Background: Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis. Methods: T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining. Results: We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions. Conclusion: The findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.

Background: Gyrodactylus salaris is a directly transmitted ectoparasite that reproduces in situ on its fish host. Wild Norwegian (East Atlantic) salmon stocks are thought to be especially susceptible to the parasite due to lack of co-adaptation, contrary to Baltic salmon stocks. This study i) identifies whether time- and density-dependent mechanisms in gyrodactylid population growth exist in G. salaris-Atlantic salmon interactions and ii) based on differences between Norwegian and Baltic stocks, determines whether the ‘Atlantic susceptible, Baltic resistant’ paradigm holds as an example of local adaptation. Methods: A total of 18 datasets of G. salaris population growth on individually isolated Atlantic salmon (12 different stocks) infected with three parasite strains were re-analysed using a Bayesian approach. Datasets included over 2000 observations of 388 individual fish. Results: The best fitting model of population growth was time-limited; parasite population growth rate declined consistently from the beginning of infection. We found no evidence of exponential population growth in any dataset. In some stocks, a density dependence in the size of the initial inoculum limited the maximum rate of parasite population growth. There is no evidence to support the hypothesis that all Norwegian and Scottish Atlantic salmon stocks are equally susceptible to G. salaris, while Baltic stocks control and limit infections due to co-evolution. Northern and Western Norwegian as well as the Scottish Shin stocks, support higher initial parasite population growth rates than Baltic, South-eastern Norwegian, or the Scottish Conon stocks, and several Norwegian stocks tested (Akerselva, Altaelva, Lierelva, Numedalslågen), and the Scottish stocks (i.e. Conon, Shin), were able to limit infections after 40–50 days. No significant differences in performance of the three parasite strains (Batnfjordselva, Figga, and Lierelva), or the two parasite mitochondrial haplotypes (A and F) were observed. Conclusions: Our study shows a spectrum of growth rates, with some fish of the South-eastern Norwegian stocks sustaining parasite population growth rates overlapping those seen on Baltic Neva and Indalsälv stocks. This observation is inconsistent with the ‘Baltic-resistant, Atlantic-susceptible’ hypothesis, but suggests heterogeneity, perhaps linked to other host resistance genes driven by selection for local disease syndromes.

Background: Taenia solium cysticercosis is a parasitic meat-borne disease that is highly prevalent in pigs and humans in Africa, but the burden is vastly underestimated due to the lack of official control along the pork commodity chain, which hampers long-term control policies. Methods: The apparent and corrected prevalences of T. solium cysticercosis were investigated in pork carcasses slaughtered and retailed in Antananarivo (Madagascar), thanks to a 12-month monitoring plan in two urban abattoirs. Results: Overall apparent prevalence was estimated at 4.6 % [4.2 – 5.0 %]. The corrected overall prevalence defined as the estimated prevalence after accounting for the sensitivity of meat inspection was 21.03 % [19.18- 22.87 %]. Significant differences among geoclimatic regions were observed only for indigenous pigs, with an apparent prevalence estimated at 7.9 % [6.0 – 9.9 %] in the northern and western regions, 7.3 % [6.0 – 8.6 %] in the central region, and 6.2 % [4.7 – 7.8 %] in the southern region. In the central region, where both exotic and indigenous pigs were surveyed, indigenous pigs were 8.5 times [6.7 – 10.7] more likely to be infected than exotic improved pigs. Urban consumers were more likely to encounter cysticercosis in pork in the rainy season, which is a major at risk period, in particular in December. Differences between abattoirs were also identified. Conclusion: Our results underline the need for improved surveillance and control programmes to limit T. solium cysticercosis in carcasses by introducing a risk-based meat inspection procedure that accounts for the origin and breed of the pigs, and the season.

E. Vannier and P. J. Krause presented an excellent article on “Babesiosis in China, an emerging threat” in the Lancet Infectious Diseases in December 2014, which updated research on human babesiosis in China. However, a neglected and emerging issue has not been mentioned in EV & PJK’s article, that is the co-infections with B. microti and P. falciparum parasites that exist in syndemic areas, spatially in the China-Myanmar border areas of Yunnan province, China. Therefore, two important issues are addressed in here, including (i) the new emerging infections with Babesia spp. which are normally ignored in malaria endemic areas due to similarities in pathogenic morphology and clinical symptoms, (ii) additional consideration on babesiosis rather than drug-resistant malaria when anti-malaria treatment for the febrile cases in clinics fails.

N. Latorre-Margalef et al.

B. P. Chan et al.

D. R. Feikin et al.

Background: Culex pipiens pipiens plays an important role in the transmission of several vector-borne pathogens such as West Nile virus (WNV) in North America. Laboratory and field studies suggest that this species is ornithophilic but because of genetic hybridization with sibling species during the active mosquito season, it may occasionally feed on mammals. Adult female Cx. p. pipiens undergo a facultative diapause and may serve as an overwintering mechanism for WNV. To determine the effect of diapause on the innate host preference of Cx. p. pipiens emerging from winter hibernation, we conducted host-choice experiments using bird and mammal hosts. Methods: Mosquitoes were reared under non-diapause induced (NDI), diapause induced (DI), and field collected from overwintering (OW) hibernaculae. They were released into a large mesh enclosure housing two lard can traps, and given a choice between feeding on a dove or a rat. Results: Host seeking Cx. p. pipiens were four times more likely to feed on the dove than the rat, regardless of experimental conditions. Under NDI conditions, Cx. p. pipiens were (p < 0.001) more attracted to the bird (79.9 % [75.6-84.1]) than the rat (20.1 [15.9-24.4]). Overwintering mosquitoes and those exposed to DI conditions were also significantly (p < 0.001) more attracted to birds (81.6 % [75.9-87.3]) than to rats (18.5 [12.7-24.2]). Conclusions: We provide new information about the innate host preference of Cx. p. pipiens emerging from diapause in temperate habitats where winter survival is crucial for disease transmission cycles. Although we showed that Cx. p. pipiens prefers an avian to a mammalian host, nearly 20 % of emerging mosquitoes in the spring could feed on mammals. Changes in host preferences may also contain valuable clues about transmission dynamics and subsequent timely interventions by vector control and public health practitioners.

M. R. Moore and C. G. Whitney

C. Dendle et al.

S. Avcin et al.

J. Heyckendorf et al.

C. Mok et al.

C. S. Arriola et al.

Background: The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. Methods: This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart™ malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. Results: A total of 188 (30.2 %) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100 %) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5 % and 96.1 %, respectively). Conclusion: The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart™ Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.

Background: Tsetse-transmitted African trypanosomes cause both nagana (African animal Trypanosomiasis-AAT) and sleeping sickness (human African Trypanosomiasis - HAT) across Sub-Saharan Africa. Vector control and chemotherapy are the contemporary methods of tsetse and trypanosomiasis control in this region. In most African countries, including Uganda, veterinary services have been decentralised and privatised. As a result, livestock keepers meet the costs of most of these services. To be sustainable, AAT control programs need to tailor tsetse control to the inelastic budgets of resource-poor small scale farmers. To guide the process of tsetse and AAT control toolkit selection, that now, more than ever before, needs to optimise resources, the costs of different tsetse and trypanosomiasis control options need to be determined. Methods: A detailed costing of the restricted application protocol (RAP) for African trypanosomiasis control in Tororo District was undertaken between June 2012 and December 2013. A full cost calculation approach was used; including all overheads, delivery costs, depreciation and netting out transfer payments to calculate the economic (societal) cost of the intervention. Calculations were undertaken in Microsoft Excel™ without incorporating probabilistic elements. Results: The cost of delivering RAP to the project was US$ 6.89 per animal per year while that of 4 doses of a curative trypanocide per animal per year was US$ 5.69. However, effective tsetse control does not require the application of RAP to all animals. Protecting cattle from trypanosome infections by spraying 25 %, 50 % or 75 % of all cattle in a village costs US$ 1.72, 3.45 and 5.17 per animal per year respectively. Alternatively, a year of a single dose of curative or prophylactic trypanocide treatment plus 50 % RAP would cost US$ 4.87 and US$ 5.23 per animal per year. Pyrethroid insecticides and trypanocides cost 22.4 and 39.1 % of the cost of RAP and chemotherapy respectively. Conclusions: Cost analyses of low cost tsetse control options should include full delivery costs since they constitute 77.6 % of all project costs. The relatively low cost of RAP for AAT control and its collateral impact on tick control make it an attractive option for livestock management by smallholder livestock keepers.

Background: Leishmania infantum is the main etiological agent of both visceral and cutaneous clinical forms of leishmaniasis in the Mediterranean area. Leishmania/HIV coinfection in this area is characterized by a chronic course and frequent recurrences of clinical episodes. The present study using Multilocus Microsatellite Typing (MLMT) analysis, a highly discriminative tool, aimed to genetically characterize L. infantum isolates taken from monitored Leishmania/HIV coinfected patients presenting successive clinical episodes. Methods: In this study, by the analysis of 20 microsatellite loci, we studied the MLMT profiles of 25 L. infantum isolates from 8 Leishmania/HIV coinfected patients who had experienced several clinical episodes. Two to seven isolates per patient were taken before and after treatment, during clinical and non-clinical episodes, with time intervals of 6 days to 29 months. Genetic diversity, clustering and phenetic analyses were performed. Results: MLMT enabled us to study the genetic characteristics of the 25 L. infantum isolates, differentiating 18 genotypes, corresponding to a genotypic diversity of 0.72. Fifteen genotypes were unique in the total sample set and only 3 were repeated, 2 of which were detected in different patients. Both clustering and phylogenetic analyses provided insights into the genetic links between the isolates; in five patients isolates showed clear genetic links: either the genotype was exactly the same or only slightly different. In contrast, the isolates of the other three patients were dispersed in different clusters and some could be the result of mixing between populations. Conclusions: Our data indicated a great MLMT variability between isolates from coinfected patients and no predominant genotype was observed. Despite this, almost all clinical episodes could be interpreted as a relapse rather than a reinfection. The results showed that diverse factors like an intrapatient evolution over time or culture bias could influence the parasite population detected in the patient, making it difficult to differentiate between relapse and reinfection.

Background: Glossina fuscipes fuscipes is the main vector of African Trypanosomiasis affecting both humans and livestock in Uganda. The human disease (sleeping sickness) manifests itself in two forms: acute and chronic. The Lake Victoria basin in Uganda has the acute form and a history of tsetse re-emergence despite concerted efforts to control tsetse. The government of Uganda has targeted the basin for tsetse eradication. To provide empirical data for this initiative, we screened tsetse flies from the basin for genetic variation at the mitochondrial DNA cytochrome oxidase II (mtDNA COII) gene with the goal of investigating genetic diversity and gene flow among tsetse, tsetse demographic history; and compare these results with results from a previous study based on microsatellite loci data in the same area. Methods: We collected 429 Gff tsetse fly samples from 14 localities in the entire Ugandan portion of the Lake Victoria coast, covering 40,000 km 2 . We performed genetic analyses on them and added data collected for 56 Gff individuals from 4 additional sampling sites in the basin. The 529pb partial mitochondrial DNA cytochrome oxidase II (mtDNA COII) sequences totaling 485 were analysed for genetic differentiation, structuring and demographic history. The results were compared with findings from a previous study based on microsatellite loci data from the basin. Results: The differences within sampling sites explained a significant proportion of the genetic variation. We found three very closely related mtDNA population clusters, which co-occurred in multiple sites. Although Φ ST (0 – 0.592; P < 0.05) and Bayesian analyses suggest some level of weak genetic differentiation, there is no correlation between genetic divergence and geographic distance (r = 0.109, P = 0.185), and demographic tests provide evidence of locality-based demographic history. Conclusion: The mtDNA data analysed here complement inferences made in a previous study based on microsatellite data. Given the differences in mutation rates, mtDNA afforded a look further back in time than microsatellites and revealed that Gff populations were more connected in the past. Microsatellite data revealed more genetic structuring than mtDNA. The differences in connectedness and structuring over time could be related to vector control efforts. Tsetse re-emergence after control interventions may be due to re-invasions from outside the treated areas, which emphasizes the need for an integrated area-wide tsetse eradication strategy for sustainable removal of the tsetse and trypanosomiasis problem from this area.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite. It has extensive host populations and is prevalent globally; T. gondii infection can cause a zoonotic parasitic disease. Microneme protein 3 (MIC3) is a secreted protein that is expressed in all stages of the T. gondii life cycle. It has strong immunoreactivity and plays an important role in the recognition, adhesion and invasion of host cells by T. gondii. This article reviews the molecular structure of MIC3, its role in the invasion of host cells by parasites, its relationship with parasite virulence, and its induction of immune protection to lay a solid foundation for an in-depth study of potential diagnostic agents and vaccines for preventing toxoplasmosis.

Background: Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Methods: Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). Results: The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ 2  = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Conclusions: Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.