Parallel Propagation of Toxoplasma gondii In Vivo, In Vitro and in Alternate Model: Towards Less Dependence on the Mice Model.

Toxoplasma gondii is an obligate intracellular protozoan. In pregnant women, it can lead to severe birth defects or intrauterine death of the fetus. Most of what is currently know on cell biology of T. gondii comes from studies relying on the RH strain propagated in mice. According to the recommendations concerning the animal welfare, we assayed in vitro/in vivo procedures to replace, or at least reduce, the demanding animal model for strain propagation. We evaluated the genetic and phenotypic stability of the RH strain throughout its parallel continuous propagation in mice, in human foreskin fibroblasts (HFF) and in an alternate fashion of these two procedures. We also assessed the virulence impact on the RH strain after different periods of its long-term propagation strictly in cells. The RH strain completely lost its virulence after long-term passage in HFF. Nevertheless, we obtained a successful outcome with the alternate passaging of the parasite in HFF and in mice as this approach enabled T. gondii to maintain the evaluated phenotypic properties, mainly its virulence potential. Also, no genetic changes were observed in genes known to be highly polymorphic or involved in pathoadaptation. In conclusion, the alternate model seems to be a feasible method for T. gondii propagation and maintenance, strongly impacting the number of sacrificed mice.

First Report of Echinococcus ortleppi in Free-Living Wild Boar (Sus scrofa) from Portugal.

Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors' knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock-wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field.

Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters.

The third-stage larvae (L3) of the Anisakidae family are parasitic nematodes with zoonotic impact and are frequently encountered in the organs and musculature of various fish intended for human consumption. Since Anisakis simplex (s.s.) and A. pegreffii are the major aetiological agents of human disease, this study aims to combine the morphological and molecular data on the recovered anisakid larvae to contribute to a simplified morphological distinction of those species and conducted a survey of anisakid larvae infection in horse mackerel (Trachurus trachurus). Here, 116 horse mackerel caught in Portuguese waters were analysed for the presence of L3 of anisakids, and 3148 larvae were collected, of which only 30% were retrieved during visual inspection. As such, visual inspection does not appear to be very effective in anisakid detection. A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were 32.1 and 27.1 parasites per fish, respectively. The morphological and molecular analyses of 196 L3 randomly chosen from the total sample of parasites demonstrated the presence of L3 of mostly Anisakis spp., with only one L3 of Hysterothylacium aduncum. Relative frequencies of 62.9% for A. pegreffii and 37.1% for A. simplex (s.s.) were obtained. The morphometry differences between these two sibling species were evaluated, and the results demonstrated significant differences between the length of the ventriculus and the length of the oesophagus. Precisely, A. simplex (s.s.) has a longer oesophagus and ventriculus than A. pegreffii. As such, these differences may be used to distinguish the two species through morphological analysis.

Towards a rapid sequencing-based molecular surveillance and mosaicism investigation of Toxoplasma gondii.

Advances in molecular epidemiology of Toxoplasma gondii are hampered by technical and cost-associated hurdles underlying the acquisition of genomic data from parasites. In order to implement an enhanced genotyping approach for molecular surveillance of T. gondii, we applied a multi-locus amplicon-based sequencing strategy to samples associated with human infection. This approach, targeting genome-dispersed polymorphic loci potentially involved in adaptation and virulence, genetically discriminated almost all 68 studied strains and revealed a scenario of marked genomic mosaicism. Two-thirds (n = 43) of all strains were classified as recombinant, although recombination seemed to be linked to the classical archetypal lineage. While 92% of the Sag2 archetype I strains revealed genetic mosaicism, only 45% of Sag2 archetype II strains were identified as recombinant. Contrarily to the virulence-associated archetype I, most type II strains (regardless of their recombination background) were non-virulent in mouse. Besides Sag2, some of the newly studied loci (namely the type I/I-like alleles of Sag1, B17, PK1, and Sag3 and type III/III-like alleles of TgM-A) constitute promising candidates to rapidly infer T. gondii mouse virulence. Our successful attempt to capture microsatellite length variation launches good perspectives for the straightforward transition from the laborious intensive historical method to more informative next-generation sequencing (NGS)/bioinformatics-based methodologies. Overall, while T. gondii whole-genome sequencing will be hardly feasible in most laboratories, this study shows that a discrete loci panel has the potential to improve the molecular epidemiology of T. gondii towards a better monitoring of circulating genotypes with clinical importance.

Molecular and virulence characterization of Toxoplasma gondii strains isolated from humans in Portugal.

Toxoplasma gondii is an apicomplexan parasite responsible for toxoplasmosis which infects all warm-blooded vertebrates, including mammals and birds. The majority of studies conducted in Europe have revealed that more than 80 % of strains isolated from human infections belong to genotype II, whereas genotypes I and III are responsible for a small number of cases. Atypical and recombinant strains are generally associated with more severe infections. In Portugal, there is a lack of data concerning genetic diversity as the classical typing studies in humans have never been performed. We aimed to determine the Sag2 and microsatellite-based (TUB2, TgM-A, W35, B17, B18) genotypes of T. gondii isolated from humans in Portugal, as well as to study their virulence in mice. We analyzed 48 strains from congenital and acquired toxoplasmosis collected during the last two decades. Sag2-based genotyping of T. gondii was achieved in all 48 strains where 35 (73 %) were classified as type II and 13 (27 %) were type I. The multilocus PCR of five microsatellites allowed the classification of 10 strains (21 %) as recombinant strains that had been previously identified as type II or I by Sag2 typing. Seven out of the 48 strains, including three type I, three recombinant, and one type I, were virulent in mice. This study constitutes the first evidence of recombinant strains circulating in Portugal in humans from congenital infection, highlighting the need for a better evaluation of these strains as their phenotype is still barely understood.

Toxoplasma gondii seroprevalence in the Portuguese population: comparison of three cross-sectional studies spanning three decades.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan infecting up to one-third of the world's population, constituting a life threat if transmitted from mother to child during pregnancy. In Portugal, there is a lack of knowledge of the current epidemiological situation, as the unique toxoplasmosis National Serological Survey was performed in 1979/1980. METHODS: We studied the seroprevalence trends in the Portuguese general population over the past 3 decades, by assessing chronological spread cross-sectional studies, with special focus on women of childbearing age, by age group, region and gender. RESULTS: The T. gondii overall seroprevalence decreased from 47% in 1979/1980 to 22% (95% CI 20% to 24%) in 2013. Generally, we observed that the prevalence of T. gondii IgG increased significantly with age and it decreased over time, both in the general population and in the childbearing women (18% prevalence in 2013). CONCLUSIONS: The scenario observed for the latter indicates that more than 80% of childbearing women are susceptible to primary infection yielding a risk of congenital toxoplasmosis and respective sequelae. Since there is no vaccine to prevent human toxoplasmosis, the improvement of primary prevention constitutes a major tool to avoid infection in such susceptible groups.

Genotyping Characterization of Toxoplasma gondii in Cattle, Sheep, Goats and Swine from the North of Portugal.

BACKGROUND: Epidemiological investigations on Toxoplasma gondii infection have found a significant association between human toxoplasmosis and consumption of raw or undercooked meat. The present study aimed to characterize genotypes of T. gondii in 20 cattle, 40 sheep, 15 goats and 16 pigs from the North of Portugal. METHODS: Nested PCR amplified the surface antigen 2 (SAG2) gene. Sequencing analysis was performed in order to assess the prevalence of SAG2 type strains (I, II and III). RESULTS: Three and 4 strains of SAG2 type II were identified in heart samples of cattle and sheep, respectively. Three SAG2 type II strains were detected in brain, diaphragm and heart of 3 pigs. Three strains detected in heart samples of 3 goats belonged to SAG2 types I or II; with the same result being observed in heart samples from 2 sheep and in 2 brain and 1 heart samples from 3 pigs. CONCLUSION: SAG2 type II has been shown for the first time to infect cattle in North of Portugal. In addition, SAG2 type II has also been confirmed as the predominant strain in sheep and pigs in the same region. This is the first molecular report of T. gondii in goats from Portugal.

Waterborne transmission of Giardia and Cryptosporidium at river beaches in Southern Europe (Portugal).

Giardia and Cryptosporidium are the most frequent enteric protozoa causing gastroenteritis in humans worldwide. Intense recreational activity at Portuguese river beaches triggered the opportunity for a 2-year seasonal survey of 19 large river basin beaches. A total of 74 samples were collected and processed according to USEPA Method 1623 to detect Cryptosporidium and Giardia (oo)cysts. Faecal indicators (thermotolerant/total coliforms, Escherichia coli, and enterococci) and physicochemical parameters were also analysed according to the EU Bath Water Directive (BWD). Results pointed to a widespread presence of these protozoa at Portuguese river beaches. The percentage of samples testing positive for Giardia and Cryptosporidium were 85 and 82% respectively, with no significant differences between wet and dry seasons (p > 0.05). Although Portuguese river beaches present a very low exposure risk for infection with Giardia and Cryptosporidium (under 10(-3)), a few particular cases revealed values over 0.2%, and were related to stormy wet events. The correlation between levels of Giardia and thermotolerant coliforms, E. coli and enterococci, was high (r ≥ 0.87, p < 0.001), suggesting the need to carry out specific procedures for the detection of Giardia and Cryptosporidium whenever the values of those faecal indicators approach the maximum allowed level of the EU BWD.

Prevalence and risk factors for Giardia duodenalis infection among children: a case study in Portugal.

BACKGROUND: Giardia duodenalis is a widespread parasite of mammalian species, including humans. The prevalence of this parasite in children residing in Portugal is currently unknown. This study intended to estimate G. duodenalis infection prevalence and identify possible associated risk factors in a healthy paediatric population living in the District of the Portuguese capital, Lisbon. METHODS: Between February 2002 and October 2008, 844 children were randomly selected at healthcare centres while attending the national vaccination program. A stool sample and a questionnaire with socio-demographic data were collected from each child. Giardia infection was diagnosed by direct examination of stools and antigen detection by ELISA. RESULTS: The population studied revealed a gender distribution of 52.8% male and 47.2% female. Age distribution was 47.4% between 0-5 years and 52.6% between 6-15 years.The prevalence of Giardia infection was 1.9% (16/844) when estimated by direct examination and increased to 6.8% (57/844) when ELISA results were added. The prevalence was higher among children aged 0-5 years (7.8%), than among older children (5.8%), and was similar among genders (6.9% in boys and 6.5% in girls). The following population-variables were shown to be associated risk factors for G. duodenalis infection: mother's educational level (odds ratio (OR)= 4.49; confidence interval (CI): 1.20-16.84), father's educational level (OR = 12.26; CI: 4.08-36.82), presence of Helicobacter pylori infection (OR = 1.82; CI: 1.05-3.15), living in houses with own drainage system (OR = 0.10; CI: 0.02-0.64) and reported household pet contact, especially with dogs (OR = 0.53; CI: 0.31-0.93). CONCLUSION: The prevalence of giardiasis in asymptomatic children residing in the region of Lisbon is high. Several risk factors were associated with Giardia prevalence and highlight the importance of parents' education and sanitation conditions in the children's well being. The association between G. duodenalis and H. pylori seems an important issue deserving further investigation in order to promote prevention or treatment strategies.

Oftalmia simpática pós-vitrectomia / Sympathetic ophthalmia following vitrectomy: report of once case

Estudo de um caso de oftalmia simpática pós-vitrectomia via pars plana, cujo quadro clínico no olho simpatizante caracterizou-se por iridociclite näo granulamatosa, turvaçäo vítrea, hiperemia do disco óptico e descolamento seroso de retina. O período de latência entre a vitrectomia e o início dos sintomas foi de 70 dias. Apresentamos, no presente caso, aspectos terapêuticos abordando a necessidade do uso de drogas imunossupressoras. Trata-se do primeiro caso, de nosso conhecimento, de oftalmia simpática pós-vitrectomia via pars plana relatado na literatura nacional

Prevalência de tracoma em bairro do município de Palmares, Estado de Pernambuco, Brasil / Trachoma prevalence in Palmares, state of Pernambuco

Tracoma tem sido diagnosticado em várias localidades brasileiras, mas os estudos de prevalência desta doença no Brasil säo raros. A presente investigaçäo, realizada na cidade de Palmares, Estado de Pernambuco, mostrou que 9,29% da populaçäo examinada no Bairro de Newton Carneiro apresentava algum sinal clínico de tracoma. A prevalência de tracoma folicular foi de 7,40% e a de tracoma cicatricial de 1,88%. Näo foram observadas triquíases e opacidades corneanas. A confirmaçäo laboratorial, através da imunofluorescência direta, ocorreu em 66,66% dos espécimes. Tracoma na populaçäo investigada em Palmares mostrou-se como doença que näo leva à cegueira