Serological evidence of Toxoplasma gondii in hunted wild boar from Portugal

Toxoplasmosis is an important parasitic zoonosis with a worldwide distribution, being the parasitic disease with the highest occurrence in Europe. Wild boar has an important role in the epidemiological cycle of Toxoplasma gondii as an intermediate host, that can potentially infect humans when the meat is consumed raw or undercooked. The purpose of this work was to determine the presence of antibodies to T. gondii in serum of hunted wild boar. During the hunting season 2011/2012, sera samples were collected from 97 wild boar and tested for IgG antibodies to T. gondii, using the modified agglutination test. Twenty out of the 97 wild boar (20.6%) were seropositive for T. gondii IgG antibodies. Multivariate logistic regression analysis showed that males and older animals were associated with T. gondii seropositivity. These results show that T. gondii has an important presence in wild boar population from Portugal, suggesting a potential zoonotic risk for humans when wild boar meat or meat products are consumed raw or undercooked.

"Portuguese Observatory of Taeniasis and Cysticercosis": A "One Health" example for epidemiological surveillance of one neglected disease in a European country

Cysticercosis results from the ingestion Taenia solium eggs directly by faecal-oral route or contaminated food or water. Human tapeworm carriers who have become infected after ingesting pork meat contaminated with cysticerci release these eggs. Cysticercosis occurs after tapeworm eggs are ingested by an intermediate host (pig or human) and then hatch, migrate, and lodge in the host's tissues, where they develop onto larval cysticerci. When they lodged in the central nervous system of humans, results in the disease condition called Neurocysticercosis (NCC), with a heterogeneous manifestations depending of the locations of cysts, number, size and their stage of evolution (1). Consequently the prognostic ranges from asymptomatic to situations leading to death in 2% to 9.8%. of cases (7) In swine’s there are few studies, but recent works have proved that animals, for the same reasons, also have neurological abnormalities, expressed by seizures, stereotypic walk in circles, chewing motions with foamy salivation included tonic muscle contractions followed by a sudden diminution in all muscle tone leading to collapse (2). Conventional domestic wastewater treatment processes may not be totally effective in inactivating parasites eggs from Taenia solium, allowing some contamination of soils and agricultural products (11). In Portugal there are some evidence of aggregation of human cysticercosis cases in specific regions, bases in ecological design studies (6). There are few information about human tapeworm carriers and social and economic factors associated with them. Success in knowledge and consequently in lowering transmission is limited by the complex network of biological and social factors that maintain the spread. Effective control of mostly zoonosis require One Health approach, after a real knowledge and transparency in the information provided by the institutions responsible for both animal and human health, allowing sustained interventions targeted at the transmission cycle's crucial nodes. In general, the model used to control, reflects a rural reality, where pigs are raised freely, poor sanitation conditions and incipient sanitary inspection. In cysticercosis, pigs are obligate intermediate hosts and so considered as first targets for control and used as sentinels to monitor environmental T. solium contamination (3). Usually environmental contamination with Taenia spp. eggs is a key issue in most of studies with landscape factors influencing presence of Taenia spp. antigens in both pigs and humans (5). Soil-related factors as well as socio-economic and behavioural factors are associated with the emergence of significant clustering human cysticercosis (4,5). However scarce studies has been produced in urban environmental and in developed countries with the finality to characterize the spatial pattern. There are still few data available regarding its prevalence and spatial distribution; Transmission patterns are likely to exhibit correlations as housing conditions, water supply, basic sanitation, schooling and birthplace of the individual or relatives, more than pigs rearing free, soil conditions (9). As a matter of fact, tapeworm carriers from endemic zones can auto-infect or transmit infection to other people or arrive already suffering NCC (as a result of travelling to or being a citizen from an endemic cysticercosis country) to a free cysticercosis country. Transmission is fecal-oral; this includes transmission through person-to-person contact, through autoinfection, or through contaminated food This has been happening in different continents as North America (5.4–18% been autochthonous), Europe and Australia (7). Recently, case reports of NCC have also emerged from Muslim countries. (10). Actually, different papers relate an epidemic situation in Spain and Portugal (7, 8). However the kind of study done does not authorize such conclusion. There are no evidence that infections were acquired in Portugal and there are not characterized the mode of transmission. Papers with these kind of information will be allow to have economic consequences resulted from artificial trade barriers with serious consequences for pig producers and pig meat trade. We need transparency in information’s that allow provide the basis to support the development and targeting of future effective control programmes (and prove we need that). So, to have a real picture of the disease, it is necessary integrate data from human, animal and environmental factors surrounding human and pig cases to characterize the pattern of the transmission. The design needs to be able to capture unexpected, and not common outcomes (routine data). We need to think “One Health” to get a genuine image of the situation.

The intermediate snail-host: an agenda for future study

The strategy for the control of schistosomiasis has shifted from one directed towards the interruption of transmission to one of reducing morbidity. As a consequence of this change, it appears prudent to reassess the role of the malacologist and malacology and identify the future direction to be taken by the discipline. The present paper addresses these concerns; first, by reviewing the role of the snail-hosts in the epidemiology and control of schistosomiasis, and, secondly, by suggesting areas for future study. The possible application of newer methods in biotechnology for the resolution of malacological problems are discussed.

Eurytrema coelomaticum (Digenea, Dicrocoeliidae): the effect of infection on carbohydrate contents of its intermediate snail host, Bradybaena similaris (Gastropoda, Xanthonychidae)

The interface Eurytrema coelomaticum/Bradybaena similaris was studied by quantifying the amount of glucose on the hemolymph and the content of glycogen in the cells of the digestive gland and the cephalopedal mass of infected and uninfected snails. Samples were analyzed on days 0, 30, 90 and 150 post-infection. The infected snails had less glucose in the hemolymph, with a reduction of 67.05 por cento at 30 days, and 62.09 por cento at 90 days post-infection. The reduction in glycogen content was 86.41 por cento in the digestive gland and 79.1 por cento in the cephalopedal mass at 30 days, and 92.71 por cento and 90.89 por cento in these organs respectively at 90 days post-infection. It is proposed that the sporocysts absorb glucose directly from the hemolymph.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

Degree of host-parasite compatibility between Schistosoma mansoni and their intermediate molluscan hosts in Brazil

The compatibility of Biomphalaria tenagophila, B. straminea and B. glabrata from Minas Gerais with different strains of Schistosoma mansoni was evaluated using the method of Frandsen (1979b) in standardized experiments. One hundred and fifty of each species of snail were individually exposed in the laboratory to 50 miracidia of S. mansoni lines LE, SJ and AL. The cercariae from the infected snails were counted and used to calculate TCP/100 indices, which were compared with those of Frandsen (1979b). For B. tenagophila the TCP/100 indices varied from 37,996 to 74,266 (class II and III). The snail was poorly compatible with LE (class II) and compatible with SJ and AL (class III). For B. straminea the indices varied from 9,484 to 20,508. The snail was not very compatible with SJ (class I) and poorly compatible with LE and AL (class II). For B. glabrata the indices varied from 588,828 to 1,039,065. The snails was extremely compatible (class VI) with the three lines of S. mansoni. These results confirm the epidemiological importance of B. glabrata in Brazil followed by B. tenagophila and B. straminea.

Molecular characterisation of intermediate snail hosts and the search for resistance genes

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

The epidemiology and control of schistosomiasis mansoni where Biomphalaria tenagophila is the snail host

The epidemiology and control of schistosomiasis mansoni in the Municipality of Pedro de Toledo (State of S. Paulo, Brazil) since 1980, has been studied. In 1980 the prevalence evaluated by stool exams (Kato-Katz method) was 22.8% and no statistical difference at 5.0% level was observed between rural and urban zones. The intensity of infection was low (58.5 eggs/g of faeces); the highest prevalence and intensity of infection rates were observed within the group of from 5 to 29 years of age, respectively. The transmission of schistosomiasis usually occurred during leisure time. The majority of the carriers of the parasite were asymptomatic. Of the B. tenagophila examined only 0.4% were found to be infected. The control programme has been intensified from 1981 on resulting in a sharp decrease in the prevalence from 22.8% in 1980 to 6% at the present time. This result shows that, in spite of the control programme there is a residual human prevalence. A beginning has been made on the investigation into the possible causes of this residual prevalence (6.0% was maintained through out 1987).

Studies on survival, biological activities and behavior of Biomphalaria glabrata, the host snail of Schistosomiasis, submitted to increased hydrostatic pressure: a technique

To study changes in survival, in biological activities and behavior of planorbids submitted to increased hydrostatic pressure, we developed a technique using two transparent chambers and a hydraulic piston. The apparatus permitted renewal of the liquid medium without substantial variations in pressure, thus eliminating excretion products and maintaining the desired O2 level and thereby permitting us to evaluate the effects of pressure independently of the occurrence of anoxia. Pressure was maintained without any contact of the liquid medium with compressed air, a situation which reproduced with relative fidelity what occurs in nature and assured the presence of the same amounts of gases in the two observation chambers (Control and Experimental). Biomphalaria glabrata was found to be able to survive at least 48 hours when submitted to 49.02 x 10**4 Pa (equivalent to a water depth of 48.8 m), continuing to day egg masses and showing few behavioral changes when compared with the control group.

Effects of Bayluscide WP 70® on the Survival and Water-leaving Behaviour of Biomphalaria straminea, Snail Host of Schistosomiasis in Northeast Brazil

The toxic and behavioural effects of niclosamide (Bayluscide WP 70®) on Biomphalaria straminea from a highly endemic area of schistosomiasis in northeastern Brazil were investigated through laboratory bioassays. The LD50 and LD90 were 0.114 mg/l and 0.212 mg/l, respectively. Water-leaving behaviour occurred among 14% to 30% of the snails in the presence of sublethal doses of niclosamide and among 16% of the controls. It was concluded that both the relatively low susceptibility to niclosamide and water-leaving behaviour of local B. straminea may be responsible for the recolonization of transmission foci after mollusciciding. It was suggested that recently improved measures of snail control, such as controlled-release formulations of niclosamide and plant molluscicides should be considered in areas where snail control is recommended

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exihibits a remarkable degree of intraespecific polymorphism. Low stringency-PCR using primers for 18S rRNA permited the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using DdeI permits the differentiation of the three intermediate hosts of Schistosoma mansoni. The molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

A potential snail host of schistosomiasis in Bolivia: Biomphalaria amazonica paraense, 1966

Biomphalaria amazonica Paraense, 1996 was collected from a permanent pond in the outskirts of the Bolivian city of Santa Cruz. Identification of the collected specimens was made by comparison with the original description of the species and with topotypic material in the collection of Instituto Oswaldo Cruz. Phylogenetic analysis confirmed that these Bolivian specimens belong to B. amazonica.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

Population dynamics of intermediate snail hosts of Fasciola hepatica and some environmental factors in San Juan y Martinez municipality, Cuba

The variation of abundances of intermediate snail hosts of Fasciola hepatica in Cuba (Fossaria cubensis and Pseudosuccinea columella) was studied during one year under natural conditions at five sampling sites in San Juan y Martinez municipality, Pinar del Rio province, Cuba. The effect of some environmental variables on the lymnaeid abundances was also studied. A canonical correspondence analysis showed that both species do not generally occur together in the same habitat and that most factors affect them in an opposite fashion, although both of them correlate positively through time to the diversity of the habitats. F. cubensis prefers the sites that are in or closer to the city whereas P. columella is more abundant in rural sites. Lymnaeid abundances are mainly affected by nitrite and nitrate concentrations as well as by the abundance of the thiarid Tarebia granifera. F. cubensis is more abundant in polluted habitats with low densities (or absence) of T. granifera whereas P. columella prefers cleaner habitats and can coexist with the thiarid, even at its higher densities. The implications of divergent preferences of the two lymnaeids for the control of fasciolosis are discussed.