Parasites and Vectors

Genetic similarities between Cyclospora cayetanensis and cecum-infecting avian Eimeria spp. in apicoplast and mitochondrial genomes

Background: Cyclospora cayetanensis is an important cause for diarrhea in children in developing countries and foodborne outbreaks of cyclosporiasis in industrialized nations. To improve understanding of the basic biology of Cyclospora spp. and development of molecular diagnostic tools and therapeutics, we sequenced the complete apicoplast and mitochondrial genomes of C. cayetanensis. Methods: The genome of one Chinese C. cayetanensis isolate was sequenced using Roche 454 and Illumina technologies. The assembled genomes of the apicoplast and mitochondrion were retrieved, annotated, and compared with reference genomes for other apicomplexans to infer genome organizations and phylogenetic relationships. Sequence variations in the mitochondrial genome were identified by comparison of two C. cayetanensis nucleotide sequences from this study and a recent publication. Results: The apicoplast and mitochondrial genomes of C. cayetanensis are 34,155 and 6,229 bp in size and code for 65 and 5 genes, respectively. Comparative genomic analysis showed high similarities between C. cayetanensis and Eimeria tenella in both genomes; they have 85.6 % and 90.4 % nucleotide sequence similarities, respectively, and complete synteny in gene organization. Phylogenetic analysis of the genomic sequences confirmed the genetic similarities between cecum-infecting avian Eimeria spp. and C. cayetanensis. Like in other coccidia, both genomes of C. cayetanensis are transcribed bi-directionally. The apicoplast genome is circular, codes for the complete machinery for protein biosynthesis, and contains two inverted repeats that differ slightly in LSU rRNA gene sequences. In contrast, the mitochondrial genome has a linear concatemer or circular mapping topology. Eight single-nucleotide and one 7-bp multiple-nucleotide variants were detected between the mitochondrial genomes of C. cayetanensis from this and recent studies. Conclusions: The apicoplast and mitochondrial genomes of C. cayetanensis are highly similar to those of cecum-infecting avian Eimeria spp. in both genome organization and sequences. The availability of sequence data beyond rRNA and heat shock protein genes could facilitate studies of C. cayetanensis biology and development of genotyping tools for investigations of cyclosporiasis outbreaks.

Comparability between insecticide resistance bioassays for mosquito vectors: time to review current methodology?

Background: Insecticides play an integral role in the control of mosquito-borne diseases. With resistance to insecticides on the rise, surveillance of the target population for optimal choice of insecticides is a necessity. The Centers for Disease Control and Prevention (CDC) bottle assay and the World Health Organization (WHO) susceptibility test are the most frequently used methods in insecticide resistance monitoring. However, the two bioassays differ in terms of insecticide delivery and how insecticide susceptibility is measured. To evaluate how equivalent data from the two assays are, we compared the two methods side-by-side. Methods: We did a literature search from 1998 to December 2014 to identify publications that performed both assays on the same mosquito population and compared the results. We then tested the WHO and CDC bioassays on laboratory strains of Aedes aegypti, Anopheles stephensi, An. gambiae and An. arabiensis with different insecticide resistance levels against permethrin, λ-cyhalothrin, DDT, bendiocarb and malathion. In addition, we also measured the relationship between time-to-knockdown and 24 h mortality. Results: Both published data and results from the present laboratory experiments showed heterogeneity in the comparability of the two bioassays. Following their standard procedures, the two assays showed poor agreement in detecting resistance at the WHO cut-off mark of 90 % (Cohen’s κ = 0.06). There was better agreement when 24 h mortality was recorded in the CDC bottle assay and compared with that of the WHO susceptibility test (Cohen’s κ = 0.5148). Time-to-knockdown was shown to be an unreliable predictor of 24 h mortality. Conclusion: Even though the two assays can detect insecticide resistance, they may not be used interchangeably. While the diagnostic dose in the WHO susceptibility test does not allow for detecting shifts at low or extreme resistance levels, time-to-knockdown measured in the CDC bottle assay is a poor predictor of 24 h mortality. Therefore, dose–response assays could provide the most flexibility. New standardized bioassays are needed that produce consistent dose–response measurements with a minimal number of mosquitoes.

Assessment of mosquito larval productivity among different land use types for targeted malaria vector control in the western Kenya highlands

Background: Mosquito larval source management (LSM) is likely to be more effective when adequate information such as dominant species, seasonal abundance, type of productive habitat, and land use type are available for targeted sites. LSM has been an effective strategy for reducing malaria morbidity in both urban and rural areas in Africa where sufficient proportions of larval habitats can be targeted. In this study, we conducted longitudinal larval source surveillance in the western Kenya highlands, generating data which can be used to establish cost-effective targeted intervention tools. Methods: One hundred and twenty-four (124) positive larval habitats were monitored weekly and sampled for mosquito larvae over the 85-week period from 28 July 2009 to 3 March 2011. Two villages in the western Kenya highlands, Mbale and Iguhu, were included in the study.After preliminary sampling, habitats were classified into four types: hoof prints (n = 21; 17 % of total), swamps (n = 32; 26 %), abandoned goldmines (n = 35; 28 %) and drainage ditches (n = 36; 29 %). Positive habitats occurred in two land use types: farmland (66) and pasture (58). No positive larval habitats occurred in shrub land or forest. Results: A total of 46,846 larvae were sampled, of which 44.1 % (20,907) were from abandoned goldmines, 30.9 % (14,469) from drainage ditches, 22.4 % (10,499) from swamps and 2.1 % (971) from hoof prints. In terms of land use types, 57.2 % (26,799) of the sampled larvae were from pasture and 42.8 % (20,047) were from farmland. Of the specimens identified morphologically, 24,583 (52.5 %) were Anopheles gambiae s.l., 11,901 (25.4 %) were Culex quinquefasciatus, 5628 (12 %) were An. funestus s.l. and 4734 (10.1 %) were other anopheline species (An. coustani, An. squamosus, An. ziemanni or An. implexus). Malaria vector dynamics varied seasonally, with An.gambiae s.s. dominating during wet season and An.arabiensis during dry season. An increased proportion of An. arabiensis was observed compared to previous studies. Conclusion: These results suggest that long-term monitoring of larval habitats can establish effective surveillance systems and tools. Additionally, the results suggest that larval control is most effective in the dry season due to habitat restriction, with abandoned goldmines, drainage ditches and swamps being the best habitats to target. Both farmland and pasture should be targeted for effective larval control. An increased proportion of An. arabiensis in the An. gambiae complex was noticed in this study for the very first time in the western Kenya highlands; hence, further control tools should be in place for effective control of An. arabiensis.

Cost and cost-effectiveness of soil-transmitted helminth treatment programmes: systematic review and research needs

Background: In this time of rapidly expanding mass drug administration (MDA) coverage and the new commitments for soil-transmitted helminth (STH) control, it is essential that resources are allocated in an efficient manner to have the greatest impact. However, many questions remain regarding how best to deliver STH treatment programmes; these include which age-groups should be targeted and how often. To perform further analyses to investigate what the most cost-effective control strategies are in different settings, accurate cost data for targeting different age groups at different treatment frequencies (in a range of settings) are essential. Methods: Using the electronic databases PubMed, MEDLINE, and ISI Web of Knowledge, we perform a systematic review of costing studies and cost-effectiveness evaluations for potential STH treatment strategies. We use this review to highlight research gaps and outline the key future research needs. Results: We identified 29 studies reporting costs of STH treatment and 17 studies that investigated its cost-effectiveness. The majority of these pertained to programmes only targeting school-aged children (SAC), with relatively few studies investigating alternative preventive chemotherapy (PCT) treatment strategies. The methods of cost data collection, analysis and reporting were highly variable among the different studies. Only four of the costing studies were found to have high applicability for use in forthcoming economic evaluations. There are also very few studies quantifying the costs of increasing the treatment frequency. Conclusions: The absence of cost data and inconsistencies in the collection and analysis methods constitutes a major research gap for STH control. Detailed and accurate costs of targeting different age groups or increasing treatment frequency will be essential to formulate cost-effective public health policy. Defining the most cost-effective control strategies in different settings is of high significance during this period of expanding MDA coverage and new resource commitments for STH control.

New insights into the immunopathology of early Toxocara canis infection in mice

Background: Nematodes of the genus Toxocara are cosmopolitan roundworms frequently found in dogs and cats. Toxocara spp. can accidentally infect humans and cause a zoonosis called human toxocariasis, which is characterized by visceral, ocular or cerebral migration of larval stages of the parasite, without completing its life cycle. In general, chronic nematode infections induce a polarized T H 2 immune response. However, during the initial phase of infection, a strong pro-inflammatory response is part of the immunological profile and might determine the outcome and/or pathology of the infection. Methods: Parasitological aspects and histopathology during larval migration were evaluated after early T. canis experimental infection of BALB/c mice, which were inoculated via the intra-gastric route with a single dose of 1000 fully embryonated eggs. Innate immune responses and systemic cytokine patterns (T H 1, T H 2, T H 17 and regulatory cytokines) were determined at different times after experimental challenge by sandwich ELISA. Results: We found that experimental infection with T. canis induced a mix of innate inflammatory/T H 17/T H 2 responses during early infection, with a predominance of the latter. The T H 2 response was evidenced by significant increases in cytokines such as IL-4, IL-5, IL-13 and IL-33, in addition to increasing levels of IL-6 and IL-17. No significant increases were observed for IL-10, TNF-α or IFN-γ levels. In parallel, parasitological analysis clearly revealed the pattern of larval migration through the mouse organs, starting from the liver in the first 24 h of infection, reaching the peak in the lungs on the 3rd day of infection and finally being found numerously in the brain after 5 days of infection. Peripheral leukocytosis, characterized by early neutrophilia and subsequent eosinophilia, was remarkable during early infection. The tissue damage induced by larvae was evidenced by histopathological analysis of the organs at different time points of infection. In all of the affected organs, larval migration induced intense inflammatory infiltrate and hemorrhage. Conclusion: In conclusion, these new insights into early T. canis infection in mice presented here enabled a better understanding of the immunopathological events that might also occur during human toxocariasis, thus contributing to future strategies of diagnosis and control.

A histochemical study of the Nras/let-60 activity in filarial nematodes

Background: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides. Methods: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy. Results: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi. Conclusion: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

A quantitative evaluation of the extent of fluralaner uptake by ticks ( Ixodes ricinus , Ixodes scapularis ) in fluralaner (Bravecto <sup>TM</sup> ) treated vs. untreated dogs using the parameters tick weight and coxal index

Background: Fluralaner is a new antiparasitic drug that was recently introduced as Bravecto TM chewable tablets for the treatment of tick and flea infestations in dogs. Most marketed tick products exert their effect via topical application and contact exposure to the parasite. In contrast, Bravecto TM delivers its acaricidal activity through systemic exposure. Tick exposure to fluralaner occurs after attachment to orally treated dogs, which induces a tick-killing effect within 12 h. The fast onset of killing lasts over the entire treatment interval (12 weeks) and suggests that only marginal uptake by ticks is required to induce efficacy. Three laboratory studies were conducted to quantify the extent of uptake by comparison of ticks’ weight and coxal index obtained from Bravecto TM -treated and negative-control dogs. Methods: Three studies were conducted using experimental tick infestation with either Ixodes ricinus or Ixodes scapularis after oral administration of fluralaner to dogs. All studies included a treated (Bravecto TM chewable tablets, MSD Animal Health) and a negative control group. Each study had a similar design for assessing vitality and weighing of ticks collected from dogs of both groups. Additionally, in one study the coxal index (I. ricinus) was calculated as a ratio of tick’s ventral coxal gap and dorsal width of scutum. Tick weight data and coxal indices from Bravecto TM -treated and negative-control groups were compared via statistical analysis. Results: Ticks collected from Bravecto TM -treated dogs weighed significantly less (p ≤ 0.0108) than ticks collected from negative-control dogs, and their coxal index was also significantly lower (p < 0.0001). The difference in tick weights was demonstrated irrespective of the tick species investigated (I. ricinus, I. scapularis). At some assessments the mean tick weights of Bravecto TM -treated dogs were significantly lower than those of unfed pre-infestation (baseline) ticks. The demonstrated tick-killing efficacy was in the range of 94.6 – 100 %. Conclusions: Tick weights and coxal indices confirm that a minimal uptake results in a sufficient exposure of ticks to fluralaner (Bravecto TM ) and consequently in a potent acaricidal effect.

Current status of invasive mosquito surveillance in the UK

Background: Non-native invasive mosquitoes have for many years made incursions into Europe, and are now established in many European countries. The continued European importation of potential vectors and their expansion within Europe increases their potential for importation and establishment in the UK. Coupled with increasing numbers of returning dengue and chikungunya infected travellers, the potential exists for transmission of vector borne disease in new regions. Methods: To ensure a cost-effective risk assessment and preparedness strategy the UK employs a multi-faceted approach to surveillance for non-native Aedes mosquitoes, including passive and active surveillance strategies at a local, regional, and national level. Passive surveillance, including a national mosquito recording scheme and local authority nuisance biting reporting, are combined with targeted active surveillance at seaports, airports, used tyre importers, and motorway service stations. Results: There is no evidence to date that any invasive Aedes species (e.g., Aedes albopictus, Aedes japonicus, Aedes aegypti) occur in the UK despite sharing many of the same routes that have been found to have facilitated their entry into other countries. Conclusions: This paper sets in context the UK approaches with other European countries and those recommended by the ECDC. It also highlights future UK strategies to enhance surveillance for non-native mosquitoes to help ensure that incursions can be managed, and these mosquitoes do not establish and public health is protected. Focus will be given to increasing the number of submissions of mosquitoes to passive surveillance schemes and maintaining active surveillance efforts at key routes of potential importation.

Characterisation of Dermanyssus gallinae glutathione S- transferases and their potential as acaricide detoxification proteins

Background: Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry. Methods: A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, −2 and −3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, −2 and −3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs. Results: 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and −3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17 % respectively. Phoxim also inhibited rDeg-2-GST (14.8 %) and rDeg-GST-3 (20.6 %) activities. Conclusions: Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Ubiquitin-specific proteases are differentially expressed throughout the Schistosoma mansoni life cycle

Background: The ubiquitination process can be reversed by deubiquitinating enzymes (DUBs). These proteases are involved in ubiquitin processing, in the recovery of modified ubiquitin trapped in inactive forms, and in the recycling of ubiquitin monomers from polyubiquitinated chains. The diversity of DUB functions is illustrated by their number and variety of their catalytic domains with specific 3D architectures. DUBs can be divided into five subclasses: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs or UBPs), ovarian tumour proteases (OTUs), Machado-Joseph disease proteases (MJDs) and JAB1/MPN/Mov34 metalloenzymes (JAMMs). Methods: Considering the role that the ubiquitin-proteasome system has been shown to play during the development of Schistosoma mansoni, our main goal was to identify and characterize SmUSPs. Here, we showed the identification of putative ubiquitin-specific proteases using bioinformatic approaches. We also evaluated the gene expression profile of representative USP family members using qRT-PCR. Results: We reported 17 USP family members in S. mansoni that present a conservation of UCH domains. Furthermore, the putative SmUSP transcripts analysed were detected in all investigated stages, showing distinct expression during S. mansoni development. The SmUSPs exhibiting high expression profiles were SmUSP7, SmUSP8, SmUSP9x and SmUSP24. Conclusion: S. mansoni USPs showed changes in expression levels for different life cycle stages indicating their involvement in cellular processes required for S. mansoni development. These data will serve as a basis for future functional studies of USPs in this parasite.

Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) - II - Egg storage and hatching

Background: Management of large quantities of eggs will be a crucial aspect of the efficient and sustainable mass production of mosquitoes for programmes with a Sterile Insect Technique component. The efficiency of different hatching media and effectiveness of long term storage methods are presented here. Methods: The effect on hatch rate of storage duration and three hatching media was analysed: deionized water, boiled deionized water and a bacterial broth, using Two-way ANOVA and Post hoc Tukey tests, and the Pearson correlation coefficient was used to find the effect on the proportion of collapsed eggs. Two long term storage methods were also tested: conventional storage (egg paper strips stored in zip lock bags within a sealed plastic box), and water storage (egg papers in a covered plastic cup with deionized water). Regression analyses were used to find the effect of water storage and storage duration on hatch rate. Results: Both species hatched most efficiently in bacterial broth. Few eggs hatched in deionized water, and pre-boiling the water increased the hatch rate of Ae. aegypti, but not Ae. albopictus. A hatch rate greater than 80 % was obtained after 10 weeks of conventional storage in Ae. aegypti and 11 weeks in Ae. albopictus. After this period, hatching decreased dramatically: no eggs hatched after 24 weeks. Storing eggs in water produced an 85 % hatch rate after 5 months in both species. A small but significant proportion of eggs hatched in the water, probably due to combined effects of natural deoxygenation of the water over time and the natural instalment hatching typical of the species. Conclusions: The demonstrated efficiency of the bacterial broth hatching medium for both Ae. albopictus and Ae. aegypti facilitates mass production of these two important vector species in the same facility, with use of a common hatching medium reducing cost and operational complexity. Similarly the increased hatch rate of eggs stored in water would allow greater flexibility of egg management in a large programme over the medium term, particularly if oxygenation of the water by bubbling oxygen through the storage tray could be applied to prevent hatching during storage.

Targeting male mosquito mating behaviour for malaria control

Malaria vector control relies heavily on the use of Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS). These, together with the combined drug administration efforts to control malaria, have reduced the death toll to less than 700,000 deaths/year. This progress has engendered real excitement but the emergence and spread of insecticide resistance is challenging our ability to sustain and consolidate the substantial gains that have been made. Research is required to discover novel vector control tools that can supplement and improve the effectiveness of those currently available. Here, we argue that recent and continuing progress in our understanding of male mating biology is instrumental in the implementation of new approaches based on the release of either conventional sterile or genetically engineered males. Importantly, further knowledge of male biology could also lead to the development of new interventions, such as sound traps and male mass killing in swarms, and contribute to new population sampling tools. We review and discuss recent advances in the behavioural ecology of male mating with an emphasis on the potential applications that can be derived from such knowledge. We also highlight those aspects of male mating ecology that urgently require additional study in the future.

Molecular analyses on host-seeking black flies (Diptera: Simuliidae) reveal a diverse assemblage of Leucocytozoon (Apicomplexa: Haemospororida) parasites in an alpine ecosystem

Background: Molecular studies have suggested that the true diversity of Leucocytozoon (Apicomplexa: Haemospororida) species well exceeds the approximately 35 currently described taxa. Further, the degree of host-specificity may vary substantially among lineages. Parasite distribution can be influenced by the ability of the parasite to infect a host, vector preferences for certain avian hosts, or other factors such as microhabitat requirements that increase the probability that vertebrate hosts and vectors are in frequent contact with each other. Whereas most studies of haemosporidians have focused on passerine hosts, sampling vectors in the same habitats may allow the detection of other lineages affecting other hosts. Methods: We sampled abundant, ornithophilic black flies (Simuliidae) across a variety of sites and habitats in the Colorado Rocky Mountains throughout the summer of 2007. Black flies were screened with PCR using Leucocytozoon-specific primers that amplify a portion of the cytochrome b gene, and the sequences were compared to the haplotypes in the MalAvi database. Infections of Leucocytozoon from birds sampled in the same area were also included. Results: We recovered 33 unique haplotypes from the black flies in this study area, which represented a large phylogenetic diversity of Leucocytozoon parasites. However, there were no clear patterns of avian host species or geography for the distribution of Leucocytozoon haplotypes in the phylogeny. Conclusions: Sampling host-seeking vectors is a useful way to obtain a wide variety of avian haemosporidian haplotypes from a given area and may prove useful for understanding the global patterns of host, parasite, and vector associations of these ubiquitous and diverse parasites.

Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome

Background: Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome. Methods: The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy. Results: The functional characterization of the TcCAT1.1 isoform was performed in heterologous expression systems. In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m  = 0.085 ± 0.04 mM) and low affinity for ornithine (K m  = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [ 3 H]-arginine displayed a 16-fold higher efflux of [ 3 H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles. Conclusions: TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.

Inhibition of the endosymbiont &#8220; Candidatus Midichloria mitochondrii&#8221; during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16SrRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that >95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM. Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

Prevalence and molecular characterization of Cryptosporidium in giant panda ( Ailuropoda melanoleuca ) in Sichuan province, China

Background: Cryptosporidium spp. have been extensively reported to cause significant diarrheal disease in humans and domestic animals. On the contrary, little information is available on the prevalence and characterization of Cryptosporidium in wild animals in China, especially in giant pandas. The aim of the present study was to detect Cryptosporidium infections and identify Cryptosporidium species at the molecular level in both captive and wild giant pandas in Sichuan province, China.FindingsUsing a PCR approach, we amplified and sequenced the 18S rRNA gene from 322 giant pandas fecal samples (122 from 122 captive individuals and 200 collected from four habitats) in Sichuan province, China. The Cryptosporidium species/genotypes were identified via a BLAST comparison against published Cryptosporidium sequences available in GenBank followed by phylogenetic analysis. The results revealed that both captive and wild giant pandas were infected with a single Cryptosporidium species, C. andersoni, at a prevalence of 15.6 % (19/122) and 0.5 % (1/200) in captive and wild giant pandas, respectively. Conclusions: The present study revealed the existence of C. andersoni in both captive and wild giant panda fecal samples for the first time, and also provided useful fundamental data for further research on the molecular epidemiology and control of Cryptosporidium infection in giant pandas.

Paratransgenesis: a promising new strategy for mosquito vector control

The three main mosquito genera, Anopheles, Aedes and Culex, transmit respectively malaria, dengue and lymphatic filariasis. Current mosquito control strategies have proved unsuccessful, and there still is a substantial number of morbidity and mortality from these diseases. Genetic control methods have now arisen as promising alternative strategies, based on two approaches: the replacement of a vector population by disease-refractory mosquitoes and the release of mosquitoes carrying a lethal gene to suppress target populations. However, substantial hurdles and limitations need to be overcome if these methods are to be used successfully, the most significant being that a transgenic mosquito strain is required for every target species, making genetically modified mosquito strategies inviable when there are multiple vector mosquitoes in the same area. Genetically modified bacteria capable of colonizing a wide range of mosquito species may be a solution to this problem and another option for the control of these diseases. In the paratransgenic approach, symbiotic bacteria are genetically modified and reintroduced in mosquitoes, where they express effector molecules. For this approach to be used in practice, however, requires a better understanding of mosquito microbiota and that symbiotic bacteria and effector molecules be identified. Paratransgenesis could prove very useful in mosquito species that are inherently difficult to transform or in sibling species complexes. In this approach, a genetic modified bacteria can act by: (a) causing pathogenic effects in the host; (b) interfering with the host’s reproduction; (c) reducing the vector’s competence; and (d) interfering with oogenesis and embryogenesis. It is a much more flexible and adaptable approach than the use of genetically modified mosquitoes because effector molecules and symbiotic bacteria can be replaced if they do not achieve the desired result. Paratransgenesis may therefore become an important integrated pest management tool for mosquito control.

Molecular characterisation of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world

Background: The morphotaxonomy of Rhipicephalus microplus complex has been challenged in the last few years and prompted many biologists to adopt a DNA-based method for distinguishing the members of this group. In the present study, we used a mitochondrial DNA analysis to characterise the genetic assemblages, population structure and dispersal pattern of R. microplus from Southeast Asia, the region where the species originated. Methods: A phylogeographic analysis inferred from the 16S rRNA and cytochrome oxidase subunit I (COI) genes was performed with five populations of R. microplus collected from cattle in Malaysia. Malaysian R. microplus sequences were compared with existing COI and 16S rRNA haplotypes reported globally in NCBI GenBank. Results: A total of seven and 12 unique haplotypes were recovered by the 16S rRNA and COI genes, respectively. The concatenated sequences of both 16S rRNA and COI revealed 18 haplotypes. Haplotype network and phylogenetic analyses based on COI+16S rRNA sequences revealed four genetically divergent groups among Malaysian R. microplus. The significantly low genetic differentiation and high gene flow among Malaysian R. microplus populations supports the occurrence of genetic admixture. In a broader context, the 16S rRNA phylogenetic tree assigned all isolates of Malaysian R. microplus into the previously described African/Americas assemblage. However, the COI phylogenetic tree provides higher resolution of R. microplus with the identification of three main assemblages: clade A sensu Burger et al. (2014) comprises ticks from Southeast Asia, the Americas and China; clade B sensu Burger et al. (2014) is restricted to ticks that originated from China; and clade C sensu Low et al. (2015) is a new genetic assemblage discovered in this study comprising ticks from India and Malaysia. Conclusions: We conclude that the R. microplus complex consisting of at least five taxa: R. australis, R. annulatus, R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014) and the new taxon, R. microplus clade C sensu Low et al. (2015). The use of COI as the standard genetic marker in discerning the genetic assemblages of R. microplus from a broad range of biogeographical regions is proposed.

Factors affecting larval tick feeding success: host, density and time

Background: Ectoparasites rely on blood-feeding to sustain activity, support development and produce offspring. Blood-feeding is also a route for transmission of diverse vector-borne pathogens. The likelihood of successfully feeding is thus an important aspect of ectoparasite population dynamics and pathogen transmission. Factors that affect blood-feeding include ectoparasite density, host defenses, and ages of the host and ectoparasite. How these factors interact to affect feeding success is not well understood. Methods: We monitored blood-feeding success of larval Rocky Mountain wood ticks (RMWTs; Dermacentor andersoni) on deer mice (Peromyscus maniculatus) in several experiments to determine how tick density, host defense, and ages of mice and ticks interact to influence feeding success. In the first experiment, tick-naive deer mice were infested with one of several densities of RMWT larvae, while a second cohort of mice were infested with 50 larvae each. Two weeks after ticks dropped off, mice in the first cohort were re-exposed to 50 larvae each and mice in the second cohort were re-exposed to varying densities of larvae. In the second experiment mice of different ages (45–374 days old) were exposed to 50 larvae each. Two weeks later mice were re-exposed to 50 larvae each. We combined data from these and several similar experiments to test the generality of the patterns we observed. Lastly, we tested whether tick feeding success was consistent on individual mice that were challenged on four occasions. Results: Mice acquired resistance such that feeding success declined dramatically from the first to the second infestation. Feeding success also declined with tick density and tick age. Mice, however, became more permissive with age. The sizes of these effects were similar and additive. Surprisingly, over successive infestations the relative resistance among mice changed among hosts within a cohort. Conclusions: We predict that larval blood-feeding success, and thus development to the nymph stage, will change due to variation in tick age and density, as well as the age and history of the host. Incorporating these biotic factors into modeling of tick population dynamics may improve predictions of tick-borne pathogen transmission.

Seasonal associations of climatic drivers and malaria in the highlands of Ethiopia

Background: The impacts of interannual climate fluctuations on vector-borne diseases, especially malaria, have received considerable attention in the scientific literature. These effects can be significant in semi-arid and high-elevation areas such as the highlands of East Africa because cooler temperature and seasonally dry conditions limit malaria transmission. Many previous studies have examined short-term lagged effects of climate on malaria (weeks to months), but fewer have explored the possibility of longer-term seasonal effects. Methods: This study assessed the interannual variability of malaria occurrence from 2001 to 2009 in the Amhara region of Ethiopia. We tested for associations of climate variables summarized during the dry (January–April), early transition (May–June), and wet (July–September) seasons with malaria incidence in the early peak (May–July) and late peak (September–December) epidemic seasons using generalized linear models. Climate variables influenced land surface temperature (LST), rainfall, actual evapotranspiration (ET), and the enhanced vegetation index (EVI). Results: We found that both early and late peak malaria incidence had the strongest associations with meteorological conditions in the preceding dry and early transition seasons. Temperature had the strongest influence in the wetter western districts, whereas moisture variables had the strongest influence in the drier eastern districts. We also found a significant correlation between malaria incidence in the early and the subsquent late peak malaria seasons, and the addition of early peak malaria incidence as a predictor substantially improved models of late peak season malaria in both of the study sub-regions. Conclusions: These findings suggest that climatic effects on malaria prior to the main rainy season can carry over through the rainy season and affect the probability of malaria epidemics during the late malaria peak. The results also emphasize the value of combining environmental monitoring with epidemiological surveillance to develop forecasts of malaria outbreaks, as well as the need for spatially stratified approaches that reflect the differential effects of climatic variations in the different sub-regions.