Background: Blastocystis species are common enteric human parasites and carriage has been linked to Irritable Bowel Syndrome (IBS), particularly diarrhoea-predominant IBS. The spectrum of immune reactivity to Blastocystis proteins has been reported previously in symptomatic patients. We investigated differences in serum immunoglobulin profiles between patients with IBS, both positive and negative for Blastocystis carriage, and healthy controls (HC). Methods: Forty diarrhoea-predominant IBS patients (26 patients positive for Blastocystis sp., 14 negative patients) and forty HC (24 positive, 16 Blastocystis-negative) were enrolled. Age, gender, ethnicity and serum immunoglobulin A (IgA) levels were recorded and faecal specimens were analysed using smear, culture and polymerase chain reaction amplification of ribosomal DNA. Sera were tested in Western blots and the reactivities compared to known targets using monoclonal antibodies Blastofluor® (Blastocystis specific antibody), MAb1D5 (cytopathicto Blastocystis cells), anti-promatrix metalloprotease-9 (anti-MMP-9) and SDS-PAGE zymograms. Results: Levels of serum IgA were significantly lower in Blastocystis carriers (p < 0.001) but had no relationship to symptoms. Western blots demonstrated serum IgG antibodies specific for Blastocystis proteins of 17,27,37,50,60-65, 75–90, 95–105 and 150 kDa MW. Reactivity to the 27, 50 and 75-95 kDa proteins were found more frequently in the IBS group compared to the HC’s (p < 0.001) and correlation was greater for Blastocystis-positive IBS patients (p < 0.001) than for negative IBS patients (p < 0.05). MAb1D5 reacted with proteins of 27 and 100 kDa, and anti-MMP-9 with 27, 50 and 75-100 kDa proteins. Bands were seen in zymograms around 100 kDa. Conclusions: Low serum IgA levels are associated with Blastocystis carriage. All IBS patients were more likely to demonstrate reactivity with Blastocystis proteins of 27 kDa (likely a cysteine protease), 50 and 75-95 kDa MW compared to HC. The presence of antibodies to these Blastocystis proteins in some Blastocystis-negative subjects suggests either prior exposure to Blastocystis organisms or antibody cross reactivities. The anti-proMMP-9 reaction at 50 and 75–100 kDa and the zymogram result suggest that metalloproteases may be important Blastocystis antigens.Trial registrationAustralian and New Zealand Clinical Trials registry ACTRN: 12611000918921

J. D. Langereis and M. I. de Jonge

S. Ménard et al.

K. K. Riemersma and N. Komar

S. Vallabhaneni et al.

W. Park et al.

W. E. Trick et al.

Background: Schistosomiasis japonica remains a major public health problem in China. Integrating molecular analyses, such as population genetic analyses, of the parasite into the on-going surveillance programs is helpful in exploring the factors causing the persistence and/or spread of Schistosoma japonicum. However, genotyping errors can seriously affect the results of such studies, unless accounted for in the analyses. Methods: We assessed the genotyping errors (missing alleles or false alleles) of seven S. japonicum microsatellites, using a pedigree data approach for schistosome miracidia, which were stored on Whatman FTA cards. Results: Among 107 schistosome miracidia successfully genotyped, resulting in a total of 715 loci calls, a total of 31 genotyping errors were observed with 25.2 % of the miracidia having at least one error. The error rate per locus differed among loci, which ranged from 0 to 9.8 %, with the mean error rate 4.3 % over loci. With the parentage analysis software Cervus, the assignment power with these seven markers was estimated to be 89.5 % for one parent and 99.9 % for a parent pair. One locus was inferred to have a high number of null alleles and a second with a high mistyping rate. Conclusion: To the authors’ knowledge, this is the first time that S. japonicum pedigrees have been used in an assessment of genotyping errors of microsatellite markers. The observed locus-specific error rate will benefit downstream epidemiological or ecological analyses of S. japonicum with the markers.

Background: Efforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level. Methods: After blood meal on a B. pahangi-harbouring cat, the Aedes togoi mosquitoes were maintained to harvest infective third stage larvae, which were then injected into male Mongolian gerbils. Subsequently, adult B. pahangi were obtained from the infected gerbil for genomic DNA extraction. Sequencing and subsequently, construction of genomic libraries were performed. This was followed by genomic analyses and gene annotation analysis. By using archived protein sequences of B. malayi and a few other nematodes, clustering of gene orthologs and phylogenetics were conducted. Results: A total of 9687 coding genes were predicted. The genome of B. pahangi shared high similarity to that B. malayi genome, particularly genes annotated to fundamental processes. Nevertheless, 166 genes were considered to be unique to B. pahangi, which may be responsible for the distinct properties of B. pahangi as compared to other filarial nematodes. In addition, 803 genes were deduced to be derived from Wolbachia, an endosymbiont bacterium, with 44 of these genes intercalate into the nematode genome. Conclusions: The reporting of B. pahangi draft genome contributes to genomic archive. Albeit with high similarity to B. malayi genome, the B. pahangi-unique genes found in this study may serve as new focus to study differences in virulence, vector selection and host adaptability among different Brugia spp.

Background: Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease.FindingsBlood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti.All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected.Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents. Conclusions: Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

Background: Borreliosis is highly prevalent in Xinjiang Uygur Autonomous Region, China. However, little is known about the presence of Borrelia pathogens in tick species in this region, in addition Borrelia pathogens have not been isolated from domestic animals. Methods: We collected adult ticks from domestic animals at 19 sampling sites in 14 counties in northern Xinjiang from 2012 to 2014. Ticks were identified to species by morphology and were molecularly analysed by sequences of mitochondrial 16S rDNA gene; 4–8 ticks of each species at every sampling site were sequenced. 112 live adult ticks were selected for each species in every county, and were used to culture Borrelia pathogens; the genotypes were then determined by sequences of the 5S-23S rRNA intergenic spacer and the outer surface protein A (ospA) gene. Results: A total of 5257 adult ticks, belonging to four genera and seven species, were collected. Compared with three decades ago, the abundance of the five common tick species during the peak ixodid tick season has changed. Certain tick species, such as Rhipicephalus turanicus (Rh. turanicus), was found at Jimusaer, Yining, Fukang, and Chabuchaer Counties for the first time. Additionally, the sequence analyses showed that the Hyalomma asiaticum (Hy. asiaticum), Haemaphysalis punctata (Ha. punctata), and Dermacentor marginatus (D. marginatus) that were collected from different sampling sites (≥3 sites) shared identical 16S rDNA sequences respectively. For the tick species that were collected from the same county, such as Hy. asiaticum from Shihezi County and Rh. turanicus from Yining County, their 16S rDNA sequences showed genetic diversity. In addition, sixteen Borrelia isolates were found in Hy. asiaticum, Ha. punctata, D. marginatus and Rh. turanicus, which infested cattle, sheep, horse and camel in Yining, Chabuchaer, Shihezi and Shawan Counties. All of the isolates were genetically identified as B. Burgdorferi sensu stricto. Conclusions: Warmer and wetter climate may have contributed to the altered distribution and abundance of the five most common ticks in northern Xinjiang. The genetic analyses showed that certain tick species, such as Hy. asiaticum or Rh. turanicus, exhibit genetic commonness or diversity. Additionally, this study is the first to isolate B. burgdorferi sensu stricto in Hy. asiaticum asiaticum, H. punctata, D. nuttalli and D. marginatus ticks from domestic animals. These ticks may transmit borreliosis among livestock.

Background: In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and in the state of Pernambuco, Northeastern region, where an isolated population has been identified. This study assessed the phylogeographic structure and size and shape differences of the wing of three Brazilian populations. Methods: Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the Cytochrome Oxidase I mitochondrial gene. Geometrics morphometry analysis of the right wing shape of the three populations was made using discriminate canonical analysis. Results: Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and the other clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations generated high indices of interpopulational divergence. Geometric morphometry analysis indicated two distinct groups between the studied populations. Canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and both populations were genetically distant from Manacapuru. Conclusion: The polymorphic sites and geometric morphometry analysis indicate that the distance, lack of continuity and environmental differences have not modified the ancestral relationship between Recife and Rio Preto da Eva populations. The genetic and morphological similarities shared by the Recife and Rio Preto da Eva populations suggest that these populations are more closely related evolutionarily. These results confirm the existence of an L. umbratilis species complex in the North and Northeast regions.

J. W. Cheng et al.

M. Munyikwa

C. Le Brun et al.

B. Breedlove and P. M. Arguin

Background: Early diagnosis of schistosomiasis, prior to egg laying, would enable earlier treatment and help interrupt the transmission cycle of the parasite and the progress of the disease. Previously we identified six novel antigens with potential as diagnostic markers for human Schistosoma japonicum infections. In this study, we evaluated these antigens as candidate biomarkers for the early diagnosis of schistosomiasis in mice and rabbits. Methods: The transcriptional profiles of the six antigens (SjSP-13, SjSP-23, SjSP-160, SjSP-164, SjSP-189 and SjSP-216) at different developmental stages were analyzed by quantitative PCR. The recombinant proteins were expressed in E. coli and purified with nickel chelate affinity chromatography. We then developed recombinant protein-based ELISA kits to analyze the kinetics of antigen-specific antibodies during the course of infection in mice and rabbits. The early diagnostic validity of the candidate SjSP-216 was further evaluated in mice and rabbits infected with S. japonicum. Results: Of the six antigens, SjSP-13, SjSP-160 and SjSP-216 were highly expressed in 21-day old young worms, while SjSP-23, SjSP-164 and SjSP-189 were highly expressed in eggs. In the mouse model, we detected a significant increase in antibodies against SjSP-13 and SjSP-216 at 3 weeks post-infection. However, in the rabbit model, only anti-SjSP-216 antibody showed a significant increase at this time point. We recorded 100 % diagnostic sensitivity and specificity of SjSP-216-based ELISA in both infected mice and rabbits, 3 weeks after infection. Conclusions: This study strongly suggests that SjSP-216, a highly expressed gene in the young worms, could serve as a potential biomarker for the early immunodiagnosis of S. japonicum infections in vertebrate hosts.

Background: Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources.FindingsHere a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30–45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5 % of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. Conclusion: The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

Background: Understanding the ecology of malaria vectors such as species composition and population dynamics is essential for developing cost-effective strategies to control mosquito vector populations. Methods: Adult mosquitoes (n = 79,567) were collected in five villages along the China-Myanmar border from April 2012 to September 2014 using the CDC light trap without bait method. Mosquito community structure, Anopheles species composition and diversity were analyzed. Results: Twenty species of Anopheles mosquitoes were identified, with An. minimus s.l. accounting for 85 % of the total collections. Mosquito densities varied from 0.05 females per trap per night (f/t/n) to 3.00 f/t/n, with strong seasonality in all sites and densities peaked from June to August. An. minimus s.l. was predominant (accounting for 54–91 % of total captures) in four villages, An. maculatus s.l. was predominant (71 %) in the high elevation village of Dao Nong, and An. culicifacies accounted for 15 % of total captures in the peri-urban area of Simsa Lawk. All 20 species have been captured in the Mung Seng Yang village, 18 and 15 species in Ja Htu Kawng and Na Bang respectively, and nine species in both Simsa Lawk and Dao Nong. Species richness peaked from April to August. Species diversity, species dominance index, and species evenness fluctuated substantially from time to time with no clear seasonality, and varied greatly amongst villages. Conclusions: Mosquitoes were abundant in the China-Myanmar bordering agricultural area with clear seasonality. Species composition and density were strongly affected by natural environments. The targeted intervention strategy should be developed and implemented so as to achieve cost-effectiveness for malaria control and elimination along the border areas.

Background: The protozoan Giardia duodenalis is a common but highly diverse human parasite that comprises a complex of seven morphologically identical genetic assemblages, further divided into sub-assemblages. There is very little information available on the diversity of Giardia sub-assemblages and multi-locus genotypes infecting people in the United Kingdom. In this study we studied the molecular epidemiology of Giardia in symptomatic patients from North West England. Methods: Whole faecal DNA was extracted from the faecal samples of 406 Giardia cases and the parasites assemblage, sub-assemblage and multi-locus genotype were determined using PCR amplification, DNA sequencing and phylogenetic analysis of the beta-giardin, glutamate dehydrogenase, triose-phosphate isomerase and small-subunit ribosomal RNA genes. Information about age, gender and self-reported clinical outcomes was also collected from the patients to check for differences associated with the infecting Giardia assemblage. Results: Our results showed a difference in the age prevalence of the two assemblages, with assemblage A being more common in older cases. Cases infected with assemblage B more often reported vomiting and a longer illness than cases infected with assemblage A. The majority of infections (64 %) were caused by assemblage B followed by assemblage A (33 %), while mixed-assemblage infections were rare (3 %). Assemblage A isolates mostly belonged to the sub-assemblage AII and showed completed identity with previously described isolates. The level of genetic sub-structuring was significantly higher in assemblage B isolates, since a higher proportion of novel assemblage B sequences was detected compared to assemblage A. A high number of assemblage B sequences showed heterogeneous nucleotide positions that prevented the unambiguous assignment to a specific sub-assemblage. Both previously described and novel multi-locus genotypes were described in both assemblages, and up to 17 different assemblage B multi-locus genotypes were found. Conclusions: We have produced the first data on the parasite multi-locus genotypes in the UK and have demonstrated that the molecular diversity of Giardia is similar to other developed countries. Furthermore, we showed that the parasite assemblages infecting humans may be associated with patients of different ages and with different clinical outcomes.