Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. METHODOLOGY To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). FINDINGS Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. CONCLUSIONS Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region.

An adult dog that lived in central British Columbia was examined because of a history of lethargy and vomiting. Histology, immunohistochemistry, and polymerase chain reaction (PCR) examination of a hepatic mass confirmed the presence of an alveolar hydatid cyst, the first description of Echinococcus multilocularis in British Columbia. We provide recommendations for case management and remind practitioners in endemic areas of western Canada that dogs can serve as definitive and, rarely, intermediate hosts for E. multilocularis.

An immunoassay for the detection of circulating antigens in human echinococcosis.

An immunoassay (double-antibody-sandwich-ELISA) was developed to detect circulating antigens (CAg) in patients with cystic (Echinococcus granulosus) echinococcosis. Echinococcus antigens derived from heterologous intermediate hosts were used to immunize rabbits and to purify the rabbit-IgG-fraction obtained by affinity-chromatography, thus avoiding major interference with host components. The purified rabbit anti-hydatid IgG was immunosorbed with bovine and human sera. One part of the resulting IgG served as coating agent in a double antibody sandwich-ELISA; the other part, coupled to alkaline phosphatase, as detecting conjugate. The specificity of the antibody reaction was demonstrated by immunoelectrophoresis. Sera of 21 patients with cystic echinococcosis were examined with this test system. In seven of the patients' sera CAg were detected in concentrations ranging between 310 ng and 680 ng protein per ml serum. Comparing pre- and postoperative serum samples obtained from nine patients operated on for cystic echinococcosis, four sera were found to be CAg-positive before and three after operation.

Echinococcus multilocularis Detection in Live Eurasian Beavers (Castor fiber) Using a Combination of Laparoscopy and Abdominal Ultrasound under Field Conditions.

Echinococcus multilocularis is an important pathogenic zoonotic parasite of health concern, though absent in the United Kingdom. Eurasian beavers (Castor fiber) may act as a rare intermediate host, and so unscreened wild caught individuals may pose a potential risk of introducing this parasite to disease-free countries through translocation programs. There is currently no single definitive ante-mortem diagnostic test in intermediate hosts. An effective non-lethal diagnostic, feasible under field condition would be helpful to minimise parasite establishment risk, where indiscriminate culling is to be avoided. This study screened live beavers (captive, n = 18 or wild-trapped in Scotland, n = 12) and beaver cadavers (wild Scotland, n = 4 or Bavaria, n = 11), for the presence of E. multilocularis. Ultrasonography in combination with minimally invasive surgical examination of the abdomen by laparoscopy was viable under field conditions for real-time evaluation in beavers. Laparoscopy alone does not allow the operator to visualize the parenchyma of organs such as the liver, or inside the lumen of the gastrointestinal tract, hence the advantage of its combination with abdominal ultrasonography. All live beavers and Scottish cadavers were largely unremarkable in their haematology and serum biochemistry with no values suspicious for liver pathology or potentially indicative of E. multilocularis infection. This correlated well with ultrasound, laparoscopy, and immunoblotting, which were unremarkable in these individuals. Two wild Bavarian individuals were suspected E. multilocularis positive at post-mortem, through the presence of hepatic cysts. Sensitivity and specificity of a combination of laparoscopy and abdominal ultrasonography in the detection of parasitic liver cyst lesions was 100% in the subset of cadavers (95%Confidence Intervals 34.24-100%, and 86.7-100% respectively). For abdominal ultrasonography alone sensitivity was only 50% (95%CI 9.5-90.6%), with specificity being 100% (95%CI 79.2-100%). For laparoscopy alone sensitivity was 100% (95% CI 34.2-100%), with specificity also being 100% (95% CI 77.2-100%). Further immunoblotting, PCR and histopathological examination revealed one individual positive for E. multilocularis, whilst the other individual was positive for Taenia martis.

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

UNLABELLED We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. IMPORTANCE The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.

DNA probes for the detection of Fasciola hepatica in snails

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

A DNA probe for the detection of Dicrocoelium dendriticum in ants of Formica spp. and Lasius spp

Repetitive DNA sequences present in the genome of Dicrocoelium dendriticum were identified by hybridization of genomic DNA that had been digested with different restriction enzymes with 32P-labeled genomic D. dendriticum DNA. DNA fragments containing repetitive sequences were isolated from PstI-digested D. dendriticum DNA and were subcloned into a plasmid vector. Plasmids containing repetitive sequences were identified by colony hybridization. One of these plasmids, designated Ddr-IV, was isolated and used as a probe in further studies. Ddr-IV is specific for D. dendriticum since it does not hybridize to DNA isolated from other trematodes. In addition, Ddr-IV was capable of detecting D. dendriticum metacercariae in ants (Formica cunicularia, F. rufibarbis, and Lasius sp.), which act as second intermediate hosts in the parasite's life cycle. Since metacercariae constitute the infectious stage of the parasite for grazing animals, Ddr-IV will provide a useful tool for epidemiology studies of dicrocoeliosis.

Parasite communities and diet of Coris Batuensis (Pisces Labridae) from Lizard Island, Great Barrier Reef

Parasite infracommunities of the wrasse Coris batuensis (Bleeker, 1857) were analysed, and the relationship between endoparasites, diet, and host body weight inferred. Thirty-two fish were collected from Lizard Island, Australia. Percentage frequency of occurrence of prey categories in the gut was determined and abundance, prevalence and species richness of parasites were calculated. Fish mainly ate snails, bivalves and crustaceans and this did not vary with body weight. Thirty-one fish were parasitised with at least one of 21 taxa of parasites (4 ectoparasite and 17 endoparasite species), with an average of 4 species and 47 individuals per host. Tetraphyllidean cestode larvae were the most common and abundant group. Parasite life cycles are not known in detail, but small crustaceans, such as copepods and amphipods, are likely to be intermediate hosts for the cestodes, nematodes and digeneans found in C. batuensis. Molluscs, although frequent in the diet, may not be transmitting any parasite species. Numbers of prey and parasite species richness were not correlated. Composition, abundance and species richness of the parasite fauna were similar in hosts with different body weight, corresponding with C. batuensis having a similar diet throughout life. © Queensland Museum.

Advances in the Diagnosis of Human Schistosomiasis

Schistosomiasis is a major neglected tropical disease that afflicts more than 240 million people, including many children and young adults, in the tropics and subtropics. The disease is characterized by chronic infections with significant residual morbidity and is of considerable public health importance, with substantial socioeconomic impacts on impoverished communities. Morbidity reduction and eventual elimination through integrated intervention measures are the focuses of current schistosomiasis control programs. Precise diagnosis of schistosome infections, in both mammalian and snail intermediate hosts, will play a pivotal role in achieving these goals. Nevertheless, despite extensive efforts over several decades, the search for sensitive and specific diagnostics for schistosomiasis is ongoing. Here we review the area, paying attention to earlier approaches but emphasizing recent developments in the search for new diagnostics for schistosomiasis with practical applications in the research laboratory, the clinic, and the field. Careful and rigorous validation of these assays and their cost-effectiveness will be needed, however, prior to their adoption in support of policy decisions for national public health programs aimed at the control and elimination of schistosomiasis.

A morphological and genetic description of pentastomid infective nymphs belonging to the family Sebekidae Sambon, 1922 in fish in Australian waters

Infective nymphal stages of the family Sebekidae Sambon, 1922 are reported from four species of fish in Australian waters for the first time. Infected fish were collected from locations in Western Australia, the Northern Territory and north Queensland. The infective nymphs of Alofia merki Giglioli in Sambon, 1922 and Sebekia purdieae Riley, Spratt et Winch, 1990 are reported and described for the first time. The remaining specimens were identified as belonging to the genus Sebekia Sambon, 1922 based on the combination of buccal cadre shape, shape and size of hooks, and overall body size, but could not be attributed to any of the other species of Sebekia already reported due to missing required morphological features. DNA sequences of members of the family Sebekidae are presented for the first time. The lack of knowledge on the pentastome fauna of wild crocodiles, and any potential intermediate hosts, in northern Australia, is also outlined.

"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.

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.

A low stringency polymerase chain reaction approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil

The low stringency-polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluated as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of genetic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at the DNA level using LS-PCR.

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.