Assessment of the circulation of Dirofilaria immitis in dogs from northern Portugal through combined analysis of antigens, DNA and parasite forms in blood.

Dirofilariasis is a vector-borne disease frequent in many countries. Not only infected dogs, but also cats and wild canids (including wolves and foxes), represent important sources of infection for mosquitoes, which are the pathogen vectors. The disease is endemic in Mediterranean countries with increasing incidence in Italy, France, Greece and Spain, but limited epidemiological data is available from Portugal regarding its distribution and impact. Aiming to clarify this, canine whole blood samples (n = 244) from the north of Portugal were tested for Dirofilaria spp. antigens by use of a commercial rapid immunomigration test. Polymerase chain reaction (PCR) and the modified Knott test were also used to assess the presence of microfilariae. Results were also compared to assess the performance of each test used. Of the 244 animals tested, 118 (48.4%) were positive for Dirofilaria immitis (heartworm) in the serological adult worm rapid antigen detection test, and 36 (14.8%) had circulating microfilariae, identified as D. immitis. A combined positivity of 51.6% (126/244) was found. Results indicate that the risk of exposure to D. immitis in dogs is high in this region of Portugal, and that prophylaxis against the parasite is advisable to decrease the occurrence of canine infection and disease. The present study highlights the diagnostic value of serological and molecular tests in determining the prevalence of D. immitis.

Crimean-Congo hemorrhagic fever virus circulating among sheep of Portugal: a nationwide serosurvey assessment.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread zoonotic pathogen that can cause mild to severe hemorrhagic disease in humans. CCHFV may be transmitted through direct contact with tissue or blood of viremic animals; however, the primary transmission route is through infected tick bites. CCHFV RNA has been detected in ticks feeding on domestic and wild animals in western Spain, suggesting an established circulation of CCHFV in Western Europe. Ruminants have been recognized as important CCHFV reservoirs and have been linked to human cases in endemic regions. Given the emergence of CCHF in neighboring Spain, and a report of two CCHFV seropositive humans in southern Portugal in 1985, we investigated the potential circulation of this virus in the country by performing a nationwide anti-CCHFV IgG serosurvey in sentinel sheep of Portugal. Sera (n = 459) randomly selected from widely distributed farms (n = 20) of Portugal were tested using a commercial double-antigen enzyme-linked immunosorbent assay, yielding an overall seroprevalence of 0.4% (95% confidence interval [CI] 0.04-1.56%). Positive sheep were from the southern region of Portugal (Alentejo region), which raise the seroprevalence of this region to 0.74% (95% CI 0.09-2.66%). This is the first study reporting the presence of CCHFV antibodies in sheep of Portugal, thus suggesting a geographical expansion of CCHFV to this country. It seems likely that CCHFV may exist focally in southern Portugal.

Dirofilaria immitis antigenemia and microfilaremia in Iberian wolves and red foxes from Portugal.

BACKGROUND: Dirofilaria immitis is a parasitic nematode endemic in the Mediterranean countries, which causes cardiopulmonary dirofilariosis in wild and domestic animals. Despite being recognized hosts of D. immitis, wild carnivores such as wolves and foxes are frequently disregarded when considering a potential role in the transmission of these zoonotic nematodes. In Portugal, studies available regarding D. immitis circulation are scarce, likely underestimating its relevance. To add knowledge on this, we sought to assess Iberian wolves (Canis lupus signatus) and red foxes (Vulpes vulpes) from northern Portugal for D. immitis antigenemia and microfilaremia. METHODS: Blood samples from 42 Iberian wolves and 19 red foxes were collected, during 2010-2012, in Peneda-Gerês National Park. Antigenemia was searched for by rapid antigen detection test kits (Uranotest Dirofilaria ®). Microfilaremia was assessed by polymerase chain reaction (PCR). Nucleic acids were extracted from blood using QIAamp® DNA Mini Kit (Qiagen), and DNA was screened for the presence of microfilaria using a conventional PCR targeting the 5.8S-internal transcribed spacer 2-28S regions, followed by bidirectional sequencing, Basic Local Alignment Search Tool analysis and phylogenetic analysis. RESULTS: Three red foxes had antigenemia, with an occurrence of 15.8% (95% confidence interval [CI] 3.4-39.6), while showing no evidence for the presence of microfilaremia. No wolf samples presented evidence for D. immitis antigenemia. Nevertheless, two wolves were positive for D. immitis microfilaremia (4.8%; 95% CI 0.6-16.2%) as revealed by PCR and confirmed by bidirectional sequencing. CONCLUSIONS: Although Dirofilaria microfilaremia in wolves does not necessarily correlate to an endangerment of the infected animal's health, positive individuals can act as a reservoir for further infection if the intermediate mosquito hosts are present. To the best of our knowledge, one single study had reported that wolves were suitable Dirofilaria hosts, but microfilaremia have never been reported.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region  region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

A 2-Year Longitudinal Seroepidemiological Evaluation of Toxoplasma gondii Antibodies in a Cohort of Autochthonous Sheep from Central Portugal.

(1) Background: Toxoplasma gondii is an important zoonosis and one of the major causes of abortion in sheep worldwide. (2) Methods: We performed a 2-year longitudinal serological anti-T. gondii IgG screening on a cohort of a spatially confined population of a Portuguese autochthonous sheep breed in central Portugal. (3) Results: From the screening of the 2015 and 2016 sera, an increase of seroprevalence was observed (57.7% (95% CI: 49.9-65.3%) versus 69.1% (95% CI: 61.5-75.9), from 2015 and 2016, respectively) (p = 0.031). (4) Conclusions The present study is the first to provide prospective data on the anti-T. gondii serological status of a sheep cohort in Portugal, showing an increase in the occurrence of T. gondii. There is a need to provide a clearer understanding of T. gondii epidemiology in Portugal, ideally by implementing monitoring programs on sentinel herds, not only due to the high impact of T. gondii on animal health but also for it being a zoonosis.