected and examined for the presence of helminths. We recorded 12 helminth species: nine Nematoda and three Cestoda. The nematodes were: Ancylostoma caninum (7.1%), Capillaria sp. (2.4%), Molineus sp. (2.4%), Pterygodermatites affinis (11.9%), Physaloptera sibirica (9.5%), Toxocara canis (9.5%), Toxascaris leonina (2.4%) and Uncinaria stenocephala (26.2%); the cestodes were: Dipylidium caninum (4.8%), Mesocestoides sp. (4.8%) and Taenia multiceps (76.2%). Physaloptera sibirica had the highest mean intensity and T. multiceps had the highest prevalence. Based on age and sex, no differences in the intensity or prevalence of helminth species were found among the hosts. Molineus sp. was recorded for the first time in wolves from the Palearctic region; P. affinis and P. sibirica are respectively reported for the first time in wolves from Europe and Italy.

Epidemiological evaluation of Leishmania infantum zoonotic transmission risk in the recently established endemic area of Northwestern Italy.

Leishmania infantum infection had been expanding into new areas due to changes in vector and host biology. Zoonotic visceral leishmaniasis has become endemic in previously unsuitable areas as vectors find favourable climatic conditions and an increasing number of reservoir dogs are moved between traditionally and new endemic areas. Monitoring vector and disease expansion in areas of recent colonization is needed to understand transmission mechanisms and patterns of disease establishment. Here, we studied the infection status of 815 human blood donors and of 803 sympatric dogs from five, newly endemic, areas in Northwestern Italy. In autochthonous dogs, the seroprevalence of anti-L. infantum antibodies, recorded by Western blot, reached 42.22%, while in humans, the seroprevalence was of 16.81%. No significant correlation between the infection status of dogs and that of their human owners was found, but L. infantum infection was recorded in the different study areas with significant levels of diversity. Restriction fragment length polymorphism showed a high genetic variability of the circulating strains and gave useful insights on patterns of disease establishment into a naïve area.

Pulmonary artery dissection causing haemothorax in a cat: potential role of Dirofilaria immitis infection and literature review.

A 7-year-old male castrated domestic short-haired cat suddenly died. Gross examination revealed severe right-sided haemothorax with blood clots, four adult filarial nematodes in the blood clots and the caudal vena cava and haemorrhage dissecting into the tunica media of the right pulmonary artery. Histopathological investigation showed fibrosis of the tunica intima and disorganization/fragmentation of the elastic fibres accompanied by fibrous tissue deposition in the tunica media of both branches of pulmonary artery. Degenerative vasculopathy (intimal fibromuscular hyperplasia and medial hypertrophy/hyperplasia) involving pulmonary arteries was also observed. The polymerase chain reaction amplification and sequencing confirmed the identification of the parasite as Dirofilaria immitis. A diagnosis of pulmonary artery dissection with haemothorax and concomitant heartworm disease was formulated. Degenerative processes of the tunica media have been reported to cause pulmonary artery dissection in both humans and animals. Pulmonary artery remodelling induced by heartworms may be considered the underlying cause in the first case of feline pulmonary artery dissection, herein described.

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.

Evaluation of a rapid device for serological diagnosis of Leishmania infantum infection in dogs as an alternative to immunofluorescence assay and Western blotting.

In this study, we compared a rapid immunochromatographic test (Speed Leish K; BVT Groupe Virbac, La Seyne sur Mer, France) with an indirect immunofluorescence assay (IFAT) and Western blotting (WB) for the detection of Leishmania infantum antibodies in dogs. A total of 250 serum samples were collected from 125 L. infantum-positive and 125 L. infantum-negative dogs. Among the positive samples, 81 were strongly positive at low IFAT dilutions, while 44 were low-reactivity sera (IFAT titers, 1:40 to 1:80). The sensitivity and specificity of the Speed Leish K were 96.3% and 100%, respectively, compared with those of the IFAT. When IFAT low-reactivity sera (titers, 1:40 or 1:80) were tested with the Speed Leish K, using WB results as a reference, the sensitivities were 93.75% for sera with a 1:80 titer and 73.33% for sera with a 1:40 titer, and the specificity was 100%. The Speed Leish K is easy to use and performs well, so it can be considered a quick and reliable tool for the diagnosis of L. infantum infection in dogs.

An investigation into alternative reservoirs of canine leishmaniasis on the endemic island of Mallorca (Spain).

The role of wild and free-roaming domestic carnivores as a reservoir of Leishmania infantum was investigated on the Mediterranean island of Mallorca (Balearic Islands, Spain), an endemic area for this disease. Serum, blood and/or spleen samples from 169 animals [48 dogs from a kennel, 86 wild-caught feral cats, 23 pine martens (Martes martes), 10 common genets (Genetta genetta) and two weasels (Mustela nivalis)] were analysed. Seroprevalence determined by Western blotting was 38% in dogs and 16% in feral cats, while the prevalence of infection determined by PCR was 44% in dogs, 26% in cats, 39% in pine martens and 10% in genets. This is the first report of infection by L. infantum in the pine marten or any other member of the Mustelidae family. Restriction fragment length polymorphism (RFLP) analysis found 33 different patterns in 23 dogs, 14 cats and three martens. Two patterns were shared by dogs and cats, two by different cats, and one by different dogs. Patterns were different to those previously reported in carnivores from peninsular Spain. No external lesions compatible with leishmaniasis were observed in any species other than the dogs. Although the dog is probably the primary reservoir of leishmaniasis in endemic areas, the prevalence and the absence of apparent signs of this disease within the island's abundant feral cat and pine marten populations could make these species potential primary or secondary hosts of L. infantum in Mallorca.