Silvana Belo

Prevalence and seasonal variations of canine dirofilariosis in Portugal.

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 At present, accurate data on the prevalence and epidemiological pattern of dirofilariosis in Portugal is scarce and outdated.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 Knotts 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.

The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype.

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Morphological Characteristics of Schistosoma mansoni PZQ-Resistant and -Susceptible Strains Are Different in Presence of Praziquantel

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.

Curcumin Generates Oxidative Stress and Induces Apoptosis in Adult Schistosoma mansoni Worms

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.

Human gastric hyperinfection by Anisakis simplex: A severe and unusual presentation and a brief review

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.

Unusual panuveitis in a child: toxocariasis associated with ocular myiasis.

Most panuveitis in children are caused by infectious agents. A detailed clinical history and clinical examination are helpful in the diagnosis, but specific techniques are sometimes required to identify the causing specimen. We report the first published case of panuveitis in a child caused by simultaneous ocular infection by Toxocara canis and a fly larva and the innovative use of immunodiffusion technique in the vitreous for the diagnosis.

Comparative Proteomics Reveals Characteristic Proteins on Praziquantel-resistance in Schistosoma mansoni

The extensive use of Praziquantel (PZQ), the only drug available to treat schistosomiasis, has brought concern about the emergence of PZQ-resistance/tolerance by Schistosoma spp., thus reaffirming an urge for the development of new treatment alternatives. Therefore, it is imperative and urgent to study this phenomenon trying to understand what is involved in its occurrence. Studies of Schistosoma spp. genome, transcriptome and proteome are crucial to better understand this situation. By stepwise drug pressure from a fully susceptible parasite strain, our group selected a S. mansoni variant strain stably resistant to PZQ and isogenic to its fully susceptible parental counterpart, except for the genetic determinants of PZQ-resistance phenotype. Based on this, the objective of this study was to compare the proteomes of both strains, identifying proteins from male and female adult worms of PZQ-resistant and PZQ-susceptible strains, exposed and not exposed to PZQ, which were separated by high-resolution two-dimensional electrophoresis and sequenced by high throughput LC-MS/MS. Likewise, this work is extremely relevant since for the first time the proteome of a S. mansoni PZQ-resistant strain is studied and compared to the proteome of the respective S. mansoni PZQ-susceptible strain. This study identified 60 S. mansoni proteins, some of which differentially expressed in either strain, which may putatively be involved in the PZQ-resistance phenomenon. This information represents substantial progress towards deciphering the worm proteome. Furthermore, these data may constitute an informative source for further investigations into PZQ-resistance and increase the possibility of identifying proteins related to this condition, possibly contributing to avoid or decrease the likelihood of development and spread of PZQ-resistance. This is an innovative study that opens doors to PZQ-resistance surveys, contributing to discover a solution to PZQ-resistance problem, as suggests new potential targets for study.

Dirofilaria immitis and Angiostrongylus vasorum: the current situation of two major canine heartworms in Portugal

Cardiopulmonary nematodes are life-threatening pet parasites increasingly reported throughout Europe, with overlapping endemic areas. Dirofilaria immitis is a mosquito-borne whilst Angiostrongylus vasorum is a snail-borne pathogen. Both adult nematodes reside in the pulmonary arteries and right cardiac ventricle of domestic and wild canids, causing a wide spectrum of clinical presentations ranging from cough, dyspnoea and exercise intolerance to severe vascular and pulmonary disease with hearth failure that may lead to death. Information about the prevalence and distribution of cardiopulmonary parasites is essential for the control of animal diseases and, in the case of D. immitis, for the control of potentially associated illnesses in humans. However, in Portugal, heartworm studies are limited to few surveys and case reports, possibly underestimating the relevance of these nematodes. The present work reviews the data on cardiopulmonary dirofilariosis and angiostrongylosis in dogs in Portugal, providing a comprehensive update of the epidemiological situation during the past 20 years.

Molecular characterization of Dirofilaria spp. circulating in Portugal

BackgroundDirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs.MethodsBlood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott’s technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species.ResultsOf the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott’s technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods.ConclusionsThe present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.