Ectopic Pregnancy and T-Cell Lymphoma in a Eurasian Red Squirrel (Sciurus vulgaris): Possible Comorbidity and a Comparative Pathology Perspective.

Ectopic pregnancy (EP) is a life-threatening disease that affects humans and other mammals. Tumors causing ruptures of the reproductive tract have been identified as possible predisposing factors in human and veterinary medicine. We here describe a case of concomitant ectopic pregnancy and lymphoma in a Eurasian red squirrel found deceased in Italy and submitted to the public health laboratory Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT) for post-mortem examination. A full-term partially mummified ectopic fetus in the abdomen and a large fibrinonecrotic tubal scar adjacent to the right ovary were observed at necropsy. The tubal scar is likely the point of tubal rupture through which the fetus displaced. Histology revealed the presence of neoplastic cells referable to lymphoma infiltrating the ovary, spleen, small intestine, heart and peripancreatic adipose tissue. The lymphoma was further characterized as T-cell-type using immunohistochemistry. We suggest that the lymphoma, by involving the ovary, played a pathogenetic role in the development of a secondary EP by altering the genital tract at the structural and hormonal levels. To the best of our knowledge, this is the first report of concomitant ovarian lymphoma and EP in animals and humans in the literature.

Characterization of prevalence and genetic subtypes of Blastocystis sp. in wild and domestic Suidae of central Italy aided by amplicon NGS.

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and transmissible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern.

Angiostrongylus vasorum in wolves in Italy: prevalence and pathological findings.

BACKGROUND: Angiostrongylus vasorum is a nematode residing in the heart and pulmonary vessels of dogs and wild carnivores. In Europe the red fox is its reservoir, while only three records from wolves have been published. Angiostrongylus vasorum has a worldwide distribution, and many pieces of evidence demonstrate that it is spreading from endemic areas to new ones. In Italy, A. vasorum was reported with increasing frequency in dogs and foxes in the last decades, and now it is considered endemic throughout the country. Angiostrongylus vasorum can be asymptomatic or cause respiratory and circulatory disorders, at times causing severe disseminated infections. METHODS: Between February 2012 and December 2016, 25 wolves found dead in central Italy were submitted to the Istituto Zooprofilattico del Lazio e della Toscana for post-mortem examination. Samples of lungs, heart, liver, spleen, kidneys, mediastinic lymph nodes and brain were collected from each animal for histological examination. When adult and larval nematodes were microscopically seen in lungs, the other organs were processed, and five histological sections for each organ were examined. To confirm parasite identification, lung samples were submitted to a PCR-sequencing protocol targeting the ITS2 region of A. vasorum. RESULTS: Seven wolves (28.0%) harboured nematode larvae in lung sections. In two of the positive wolves, adult nematodes were visible in pulmonary arteries, in four animals larvae were also detected in other organs. DNA sequencing reactions confirmed parasite identification as A. vasorum in all the cases. CONCLUSIONS: As a result of the high prevalence of A. vasorum reported in wolves in the present study, a focus of high circulation could be hypothesised in central Italy. Nevertheless, the similarly high prevalence in foxes originating from the same areas were reported in previous papers. Histopathological evidence highlights the pathogenic potential of A. vasorum in the wolf, especially in juvenile animals.

Human exposure to piroplasms in Central and Northern Italy.

TA serosurvey has been conducted in Northern and Central Italy to investigate the presence in humans of antibodies against zoonotic Babesia and Theileria species. The study focused on a total of 432 volunteers, of which 290 were persistently exposed to tick bites because of their jobs (forester employees, livestock keepers, veterinary practitioners, farmers and hunters) and 142 resident in the same area less frequently exposed. An indirect fluorescent antibody test (IFAT) for humans was used to detect antibodies to Babesia microti, IFAT tests for veterinary use were modified to detect reactivity to Babesia bovis, Babesia canis and Theileria equi. A laboratory-derived ELISA was employed to detect antibodies to Babesia divergens. Both reactive and 10 negative sera were analysed against plasmodial antigens to evaluate possible aspecificity. A high reactivity to piroplasm antigens was found, showing significant difference between the sera of the two groups of volunteers (24% vs 7.%; p<0.001). No cross-reactivity was observed, while each professional group showed reactivity that would fit with the professional risk exposure. In particular, a high reactivity to B. microti and B. divergens antigens was observed in foresters and hunters (32% and 12%, respectively). This is the first report on the human seroreactivity to piroplasms in Italy; it also provides additional epidemiological information on these tick-borne zoonoses in Europe. Our findings suggest the possible occurrence of piroplasm infections in Italy and alert physicians to consider these otherwise neglected parasitic diseases when dealing with any febrile illness, especially in subjects exposed to tick bites.

Parasitological and molecular observations on a little family outbreak of human fasciolosis diagnosed in Italy.

In the year 2010, three children who were born in a Romanian cattle farmer family went to Italy to join their mother. One of them was admitted to an Italian pediatric hospital for severe anemia that, when she was in her country, had been treated with blood transfusion. Blood tests and an abdominal ultrasound study triggered the suspicion of biliary parasitosis. The child underwent a cholangiopancreatography that caused the release of parasitic material microscopically identified as Fasciola hepatica. All children and their mother were submitted to coproparasitological analyses, which identified F. hepatica eggs only in the patient and in her twin sister. Parasitic materials recovered and flatworm specimens by us ad hoc obtained from Italian and Romanian cattle were genetically (ITS and COI genes) analyzed, and their sequences were compared with those deposited in GenBank. Specimens from children clustered with the Romanian strain examined and showed remarkable genetic differences with flatworm specimens from Italy. Anamnesis, parasite biology, and genetic data strongly suggest that twin sisters became infected in Romania; however, human fasciolosis is an emerging sanitary problem, favored by climate changes and global drivers; therefore, it deserves more attention on behalf of physicians working in both developing and developed countries.

Is the goat a new host for the G3 Indian buffalo strain of Echinococcus granulosus?

Four goats bred in Central Italy (province of Rieti) revealed, in the liver, metacestodes of Echinococcus granulosus. The cysts, unilocular and fertile, were examined by microscopy and molecular diagnostics. Morphological data on the rostellar hooks are in agreement with the original description of the strain found in buffaloes and are largely compatible with those reported in Europe for cattle and humans. Specific PCR followed by DNA sequencing of the mitochondrial cox1 gene revealed for all the isolates 99.5% identity to the reference strain G3 genotype and 99.3% and 99.1% to G2 and G1, respectively. Further genetic markers (nad1 and 12S rRNA) confirmed the identity of the goat isolates to the G3 strain. This genotype, here reported for the first time in goats, proved to have a wider than previously supposed host range, therefore its relevance in human hydatidosis is expected to be more often evidenced.

The first report of Hepatozoon canis identified in Vulpes vulpes and ticks from Italy.

This is the first report on the presence of Hepatozoon canis in Vulpes vulpes in Italy. During the years 2005 and 2006, a total of 119 foxes were collected and their spleen tissues were screened by microscopy, polymerase chain reaction, and sequencing. In the same area, 290 ticks were picked off from dogs or collected from the environment. Microscopy detected inclusion bodies regarded as belonging to the genus Hepatozoon in four samples, whereas molecular diagnostics evidenced 16 foxes (13.4%) and 6 ticks (2.1%) positive to H. canis. The H. canis isolates we found in foxes, compared with the strains we previously detected in dogs from the same area and with the strains found in foxes from other European countries, show a certain genetic heterogeneity. In fact, seven isolates cluster with the Italian dog strain and nine isolates cluster with the fox strain found in Spain and Slovakia; moreover, the dog's strain is closely related to one tick's isolate, and the strain found in three Rhipicephalus sanguineus and in one Ixodes ricinus collected from the environment cluster with the aforementioned Spanish and Slovak fox strains. Our findings confirm the importance of R. sanguineus as final host and suggest that I. ricinus might also be implicated in parasite transmission, explaining in that way the occurrence of hepatozoonosis in areas considered R. sanguineus-free. The peridomestic habits of V. vulpes and the increasing global temperature are expected to amplify the impact of this vector-borne disease and to enforce the transmission of Hepatozoon to domestic animals.

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.

Investigation on the occurrence of Echinococcus multilocularis in Central Italy.

BACKGROUND: Recent studies on geographic distribution of Echinococcus multilocularis in Europe show that it has a wider range than previously thought. It is unclear, however, if the wider distribution is due to its recent spreading or to a lack of previous data from the new areas. Italy, previously considered E. multilocularis-free, is now part of these new areas: infected foxes (the main definitive host of the tapeworm) have been observed in a Northern Alpine territory. Thus, more surveys need to be done in other Italian regions in order to monitor the spreading of E. multilocularis. The aim of the present study was to look for this parasite in 283 foxes collected in an Apennine area of Central Italy by different diagnostic methods. RESULTS: The foxes were heavily parasitized by 11 helminthic genera, but none of the animals was infected by E. multilocularis neither by E. granulosus (harboured adult worms or their DNA). Low specificity was observed in commercially available ELISA kits for the detection of E. multilocularis antigens in the faeces. Molecular diagnostics were sensitive and specific for the detection and identification of tapeworm eggs in faeces, but less sensitive, although specific, to adult tapeworms in the intestinal content. CONCLUSION: Preliminarily, we can say that no E. multilocularis could be found in the study area. These data will enable us to follow temporal changes of the spatial distribution of the parasite in the study area of the Central Apennines. Due to its low specificity the ELISA kit for E. multilocularis coproantigens is not suitable for epidemiological surveys, whereas molecular diagnostics applied to faecal samples give useful results. Finally, absence of E. granulosus in foxes living in the endemic areas studied confirms the thought that this tapeworm prefers a different definitive host.

Vulpes vulpes: a possible wild reservoir for zoonotic filariae.

Foxes (Vulpes vulpes, n = 132) killed during the hunting seasons 2005-2006 in Central Italy (Tuscany region) were examined in order to investigate the possible importance of this animal as a wild reservoir for zoonotic filariae. In each specimen adult worms of Dirofilaria immitis and hematic microfilariae were searched for. Species identification was performed by morphology, morphometry, the Barka staining technique applied to pulmonary and splenic blood smears, and, finally, by molecular diagnostics -- polymerase chain reaction (PCR) and sequencing. Twenty-three subjects (17.4%) proved to be positive for filarial parasites. Infection by Acanthocheilonema was more widespread than by Dirofilaria. Briefly, 8 foxes harbored mature adults of D. immitis; two of them (25%) also had microfilariae that in one case were mixed with the microfilariae of D. repens. Twelve subjects had microfilariae of Acanthocheilonema reconditum, and 3 harbored microfilariae of A. dracunculoides. Molecular diagnostics confirmed all results. Our findings, drawn by the examination of a few microliters of blood obtained from foxes approximately <2 years of age, support the hypothesis that this animal may be an abundant source of infection for ticks that transmit Acanthocheilonema parasites and for mosquitoes that act as vectors for dirofilarial nematodes. Therefore foxes, contributing to the parasite circulation in areas where dogs usually undergo prophylactic treatment, have to be considered an important wild reservoir for filarial parasites that can be transmitted to companion animals and people.