Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

High Levels of Genetic Diversity within Nilo-Saharan Populations: Implications for Human Adaptation.

Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle.

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Evidence of host-associated populations of Cryptosporidium parvum in Italy.

Recent studies have revealed extensive genetic variation among isolates of Cryptosporidium parvum, an Apicomplexan parasite that causes gastroenteritis in both humans and animals worldwide. The parasite's population structure is influenced by the intensity of transmission, the host-parasite interaction, and husbandry practices. As a result, C. parvum populations can be panmictic, clonal, or even epidemic on both a local scale and a larger geographical scale. To extend the study of C. parvum populations to an unexplored region, 173 isolates of C. parvum collected in Italy from humans and livestock (calf, sheep, and goat) over a 10-year period were genotyped using a multilocus scheme based on 7 mini- and microsatellite loci. In agreement with other studies, extensive polymorphism was observed, with 102 distinct multilocus genotypes (MLGs) identified among 173 isolates. The presence of linkage disequilibrium, the confinement of MLGs to individual farms, and the relationship of many MLGs inferred using network analysis (eBURST) suggest a predominantly clonal population structure, but there is also evidence that part of the diversity can be explained by genetic exchange. MLGs from goats were found to differ from bovine and sheep MLGs, supporting the existence of C. parvum subpopulations. Finally, MLGs from isolates collected between 1997 and 1999 were also identified as a distinct subgroup in principal-component analysis and eBURST analysis, suggesting a continuous introduction of novel genotypes in the parasite population.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

Theileria parva genetic diversity and haemoparasite prevalence in cattle and wildlife in and around Lake Mburo National Park in Uganda.

Wildlife, especially Cape buffalo (Syncerus caffer), are thought to act as a reservoir for many of the important tick-borne pathogens of cattle. In this study, we have determined the prevalence of the most significant tick-borne haemoparasites in wildlife (buffalo, impala, eland and bushbuck) as well as in cattle grazing inside and neighbouring Lake Mburo National Park (LMNP) in Uganda. A high percentage of buffalo were carriers of Theileria parva, Theileria mutans, Theileria velifera, Theileria buffeli and Theileria sp. (buffalo) as well as Anaplasma marginale and Anaplasma centrale. The majority of impala sampled were carriers of A. centrale, and all were carriers of an unidentified Babesia/Theileria species. The eland and bushbuck sampled were all carriers of Theileria taurotragi and Theileria buffeli, and the majority were carriers of T. mutans. The bushbuck sampled were also carriers for Erhlichia bovis. There were some differences in the prevalence of haemoparasites between the calves sampled inside and neighbouring LMNP. In order to address the question of whether there is evidence for interbreeding between buffalo-associated and cattle-associated T. parva populations, multi-locus genotypes (MLGs) of T. parva (based on micro-satellite markers) from buffalo and from calves grazing inside and outside LMNP were compared, and the results revealed that buffalo and cattle gene pools were distinct, showing no evidence for transmission of buffalo-derived T. parva genotypes to the cattle population.

Role for parasite genetic diversity in differential host responses to Trypanosoma brucei infection.

The postgenomic era has revolutionized approaches to defining host-pathogen interactions and the investigation of the influence of genetic variation in either protagonist upon infection outcome. We analyzed pathology induced by infection with two genetically distinct Trypanosoma brucei strains and found that pathogenesis is partly strain specific, involving distinct host mechanisms. Infections of BALB/c mice with one strain (927) resulted in more severe anemia and greater erythropoietin production compared to infections with the second strain (247), which, contrastingly, produced greater splenomegaly and reticulocytosis. Plasma interleukin-10 (IL-10) and gamma interferon levels were significantly higher in strain 927-infected mice, whereas IL-12 was higher in strain 247-infected mice. To define mechanisms underlying these differences, expression microarray analysis of host genes in the spleen at day 10 postinfection was undertaken. Rank product analysis (RPA) showed that 40% of the significantly differentially expressed genes were specific to infection with one or the other trypanosome strain. RPA and pathway analysis identified LXR/RXR signaling, IL-10 signaling, and alternative macrophage activation as the most significantly differentially activated host processes. These data suggest that innate immune response modulation is a key determinant in trypanosome infections, the pattern of which can vary, dependent upon the trypanosome strain. This strongly suggests that a parasite genetic component is responsible for causing disease in the host. Our understanding of trypanosome infections is largely based on studies involving single parasite strains, and our results suggest that an integrated host-parasite approach is required for future studies on trypanosome pathogenesis. Furthermore, it is necessary to incorporate parasite variation into both experimental systems and models of pathogenesis.

Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata.

BACKGROUND: Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. RESULTS: Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. CONCLUSIONS: This analysis provides evidence that two distinct secretome genes families have evolved under contrasting selective pressures. The data supports current hypotheses regarding the biological role of TashAT family proteins in the management of host cell phenotype that may have evolved to allow adaptation of T. annulata to a specific host cell lineage. We provide new evidence of extensive allelic diversity in representative members of the enigmatic SVSP gene family, which supports a putative role for the encoded products in subversion of the host immune response.

The population structure of the Cryptosporidium parvum population in Scotland: a complex picture.

We genotyped 297 Scottish C. parvum samples using micro- and minisatellites. Treated as a single population, the population structure was epidemic. When regional populations were analysed, there was evidence of sub-population structure variations. This was dependent upon excluding sub-groups exhibiting significant genetic distance from the main population, implying genetic sub-structuring. We tested the hypothesis that these sub-groups originated outside the UK and demonstrated that one sub-group clustered with Peruvian samples. A geographically comprehensive panel of isolates would fully confirm this result. These data indicate limited sub-structuring within a small geographical area, but substantial sub-structuring over larger geographical distances. Host movement influences parasite diversity and population structure, evidenced by strong correlation (r(2) = 0.9686) between cattle movements and parasite diversity. Thus, the population structure of C. parvum is complex, with sub-populations differing in structure and being influenced by host movements, including the introduction of novel multilocus genotypes from geographically distinct regions.

Trypanosoma brucei gambiense Type 1 populations from human patients are clonal and display geographical genetic differentiation.

We have rigorously tested the hypothesis that Trypanosoma brucei gambiense Type 1 is composed of genetically homogenous populations by examining the parasite population present in Human African Trypanosomiasis (HAT) patients from the Democratic Republic of Congo (DRC) and Cameroon (CAM). We amplified eight microsatellite markers by PCR directly from blood spots on FTA filters, thereby avoiding the significant parasite selection inherent in the traditional isolation techniques of rodent inoculation or in vitro culture. All microsatellite markers were polymorphic, although for four markers there was only polymorphism between the DRC and CAM populations, not within populations, suggesting very limited genetic exchange. Within the largest population from the DRC, Hardy-Weinberg equilibrium is not evident at any loci. This evidence suggests a clonal population. However, there was significant sub-structuring between the DRC and CAM samples (F(ST) = 0.32), indicating that Trypanosoma brucei gambiense Type 1 has genetically distinct clades. The data combine to indicate that genetic exchange plays a very limited role. The finding of distinct clades in different places suggests the possibility that samples from humans with clinical signs represent clonal expansions from an underlying population that requires identifying and characterising.

Trypanosomosis in The Gambia: prevalence in working horses and donkeys detected by whole genome amplification and PCR, and evidence for interactions between trypanosome species.

BACKGROUND: The Gambia has an increasing population of equidae largely used for agriculture and transportation. A review of cases at The Gambian Horse and Donkey Trust (GHDT) indicated that a common reason for presentation is a poorly defined medical condition often attributed to trypanosomosis. There are few reports describing the prevalence or the range of clinical signs associated with infection with different species of trypanosomes in horses and donkeys, but given the importance of these animals, the role of trypanosomosis requires investigation. RESULTS: In total 241 animals from the Central River Division in The Gambia (183 horses and 58 donkeys) were screened using Whole Genome Amplification (WGA) followed by trypanosome species identification using polymerase chain reaction (PCR). The results indicated overall trypanosome prevalence of 91%; with an infection rate of 31% for Trypanosoma congolense Savannah, 87% for Trypanosoma vivax and 18% for Trypanosoma brucei sp. Multiple species were present in 43% of infections. Microscopy had a good specificity (100%) and positive predictive value (100%) for trypanosome detection, but the sensitivity (20%) and negative predictive value (10.5%) were low relative to PCR-based diagnosis. Infection with T congolense showed the greatest negative effect on packed cell volume (PCV), while infection with T. brucei sp also had a significant, although lesser, negative effect on PCV. In addition, cases positive by microscopy were associated with significantly lower PCV. However, concurrent infection with T. vivax appeared to cause less effect on PCV, compared to animals infected with T. congolense alone. CONCLUSION: The prevalence of Trypanosomosis was high in both horses and donkeys. Infection with T. congolense appeared to have the greatest clinical significance, while T. vivax infection may be of limited clinical significance in this population. Indeed, there is evidence of T. vivax co-infection ameliorating the pathology caused by T. congolense. WGA and PCR allowed a more comprehensive analysis of field infections with the detection of infections below the threshold of microscopy, and provided indications of interactions between parasite species that would otherwise remain undetected. The study raises important questions about the epidemiology of trypanosome infection in relation to disease that require a full scale longitudinal analysis.

A Genome Resequencing-Based Genetic Map Reveals the Recombination Landscape of an Outbred Parasitic Nematode in the Presence of Polyploidy and Polyandry.

The parasitic nematode Haemonchus contortus is an economically and clinically important pathogen of small ruminants, and a model system for understanding the mechanisms and evolution of traits such as anthelmintic resistance. Anthelmintic resistance is widespread and is a major threat to the sustainability of livestock agriculture globally; however, little is known about the genome architecture and parameters such as recombination that will ultimately influence the rate at which resistance may evolve and spread. Here, we performed a genetic cross between two divergent strains of H. contortus, and subsequently used whole-genome resequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterizes these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudotestcross, we characterized linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map. We exploited this map to reveal the recombination landscape, the first for any helminth species, demonstrating extensive variation in recombination rate within and between chromosomes. Analyses of these data also revealed the extent of polyandry, whereby at least eight males were found to have contributed to the genetic variation of the progeny analyzed. Triploid offspring were also identified, which we hypothesize are the result of nondisjunction during female meiosis or polyspermy. These results expand our knowledge of the genetics of parasitic helminths and the unusual life-history of H. contortus, and enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms and for related species that infect livestock and humans throughout the world. This study also demonstrates the feasibility of using whole-genome resequencing data to directly construct a genetic map in a single generation cross from a noninbred nonmodel organism with a complex lifecycle.

Use of multiple displacement amplification to increase the detection and genotyping of trypanosoma species samples immobilized on FTA filters.

Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterized by low parasitemias. Multiple displacement amplification (MDA) amplifies DNA with a simple in vitro step and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a 20-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multicopy ribosomal ITS region or 177-bp repeats, and a 20-fold increase in sensitivity over nested PCR against a single-copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.

The genetic map and comparative analysis with the physical map of Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and contributes to the debilitating disease 'Nagana' in cattle. To date we know little about the genes that determine drug resistance, host specificity, pathogenesis and virulence in these parasites. The availability of the complete genome sequence and the ability of the parasite to undergo genetic exchange have allowed genetic investigations into this parasite and here we report the first genetic map of T.brucei for the genome reference stock TREU 927, comprising of 182 markers and 11 major linkage groups, that correspond to the 11 previously identified chromosomes. The genetic map provides 90% probability of a marker being 11 cM from any given locus. Its comparison to the available physical map has revealed the average physical size of a recombination unit to be 15.6 Kb/cM. The genetic map coupled with the genome sequence and the ability to undertake crosses presents a new approach to identifying genes relevant to the disease and its prevention in this important pathogen through forward genetic analysis and positional cloning.

Molecular surveillance of Theileria parasites of livestock in Oman.

BACKGROUND: Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian Peninsula, and causes high rates of mortality and morbidity in sheep and cattle. However, there is a paucity of information on the distribution of Theileria spp. over the whole region and their impact on different hosts. The present study carried out a country-wide molecular survey for Theileria spp. of livestock in Oman across four governorates. The aim of the survey was to define the prevalence of Theileria spp. in cattle, sheep and goats, highlight risk factors for infection and identify the main tick species involved in parasite transmission. MATERIAL AND METHODS: A total of 2020 animals were examined in the survey consisting of sheep [n=592], goats [n=981] and cattle [n=447]. All three species were raised and co-grazed on the same farms. Theileria parasites were detected using PCR-RFLP and RLB of the 18S rRNA gene. Cloning and sequencing of the 18S rRNA was carried out on 11 T. lestoquardi isolates from Ash-Sharqiyah, and Ad-Dhahira governorates, and phylogenetic relationships were inferred using additional sequences of T. lestoquardi, T. annulata and T. ovis available in GenBank. RESULTS: Theileria spp. prevalence was 72.3%, 36.7% and 2.7% among cattle, sheep and goats, respectively. Strong similarity in results was obtained using RLB and PCR-RFLP for detection of Theileria spp. however, RLB detected a higher rate of mixed infection than PCR-RFPL (P<0.001). Theileria annulata was the only parasite detected in cattle, while sheep and goats carried T. ovis, T. lestoquardi and T. annulata as well as Theileria spp. OT1. Of the four Theileria spp. detected in small ruminants, overall T. ovis was most prevalent (sheep [33.4%], goats [2.0%]), whereas T. lestoquardi was less prevalent (sheep [22.0%], goats [0.5%]). A large proportion of infected sheep (19%) carried mixed infection of T. ovis and T. lestoquardi. However, single T. lestoquardi infections (3.0%) were less prevalent than T. ovis infections (14.5%). Risk of Theileria spp. infection was significantly higher for exotic breeds, relative to native breeds, of cattle (p=0.00002) and sheep (p=0.005). Phylogenetic analysis placed T. lestoquardi in Oman in the same clade as other T. lestoquardi strains isolated from the same regional area (Iraq and Iran). The main tick species, identified on the examined animals, Hyalomma anatolicum, was widely distributed and was found in all of the surveyed governorates. CONCLUSION: Theileria spp. are widespread in Oman with variable prevalence detected in different regions. Two economically important hosts, cattle and sheep are at high risk from virulent T. annulata and T. lestoquardi, respectively. The survey indicates extensive exposure to ticks and transmission of infection that has a significant economic impact. The higher prevalence of T. lestoquardi as mixed rather than single infection requires further investigation.

Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans.

Cryptosporidiosis and giardiasis are major public health concerns. The role of water and food in the epidemiology of these diseases is now well recognized. Molecular techniques are available to determine the species and genotypes of Cryptosporidium and Giardia and to distinguish human from non-human pathogens. Validated methods to determine the species, genotype and subgenotype that are present in heterologous mixtures should be applied to environmental samples to enable the monitoring and characterization of infection sources, disease tracking and the establishment of causative links to both waterborne and foodborne outbreaks. Meaningful interpretation of population structures and occurrence-prevalence baselines can be performed only by analysing a well-planned set of samples from all possible sources taken regularly over time, rather than focusing on outbreak investigations. For food, this includes such analyses in the country of origin.

Analysis of putative resistance gene loci in UK field populations of Haemonchus contortus after 6years of macrocyclic lactone use.

Sheep farmers in the UK rely on strategic anthelmintic use to treat and control gastrointestinal roundworms in their flocks. However, resistance to these drugs is now widespread and threatens the sustainability of sheep production. The mechanisms underlying resistance to the most commonly used class, the macrocyclic lactones, are not known and sensitive diagnostic tools based on molecular markers are not currently available. This prohibits accurate surveillance of resistance or assessment of strategies aimed at controlling its spread. In this study, we examined four UK field populations of Haemonchus contortus, differing in macrocyclic lactone treatment history, for evidence of selection at 'candidate gene' loci identified as determining macrocyclic lactone resistance in previously published research. Individual worms were genotyped at Hc-lgc-37, Hc-glc-5, Hc-avr-14 and Hc-dyf-7, and four microsatellite loci. High levels of polymorphism were identified at the first three candidate gene loci with remarkably little polymorphism at Hc-dyf-7. While some between-population comparisons of individual farms with and without long-term macrocyclic lactone use identified statistically significant differences in allele frequency and/or fixation index at the Hc-lgc-37, Hc-glc-5 or Hc-avr-14 loci, we found no consistent evidence of selection in other equivalent comparisons. While it is possible that different mechanisms are important in different populations or that resistance may be conferred by small changes at multiple loci, our findings suggest that these are unlikely to be major loci conferring macrocyclic lactone resistance on UK farms or suitable for diagnostic marker development. More powerful approaches, using genome-wide or whole genome sequencing, may be required to define macrocyclic lactone resistance loci in such genetically variable populations.

Theileria lestoquardi displays reduced genetic diversity relative to sympatric Theileria annulata in Oman.

The Apicomplexan parasites, Theileria lestoquardi and Theileria annulata, the causative agents of theileriosis in small and large ruminants, are widespread in Oman, in areas where cattle, sheep and goats co-graze. Genetic analysis can provide insight into the dynamics of the parasite and the evolutionary relationship between species. Here we identified ten genetic markers (micro- and mini-satellites) spread across the T. lestoquardi genome, and confirmed their species specificity. We then genotyped T. lestoquardi in different regions in Oman. The genetic structures of T. lestoquardi populations were then compared with previously published data, for comparable panels of markers, for sympatric T. annulata isolates. In addition, we examined two antigen genes in T. annulata (Tams1 and Ta9) and their orthologues in T. lestoquardi (Tlms1 and Tl9). The genetic diversity and multiplicity of infection (MOI) were lower in T. lestoquardi (He=0.64-0.77) than T. annulata (He=0.83-0.85) in all populations. Very limited genetic differentiation was found among T. lestoquardi and T. annulata populations. In contrast, limited but significant linkage disequilibrium was observed within regional populations of each species. We identified eight T. annulata isolates in small ruminants; the diversity and MOI were lower among ovine/caprine compared to bovine. Sequence diversity of the antigen genes, Tams1 and Ta9 in T. annulata (π=0.0733 and π=0.155 respectively), was 10-fold and 3-fold higher than the orthologous Tlms1 and Tl9 in T. lestoquardi (π=0.006 and π=0.055, respectively). Despite a comparably high prevalence, T. lestoquardi has lower genetic diversity compared to sympatric T. annulata populations. There was no evidence of differentiation among populations of either species. In comparison to T. lestoquardi, T. annulata has a larger effective population size. While genetic exchange and recombination occur in both parasite species, the extent of diversity, overall, is less for T. lestoquardi. It is, therefore, likely that T. lestoquardi evolved from an ancestor of present day T. annulata and that this occurred either once or on a limited number of occasions.