Background: The estimation of the prevalence and zoonotic potential of Cryptosporidium parvum cycling in bovine populations requires the use of genotyping as several morphologically similar non-parvum variants of unproven clinical and public health impact are found in cattle. Robust C. parvum prevalence estimates in cattle are lacking and comparative data of bovine and human parasites collected from the same regions are scarce. Thus, the relative contribution of the oocysts released by farmed animals to human cryptosporidiosis burden is, in general, poorly understood. Methods: The New Zealand farm-level C. parvum prevalence was estimated using a cross-sectional sample of 1283 faecal specimens collected from newborn calves on 97 dairy farms. Faeces were analysed by immunofluorescence and the Cryptosporidium parasites were genetically identified. Finally, bovine C. parvum were genetically compared with historical human clinical isolates using a bilocus subtyping scheme. Results: Immunofluoresence-positive faeces were found in 63/97 (65%) farms. C. parvum was identified in 49 (50.5%) farms, C. bovis in 6 (6.1%) farms, and on 8 (8.2%) farms the species could not be identified. The dominant C. parvum genetic variants were geographically widespread and found in both host populations, but several variants were found in humans only. Conclusions: Phenotypic tests offered by New Zealand veterinary diagnostic laboratories for the diagnosis of C. parvum may have moderate to high positive predictive values. The genetic similarities observed between human and bovine C. parvum support a model considering calves as significant amplifiers of zoonotic C. parvum in New Zealand. However, data suggest that transmission routes not associated with dairy cattle should also be taken into account in future source-attribution studies of human cryptosporidiosis.

Background: The response of Culicoides biting midges (Diptera: Ceratopogonidae) to artificial light sources has led to the use of light-suction traps in surveillance programmes. Recent integration of light emitting diodes (LED) in traps improves flexibility in trapping through reduced power requirements and also allows the wavelength of light used for trapping to be customized. This study investigates the responses of Culicoides to LED light-suction traps emitting different wavelengths of light to make recommendations for use in surveillance. Methods: The abundance and diversity of Culicoides collected using commercially available traps fitted with Light Emitting Diode(LED) platforms emitting ultraviolet (UV) (390 nm wavelength), blue (430 nm), green (570 nm), yellow (590 nm), red (660 nm) or white light (425 nm – 750 nm with peaks at 450 nm and 580 nm) were compared. A Centre for Disease Control (CDC) UV light-suction trap was also included within the experimental design which was fitted with a 4 watt UV tube (320-420 nm). Generalised linear models with negative binomial error structure and log-link function were used to compare trap abundance according to LED colour, meteorological conditions and seasonality. Results: The experiment was conducted over 49 nights with 42,766 Culicoides caught in 329 collections. Culicoides obsoletus Meigen and Culicoides scoticus Downes and Kettle responded indiscriminately to all wavelengths of LED used with the exception of red which was significantly less attractive. In contrast, Culicoides dewulfi Goetghebuer and Culicoides pulicaris Linnaeus were found in significantly greater numbers in the green LED trap than in the UV LED trap. The LED traps collected significantly fewer Culicoides than the standard CDC UV light-suction trap. Conclusions: Catches of Culicoides were in LED traps when compared to the standard CDC UV trap, however, their reduced power requirement and small size fulfils a requirement for trapping in logistically challenging areas or where many traps are deployed at a single site. Future work should combine light wavelengths to improve trapping sensitivity and potentially enable direct comparisons with collections from hosts, although this may ultimately require different forms of baits to be developed.

Background: The bacteria Orientia tsutsugamushi is the causative agent of scrub typhus, mite-borne disease, which causes an acute febrile illness in patients. An epidemiologic study was conducted to understand the characteristics of scrub typhus in South Korea.FindingsReporting of tsutsugamushi disease is mandatory in South Korea since 1994. To investigate the prevalence of tsutsugamushi disease from 2001 to 2013, medical records from the Korea Center for Disease Control and Prevention were reviewed. In total, 70,914 cases were reported during 2001–2013. Of these, 37.16% (26,349) were male and 62.84% (44,565) were female. The highest number of cases was in the 60–69-year-old age group (19,484; 27.48%), and 72.22% (51,212) were in the 50–79-year-old age group. There were 65,100 cases (91.80%) reported during October (24,964; 35.20%) and November (40,136; 56.60%). An almost four-fold increase in the number of patients was observed in 2013 (10,485 cases) compared to 2001 (2,637 cases). The highest number of patients was reported in the Jeonbuk (9,425; 13.29%) and lowest in the Jeju (362; 0.51%). Conclusions: A rapid increase in the incidence of patients with tsutsugamushi disease was observed in most areas from 2001 to 2013, with the majority of cases reported in the western and southern coast.

Background: Aedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit dengue fever and West Nile virus diseases, respectively. This study was conducted to determine the toxicity and mechanism of action of four flavonoids and two fatty acids from Millettia pinnata (Fabaceae) seed as well as six pure fatty acids and four fatty acid esters toward third instar larvae from insecticide-susceptible C. pipiens pallens and A. aegypti as well as wild A. albopictus. Efficacy of 12 experimental liquid formulations containing M. pinnata seed methanol extract and hydrodistillate (0.5–10.0% liquids) was also assessed. Methods: The contact toxicities of all compounds and 12 formulations were compared with those of two larvicides, temephos and fenthion and the commercial temephos 200 g/L emulsifiable concentrate (EC). The possible mode of larvicidal action of the constituents was elucidated using biochemical methods. Larval mortality and cAMP level were analyzed by the Bonferroni multiple-comparison method. Results: Potent toxicity was produced by karanjin, oleic acid, karanjachromene, linoleic acid, linolenic acid, pongamol, pongarotene, and elaidic acid toward C. pipiens pallens larvae (24 h LC50, 14.61–28.22 mg/L) and A. aegypti larvae (16.13–37.61 mg/L). Against wild A. albopictus larvae, oleic acid (LC50, 18.79 mg/L) and karanjin (35.26 mg/L) exhibited potent toxicity. All constituents were less toxic than either temephos or fenthion. Structure–activity relationship indicates that the degree of saturation, the side chain length, and the geometric isomerism of fatty acids appear to play a role in determining the fatty acid toxicity. Acetylcholinesterase (AChE) is the main site of action of the flavonoids, oleic acid, and palmitic acid. The mechanism of larvicidal action of elaidic acid, arachidic acid, and behenic acid might be due to interference with the octopaminergic system. Linoleic acid and linolenic acid might act on both AChE and octopaminergic receptor. M. pinnata seed extract or hydrodistillate applied as 10% liquid provided 100% mortality toward the three mosquito species larvae and the efficacy of the liquids was comparable to that of temephos 200 g/L EC. Conclusion: Further studies will warrant possible applications of M. pinnata seed-derived products as potential larvicides for the control of mosquito populations.

Background: Human African Trypanosomiasis (HAT) is an important neglected tropical disease caused by Trypanosoma spp. parasites transmitted by species of tsetse fly (Glossina spp). The most important vectors of HAT are riverine tsetse and these can be controlled by attracting them to stationary baits such as insecticide-impregnated traps or targets deployed along the banks of rivers. However, the geographical nature of some riverine habitats, particularly mangroves but also extensive lake and river networks, makes deployment of baits difficult and limits their efficacy. It is known that tsetse are attracted by the movement of their hosts. Our hypothesis was that mounting a target on canoes typically used in Africa (‘pirogues’) would produce an effective means of attracting-and-killing riverine tsetse in extensive wetland habitats. Methods: In Folonzo, southern Burkina Faso, studies were made of the numbers of tsetse attracted to a target (75 × 50 cm) of blue cloth and netting mounted on a pirogue moving along a river, versus the same target placed on the riverbank. The targets were covered with a sticky film which caught tsetse as they contacted the target. Results: The pirogue-mounted target caught twice as many G. tachinoides and G. p. gambiensis, and 8 times more G. morsitans submorsitans than the stationary one (P < 0.001). Conclusion: Pirogues are common vehicle for navigating the rivers, lakes and swamps of West Africa. The demonstration that tsetse can be attracted to targets mounted on such boats suggests that pirogues might provide a cost-effective and convenient platform for deploying targets to control tsetse in the mangrove systems of West Africa where HAT persists. Further studies to assess the impact of pirogue-mounted targets on tsetse populations in HAT foci and the protective value of targets for pirogue passengers are recommended.

Background: Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep. Methods: Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes. Results: Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection. Conclusions: Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.

Background: It has been well accepted that glycans present in schistosomes are highly antigenic. However, it is not clear what kind of worm glycans can affect the infected host to mount IgG responses and whether mounted anti-glycan IgG responses are protective. Methods: The contribution of antigenicity by glycans was measured by using competitive ELISA assay in sera from infected mice and humans. Monoclonal antibodies towards soluble Schistosoma japonicum egg antigens (SjEA) were generated from SjEA immunizated mice. The expression of glycans on surfaces of cercaria or young worm and their distributions were examined by immunofluorescence assay. The protective roles of glycans-specific mAbs were assayed by determination of the worm and egg burden in infected mice. Results: Both periodate-resistant glycans and periodate-sensitive glycans are antigenic in schistosome infections. When monoclonal antibodies against either periodate-sensitive or periodate-resistant glycans were administered prior to schistosome infections in mice, both kinds of anti-glycan antibodies were found to successfully provide protective immunity to infected mice. Conclusions: Both periodate-resistant and periodate-sensitive glycans are antigenic, and dominant anti-glycan IgG responses can play important roles in protective immunity in schistosome infected hosts.

Background: The genus Evandromyia is widely found in Brazil, but occurs mainly in Brazilian savannah. To date 13 species have been described in the subgenus Aldamyia. Here we described a new species of Evandromyia (Aldamyia) collected in the State of Mato Grosso do Sul, Brazil. Methods: Measurements were made using a micrometer eyepiece on an Olympus CH-2 binocular microscope and drawings were executed with the aid of a camera lucida. Results: The new species, Evandromyia orcyi sp. nov., is closely related to Evandromyia lenti, Evandromyia carmelinoi and Evandromyia evandroi, however, characteristics of the male terminalia and female spermathecae distinguish it from other species of the genus Evandromyia. Conclusion: With the description of Evandromyia orcyi sp. nov., six species of the subgenus Aldamyia have been reported from the State of Mato Grosso do Sul.

Background: Autochthonous populations of Dermacentor reticulatus ticks in the Netherlands were discovered after fatal cases of babesiosis occurred in resident dogs in 2004. The presence of D. reticulatus in the Netherlands has also linked with the emergence of piroplasmosis in the resident horse population. The aim of this study was to put together results of continued surveillance of field sites and hosts for this tick in the Netherlands and also in Belgium and determine their infection status for Babesia and Theileria species. Methods: Ticks were collected from the vegetation at 11 locations between 2011 and 2013. D. reticulatus ticks were also collected from different hosts between 2007 and 2013. Ticks were screened by PCR and reverse line blot (RLB). Results: A total of 1368 D. reticulatus ticks were collected from 4 previously known field locations and from 5 new locations in the Netherlands and from 2 sites in Belgium (one old and one new location). A total of 855 ticks collected from 8 locations in the Netherlands and 2 locations in Belgium were tested. Fourteen ticks (1,64%) collected at 4 field locations (Dintelse Gorzen, Rozenburg, Slikken van de Heen and St. Philipsland) were positive for Babesia canis, whereas two ticks were positive for Babesia caballi, one tick in the Dintelse Gorzen in the Netherlands and one tick was found positive in De Panne in Belgium.A further 1092 D. reticulatus ticks were collected between 2007 and 2013 from 40 dogs (132 ticks), two ticks from two humans, 51 ticks from 15 horses, two ticks from two cats, one tick from a roe deer, whereas most ticks (904) were collected from cattle (n = 25). Ticks were found throughout the year on dogs in nearly all provinces of the Netherlands. None of the ticks collected from these hosts were infected. Conclusions: D. reticulatus is continuing its spread into novel areas. The finding that some autochthonous ticks are infected with B. canis and B. caballi poses a threat to the resident dog and horse population and justifies year-round tick control measures.

Background: Tunisia is a hyper endemic country for human echinococcosis. The infection is transmitted via the eggs of Echinococcus granulosus which are passed in the faeces of the definitive canid host. Methods: This study evaluated the contamination rate of the dog faeces in different climatic conditions at eight different geographic regions throughout Tunisia. Dog faecal samples were collected from the soil and the Echinococcus eggs were identified using microscopic and molecular (Eg1121/1122 PCR, Egss1 PCR and Nad1 PCR-RFLP) tools. Results: The contamination index of dog faeces by E. granulosus eggs ranged from 8.3% to 41.3% depending on the region. Comparisons of the dog faecal contamination rate against human incidence found them to be independent. Neither human prevalence nor dog contamination index appeared to be related to climatic conditions or geographic characteristics. The genetic variability of E. granulosus samples was different within each region but was not related to geographic distance which is indicative of local divergent evolutions rather than isolation by distance. Conclusions: A high environmental dog contamination index does not necessarily correspond to high prevalence in humans as transmission is strongly linked to human behavior and hygiene.

Background: Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Methods: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 μl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. Results: The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. Conclusions: The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

Background: Theileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries. This paper provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite. Methods: To identify possible vectors for disease, ticks, biting flies and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife, and mosquitoes and biting flies were collected in traps in close proximity to outbreak herds. Ticks were identified via DNA barcoding of the mitochondrial cytochrome oxidase I gene. Barcoded ticks were pooled according to species or phylogenetic group and tested for the presence of T. orientalis and the genotypes Ikeda, Chitose and Buffeli using real-time PCR. Results: DNA barcoding and phylogenetic analysis identified ticks from the following species: Haemaphysalis longicornis, Ixodes holocyclus, Ixodes cornuatus, Ixodes hirsti, and Bothriocroton concolor. Additional Haemaphysalis, Ixodes and Bothriocroton spp. were also identified. Of the ticks investigated, only H. longicornis ticks from cattle carried theilerial DNA, with the genotypes Ikeda, Chitose and Buffeli represented. Mosquitoes collected in close proximity to outbreak herds included; Aedes camptorhynchus, Aedes notoscriptus, Coquillettidia linealis, Culex australicus, and Culex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquitoes. The haematophagous flies tested negative. Conclusions: This is the first demonstration of a potential vector for T. orientalis in the current Australasian disease outbreak.

Background: Recent studies revealed expansion of filarioid nematodes into northern Finland. In addition to Setaria tundra, an abundant filarioid, Rumenfilaria andersoni, was found inhabiting the lymphatic vessels of reindeer. Our study explores the dynamics of the rapid geographic expansion of R. andersoni, defining prevalence and density of microfilariae among 4 new cervid host species in Finland while developing a context for host-parasite ecology in Fennoscandia and more broadly in the Arctic and boreal regions. Methods: Blood samples were evaluated for presence of microfilariae from 1576 semi-domesticated reindeer, 8 captive reindeer, and free-ranging cervids including 105 wild forest reindeer, 862 moose, 114 white tailed deer and 73 roe deer in 2003–2006 (−2010). Additionally, the prepatent period and the efficacy of ivermectin treatment were investigated. Results: Rumenfilaria andersoni was found to be a common and abundant parasite in reindeer (0-90%) and wild forest reindeer (41-100%). Also moose (0-12%), white-tailed deer (15-22%) and roe deer (3%) were revealed as definitive hosts. Ivermectin was not effective against adult parasites. The prepatent period was estimated to be about five months. Conclusions: Rumenfilaria andersoni was identified in 3 endemic cervid species and the introduced white-tailed deer, all constituting previously unrecognized host species in the Palearctic. Among moose, the prevalence and intensity were substantially lower than levels observed among subspecies of reindeer. White-tailed deer had a relatively high prevalence and density of R. andersoni microfilariae (rmf), whereas our limited data for roe deer indicated that the nematode may not have been abundant. Density and prevalence of rmf in moose and white tailed deer suggests the nematode may be adapted to these species, and that these cervids may be among the primary hosts of R. andersoni and reservoirs for transmission in Finland. Our current data suggest that R. andersoni became established in Finland recently, coincidental with introduction of white-tailed deer from North America in 1935; subsequent invasion and emergence in the past 70–80 years appears driven by climate-related factors. An alternative hypothesis for a temporally deeper occurrence for R. andersoni in Fennoscandia, representing post-Pleistocene range expansion with moose tracking deglaciation, is not firmly supported.

A. Maheshwari et al.

D. Otranto et al.

A. J. Lambert et al.

Background: Certain municipalities in the Belo Horizonte Metropolitan Area (BHMA), Minas Gerais, Brazil, have the highest human visceral leishmaniasis (VL) mortality rates in the country and also demonstrate high canine seropositivity. In Brazil, the etiologic agent of VL is Leishmania (Leishmania) infantum. The aim of this study was to evaluate the intraspecific genetic variability of parasites from humans and from dogs with different clinical forms of VL in five municipalities of BHMA using PCR-RFLP and two target genes: kinetoplast DNA (kDNA) and gp63. Methods: In total, 45 samples of DNA extracted from clinical samples (n = 35) or L. infantum culture (n = 10) were evaluated. These samples originated from three groups: adults (with or without Leishmania/HIV co-infection; n = 14), children (n = 18) and dogs (n = 13). The samples were amplified for the kDNA target using the MC1 and MC2 primers (447 bp), while the Sg1 and Sg2 (1330 bp) primers were used for the gp63 glycoprotein target gene. Results: The restriction enzyme patterns of all the samples tested were monomorphic. Conclusions: These findings reveal a high degree of genetic homogeneity for the evaluated gene targets among L. infantum samples isolated from different hosts and representing different clinical forms of VL in the municipalities of BHMA studied.

Background: Endoparasites with complex life cycles are faced with several biological challenges, as they need to occupy various ecological niches throughout their development. Host phenotypes that increase the parasite’s transmission rate to the next host have been extensively described, but few mechanistic explanations have been proposed to describe their proximate causes. In this study we explore the possibility that host phenotypic changes are triggered by the production of mimicry proteins from the parasite by using an ecological model system consisting of the infection of the threespine stickleback (Gasterosteus aculeatus) by the cestode Schistocephalus solidus.MethodUsing RNA-seq data, we assembled 9,093 protein-coding genes from which ORFs were predicted to generate a reference proteome. Based on a previously published method, we built two complementary analysis pipelines to i) establish a general classification of protein similarity among various species (pipeline A) and ii) identify candidate mimicry proteins showing specific host-parasite similarities (pipeline B), a key feature underlying the possibility of molecular mimicry. Results: Ninety-four tapeworm proteins showed high local sequence homology with stickleback proteins. Four of these candidates correspond to secreted or membrane proteins that could be produced by the parasite and eventually be released in or be in contact with the host to modulate physiological pathways involved in various phenotypes (e.g. behaviors). One of these candidates belongs to the Wnt family, a large group of signaling molecules involved in cell-to-cell interactions and various developmental pathways. The three other candidates are involved in ion transport and post-translational protein modifications. We further confirmed that these four candidates are expressed in three different developmental stages of the cestode by RT-PCR, including the stages found in the host. Conclusion: In this study, we identified mimicry candidate peptides from a behavior-altering cestode showing specific sequence similarity with host proteins. Despite their potential role in modulating host pathways that could lead to parasite-induced phenotypic changes and despite our confirmation that they are expressed in the developmental stage corresponding to the altered host behavior, further investigations will be needed to confirm their mechanistic role in the molecular cross-talk taking place between S. solidus and the threespine stickleback.