Canine dirofilariasis due to Dirofilaria immitis is known to be endemic in continental Portugal. However, information about the transmitting mosquito species is still scarce, with only Culex theileri identified to date, albeit with L1-2, through dissection. This study was carried out to investigate the potential vectors of Dirofilaria spp. in continental Portugal. Mosquitoes were collected in three distinct seasons (Summer, Autumn and Spring), 2011–2013, in three districts. CDC traps and indoor resting collections were carried out in the vicinity of kennels. Mosquitoes were kept under controlled conditions for 7 days to allow the development of larval stages of Dirofilaria spp.. DNA extraction was performed separately for both head+thorax and abdomen in order to differentiate infective and infected specimens, respectively, in pools, grouped according to the species and collection site (1–40 specimen parts/pool), and examined by PCR using pan-filarial specific primers. Mosquito densities were compared using non-parametric tests. Dirofilaria development units (DDU) were estimated. In total, 9156 female mosquitoes, from 11 different species, were captured. Mosquito densities varied among the 3 districts, according to capture method, and were generally higher in the second year of collections. From 5866 specimens screened by PCR, 23 head+thorax and 41 abdomens pools, corresponding to 54 mosquitoes were found positive for D. immitis DNA. These belonged to 5 species: Culex (Cux) theileri (estimated rate of infection (ERI)=0.71%), Cx. (Cux) pipiens f. pipiens and f. molestus (ERI=0.5%), Anopheles (Ano) maculipennis s.l. (ERI=3.12%), including An. (Ano) atroparvus, Aedes (Och) caspius (ERI=3.73%) and Ae. (Och) detritus s.l. (ERI=4.39%). All but Cx. pipiens, had at least one infective specimen. No D. repens infected specimens were found. Infection rates were: 3.21% in Coimbra, 1.22% in Setúbal and 0.54% in Santarém. DDU were at least 117/year in the study period. Culex theileri, Cx. pipiens, An. maculipennis s.l. An. atroparvus, Ae.caspius and Ae. detritus s.l. were identified as potential vectors of D. immitis in three districts of Portugal, from Spring to Autumn, in 5 of the 6 collection dates in 2011–2013. Implications for transmission, in the context of climate changes, and need for prophylactic measures, are discussed.

Schistosomiasis is one of the most common human parasitic diseases whose socioeconomic impact is only surpassed by malaria. Praziquantel (PZQ) is the only drug commercially available for the treatment of all schistosome species causing disease in humans. However, there has been stronger evidences of PZQ-resistance on Schistosoma mansoni and thus it is very important to study the phenotypic characteristics associated with it. The aim of this study was to evaluate morphological alterations in S. mansoni PZQ-resistant adult worms and eggs, by comparing a PZQ-resistant strain obtained under PZQ drug pressure with a PZQ-susceptible strain. For this, scanning electronic microscopy was used to assess tegumental responsiveness of both strains under PZQ exposure, and optical microscopy allowed the monitoring of worms and eggs in the presence of the drug. Those assays showed that PZQ-susceptible worms exposed to the drug had more severe tegumental damages than the resistant one, which had only minor alterations. Moreover, contrary to what occurred in the susceptible strain, resistant worms were viable after PZQ exposure and gradually regaining full motility after removal of the drug. Eggs from resistant strain parasites are considerably smaller than those from susceptible strain. Our results suggest that there might be a difference in the tegument composition of the resistant strain and that worms are less responsive to PZQ. Changes observed in egg morphology might imply alterations in the biology of schistosomes associated to PZQ-resistance, which could impact on transmission and pathology of the disease. Moreover, we propose a hypothetical scenario where there is a different egg tropism of the S. mansoni resistant strain. This study is the first comparing two strains that only differ in their resistance characteristics, which makes it a relevant step in the search for resistance determinants.

Anisakiasis is an emerging marine food-borne zoonosis resulting from the accidental ingestion of Anisakis larvae, through the consumption of raw or undercooked infected seafood products. The first case of human gastric hyperinfection by Anisakis simplex with an unusual and severe presentation, occurring in a Portuguese woman, is described in this article. Over 140 anisakid larvae were removed by gastroscopy. Massive infection is uncommon in areas where the consumption of raw fish is not part of the traditional diet, as is the case in Portugal. The increased consumption of raw seafood products is considered a health determinant in the rise in cases of anisakiasis. However, clinicians should be aware of the emergence of these infections, not only because of the new dietary habits of the population, but also because of the high prevalence of Anisakis larvae in the different fish species usually consumed by the population, collected on the Portuguese coast.

Inducing apoptosis is an interesting therapeutic approach to develop drugs that act against helminthic parasites. Researchers have investigated how curcumin (CUR), a biologically active compound extracted from rhizomes of Curcuma longa, affects Schistosoma mansoni and several cancer cell lines. This study evaluates how CUR influences the induction of apoptosis and oxidative stress in couples of adult S. mansoni worms. CUR decreased the viability of adult worms and killed them. The tegument of the parasite suffered morphological changes, the mitochondria underwent alterations, and chromatin condensed. Different apoptotic parameters were determined in an attempt to understand how CUR affected adult S. mansoni worms. CUR induced DNA damage and fragmentation and increased the expression of SmCASP3/7 transcripts and the activity of Caspase 3 in female and male worms. However, CUR did not intensify the activity of Caspase 8 in female or male worms. Evaluation of the superoxide anion and different antioxidant enzymes helped to explore the mechanism of parasite death further. The level of superoxide anion and the activity of Superoxide Dismutase (SOD) increased, whereas the activity of Glutathione-S-Transferase (GST), Glutathione reductase (GR), and Glutathione peroxidase (GPX) decreased, which culminated in the oxidation of proteins in adult female and male worms incubated with CUR. In conclusion, CUR generated oxidative stress followed by apoptotic-like-events in both adult female and male S. mansoni worms, ultimately killing them.

Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug. Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR. This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

Dirofilariosis is a severe vector-borne emergent disease that is spreading worldwide and becoming a serious threat to human and veterinary public health. Portugal, a Mediterranean country, has favorable climate conditions for mosquito development and survival. At present, accurate data on the prevalence and epidemiological pattern of dirofilariosis in Portugal is scarce and outdated. To study these trends, a project was developed to assess the current prevalence of Dirofilaria immitis infection in shelter dogs as well as the prevalence of Dirofilaria species present in three coastal regions in central Portugal: Coimbra, Santarém, and Setúbal. Blood samples were collected from 696 shelter dogs during three consecutive years: 2011, 2012, and 2013. A rapid immunomigration technique was performed to detect female D. immitis antigens. Concurrently, to detect and identify circulating microfilariae, a modified Knott's technique and acid phosphatase histochemical staining were also performed. Of the 696 dogs sampled, 105 were positive for D. immitis, with an overall prevalence of 15.1%. Forty of the 105 dogs were antigen negative but were positive for D. immitis microfilariae. Three animals were co-infected with D. immitis and Acanthocheilonema dracunculoides, and there was also one dog infected only with A. dracunculoides, all confirmed by polymerase chain reaction. The highest prevalence of canine heartworm during the 3-y period was in Setúbal (24.8%), followed by Coimbra (13.8%), and Santarém (13.2%), with significant inter-district differences. Our results demonstrate a higher prevalence of dirofilariosis compared with findings of previous studies and show an increasing rate of infection in the southern areas of Portugal attributed, at least in part, to bioclimatic and ecological factors. The present study updates the epidemiological situation and correlates the risk of dirofilariosis transmission within each region. These findings are highly relevant to both human and veterinary public health, contributing to the general awareness of pet owners and veterinarian practitioners and reinforcing the need for effective control measures against vectors and preventive therapy in companion animals.