Parasites and Vectors

Invaders, natives and their enemies: distribution patterns of amphipods and their microsporidian parasites in the Ruhr Metropolis, Germany

Background: The amphipod and microsporidian diversity in freshwaters of a heterogeneous urban region in Germany was assessed. Indigenous and non-indigenous host species provide an ideal framework to test general hypotheses on potentially new host-parasite interactions, parasite spillback and spillover in recently invaded urban freshwater communities. Methods: Amphipods were sampled in 17 smaller and larger streams belonging to catchments of the four major rivers in the Ruhr Metropolis (Emscher, Lippe, Ruhr, Rhine), including sites invaded and not invaded by non-indigenous amphipods. Species were identified morphologically (hosts only) and via DNA barcoding (hosts and parasites). Prevalence was obtained by newly designed parasite-specific PCR assays. Results: Three indigenous and five non-indigenous amphipod species were detected. Gammarus pulex was further distinguished into three clades (C, D and E) and G. fossarum more precisely identified as type B. Ten microsporidian lineages were detected, including two new isolates (designated as Microsporidium sp. nov. RR1 and RR2). All microsporidians occurred in at least two different host clades or species. Seven genetically distinct microsporidians were present in non-invaded populations, six of those were also found in invaded assemblages. Only Cucumispora dikerogammari and Dictyocoela berillonum can be unambiguously considered as non-indigenous co-introduced parasites. Both were rare and were not observed in indigenous hosts. Overall, microsporidian prevalence ranged from 50 % (in G. roeselii and G. pulex C) to 73 % (G. fossarum) in indigenous and from 10 % (Dikerogammarus villosus) to 100 % (Echinogammarus trichiatus) in non-indigenous amphipods. The most common microsporidians belonged to the Dictyocoela duebenum- /D. muelleri- complex, found in both indigenous and non-indigenous hosts. Some haplotype clades were inclusive for a certain host lineage. Conclusions: The Ruhr Metropolis harbours a high diversity of indigenous and non-indigenous amphipod and microsporidian species, and we found indications for an exchange of parasites between indigenous and non-indigenous hosts. No introduced microsporidians were found in indigenous hosts and prevalence of indigenous parasites in non-indigenous hosts was generally low. Therefore, no indication for parasite spillover or spillback was found. We conclude that non-indigenous microsporidians constitute only a minimal threat to the native amphipod fauna. However, this might change e.g. if C. dikerogammari adapts to indigenous amphipod species or if other hosts and parasites invade.

First report outside Eastern Europe of West Nile virus lineage 2 related to the Volgograd 2007 strain, northeastern Italy, 2014

Background: West Nile virus (WNV) is a Flavivirus transmitted to vertebrate hosts by mosquitoes, maintained in nature through an enzootic bird-mosquito cycle. In Europe the virus became of major public health and veterinary concern in the 1990s. In Italy, WNV re-emerged in 2008, ten years after the previous outbreak and is currently endemic in many areas of the country. In particular, the northeastern part of Italy experience continuous viral circulation, with human outbreaks caused by different genovariants of WNV lineage 1, Western-European and Mediterranean subcluster, and WNV lineage 2, Hungarian clade. Alongside the WNV National Surveillance Program that has been in place since 2002, regional surveillance plans were implemented after 2008 targeting mosquitoes, animals and humans.FindingsIn July and September 2014, West Nile virus lineage 2 was detected in pools of Culex pipiens s.l. mosquitoes from northeastern Italy. Whole genome sequencing and phylogenetic analysis of two representative samples identified the presence of WNV lineage 2 related to the Volgograd 2007 strain (99.3 % nucleotide sequence identity), in addition to WNV lineage 2 Hungarian clade. Conclusions: This is the first evidence of the circulation of a WNV lineage 2 strain closely related to the Volgograd 2007 outside Eastern Europe, where it has caused large human outbreaks. This strain may pose a new threat to animal and human health in Italy.

Repurposing of anticancer drugs: in vitro and in vivo activities against Schistosoma mansoni

Background: Drug discovery for the neglected tropical disease schistosomiasis has a high priority. Anticancer drugs, especially protein kinase inhibitors, might serve as a starting point for drug discovery owing to the importance of protein kinases in helminth growth and development. Furthermore, the Schistosoma mansoni genome encodes several genes for targets of drugs marketed for human use, including several anticancer drugs. Methods: In this study, we screened the approved oncology drug set of the National Cancer Institute’s Developmental Therapeutic Program for antischistosomal activity. Drugs were tested in vitro against the larval and adult stage of S. mansoni. IC 50 values and albumin binding were determined for active compounds. Lead compounds were tested in the chronic S. mansoni mouse model. Results: Eleven of the 114 compounds tested revealed IC 50 values ≤ 10 μM against both S. mansoni stages. Five of these lost activity against adult S. mansoni in the presence of serum albumin. Of 6 compounds studied in vivo, the highest activity was observed from two kinase inhibitors trametinib, and vandetanib, which reduced worm burden by 63.6 and 48.1 % respectively, after a single oral dose of 400 mg/kg body weight. Conclusion: Our study has confirmed that oncology drugs possess antischistosomal activity. There is space for further investigation, including elucidation of the mechanisms of action of schistosome-active cancer drugs, application of different treatment courses, and structure-activity relationship studies for improving drug potency.

Efficacy assessment of biocides or repellents for the control of Sarcoptes scabiei in the environment

Background: Sarcoptes scabiei infection is a contagious disease affecting both humans and animals. The transmission occurs either by direct contact or from the environment where mites could survive several days remaining infective. The number of products available for environmental control of S. scabiei is very limited. The objective of the present study was to assess the efficacy of biocides or repellents against S. scabiei var suis. Methods: Tested products included pyrethroids: permethrin, esdepallethrin and bioresmethrin, bifenthrin, cypermethrin and imiprothrin, cyfluthrin, tetramethrin and sumithrin. We also tested repellents: DEET, icaridin and IR3535. Sarcoptes scabiei var suis mites were collected from experimentally-infected pigs. For each test, 20 live mites of all motile stages were placed in a plastic Petri dish and sprayed uniformly by each product. Control mites were sprayed by distilled water. The study was performed in triplicate under room conditions and the mites were inspected under a stereomicroscope at intervals (5, 10, 15, 20, 25, 30, 40, 50, 60 min, 2, 3, 4, 5 and 24 h) after exposure to the products. Results: All the products, except the combination of tetramethrin and sumithrin (A-PAR®), were able to kill all mites within 24 h. The median survival time was 50 ± 30.4 min, 120 ± 309 min, 10 ± 5.9 min, 40 ± 36.8 min, 15 ± 7.3 min, 180 ± 417 min and 1440 ± 600 min when mites were exposed to permethrin 4 %, permethrin 0.6 %, esdepallethrin and bioresmethrin, bifenthrin, cypermethrin and imiprothrin, cyfluthrin, tetramethrin and sumithrin, respectively. The median survival time was 20 ± 6.5 min, 15 ± 4.3 min, 30 ± 42.1 min and 15 ± 4.9 min for DEET 25, DEET 50, icaridin 20 and IR3535 20 %, respectively. Conclusions: The results of the present study could support evidence-based use of biocides and repellents in households, hospitals and farms.

Parasitological, serological and molecular survey of Trypanosoma evansi infection in dromedary camels from Cholistan Desert, Pakistan

Background: Surra, a vector borne disease caused by Trypanosoma (T.) evansi, affects the health, productivity and working capacity of camels. Since clinical signs are not pathognomonic, diagnosis must be confirmed by laboratory methods. This is a first study on the prevalence of surra in Cholistan Desert, Pakistan using a broad variety of diagnostic tests thereby emphasizing it as a risk for the dromedaries of Pakistan. Methods: In a cross sectional study, 1005 dromedary camels from three districts in the Cholistan Desert were sampled to assess the prevalence of trypanosomosis due to T. evansi by means of parasitological (Giemsa stained thin smear), serological (formol gel test, CATT/T. evansi, ELISA/VSG RoTat 1.2, immune trypanolysis) and molecular tests (TBR1/2 PCR and RoTat 1.2 PCR). Kappa was calculated to assess the degree of agreement between different tests whereas chi-square test along with odds ratios and their 95 % confidence intervals were used to study influence of breed, gender, age and locality on disease prevalence. Results: Overall prevalence was 0.7 % with Giemsa stained thin smears (GST), 40.1 % with formol gel test (FGT), 47.7 % with CATT/T. evansi, 44.2 % with ELISA/VSG RoTat 1.2, 39.9 % with immune trypanolysis (TL), 31.9 % with TBR1/2 PCR and 30.5 % with RoTat1.2 PCR. Based on these results, the Cholistan Desert appears to be a high risk area for surra. According to TL and TBR1/2 PCR, camels at Bahawalpur are approximately two times more likely to be infected than those in Bahawalnagar (OR = 1.8; 95 % CI: 1.38-2.42) and Rahim Yar Khan (OR = 1.9; 95 % CI: 1.30-2.75). Test agreement of TL was moderate with CATT/T. evansi (k = 0.43; 95 % CI: 0.378-0.489) and ELISA/VSG RoTat 1.2 (k = 0.54; 95 % CI: 0.489-0.594) and poor with the other tests. Test agreement between TBR1/2 PCR and RoTat1.2 PCR was almost perfect (k = 0.96; 95 % CI: 0.950-0.984). We didn't find evidence for the presence of T. evansi type B in the studied population. Conclusion: Our study supports using antibody detection tests, rather than parasitological and molecular examination, to assess surra prevalence in camels. It also calls for implementation of measures to control surra in the Cholistan Desert.

Engaging scientists: An online survey exploring the experience of innovative biotechnological approaches to controlling vector-borne diseases

Abstract: Background: Pioneering technologies (e.g., nanotechnology, synthetic biology or climate engineering) are often associated with potential new risks and uncertainties that can become sources of controversy. The communication of information during their development and open exchanges between stakeholders is generally considered a key issue in their acceptance. While the attitudes of the public to novel technologies have been widely considered there has been relatively little investigation of the perceptions and awareness of scientists working on human or animal diseases transmitted by arthropods. Methods: Consequently, we conducted a global survey on 1889 scientists working on aspects of vector-borne diseases, exploring, under the light of a variety of demographic and professional factors, their knowledge and awareness of an emerging biotechnology that has the potential to revolutionize the control of pest insect populations. Results: Despite extensive media coverage of key developments (including releases of manipulated mosquitoes into human communities) this has in only one instance resulted in scientist awareness exceeding 50 % on a national or regional scale. We document that awareness of pioneering releases significantly relied on private communication sources that were not equally accessible to scientists from countries with endemic vector-borne diseases (dengue and malaria). In addition, we provide quantitative analysis of the perceptions and knowledge of specific biotechnological approaches to controlling vector-borne disease, which are likely to impact the way in which scientists around the world engage in the debate about their value. Conclusions: Our results indicate that there is scope to strengthen already effective methods of communication, in addition to a strong demand by scientists (expressed by 79.9 % of respondents) to develop new, creative modes of public engagement.

The effect of IFN-γ and TGF-β in the functional activity of mononuclear cells in the presence of Entamoeba histolytica

Background: Entamoeba histolytica (E. histolytica) causes amoebiasis, which is a disease with significant morbidity and mortality. Phagocytic cells and cytokines appear to be important in amoebiasis, but very little is known about the influence of these cells and cytokines in protozoan infections. The aim of this study was to analyse the supernatant of cultures of mononuclear (MN) cells with E. histolytica to determine: 1) the levels of the cytokines IFN-γ and TGF-β, and 2) the amoebicidal activity of MN cells after incubation with cytokines. Methods: Blood samples were collected from 30 volunteer donors. The cytokine concentrations in MN cells culture supernatants, superoxide release, leukophagocytosis, amoebicide activity, intracellular calcium release and apoptosis were analysed. Results: The IFN-γ concentrations were 6.22 ± 0.36 and TGF-β concentrations were 17.01 ± 2.21 in cells–trophozoite culture supernatants. MN cells, independently of cytokines, in the presence of amoeba increase the superoxide release. In the absence of cytokines, the ingestion of MN cells by amoebae was higher. In the presence of IFN- γ or TGF- β, a lower ingestion of MN cells was observed by amoebae. MN cells treated with cytokines exhibited higher amoebicide and apoptosis indexes. The incubation of cytokines increased the intracellular calcium release by MN cells. Conclusions: These results suggest that cytokines play a beneficial role for the host by activating MN cells against E. histolytica. The increased death of amoebae during the leukophagocytosis suggests that both cytokines (IFN-γ and TGF-β) can modulate the functional activity of MN cells and that these cytokines probably are important in the control of amoebic infections.

Associations between school- and household-level water, sanitation and hygiene conditions and soil-transmitted helminth infection among Kenyan school children

Background: Soil-transmitted helminths, a class of parasitic intestinal worms, are pervasive in many low-income settings. Infection among children can lead to poor nutritional outcomes, anaemia, and reduced cognition. Mass treatment, typically administered through schools, with yearly or biannual drugs is inexpensive and can reduce worm burden, but reinfection can occur rapidly. Access to and use of sanitation facilities and proper hygiene can reduce infection, but rigorous data are scarce. Among school-age children, infection can occur at home or at school, but little is known about the relative importance of WASH in transmission in these two settings. Methods: We explored the relationships between school and household water, sanitation, and hygiene conditions and behaviours during the baseline of a large-scale mass drug administration programme in Kenya. We assessed several WASH measures to quantify the exposure of school children, and developed theory and empirically-based parsimonious models. Results: Results suggest mixed impacts of household and school WASH on prevalence and intensity of infection. WASH risk factors differed across individual worm species, which is expected given the different mechanisms of infection. Conclusions: No trend of the relative importance of school versus household-level WASH emerged, though some factors, like water supply were more strongly related to lower infection, which suggests it is important in supporting other school practices, such as hand-washing and keeping school toilets clean.

Cyclospora cayetanensis infection in transplant traveller: a case report of outbreak

Background: Cyclospora cayetanensis is a protozoan parasite causing intestinal infections. A prolonged course of infection is often observed in immunocompromised individuals. In Europe, less than 100 cases of C. cayetanensis infection have been reported to date, almost all of which being diagnosed in individuals after travelling abroad.FindingsWe described cases of three businessmen who developed acute traveller’s diarrhoea after they returned to Poland from Indonesia. One of the travellers was a renal transplant recipient having ongoing immunosuppressive treatment. In each case, acute and prolonged diarrhoea and other intestinal disorders occurred. Oocysts of C. cayetanensis were identified in faecal smears of two of the travellers (one immunosuppressed and one immunocompetent). Diagnosis was confirmed by the successful amplification of parasite DNA (18S rDNA). A co-infection with Blastocystis hominis was identified in the immunocompetent man. Conclusions: Infection of C. cayetanensis shall be considered as the cause of prolonged acute diarrhoea in immunocompromised patients returning from endemic regions.

Study of the snail intermediate hosts of urogenital schistosomiasis in Niakhar, region of Fatick, West central Senegal

Background: Schistosoma haematobium is the most widespread schistosome species in Senegal and occurs in several regions of the country especially in the Sudan-Sahelian zone. The aims of the study were i) to determine the freshwater species ii) to ascertain the role of the identified snail species in the transmission of S. haematobium and iii) to study the impact of drought on the snails. Methods: Snails were sampled each year in 17 sites from July to November-December 2012 and 2013. At each snail survey, snails were grouped by village, counted, identified according to shell morphology and the rates of schistosome cercarial shedding recorded. The shell height of the snails collected in July was measured and classified into four groups according to their size in order to determine those that are open to aestivation. Results: B. senegalensis and B. umbilicatus were the only snails intermediate hosts collected in the Niakhar study area. B. senegalensis is found in all the 17 sampling sites while B. umbilicatus was only found in one site out of the many surveyed. The total number of B. senegalensis collected in 2012 and 2013 was 1032 and 8261 respectively. A total of 901 and 6432 B. senegalensis were tested for Schistosoma spp. infestation in 2012 and 2013 respectively. For B. umbilicatus, 58 snails were collected and tested in 2012. In 2013, 290 were collected and 281 tested. The overall rates of schistosome cercarial shedding were 0 % in 2012 and 0.12 % in 2013 for B. senegalensis and 13.79 and 4.98 % in 2012 and 2013 respectively for B. umbilicatus. For both species collected in July, size group 3 individuals (7–9.9 mm) were the most numerous, 63.6 and 57.8 % for B. senegalensis and B. umbilicatus respectively. B. umbilicatus was reported for the first time in the region of Fatick located in the old ecological zone of Sine-Saloum, is able to maintain Schistosoma spp. larvae during 7 months of drought and may transmit the disease in early July, increasing the period and the risk of transmission. Conclusion: This study recommends an adaptation of snail control strategies at pond cycles and ecology of the snails in these seasonal foci. Malacological control strategies must take into account these phenomena of drought resistance and the capacity of some snails to maintain parasite during aestivation. The treatment of ponds with Bayluscide at the end of the rainy season in November and upon onset of rains in July would be more advantageous to the control of snails thereby reducing transmission of urogenital schistosomiasis in the Niakhar area.

The first morphometric and phylogenetic perspective on molecular epidemiology of Echinococcus granulosus sensu lato in stray dogs in a hyperendemic Middle East focus, northwestern Iran

Background: Hydatidosis is considered to be a neglected cyclo-zoonotic disease in Middle East countries particularly northwestern Iran which is caused by metacestode of tapeworm Echinococcus granulosus sensu lato. Human hydatidosis is a high public health priority in the area, however there is little known from a morphometric and phylogenetic perspective on molecular epidemiology of adult Echinococcus spp. in Iranian stray dogs. Methods: 80 dogs (38 males and 42 females) were collected during June 2013 to April 2014 in northwestern Iran. The isolated parasites from each dog were distinguished by morphometric keys including small, large hook length and blade length. Subsequently, isolates were confirmed by sequencing of mitochondrial cytochrome oxidase subunit 1 gene. Results: 16 (8 males and 8 females) (Prevalence 20 %) out of 80 dogs were infected to genus Echinococcus. With regard to demographic factors, the frequency of parasitism in both male, female adults and their age groups showed no difference (P > 0.05). The phylogenetic analyses of cox1 sequences firmly revealed the 13 sheep strains (G1), one buffalo strain (G3), one camel strain (G6) and one mixed infection. The findings of rostellar hook morphology show an intraspecies variation range among G1 isolates. However, hook measurements in Echinococcus derived from G1 (sheep strain) were not a significant difference from those G6 and G3 strains. Six unique haplotypes were identified containing a high range of diversity (Haplotype diversity 0.873 vs. Nucleotide diversity 0.02). Conclusions: First presence of camel strain (G6) in this region seems to indicate that potential intermediate hosts play a secondary role in the maintenance of camel-dog biology. Current findings have heightened our knowledge about determination of Echinococcus prevalence, strains of taxonomy and genotypic trait of parasite in Iranian stray dogs which will also help in the development of strategies for monitoring and control of infected stray dogs in the area.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

Background: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths. Methods: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1. Results: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC 50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time. Conclusions: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Development of advantus™(imidacloprid) soft chewable tablets for the treatment of Ctenocephalides felis infestations on dogs

Background: Studies reported here were conducted to evaluate the safety and effectiveness of advantus™ (imidacloprid) soft chewable tablets for the treatment of flea (Ctenocephalides felis) infestations on dogs and puppies 10 weeks of age or older and weighing 4 pounds or greater. Methods: A pharmacokinetic study was conducted to evaluate parameters of orally administered imidacloprid. A dose confirmation study was conducted to confirm the efficacy of 0.75 mg/kg at 8, 12 or 24 hours post-treatment. A knockdown and speed of kill study was conducted to confirm the efficacy of 0.75 mg/kg dose at 0.5, 1, 4 or 24 hours post-treatment. The safety of a daily dose administered for six months at approximately 1, 3, and 5 times the maximum exposure dose of 3.75 mg/kg was evaluated in puppies. A field study was conducted to evaluate the safety and efficacy of a daily oral dose of 0.75 mg/kg for 14 days in client-owned dogs. Results: The pharmacokinetic parameters of the final imidacloprid oral formulation were; T max 1.31 hours, C max 690.0 ng/mL, AUC 2615.5 h*ng/mL and half-life was 2.2 hours.The efficacy of 0.75 mg/kg BW was 98.6 %, 99.9 % and 100 % at 8, 12 and 24 hours post-treatment, respectively. The live flea counts were significantly different (p < 0.0001) and the treatment was well tolerated.The flea counts at 1 hour post-treatment were significantly lower in the treated group and the speed of kill efficacy was 96.6 % at 4 hours post-treatment in the knockdown and speed of kill study.The target animal safety study showed that the advantus™ soft chewable tablets administered orally to 10-week-old puppies once daily for 6 months at approximately 1, 3 and 5 times the maximum dose of 3.75 mg/kg was well tolerated and did not produce clinically relevant findings in Beagles.In the field study, efficacy of the soft chewable tablets administered daily for 14 days to flea-infested dogs was 98.2 %. Conclusion: Imidacloprid administered orally as a soft chewable tablet for the treatment of fleas on dogs was safe and highly effective with a rapid knockdown effect and rapid elimination.

Impact of habitat fragmentation on tsetse populations and trypanosomosis risk in Eastern Zambia

Background: Fragmentation of tsetse habitat in eastern Zambia is largely due to encroachments by subsistence farmers into new areas in search of new agricultural land. The impact of habitat fragmentation on tsetse populations is not clearly understood. This study was aimed at establishing the impact of habitat fragmentation on physiological and demographic parameters of tsetse flies in order to enhance the understanding of the relationship between fragmentation and African animal trypanosomosis (AAT) risk. Methods: A longitudinal study was conducted to establish the age structure, abundance, proportion of females and trypanosome infection rate of Glossina morsitans morsitans Westwood (Diptera: Glossinidae) in areas of varying degrees of habitat fragmentation in Eastern Zambia. Black screen fly rounds were used to sample tsetse populations monthly for 1 year. Logistic regression was used to analyse age, proportion of females and infection rate data. Results: Flies got significantly older as fragmentation increased (p < 0.004). The proportion of old flies, i.e. above ovarian category four, increased significantly (P < 0.001) from 25.9 % (CI 21.4–31.1) at the least fragmented site (Lusandwa) to 74.2 % (CI 56.8–86.3) at the highly fragmented site (Chisulo). In the most fragmented area (Kasamanda), tsetse flies had almost disappeared. In the highly fragmented area a significantly higher trypanosome infection rate in tsetse (P < 0.001) than in areas with lower fragmentation was observed. Consequently a comparatively high trypanosomosis incidence rate in livestock was observed there despite lower tsetse density (p < 0.001). The overall proportion of captured female flies increased significantly (P < 0.005) as fragmentation reduced. The proportion increased from 0.135 (CI 0.10–0.18) to 0.285 (CI 0.26–0.31) at the highly and least fragmented sites, respectively. Conclusions: Habitat fragmentation creates conditions to which tsetse populations respond physiologically and demographically thereby affecting tsetse-trypanosome interactions and hence influencing trypanosomosis risk. Temperature rise due to fragmentation coupled with dominance of old flies in populations increases infection rate in tsetse and hence creates high risk of trypanosomosis in fragmented areas. Possibilities of how correlations between biological characteristics of populations and the degree of fragmentation can be used to structure populations based on their well-being, using integrated GIS and remote sensing techniques are discussed.

Influence of host nutritional condition on post-infection traits in the association between the manipulative acanthocephalan Pomphorhynchus laevis and the amphipod Gammarus pulex

Background: Several parasites with complex life-cycles induce phenotypic alterations in their intermediate hosts. According to the host manipulation hypothesis, such phenotypic alterations are supposed to increase the fitness of the parasite at the expense of that of its intermediate hosts through increasing the probability of transmission to next hosts. Although the phenomenon has received a large attention, the proximate factors modulating the occurrence and intensity of host manipulation remain poorly known. It has however, been suggested that the amount of energy reserves in the intermediate host might be a key parameter, although its precise influence on the intensity of manipulation remains unclear. Dietary depletion in the host may also lead to compromise with other parasite traits, such as probability of establishing or growth or virulence. Methods: Here, we address the question through performing experimental infections of the freshwater amphipod Gammarus pulex with two different populations of the acanthocephalan fish parasite Pomphorhynchus laevis, and manipulation of host nutritional condition. Following exposure, gammarids were given either a “standard” diet (consisting of elm leaves and chironomid larvae) or a “deprived” food treatment (deprived in proteins), and infection parameters were recorded. Once parasites reached the stage at which they become infective to their definitive host, refuge use (a behavioural trait presumably implied in trophic transmission) was assessed, and metabolic rate was measured. Results: Infected gammarids exposed to the deprived food treatment showed a lower metabolic rate, indicative of a lower body condition, compared to those exposed to the standard food treatment. Parasite size was smaller, and, depending on the population of origin of the parasites, intensity of infection was lower or mortality was higher in deprived hosts. However, food treatment had no effect on either the timing or intensity of behavioural modifications. Conclusions: Overall, while our results suggest that acanthocephalan parasites develop better in hosts in good condition, no evidence was found for an influence of host nutritional condition on host manipulation by parasites.

First report of the invasive mosquito species Aedes koreicus in the Swiss-Italian border region

Background: In 2012 and 2013, an entomological survey of Aedes albopictus, the Asian tiger mosquito, was carried out in the border region of southern Switzerland and northern Italy, using ovitraps. In July 2013, besides A. albopictus already known to the region several unusual eggs were recovered.FindingsA total of 548 seemingly different eggs were found within three communities: Chiasso (Switzerland), and Como and Brunate (Italy). Proteomic diagnostics based on matrix-assisted laser desorption/ionization mass-spectrometry (MALDI-TOF MS) and morphological identification of one reared adult revealed the presence of at least 18 A. (Finlaya) koreicus (Edwards, 1917) specimens. A. koreicus is a species native to Southeast Asia and is competent to transmit Japanese encephalitis and potentially other arboviruses, as well as the dog heartworm Dirofilaria immitis. While new to Switzerland, this invasive species has previously been reported from Belgium, north-eastern Italy and European Russia. Conclusions: This is the first report of the introduction of this exotic mosquito species into Switzerland and Lombardy, Italy, suggesting the range of A. koreicus is expanding in Central Europe. As A. koreicus is competent to vector pathogens its establishment imposes a risk to public and veterinary health. From a technical point of view, the presence of A. koreicus alongside A. albopictus requires careful analysis and reliable diagnostics. As a diagnostic tool the use of the recently developed MALDI-TOF MS approach has proofed to be a very useful approach, particularly since hatching rates of A. koreicus seem to be low, making identification by classic morphology difficult, if not impossible.

Re-evaluation of the species of hookworms infecting dogs in Central Vietnam

Background: Differentiation of canine hookworm species is crucial from both a veterinary and public health standpoint. In Vietnam, three hookworm species, namely Ancylostoma caninum, Ancylostoma braziliense and Uncinaria stenocephala are reported to infect dogs. In light of the emerging distribution of A. ceylanicum in Asia, this study aims to re-evaluate the status of Ancylostoma in dogs in Vietnam. Methods: Faecal samples collected from 200 community dogs in Dak Lak province were subjected to faecal floatation for the detection of hookworm eggs. Hookworm-positive samples were subjected to a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay targeting the internal transcribed spacer (ITS) region of rDNA for hookworm species identification. A subset of hookworm-positive samples was also subject to haplotype characterisation at the cytochrome oxidase-1 (COX-1) gene. Detailed morphological criteria were utilised in addition to molecular markers, to identify adult hookworms recovered from necropsied dogs. Results: Of 200 canine faecal samples, 111 (55.5 %) were positive for hookworm eggs on faecal flotation. Of these, 94/111 (84.7 %) were successfully amplified and assigned species status by PCR-RFLP targeting the ITS region. In total, 54.3 % (51/94) dogs harboured single infections with A. ceylanicum, 33.0 % (31/94) with A. caninum, and 12.7 % (12/94) harboured mixed infections with both A. ceylanicum and A. caninum. Adult worms recovered from necropsied dogs matched morphological description provided for A. ceylanicum, Looss (1911) for which the mediolateral and posteriolateral rays are parallel. Characterisation of the COX-1 gene placed all Vietnamese canine isolates of A. ceylanicum within the ‘zoonotic’ haplotype. Conclusion: Based on this information, it is apparent that the hookworms present in dogs in Vietnam are those of A. ceylanicum and not A. braziliense. Owing to the endemic nature of this significant zoonosis in dogs, the study strongly advocates for specific identification of this hookworm in human hookworm surveys.

Antibody response of definitive hosts against antigens of two life stages of the neuropathogenic schistosome Trichobilharzia regenti

Background: The nasal avian schistosome Trichobilharzia regenti spends part of its intravertebrate period of life within the central nervous system. Migration of the parasites can be accompanied by neuromotor disorders or paralysis in natural definitive hosts (ducks) and even in laboratory mammals. Cercariae are also able to penetrate human skin and induce cercarial dermatitis. While the cellular and antibody responses against cercariae and migrating schistosomula have been investigated in mice, little is known about immune reactions in birds. This study first describes the dynamics of antibody response in infected ducks and identifies frequently recognized antigens that may serve as diagnostic markers of infection by T. regenti. Methods: Groups of 35 domestic ducks and 10 mallards were exposed to different doses of T. regenti cercariae. Sera were collected at predefined time intervals and tested by ELISA for the presence of specific anti-cercarial IgY and IgM. Antigens recognized by the antibodies were identified on Western blots of cercariae and schistosomula. The applicability in immunodiagnostics was statistically evaluated by expression of specificity and sensitivity values for individual antigens. Results: In ELISA, the levels of anti-cercarial IgM peaked on day 15 pi. Increased production of IgY associated with the later phases of infection was observed in most individuals around 20 dpi and culminated 30 dpi. The time course of antibody response did not differ among experimental groups, variations were only observed in the levels of specific IgY which depended rather on the age of ducks at the time of infection than on the infectious dose. On Western blots, 40 cercarial and 7 schistosomular antigens were recognized by IgY from infected ducks. Among them, 4 cercarial antigens of 50, 47, 32 and 19 kDa provided the most sensitive and specific reactions. Conclusions: Antigens of cercariae and schistosomula elicited distinct antibody response in ducks, which correlated positively with the age of animals at the time of infection. Several antigens originating in cercariae and fewer in schistosomula were recognized by IgY with diverse sensitivity and specificity; only a few seemed to be common to both stages. Four of them were considered as the most promising candidates for immunodiagnostics.

ANISERP: a new serpin from the parasite Anisakis simplex

Background: Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). Methods: The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. Results: The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. Conclusions: Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.