Parasites and Vectors

Saliva from nymph and adult females of Haemaphysalis longicornis : a proteomic study

Background: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. Methods: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. Results: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. Conclusions: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

What’s in SWAP? Abundance of the principal constituents in a soluble extract of Schistosoma mansoni revealed by shotgun proteomics

Background: The soluble antigen preparation of adult schistosomes (SWAP) has often been used to probe host responses against these blood-dwelling parasites. Despite its long-established use there is limited knowledge about its composition. The information we provide here on the molecular composition of SWAP may contribute as a guide for a rational selection of antigenic targets. Methods: Label-free quantitative shotgun proteomics was employed to determine the composition and abundance of SWAP constituents. Briefly, paired adult Schistosoma mansoni worms were sonicated in PBS pH 7.2 and ultracentrifuged for recovery of the soluble supernatant. An aliquot was subjected to trypsin digestion. Resulting peptides were separated under ultra-high performance liquid chromatography and analysed using an orbitrap mass spectrometer. Spectral data were interrogated using SequestHT against an in-house S. mansoni database. Proteins were quantified by recording the mean area under curve obtained for up to three most intense detected peptides. Proteins within the 90 th percentile of the total SWAP mass were categorized according to their sub-cellular/tissue location. Results: In this work we expanded significantly the list of known SWAP constituents. Through application of stringent criteria, a total of 633 proteins were quantitatively identified. Only 18 proteins account to 50 % of the total SWAP mass as revealed by their cumulative abundance. Among them, none is predicted as a secreted molecule reinforcing the point that SWAP is dominated by cytosolic and cytoskeletal proteins. In contrast, only 3 % of the mass comprised proteins proposed to function at the host-parasite interfaces (tegument and gut), which could conceivably represent vulnerable targets of a protective immune response. Paradoxically, at least 11 SWAP proteins, comprising ~25 % of its mass, have been tested as vaccine candidates. Conclusions: Our data suggest that use of SWAP to probe host responses has greatest value for diagnostic purposes or assessing intensity of infection. However, the preparation is of limited utility as an antigen source for investigating host responses to proteins expressed at or secreted from worm-host interfaces. The data also pose the question as to why vaccination with SWAP, containing so many proposed vaccine candidates, has no additive or even synergistic effects on the induction of protection.

Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens

Background: The genus Diplostomum (Platyhelminthes: Trematoda: Diplostomidae) is a diverse group of freshwater parasites with complex life-cycles and global distribution. The larval stages are important pathogens causing eye fluke disease implicated in substantial impacts on natural fish populations and losses in aquaculture. However, the problematic species delimitation and difficulties in the identification of larval stages hamper the assessment of the distributional and host ranges of Diplostomum spp. and their transmission ecology. Methods: Total genomic DNA was isolated from adult worms and shotgun sequenced using Illumina MiSeq technology. Mitochondrial (mt) genomes and nuclear ribosomal RNA (rRNA) operons were assembled using established bioinformatic tools and fully annotated. Mt protein-coding genes and nuclear rRNA genes were subjected to phylogenetic analysis by maximum likelihood and the resulting topologies compared. Results: We characterised novel complete mt genomes and nuclear rRNA operons of two closely related species, Diplostomum spathaceum and D. pseudospathaceum. Comparative mt genome assessment revealed that the cox1 gene and its ‘barcode’ region used for molecular identification are the most conserved regions; instead, nad4 and nad5 genes were identified as most promising molecular diagnostic markers. Using the novel data, we provide the first genome wide estimation of the phylogenetic relationships of the order Diplostomida, one of the two fundamental lineages of the Digenea. Analyses of the mitogenomic data invariably recovered the Diplostomidae as a sister lineage of the order Plagiorchiida rather than as a basal lineage of the Diplostomida as inferred in rDNA phylogenies; this was concordant with the mt gene order of Diplostomum spp. exhibiting closer match to the conserved gene order of the Plagiorchiida. Conclusions: Complete sequences of the mt genome and rRNA operon of two species of Diplostomum provide a valuable resource for novel genetic markers for species delineation and large-scale molecular epidemiology and disease ecology studies based on the most accessible life-cycle stages of eye flukes.

Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora , Dictyocaulus viviparus and Fasciola hepatica in cattle

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100 % for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. Conclusions: A versatile bead-based assay using fluorescence detection (xMAP® technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

Differential alterations in the small intestine epithelial cell turnover during acute and chronic infection with Echinostoma caproni (Trematoda)

Background: The intestinal epithelium plays a multifactorial role in mucosal defense. In this sense, augmented epithelial cell turnover appears as a potential effector mechanism for the rejection of intestinal-dwelling helminths. Methods: A BrdU pulse-chase experiment was conducted to investigate the infection-induced alterations on epithelial cell kinetics in hosts of high (mouse) and low (rat) compatibility with the intestinal trematode Echinostoma caproni. Results: High levels of crypt-cell proliferation and tissue hyperplasia were observed in the ileum of infected mice, coinciding with the establishment of chronic infections. In contrast, the cell migration rate was about two times higher in the ileum of infected rats compared with controls, with no changes in tissue structure, indicating that an accelerated cell turnover is associated with worm expulsion. Conclusion: Our results indicate that E. caproni infection induces a rapid renewal of the intestinal epithelium in the low compatible host that may impair the establishment of proper, stable host-parasite interactions, facilitating worm clearance.

Knockdown resistance ( kdr ) mutations in Indian Anopheles culicifacies populations

Background: Anopheles culicifacies s.l. is one of the primary vectors of malaria in India responsible for the highest number of malaria cases. This vector is resistant to DDT in most parts of the country with indication of emerging resistance to pyrethroids. Since knockdown resistance (kdr) is known to confer cross-resistance between DDT and pyrethroids owing to a common target site of action, knowledge of prevalence of knockdown resistance (kdr) alleles is important from insecticide resistance management point of view. Methods: Nine populations of An. culicifacies belonging to five states of India, representing northern, western and central-east India, were screened for the presence of two alternative kdr mutations L1014F and L1014S using PCR-based assays. Dead and alive mosquitoes, following WHO standard insecticide susceptibility test against deltamethrin and DDT, were tested for allelic association. Results: L1014F mutation was recorded in all populations studied except from Haryana and Rajasthan states in northern India, with low frequencies ranging between 0.012 and 0.076; whereas presence of L1014S mutation was recorded in five populations only belonging to central-east India, with allelic frequencies ranging between 0.010 and 0.046. Both the kdr mutant alleles were found mostly in heterozygous condition without deviating from Hardy-Weinberg equilibrium. Both mutations showed protection against deltamethrin whereas only L1014S mutation showed protection against DDT when tested using additive model. Conclusions: The two L1014-kdr mutations, L1014F and L1014S, co-occurred in five populations belonging to Chhattisgarh and Odisha states of India whereas L1014F was present in all populations studied except populations from northern states. Both kdr mutations were found with very low allelic frequencies mostly in heterozygous condition and exhibited protection against deltamethrin.

Tongkat Ali ( Eurycoma longifolia ): a possible therapeutic candidate against Blastocystis sp.

Background: In the local Malaysian context, herbal plants such as Eurycoma longifolia (Tongkat Ali), Orthosiphon stamineus (MisaiKucing), Ficus deltoidea (Mas Cotek), Zingiber officinale (Halia Bara) and Barringtonia racemosa (Putat) are known and widely used for its therapeutic properties. The first part of this study aims to screen for the anti-protozoal activity of these herbal plant extracts against Blastocystis sp. isolate subtype (ST) 3. Herbal extract with the highest efficacy was further fractionized into water and ethyl acetate fractions and tested against ST1, ST3 and ST5 Blastocystis sp. isolates. These isolates were also exposed to allopathic drugs, Metronidazole (MTZ), Tinidazole, Trimethoprim-sulfamethoxazole(TMP-SMX), Ketoconazole and Nitazoxanide for comparison purpose. Methods: Blastocystis sp. isolates from human-derived stool samples were exposed to herbal extracts and allopathic drugs at a concentration of 0.1 mg/ml and 1.0 mg/ml and were incubated at 37 °C. Growth profile studies were carried out. After 72 h of treatment, the viability of Blastocystis sp. as a result of the effects of the drugs and herbal extracts were assessed. Results: Based on the screening process, amongst all the extracts, Tongkat Ali exhibited the highest anti-protozoal activity at 1.0 mg/ml. Between the water and ethyl acetate fractions of Tongkat Ali, the ethyl acetate fraction exhibited a slightly higher percentage of anti-protozoal activity at 1.0 mg/ml across subtypes, ST1 (94.9 %), ST3 (95.1 %) and ST5 (94.3 %). When tested with allopathic drugs, at the same concentration, MTZ exhibited the highest anti-protozoal activity across subtypes, ST1 (95.8 %), ST3 (93.4 %) and ST5 (90.8 %). Conclusion: This study is the first to describe the anti-protozoal properties of Tongkat Ali against Blastocystis sp. isolates. Ethyl acetate fraction of Tongkat Ali demonstrated the highest anti-protozoal activity against Blastocystis sp. isolates and showed a sizeable reduction in the cell count which was comparable with MTZ. Tongkat Ali also demonstrated a more uniformed sensitivity across subtypes in comparison to the allopathic drugs.

A comparative evaluation of the performance of commercially available rapid immunochromatographic tests for the diagnosis of visceral leishmaniasis in Bangladesh

Background: Accurate and early diagnosis of Visceral Leishmaniasis (VL) is a prerequisite for proper treatment and restricting disease propagation in endemic foci. An rK39 antigen-based immunochromatographic test is now recommended for its diagnostic accuracy and operational feasibility at point of care. In endemic regions of Bangladesh, rK39 or rKE16 antigen-based Rapid Diagnostic Tests (RDTs) are routinely performed on whole blood for diagnosis of VL. However, manufacturer’s instructions require use of serum. Therefore, we wanted to assess whether the diagnostic accuracy of these RDTs is as good on whole blood as on serum. Methods: We evaluated and compared the sensitivity and specificity of five different commercially available RDTs on whole blood and on serum. We enrolled 30 VL patients, 35 endemic healthy controls and 30 Tuberculosis (TB) patients in our study from Mymensingh, a hyper-endemic region in Bangladesh. Results: The sensitivity of all RDTs ranged between 96.67 % (95%CI: 82.72-99.44 %) and 100 % (95%CI: 96.34-100 %). The specificity ranged between 93.85 % (95%CI: 84.97-98.26 %) and 98.46 % (95%CI: 91.69-99.74 %), except for the Onsite leishmania Ab (Rev B) kit which showed markedly lower specificity (31.25-58.46 %). There was no significant difference in sensitivity and specificity between blood and serum. The Cohen kappa index (k >0.97) indicated excellent agreement. Conclusions: We conclude from the study that the use of blood for RDT in lieu of serum is appropriate for diagnosis of VL in peripheral endemic regions provided the manufacturer recommendations are followed and the RDT is of good quality.

A narrative review of visceral leishmaniasis in Armenia, Azerbaijan, Georgia, Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, Uzbekistan, the Crimean Peninsula and Southern Russia

There is an extensive body of medical and scientific research literature on visceral leishmaniasis (VL) in the Caucasus, Central Asia, the Crimean Peninsula and the southern part of The Russian Federation that is written in Russian, making it inaccessible to the majority of people who are interested in the leishmaniases in general and VL in particular. This review and summary in English of VL in what was Imperial Russia, which then became the Soviet Union and later a number of different independent states intends to give access to that majority. There are numerous publications in Russian on VL and, mostly, those published in books and the main scientific journals have been included here. The vast geographical area encompassed has been subdivided into four main parts: the southern Caucasus, covering Armenia, Azerbaijan and Georgia; Central Asia, covering Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan; the Crimean Peninsula and the northern Caucasus, which is part of The Russian Federation. Only rare cases of VL have been recorded in the northern Caucasus and Crimean Peninsula. In the other countries mentioned, human VL has been more intense but epidemics like those associated with L. donovani in India and East Africa have not occurred. For most of the countries, there are sections on the distribution, clinical aspects, the causative agent, the reservoirs and the vectors. Serological surveys and research into therapy are also covered. Recent studies on VL in Uzbekistan covered the application of serological, biochemical and molecular biological methods to diagnose human and canine VL, to identify the leishmanial parasites causing them in Uzbekistan and neighbouring Tajikistan and the epidemiology of VL in the Namangan Region of the Pap District, Eastern Uzbekistan. More recently, two studies were carried out in Georgia investigating the prevalence of human and canine VL, and the species composition of phlebotomine sand flies and their rates of infection with what was probably L. infantum in Tbilisi, eastern Georgia and Kutaisi, a new focus, in western Georgia. Though published in English, summaries of this information have been included where relevant to update the parts on VL in Uzbekistan and Georgia.

Asymmetric effects of native and exotic invasive shrubs on ecology of the West Nile virus vector Culex pipiens (Diptera: Culicidae)

Background: Exotic invasive plants alter the structure and function of native ecosystems and may influence the distribution and abundance of arthropod disease vectors by modifying habitat quality. This study investigated how invasive plants alter the ecology of Culex pipiens, an important vector of West Nile virus (WNV) in northeastern and midwestern regions of the United States. Methods: Field and laboratory experiments were conducted to test the hypothesis that three native leaf species (Rubus allegheniensis, blackberry; Sambucus canadensis, elderberry; and Amelanchier laevis, serviceberry), and three exotic invasive leaf species (Lonicera maackii, Amur honeysuckle; Elaeagnus umbellata, autumn olive; and Rosa multiflora, multiflora rose) alter Cx. pipiens oviposition site selection, emergence rates, development time, and adult body size. The relative abundance of seven bacterial phyla in infusions of the six leaf species also was determined using quantitative real-time polymerase chain reaction to test the hypothesis that variation in emergence, development, and oviposition site selection is correlated to differences in the diversity and abundance of bacteria associated with different leaf species, important determinants of nutrient quality and availability for mosquito larvae. Results: Leaf detritus from invasive honeysuckle and autumn olive yielded significantly higher adult emergence rates compared to detritus from the remaining leaf species and honeysuckle alleviated the negative effects of intraspecific competition on adult emergence. Conversely, leaves of native blackberry acted as an ecological trap, generating high oviposition but low emergence rates. Variation in bacterial flora associated with different leaf species may explain this asymmetrical production of mosquitoes: emergence rates and oviposition rates were positively correlated to bacterial abundance and diversity, respectively. Conclusions: We conclude that the displacement of native understory plant species by certain invasive shrubs may increase production of Cx. pipiens with potential negative repercussions for human and wildlife health. These findings may be relevant to mosquito control and invasive plant management practices in the geographic range of Cx. pipiens. Further, our discovery of a previously unknown ecological trap for an important vector of WNV has the potential to lead to novel alternatives to conventional insecticides in mosquito control by exploiting the apparent “attract-kill” properties of this native plant species.

Preliminary expression profile of cytokines in brain tissue of BALB/c mice with Angiostrongylus cantonensis infection

Background: Angiostrongylus cantonensis (A. cantonensis) infection can result in increased risk of eosinophilic meningitis. Accumulation of eosinophils and inflammation can result in the A. cantonensis infection playing an important role in brain tissue injury during this pathological process. However, underlying mechanisms regarding the transcriptomic responses during brain tissue injury caused by A. cantonensis infection are yet to be elucidated. This study is aimed at identifying some genomic and transcriptomic factors influencing the accumulation of eosinophils and inflammation in the mouse brain infected with A. cantonensis. Methods: An infected mouse model was prepared based on our laboratory experimental process, and then the mouse brain RNA Libraries were constructed for deep Sequencing with Illumina Genome Analyzer. The raw data was processed with a bioinformatics’ pipeline including Refseq genes expression analysis using cufflinks, annotation and classification of RNAs, lncRNA prediction as well as analysis of co-expression network. The analysis of Refseq data provides the measure of the presence and prevalence of transcripts from known and previously unknown genes. Results: This study showed that Cys-Cys (CC) type chemokines such as CCL2, CCL8, CCL1, CCL24, CCL11, CCL7, CCL12 and CCL5 were elevated significantly at the late phase of infection. The up-regulation of CCL2 indicated that the worm of A. cantonensis had migrated into the mouse brain at an early infection phase. CCL2 could be induced in the brain injury during migration and CCL2 might play a major role in the neuropathic pain caused by A. cantonensis infection. The up-regulated expression of IL-4, IL-5, IL-10, and IL-13 showed Th2 cell predominance in immunopathological reactions at late infection phase in response to infection by A. cantonensis. These different cytokines can modulate and inhibit each other and function as a network with the specific potential to drive brain eosinophilic inflammation. The increase of ATF-3 expression at 21 dpi suggested the injury of neuronal cells at late phase of infection. 1217 new potential lncRNA were candidates of interest for further research. Conclusions: These cytokine networks play an important role in the development of central nervous system inflammation caused by A. cantonensis infection.

Karyotypic assignment of Sri Lankan Anopheles culicifacies species B and E does not correlate with cytochrome oxidase subunit I and microsatellite genotypes

Background: The identification of species B and E in the Anopheles culicifacies complex in the Indian subcontinent has been based on Y-chromosome karyotype. Since no detectable variations were previously found in DNA markers commonly used for sibling species identification, further molecular characterization using cytochrome oxidase subunit I (COI) and microsatellite markers was carried out on Y-chromosome karyotyped Anopheles culicifacies specie B and E from Unnichchai, Kallady and Ranawarunawa in Sri Lanka .FindingsCOI sequence analysis (n = 22) revealed the presence of nine unique haplotypes with six in each species. Three haplotypes were shared by both species. The two sibling species had a pairwise FST value of 1.338 (p < 0.05) with the number of migrants (Nm) value <1. The genetic structure analysis resulted in two genetic clusters not 100 % associated with karyotypes. While none of the species B were incorrectly assigned two were inconclusive. Five out of 26 specimens karyotyped as species E were incorrectly assigned, while further 9 were inconclusive. Conclusions: The new molecular data support the existence of two genetically different populations of the Culicifacies Complex in Sri Lanka that are not associated with the Y-chromosome karyotype. Detailed analysis with more microsatellite markers and assortative mating experiments are needed to establish the presence of the two genetically distinct populations and relate them to Y-chromosome morphology.

L-Arginine supplementation in mice enhances NO production in spleen cells and inhibits Plasmodium yoelii transmission in mosquitoes

Background: The life cycle of Plasmodium is complex, requiring invasion of two different hosts, humans and mosquitoes. In humans, initiation of an effective Th1 response during early infection is critical for the control of parasite multiplication. In mosquitoes, inhibition of the development of sexual-stage parasites interrupts the parasite transmission. In this study, we aim to investigate whether dietary supplementation of L-arginine (L-Arg) in mice affects Plasmodium yoelii 17XL (Py17XL) transmission in mosquitoes. Methods: BALB/c mice were orally administered with 1.5 mg/g L-Arg daily for 7 days and infected with Py17XL. The mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 in spleen cells were determined by real-time RT-PCR. The amount of nitric oxide (NO) released by spleen cells in vitro was determined by the Griess method. The effect of L-Arg supplementation on subsequent development of P. yoelii gametocytes was evaluated by an in vitro ookinete culture assay and mosquito feeding assay. Results: Pretreatment of mice with L-Arg significantly increased the transcript level of iNOS in spleen cells and the amount of NO synthesized. Dietary L-Arg supplementation also significantly reduced the number of zygotes and ookinetes formed during in vitro culture and the number of oocysts formed on mosquito midguts after blood feeding. Conclusions: L-Arg enhances host immunity against blood-stage parasites as well as suppressing subsequent parasite development in mosquitoes. L-Arg as an inexpensive and safe supplement may be used as a novel adjunct treatment against malarial infection.

A field trial of spinosad for the treatment and prevention of flea infestation in shepherd dogs living in close proximity to flea-infested sheep

Background: Three flea species, Pulex irritans, Ctenocephalides canis and C. felis parasitize shepherd dogs living on sheep farms in Greece. The aim of this randomized, blinded, placebo-controlled trial was to examine the efficacy of spinosad, when administered three times every 4 weeks, as the only intervention to treat and prevent flea infestations in shepherd dogs living on sheep farms. Methods: Thirty dogs living on sheep farms and infested by at least 24 fleas were randomly allocated into equal groups. Group A dogs received spinosad (45–70 mg/kg body weight), every 4 weeks for three administrations, whereas Group B dogs were placebo-treated. Flea counting was performed at the beginning of the trial (day 0) and after 14, 28, 56 and 84 days. The first five fleas from each dog and 2–6 fleas collected from 5–11 sheep were used for species identification. Results: The percentage of dogs with zero flea counts was significantly higher in group A than in group B at days 14, 28, 56 and 84 and flea counts were significantly lower in group A than in group B at days 14, 28, 56 and 84. In group A, flea counts were significantly lower at days 14, 28, 56 and 84 compared to day 0 whereas there were no changes in flea counts of group B dogs. The percent efficacy of spinosad for the treatment and prevention of flea infestation was higher than 98 % (arithmetic means) or higher than 99 % (geometric means) throughout the study. No adverse reactions were recorded.C. canis was the predominant flea species of dogs at day 0. In group A the relative abundance of C. felis increased at day14 whereas in group B the relative abundance of P. irritans increased at days 14, 28, 56 and 84. Conclusions: Spinosad is safe and effective for the treatment of C. canis and C. felis infestations and for the prevention of P. irritans, C. canis and C. felis infestations in shepherd dogs living in close proximity to sheep.

Taenia solium taeniosis/cysticercosis and the co-distribution with schistosomiasis in Africa

Background: This study aimed to map the distribution of Taenia solium taeniosis/cysticercosis and the co-distribution with schistosomiasis in Africa. These two major neglected tropical diseases are presumed to be widely distributed in Africa, but currently the level of co-distribution is unclear. Methods: A literature search on T. solium taeniosis/cysticercosis was performed to compile all known studies on the presence of T. solium and apparent prevalence of taeniosis and porcine cysticercosis in Africa. Studies were geo-referenced using an online gazetteer. A Bayesian framework was used to combine the epidemiological data on the apparent prevalence with external information on test characteristics to estimate informed district-level prevalence of taeniosis and porcine cysticercosis. Districts with T. solium taeniosis/cysticercosis presence were cross-referenced with the Global Neglected Tropical Diseases Database for schistosomiasis presence. Results: The search strategies identified 141 reports of T. solium in Africa from 1985 to 2014 from a total of 476 districts in 29 countries, 20 with porcine cysticercosis, 22 with human cysticercosis, and 16 with taeniosis, in addition to 2 countries identified from OIE reports. All 31 countries were considered, on national scale, to have co-distribution with schistosomiasis. Presence of both parasites was confirmed in 124 districts in 17 countries. The informed prevalence of taeniosis and porcine cysticercosis were estimated for 14 and 41 districts in 10 and 13 countries, respectively. Conclusions: With the paucity of data, T. solium infection is grossly under-reported and expected to be more widespread than this study suggests. In areas where co-distribution occurs there is a need for increased emphasis on evaluation of integrated intervention approaches for these two helminth infections and allocation of resources for evaluating the extent of adverse effects caused by mass drug administration.

Evaluation of the toxicity and repellence of an organic fatty acids mixture (C8910) against insecticide susceptible and resistant strains of the major malaria vector Anopheles funestus Giles (Diptera: Culicidae)

Background: Malaria vector control relies principally on the use of insecticides, especially pyrethroids. Because of the increasing occurrence of insecticide resistance in target vector populations, the development of new insecticides, particularly those with novel modes of action, is particularly important, especially in terms of managing insecticide resistance. The C8910 formulation is a patented mixture of compounds comprising straight-chain octanoic, nonanoic and decanoic saturated fatty acids. This compound has demonstrated toxic and repellent effects against several arthropod species. The aims of this study were to measure the insecticidal effects of C8910 against an insecticide susceptible (FANG) and a pyrethroid resistant (FUMOZ-R) laboratory strain of An. funestus as well as against wild-caught An. funestus material from Zambia (ZamF), and to investigate the repellent effects of two formulations of C8910 against these strains. Methods: Toxicity against adult females was assessed using a range of concentrations based on the CDC bottle bioassay method and repellence of three different C8910 formulations was assessed using standard choice-chamber bioassays. Results: C8910 proved equally toxic to adult females of the FUMOZ-R and FANG laboratory strains, as well as to adult females of the wild-caught (ZamF) sample. None of the C8910 formulations tested gave any conclusive indication of repellence against any of the strains. Conclusion: C8910 is equally effective as an adulticide against pyrethroid resistant and insecticide susceptible An. funestus. However, the formulations tested did not show any consistent repellence against laboratory reared and wild-caught female samples of this species. Nevertheless, C8910 shows potential as an adulticide that can be used for malaria vector control, particularly in those instances where insecticide resistance management is required.

Serological and molecular analysis of feline vector-borne anaplasmosis and ehrlichiosis using species-specific peptides and PCR

Background: With the exception of Bartonella spp. or Cytauxzoon felis, feline vector-borne pathogens (FVBP) have been less frequently studied in North America and are generally under-appreciated as a clinical entity in cats, as compared to dogs or people. This study investigated selected FVBP seroreactivity and PCR prevalence in cats using archived samples. Methods: Feline blood samples submitted to the Vector Borne Diseases Diagnostic Laboratory (VBDDL) at North Carolina State University College of Veterinary Medicine (NCSU-CVM) between 2008 and 2013 were tested using serological assays and PCR. An experimental SNAP® Multi-Analyte Assay (SNAP® M-A) (IDEXX Laboratories, Inc. Westbrook, Maine, USA) was used to screen all sera for antibodies to Anaplasma and Ehrlichia genus peptides and A.phagocytophilum, A.platys, B.burgdorferi, E.canis, E.chaffeensis, and E.ewingii species-specific peptides. PCR assays were used to amplify Anaplasma or Ehrlichia DNA from extracted ethylenediaminetetraacetic acid (EDTA)-anti-coagulated blood samples. Amplicons were sequenced to identify species. Results: Overall, 7.8 % (56/715) of cats were FVBP seroreactive and 3.2 % (13/406) contained Anaplasma or Ehrlichia DNA. Serologically, B.burgdorferi (5.5 %) was the most prevalent FVBP followed by A.phagocytophilum (1.8 %). Ehrlichia spp. antibodies were found in 0.14 % (12/715) of cats with species-specific seroreactivity to E.canis (n = 5), E.ewingii (n = 2) and E.chaffeensis (n = 1). Of seropositive cats, 16 % (9/56) were exposed to more than one FVBP, all of which were exposed to B.burgdorferi and either A.phagocytophilum (n = 7) or E.ewingii (n = 2). Based upon PCR and DNA sequencing, 4, 3, 3, 2, and 1 cat were infected with A.phagocytophilum, A.platys, E. ewingii, E. chaffeensis and E.canis, respectively. Conclusions: Cats are exposed to and can be infected with vector-borne pathogens that commonly infect dogs and humans. To our knowledge, this study provides the first evidence for E.chaffeensis and E.ewingii infection in naturally-exposed cats in North America. Results from this study support the need for regional, serological and molecular FVBP prevalence studies, the need to further optimize serodiagnostic and PCR testing for cats, and the need for prospective studies to better characterize clinicopathological disease manifestations in cats infected with FVBP.

Relationship between insecticide resistance and kdr mutations in the dengue vector Aedes aegypti in Southern China

Background: Aedes aegypti is an important vector for dengue virus and thus has been targeted with pyrethroid insecticides in many areas of the world. As such, resistance has been detected to several of these insecticides, including in China, but the mechanisms of the resistance are not well understood in this country. Methods: Using the World Health Organization larval mosquito bioassay, five field populations of Aedes aegypti from Southern China were characterized for their resistance to cypermethrin and cyhalothrin. RNA extraction with PCR amplification, cloning and sequencing of the sodium channel gene was followed by comparisons of susceptible and wild mosquito strains Additionally, genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the sodium channel genes to detect S989P, V1016G and F1534C mutations and allow for correlation analysis of resistance expression for the different mutations. Results: All wild strains expressed resistance to cypermethrin and cyhalothrin and the resistance expression between the two insecticides was highly correlated suggesting cross-resistance between these two pyrethroids. The AS-PCR technique effectively distinguished individual genotypes for all three mutations. Among the five wild strains tested, two strains carried all three mutations. Although the S989P and V1016G mutations were positively correlated to resistance expression of both pyrethroids, the F1534C mutation was negatively correlated. Conclusions: Our methodology proved highly reliable and will aid future detection of kdr mutations. The three sodium channel mutations were common in the Ae. aegypti strains sampled from Southern China. The V1016G mutation appears to be the most important kdr mutation in Ae. aegypti strains in Southern China.

Impregnating hessian strips with the volatile pyrethroid transfluthrin prevents outdoor exposure to vectors of malaria and lymphatic filariasis in urban Dar es Salaam, Tanzania

Background: Semi-field trials using laboratory-reared Anopheles arabiensis have shown that, delivering the volatile pyrethroid transfluthrin by absorption into hessian strips, consistently provided > 99 % human protective efficacy against bites for 6 months without retreating. Here the impact of this approach upon human exposure to wild populations of vectors for both malaria and filariasis under full field conditions is assessed for the first time. Methods: Transfluthrin-treated and untreated strips were placed around human volunteers conducting human landing catch in an outdoor environment in urban Dar es Salaam, where much human exposure to malaria and filariasis transmission occurs outdoors. The experiment was replicated 9 times at 16 outdoor catching stations in 4 distinct locations over 72 working nights between May and August 2012. Results: Overall, the treated hessian strips conferred 99 % protection against An. gambiae (1 bite versus 159) and 92 % protection against Culex spp. (1478 bites versus 18,602). No decline in efficacy over the course of the study could be detected for the very sparse populations of An. gambiae (P = 0.32) and only a slow efficacy decline was observed for Culex spp. (P < 0.001), with protection remaining satisfactory over 3 months after strip treatment. Diversion of mosquitoes to unprotected humans in nearby houses was neither detected for An. gambiae (P = 0.152) nor for Culex spp. (Relative rate, [95 % CI] = 1.03, [0.95, 1.11], P = 0.499). Conclusion: While this study raises more questions than it answers, the presented evidence of high protection over long periods suggest this technology may have potential for preventing outdoor transmission of malaria, lymphatic filariasis and other vector-borne pathogens.

Serological and molecular diagnostic surveys combined with examining hematological profiles suggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt

Background: Babesiosis threatens the development of the cattle and buffaloes industries in Egypt and improved control is needed. The main objectives of this study are surveying the presence of bovine babesiosis in distinct selected bovine and buffalo populations in Egypt using novel molecular and previously validated serological methods, while also comparing the occurrence of hematological alterations among Babesia infected cattle and buffalos. Methods: A total of 253 and 81 blood samples from apparently healthy cattle and buffaloes, respectively, were randomly collected from diverse locations in Egypt. All samples were tested for Babesia bovis and B. bigemina infection using blood film examination, competitive ELISA (cELISA) and PCR. Novel semi-nested and nested PCR assays for the detection of B. bovis and B. bigemina respectively, were developed and used to analyze DNA extracted from bovine and buffalo samples. Hematological profiles were studied using a hematological analyzer. Results: Blood films examination revealed 13.8 % and 7.4 % Babesia infection rates in cattle and buffaloes, respectively. However, in cattle, the cELISA detected 32.8 %, 21.3 % and 10.7 % infection rates with B. bigemina, B. bovis and mixed infection, respectively. In addition, cELISA identified 22.2 %, 22.2 % and 6.2 % infection rates with B. bigemina, B. bovis and mixed infection, respectively in buffaloes. The semi-nested PCR assay showed that 15 % of the tested samples were positive for B. bovis in cattle, but just 3 % in buffaloes. Infections with B. bigemina were also found in cattle (32.4 %), but not in buffaloes upon nested PCR analysis. Sequencing analysis confirmed the identity of the PCR amplicons and showed that Egyptian genotypes of B. bigemina and B. bovis highly resemble sequences previously deposited in GenBank. Hemograms performed on the sampled animals revealed macrocytic hypochromic anemia associated with reduced platelet counts in infected cattle with babesiosis. In addition, marked increases in total leukocyte and granulocytic counts and decreases in lymphocytic counts were found in infected cattle. In contrast, no such hematological anomalies were found in presumably Babesia-infected buffaloes. Conclusions: Frequent occurrence of babesiosis among apparently healthy bovines in Egypt, suggests the need for appropriately designed prevalence studies in this country. Infected bovine, but not buffalo, populations often present hematological disorders compatible with intravascular hemolysis and thrombocytopenia.