High prevalence of Schistosoma haematobium × Schistosoma bovis hybrids in schoolchildren in Côte d'Ivoire.

Schistosomiasis is a neglected tropical disease, though it is highly prevalent in many parts of sub-Saharan Africa. While Schistosoma haematobium-bovis hybrids have been reported in West Africa, no data about Schistosoma hybrids in humans are available from Côte d'Ivoire. This study aimed to identify and quantify S. haematobium-bovis hybrids among schoolchildren in four localities of Côte d'Ivoire. Urine samples were collected and examined by filtration to detect Schistosoma eggs. Eggs were hatched and 503 miracidia were individually collected and stored on Whatman® FTA cards for molecular analysis. Individual miracidia were molecularly characterized by analysis of mitochondrial cox1 and nuclear internal transcribed spacer 2 (ITS 2) DNA regions. A mitochondrial cox1-based diagnostic polymerase chain reaction was performed on 459 miracidia, with 239 (52.1%) exhibiting the typical band for S. haematobium and 220 (47.9%) the S. bovis band. The cox1 and ITS 2 amplicons were Sanger sequenced from 40 randomly selected miracidia to confirm species and hybrids status. Among the 33 cox1 sequences analysed, we identified 15 S. haematobium sequences (45.5%) belonging to seven haplotypes and 18 S. bovis sequences (54.5%) belonging to 12 haplotypes. Of 40 ITS 2 sequences analysed, 31 (77.5%) were assigned to pure S. haematobium, four (10.0%) to pure S. bovis and five (12.5%) to S. haematobium-bovis hybrids. Our findings suggest that S. haematobium-bovis hybrids are common in Côte d'Ivoire. Hence, intense prospection of domestic and wild animals is warranted to determine whether zoonotic transmission occurs.

Prevalence and distribution of livestock schistosomiasis and fascioliasis in Côte d'Ivoire: results from a cross-sectional survey.

BACKGROUND: Schistosoma and Fasciola are zoonotic parasites of public health and veterinary importance. However, while the epidemiology of schistosomiasis in humans is well studied, little is known about fascioliasis and schistosomiasis in livestock in Côte d'Ivoire. This study aimed to determine the prevalence and the distribution of livestock schistosomiasis and fascioliasis across Côte d'Ivoire. In 2018, we conducted a cross-sectional survey in abattoirs and farms in 13 departments of Côte d'Ivoire. In abattoirs, the mesenteric veins and livers of slaughtered cattle, sheep and goats were examined for adult Schistosoma and Fasciola flukes. Faeces from live cattle, goats and sheep were collected and examined for Schistosoma and Fasciola eggs using a sedimentation technique. RESULTS: A total of 386 cattle, 174 goats and 151 sheep from abattoirs and 435 cattle, 22 goats and 176 sheep from farms were sampled. The observed prevalence of schistosomiasis was higher in slaughtered animals. Fascioliasis was more prevalent in farm animals. The prevalence of schistosomiasis in slaughtered cattle varied between 5.9% (95% confidence interval (CI): 0.7-19.7%) and 53.3% (95% CI: 37.9-68.3%) with the highest prevalence observed in Ouangolodougou in the North. Cattle from farms had a relatively low prevalence of schistosomiasis, with the highest prevalence found in Ouangolodougou (2.4%, 95% CI: 0.7-6.1%). The prevalence of fascioliasis varied considerably from one department to another, ranging from nil (95% CI: 0.0-18.5%) to 50.8% (95% CI: 43.4-58.2%), with the highest prevalence found in farm cattle in Dikodougou in the North. Sheep and goats had a lower prevalence of schistosomiasis and fascioliasis than cattle. In slaughtered animals, cattle aged 4 years and older were at highest risk for schistosomiasis (odds ratio (OR): 2.4; 95% CI: 1.0-5.6) and fascioliasis (OR: 2.1; 95% CI: 1.1-3.9). In farm animals, male cattle had higher odds of being infected with Schistosoma (OR: 4.3; 95% CI: 0.7-26.9) than females. CONCLUSIONS: Our study confirms that schistosomiasis and fascioliasis are endemic in livestock across Côte d'Ivoire. A strategic control programme should be considered, especially for cattle, including providing drinking water in troughs to reduce faecal contamination of water sources by cattle.

Accuracy of the sedimentation and filtration methods for the diagnosis of schistosomiasis in cattle.

Infection with blood flukes of the genus Schistosoma causes considerable human and animal morbidity, mortality and economic loss to the livestock industry. Current diagnostic tools have limitations. In this study, we compared the sedimentation and filtration methods for the diagnosis of schistosomiasis in livestock. A total of 196 faecal samples from cattle in Côte d'Ivoire were subjected to sedimentation and filtration for the diagnosis of schistosomiasis and other intestinal parasite infections. Schistosoma eggs or miracidia were discovered in 32 samples: 15 by filtration only, seven by sedimentation only, six concurrently by both methods and four by observing miracidia swimming on the sedimentation slide. The sensitivity of sedimentation and filtration was 41% and 66%, respectively. Cases with no Schistosoma eggs identified in the sediment but miracidia swimming on the slide indicate that eggs had hatched before microscopy. More accurate diagnostic are required for livestock schistosomiasis, in order to better understand the epidemiology and inform control and elimination efforts in livestock and human populations.

Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

Molecular Characterization of Canine Rabies Virus, Mali, 2006-2013.

We genetically characterized 32 canine rabies viruses isolated in Mali during 2006-2013 and identified 3 subgroups that belonged to the Africa 2 lineage. We also detected subgroup F rabies virus. This information should be useful for development of mass vaccination campaigns for dogs and eventual large-scale control programs in this country.

Genetic characterization of schistosome species from cattle in Côte d'Ivoire.

BACKGROUND: Schistosomiasis is a water-based parasitic disease that affects humans, livestock and wild animals. While considerable resources are dedicated to the surveillance, disease mapping, control and elimination of human schistosomiasis, this is not the case for livestock schistosomiasis. Indeed, there are important data and knowledge gaps concerning the species present, population genetic diversity, infection prevalence, morbidity and economic impact. This study aimed to identify circulating schistosome species in cattle across Côte d'Ivoire and to investigate their population diversity and structuring. METHODS: Overall, 400 adult schistosomes were collected from slaughtered cattle at six sites across Côte d'Ivoire. Additionally, 114 miracidia were collected from live cattle at one site: Ferkessédougou, in the northern part of Côte d'Ivoire. DNA from all specimens was extracted and the cox1 and ITS1/2 regions amplified and analysed to confirm species. The genetic diversity and structuring of the schistosome populations were investigated using 12 microsatellite markers. RESULTS: All adult schistosomes and miracidia presented Schistosoma bovis mitochondrial cox1 profile. Nuclear ITS1/2 data were obtained from 101 adult schistosomes and four miracidia, all of which presented an S. bovis profile. Genetic diversity indices revealed a deficiency of heterozygotes and signals of inbreeding across all sites, while structure analyses displayed little geographic structuring and differentiation. Cattle in Côte d'Ivoire thus appear to be mono-species infected with S. bovis. Hybrids of Schistosoma haematobium × S. bovis have not been identified in this study. Cattle schistosomes appear to be panmictic across the country. CONCLUSIONS: Our results contribute to a deeper understanding of schistosome populations in Ivorian cattle and emphasize a One Health approach of joint human and animal surveillance and prevention and control programmes for schistosomiasis.

Prevalence and risk factors of schistosomiasis and hookworm infection in seasonal transmission settings in northern Côte d'Ivoire: A cross-sectional study.

BACKGROUND: Schistosomiasis and hookworm infection remain public health problems in large parts of sub-Saharan Africa. The epidemiology of schistosomiasis and hookworm was studied in seasonal transmission settings in the northern part of Côte d'Ivoire. METHODOLOGY: In August 2018, a cross-sectional study was conducted. Urine and stool samples were collected from 742 individuals aged 6-96 years in 16 localities from four departments in northern Côte d'Ivoire. Urine samples were examined by a filtration method for quantification of Schistosoma haematobium eggs. Stool samples were subjected to duplicate Kato-Katz thick smears and eggs of Schistosoma mansoni and soil-transmitted helminths (STHs) were counted. Additionally, a questionnaire was administered to determine demographic characteristics and to identify risk factors of schistosomiasis and STHs. Malacologic surveys were carried out at water points that are contacted by humans and animals. PRINCIPAL FINDINGS: The prevalence of schistosomiasis was very low. Only two cases of S. mansoni were found (0.3%, 95% confidence interval [CI]: 0.1-1.0%). The distribution of S. haematobium was focal, with cases found only in two departments; Ferkessédougou (5.4%, 95% CI: 2.5-9.9%) and Ouangolodougou (2.7%, 95% CI: 0.9-6.3%). Hookworm was the only STH species observed with a prevalence of 1.5% (95% CI: 0.8-2.8%). A higher risk of S. haematobium infection was observed in males compared to females, but the difference was not statistically significant (2.3% versus 1.3%, odds ratio [OR]: 1.5, 95% CI: 0.8-2.7). Participants aged 16-20 years showed the highest prevalence of S. haematobium. A total of 111 human- and animal-water contact points were identified at 47 water sources. Three potential intermediate host snails of schistosomes were collected; namely, Bulinus forskalii (n = 761), Bulinus truncatus (n = 205), and Biomphalaria pfeifferi (n = 1). Yet, only one specimen of Bu. truncatus was found to be shedding schistosome cercariae. CONCLUSIONS/SIGNIFICANCE: This study confirms very low transmission of schistosomiasis and hookworm in northern Côte d'Ivoire. The establishment and rigorous implementation of integrated surveillance-response systems could lead to the elimination of schistosomiasis and hookworm in this part of Côte d'Ivoire.

Bottlenecks and the maintenance of minor genotypes during the life cycle of Trypanosoma brucei.

African trypanosomes are digenetic parasites that undergo part of their developmental cycle in mammals and part in tsetse flies. We established a novel technique to monitor the population dynamics of Trypanosoma brucei throughout its life cycle while minimising the confounding factors of strain differences or variation in fitness. Clones derived from a single trypanosome were tagged with short synthetic DNA sequences in a non-transcribed region of the genome. Infections were initiated with mixtures of tagged parasites and a combination of polymerase chain reaction and deep sequencing were used to monitor the composition of populations throughout the life cycle. This revealed that a minimum of several hundred parasites survived transmission from a tsetse fly to a mouse, or vice versa, and contributed to the infection in the new host. In contrast, the parasites experienced a pronounced bottleneck during differentiation and migration from the midgut to the salivary glands of tsetse. In two cases a single tag accounted for > or =99% of the population in the glands, although minor tags could be also detected. Minor tags were transmitted to mice together with the dominant tag(s), persisted during a chronic infection, and survived transmission to a new insect host. An important outcome of the bottleneck within the tsetse is that rare variants can be amplified in individual flies and disseminated by them. This is compatible with the epidemic population structure of T. brucei, in which clonal expansion of a few genotypes in a region occurs against a background of frequent recombination between strains.

Population genetic structure of Schistosoma haematobium and Schistosoma haematobium × Schistosoma bovis hybrids among school-aged children in Côte d'Ivoire.

While population genetics of Schistosoma haematobium have been investigated in West Africa, only scant data are available from Côte d'Ivoire. The purpose of this study was to analyze both genetic variability and genetic structure among S. haematobium populations and to quantify the frequency of S. haematobium × S. bovis hybrids in school-aged children in different parts of Côte d'Ivoire. Urine samples were subjected to a filtration method and examined microscopically for Schistosoma eggs in four sites in the western and southern parts of Côte d'Ivoire. A total of 2692 miracidia were collected individually and stored on Whatman® FTA cards. Of these, 2561 miracidia were successfully genotyped for species and hybrid identification using rapid diagnostic multiplex mitochondrial cox1 PCR and PCR Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the nuclear ITS2 region. From 2164 miracidia, 1966 (90.9%) were successfully genotyped using at least 10 nuclear microsatellite loci to investigate genetic diversity and population structure. Significant differences were found between sites in all genetic diversity indices and genotypic differentiation was observed between the site in the West and the three sites in the East. Analysis at the infrapopulation level revealed clustering of parasite genotypes within individual children, particularly in Duekoué (West) and Sikensi (East). Of the six possible cox1-ITS2 genetic profiles obtained from miracidia, S. bovis cox1 × S. haematobium ITS2 (42.0%) was the most commonly observed in the populations. We identified only 15 miracidia (0.7%) with an S. bovis cox1 × S. bovis ITS2 genotype. Our study provides new insights into the population genetics of S. haematobium and S. haematobium × S. bovis hybrids in humans in Côte d'Ivoire and we advocate for researching hybrid schistosomes in animals such as rodents and cattle in Côte d'Ivoire.

Title: Structuration génétique des populations de Schistosoma haematobium et des hybrides Schistosoma haematobium × Schistosoma bovis chez les enfants d'âge scolaire en Côte d'Ivoire. Abstract: Alors que la génétique des populations de Schistosoma haematobium a été étudiée en Afrique de l'Ouest, seules quelques données sont disponibles pour la Côte d'Ivoire. Le but de cette étude était d'analyser à la fois la variabilité génétique et la structure génétique des populations de S. haematobium et de quantifier la fréquence des hybrides S. haematobium × S. bovis chez les enfants d'âge scolaire dans différentes régions de la Côte d'Ivoire. Des échantillons d'urine ont été soumis à une méthode de filtration et examinés au microscope pour les œufs de Schistosoma dans quatre sites de l'ouest et du sud de la Côte d'Ivoire. Au total, 2 692 miracidia ont été collectés individuellement et stockés sur des cartes Whatman® FTA. Parmi ceux-ci, 2 561 miracidia ont été génotypés avec succès pour l'identification des espèces et des hybrides à l'aide de la PCR multiplex de diagnostic rapide du cox1 mitochondrial et d'une analyse du polymorphisme de longueur des fragments de restriction de PCR (PCR-RFLP) de la région ITS2 de l'ADN nucléaire. Sur 2 164 miracidia, 1 966 (90,9 %) ont été génotypés avec succès en utilisant au moins 10 loci microsatellites nucléaires pour étudier la diversité génétique et la structure de la population. Des différences significatives ont été trouvées entre les sites dans tous les indices de diversité génétique et une différenciation génotypique a été observée entre le site de l'Ouest et les trois sites de l'Est. L'analyse au niveau de l'infrapopulation a révélé un regroupement des génotypes de parasites au sein de chaque enfant, en particulier à Duekoué (Ouest) et Sikensi (Est). Parmi les six profils génétiques cox1-ITS2 possibles obtenus à partir de miracidia, S. bovis cox1 × S. haematobium ITS2 (42,0 %) était le plus fréquemment observé dans les populations. Nous avons identifié seulement 15 miracidia (0,7 %) avec un génotype S. bovis cox1 × S. bovis ITS2. Notre étude apporte de nouvelles connaissances sur la génétique des populations de S. haematobium et des hybrides S. haematobium × S. bovis chez l'homme en Côte d'Ivoire et nous plaidons pour la recherche de schistosomes hybrides chez les animaux (rongeurs et bovins) en Côte d'Ivoire.

Population genetic structure of Aldabra giant tortoises.

Evolution of population structure on islands is the result of physical processes linked to volcanism, orogenic events, changes in sea level, as well as habitat variation. We assessed patterns of genetic structure in the giant tortoise of the Aldabra atoll, where previous ecological studies suggested population subdivisions as a result of landscape discontinuity due to unsuitable habitat and island separation. Analysis of mitochondrial DNA (mtDNA) control region sequences and allelic variation at 8 microsatellite loci were conducted on tortoises sampled in 3 locations on the 2 major islands of Aldabra. We found no variation in mtDNA sequences. This pattern corroborated earlier work supporting the occurrence of a founding event during the last interglacial period and a further reduction in genetic variability during historical time. On the other hand, significant population structure recorded at nuclear loci suggested allopatric divergence possibly due to geographical barriers among islands and ecological partitions hindering tortoise movements within islands. This is the first attempt to study the population genetics of Aldabra tortoises, which are now at carrying capacity in an isolated terrestrial ecosystem where ecological factors appear to have a strong influence on population dynamics.

Efficacy of triclabendazole and albendazole against Fasciola spp. infection in cattle in Côte d'Ivoire: a randomised blinded trial.

Triclabendazole is the anthelminthic of choice for the treatment of fascioliasis, however, it is only registered in a few countries. We investigated the efficacy of a single-dose of triclabendazole (12 mg/kg) or albendazole (15 mg/kg) against Fasciola spp. infection in cattle on farms in the northern part of Côte d'Ivoire in a randomized clinical trial. Faecal samples were obtained from 196 cattle, of which 155 (79.1%) were found positive for Fasciola spp. by the sedimentation technique. Cattle infected with Fasciola spp. were randomly allocated (3:3:1) to receive triclabendazole (n = 66), albendazole (n = 67) or left untreated to serve as control (n = 22). Follow-up faecal samples were collected on days 21, 28, 90 and 188 post-treatment. No adverse events were observed as reported by farmers in any of the treatment groups. The proportion of non-egg shedding cattle, assessed at day 21 (primary outcome), was significantly higher in cattle treated with triclabendazole (95.4%) compared to those receiving albendazole (70.3%; odds ratio [OR] 8.73, 95% confidence interval [CI] 2.43-31.28, p <0.001). The egg reduction rate (ERR) expressed as number of eggs per gram of faeces, a secondary endpoint assessed at day 21 post-treatment, was significantly higher in the triclabendazole arm (arithmetic mean (AM) ERR = 99.8%) than in the albendazole arm (AM ERR = 92.2%), with a difference of 7.6%-points (95% CI: 0.9-14.5%-points, p=0.026). This is the first report of efficacy of triclabendazole against Fasciola spp. in naturally infected cattle in Côte d'Ivoire. Our results confirm that triclabendazole is the most effective treatment of fascioliasis and therefore, should be considered for the control of livestock fascioliasis; if resources allow in combination with intermediate host snail control and raising farmers awareness of pasture and livestock management to avoid reinfection.

Molecular Confirmation of a Fasciola gigantica × Fasciola hepatica Hybrid in a Chadian Bovine.

Fascioliasis is a zoonotic infection of humans and, more commonly, ruminants. It is caused by 2 liver fluke species, Fasciola hepatica and Fasciola gigantica, which differ in size. The traditional morphological methods used to distinguish the 2 species can be unreliable, particularly in the presence of hybrids between the 2 species. The development of advanced molecular methods has allowed for more definitive identification of Fasciola species, including their hybrids. Hybrids are of concern, as it is thought that they could acquire advantageous traits such as increased pathogenicity and host range. In 2013, we collected flukes from Fasciola-positive cattle, sheep, and goats slaughtered in 4 Chadian abattoirs. DNA from 27 flukes was extracted, amplified, and analyzed to identify species using the ITS1+2 locus. Twenty-six of the 27 flukes were identified as F. gigantica, while the remaining fluke showed heterozygosity at all variable sites that distinguish F. hepatica and F. gigantica. Cloning and sequencing of both alleles confirmed the presence of 1 F. hepatica and 1 F. gigantica allele. To our knowledge, this is the first unambiguous, molecular demonstration of the presence of such a hybrid in a bovine in sub-Saharan Africa.

Intraspecific competition between co-infecting parasite strains enhances host survival in African trypanosomes.

It is becoming increasingly clear that under natural conditions parasitic infections commonly consist of co-infections with multiple conspecific strains. Multiple-strain infections lead to intraspecific interactions and may have important ecological and evolutionary effects on both hosts and parasites. However, experimental evidence on intraspecific competition or facilitation in infections has been scarce because of the technical challenges of distinguishing and tracking individual co-infecting strains. To overcome this limitation, we engineered transgenic strains of the protozoan parasite Trypanosoma brucei, the causal agent of human African sleeping sickness. Different strains were transfected with fluorescence genes of different colors to make them visually distinguishable in order to investigate the effects of multiple-strain infections on parasite population dynamics and host fitness. We infected mice either with each strain alone or with mixes of two strains. Our results show a strong mutual competitive suppression of co-infecting T. brucei strains very early in infection. This mutual suppression changes within-host parasite dynamics and alleviates the effects of infection on the host. The strength of suppression depends on the density of the co-infecting strain, and differences in life-history traits between the strains determine the consequences of strain-strain competition for the host. Unexpectedly, co-infection with a less virulent strain significantly enhances host survival (+15%). Analysis of the strain dynamics reveals that this is due to the suppression of the density of the more virulent strain (-33%), whose degree of impact ultimately determines the physical condition of the host. The competitive suppression is likely caused by allelopathic interference or by apparent competition mediated by strain-specific immune responses. These findings highlight the importance of intraspecific variation for parasite-parasite and parasite-host interactions. To fully understand parasite and disease dynamics, the genetic diversity of infections must be taken into account. Through changes in parasite dynamics, intraspecific variation may further affect transmission dynamics and select for increased virulence of each strain. The precise mechanisms underlying mutual suppression are not yet understood but may be exploitable to fight this devastating parasite. Our results are therefore not only of basic ecological interest investigating an important form of intraspecific competition, but may also have applied relevance for public health.

Prevalence and Risk Factors for Schistosomiasis among Schoolchildren in two Settings of Côte d'Ivoire.

Schistosomiasis is a parasitic disease affecting more than 250 million people, primarily in sub-Saharan Africa. In Côte d'Ivoire both Schistosoma haematobium (causing urogenital schistosomiasis) and Schistosoma mansoni (causing intestinal schistosomiasis) co-exist. This study aimed to determine the prevalence of S. haematobium and S. mansoni and to identify risk factors among schoolchildren in the western and southern parts of Côte d'Ivoire. From January to April 2018, a cross-sectional study was carried out including 1187 schoolchildren aged 5-14 years. Urine samples were examined by a filtration method to identify and count S. haematobium eggs, while stool samples were subjected to duplicate Kato-Katz thick smears to quantify eggs of S. mansoni and soil-transmitted helminths. Data on sociodemographic, socioeconomic, and environmental factors were obtained using a pretested questionnaire. Multivariate logistic regression was employed to test for associations between variables. We found a prevalence of S. haematobium of 14.0% (166 of 1187 schoolchildren infected) and a prevalence of S. mansoni of 6.1% (66 of 1089 schoolchildren infected). In the southern part of Côte d'Ivoire, the prevalence of S. haematobium was 16.1% with a particularly high prevalence observed in Sikensi (35.6%), while S. mansoni was most prevalent in Agboville (11.2%). Swimming in open freshwater bodies was the main risk factor for S. haematobium infection (adjusted odds ratio (AOR) = 127.0, 95% confidence interval (CI): 25.0-634.0, p < 0.001). Fishing and washing clothes in open freshwater bodies were positively associated with S. haematobium and S. mansoni infection, respectively. Preventive chemotherapy using praziquantel should be combined with setting-specific information, education, and communication strategies in order to change children's behavior, thus avoiding contact with unprotected open freshwater.

New fluorescence markers to distinguish co-infecting Trypanosoma brucei strains in experimental multiple infections.

Multiple-genotype infections are increasingly recognized as important factors in disease evolution, parasite transmission dynamics, and the evolution of drug resistance. However, the distinction of co-infecting parasite genotypes and the tracking of their dynamics have been difficult with traditional methods based on various genotyping techniques, leaving most questions unaddressed. Here we report new fluorescence markers of various colours that are inserted into the genome of Trypanosoma brucei to phenotypically label live parasites of all life cycle stages. If different parasite strains are labelled with different colours they can be easily distinguished from each other in experimental studies. A total of 10 T. brucei strains were successfully transfected with different fluorescence markers and were monitored in culture, tsetse flies and mice, to demonstrate stability of marker expression. The use of fluorescence activated cell sorting (FACS) allowed rapid and accurate identification of parasite strains labelled with different markers. Cell counts by FACS were virtually identical to counts by traditional microscopy (n=75, Spearman's rho: 0.91, p<0.0001) but were considerably faster and had a significantly lower sampling error (66% lower, d.f.=73, t=-17.1, p<0.0001). Co-infecting strains transfected with fluorescence genes of different colour were easily distinguished by eye and their relative and absolute densities were reliably counted by FACS in experimental multiple infections in mice. Since the FACS can simultaneously determine the population sizes of differently labelled T. brucei strains or subspecies it allows detailed and efficient tracking of multiple-genotype infections within a single host or vector individual, enabling more powerful studies on parasite dynamics. In addition, it also provides a simple way to separate genotypes after experimental mixed infections, to measure responses of the single strains to an applied treatment, thus eliminating the need for laborious cloning steps. The markers presented broaden the spectrum of tools available for experimental studies on multiple-genotype infections. They are fundamentally different from isoenzyme analysis and other genotyping approaches in that they allow the distinction of parasite genotypes based on an easily recognizable phenotypic trait. They will be of specific interest to researches addressing ecological, evolutionary and epidemiological questions using trypanosomes as an experimental system.

De Novo Genome Assembly Shows Genome Wide Similarity between Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense.

BACKGROUND: Trypanosoma brucei is a eukaryotic pathogen which causes African trypanosomiasis. It is notable for its variant surface glycoprotein (VSG) coat, which undergoes antigenic variation enabled by a large suite of VSG pseudogenes, allowing for persistent evasion of host adaptive immunity. While Trypanosoma brucei rhodesiense (Tbr) and T. b gambiense (Tbg) are human infective, related T. b. brucei (Tbb) is cleared by human sera. A single gene, the Serum Resistance Associated (SRA) gene, confers Tbr its human infectivity phenotype. Potential genetic recombination of this gene between Tbr and non-human infective Tbb strains has significant epidemiological consequences for Human African Trypanosomiasis outbreaks. RESULTS: Using long and short read whole genome sequencing, we generated a hybrid de novo assembly of a Tbr strain, producing 4,210 scaffolds totaling approximately 38.8 megabases, which comprise a significant proportion of the Tbr genome, and thus represents a valuable tool for a comparative genomics analyses among human and non-human infective T. brucei and future complete genome assembly. We detected 5,970 putative genes, of which two, an alcohol oxidoreductase and a pentatricopeptide repeat-containing protein, were members of gene families common to all T. brucei subspecies, but variants specific to the Tbr strain sequenced in this study. Our findings confirmed the extremely high level of genomic similarity between the two parasite subspecies found in other studies. CONCLUSIONS: We confirm at the whole genome level high similarity between the two Tbb and Tbr strains studied. The discovery of extremely minor genomic differentiation between Tbb and Tbr suggests that the transference of the SRA gene via genetic recombination could potentially result in novel human infective strains, thus all genetic backgrounds of T. brucei should be considered potentially human infective in regions where Tbr is prevalent.

Apolipoprotein L1 Variant Associated with Increased Susceptibility to Trypanosome Infection.

UNLABELLED: African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE: Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis.

Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

Comparative genomics reveals multiple genetic backgrounds of human pathogenicity in the Trypanosoma brucei complex.

The Trypanosoma brucei complex contains a number of subspecies with exceptionally variable life histories, including zoonotic subspecies, which are causative agents of human African trypanosomiasis (HAT) in sub-Saharan Africa. Paradoxically, genomic variation between taxa is extremely low. We analyzed the whole-genome sequences of 39 isolates across the T. brucei complex from diverse hosts and regions, identifying 608,501 single nucleotide polymorphisms that represent 2.33% of the nuclear genome. We show that human pathogenicity occurs across a wide range of parasite genotypes, and taxonomic designation does not reflect genetic variation across the group, as previous studies have suggested based on a small number of genes. This genome-wide study allowed the identification of significant host and geographic location associations. Strong purifying selection was detected in genomic regions associated with cytoskeleton structure, and regulatory genes associated with antigenic variation, suggesting conservation of these regions in African trypanosomes. In agreement with expectations drawn from meiotic reciprocal recombination, differences in average linkage disequilibrium between chromosomes in T. brucei correlate positively with chromosome size. In addition to insights into the life history of a diverse group of eukaryotic parasites, the documentation of genomic variation across the T. brucei complex and its association with specific hosts and geographic localities will aid in the development of comprehensive monitoring tools crucial to the proposed elimination of HAT by 2020, and on a shorter term, for monitoring the feared merger between the two human infective parasites, T. brucei rhodesiense and T. b. gambiense, in northern Uganda.

Noncrop flowering plants restore top-down herbivore control in agricultural fields.

Herbivore populations are regulated by bottom-up control through food availability and quality and by top-down control through natural enemies. Intensive agricultural monocultures provide abundant food to specialized herbivores and at the same time negatively impact natural enemies because monocultures are depauperate in carbohydrate food sources required by many natural enemies. As a consequence, herbivores are released from both types of control. Diversifying intensive cropping systems with flowering plants that provide nutritional resources to natural enemies may enhance top-down control and contribute to natural herbivore regulation. We analyzed how noncrop flowering plants planted as "companion plants" inside cabbage (Brassica oleracea) fields and as margins along the fields affect the plant-herbivore-parasitoid-predator food web. We combined molecular analyses quantifying parasitism of herbivore eggs and larvae with molecular predator gut content analysis and a comprehensive predator community assessment. Planting cornflowers (Centaurea cynanus), which have been shown to attract and selectively benefit Microplitis mediator, a larval parasitoid of the cabbage moth Mamestra brassicae, between the cabbage heads shifted the balance between trophic levels. Companion plants significantly increased parasitism of herbivores by larval parasitoids and predation on herbivore eggs. They furthermore significantly affected predator species richness. These effects were present despite the different treatments being close relative to the parasitoids' mobility. These findings demonstrate that habitat manipulation can restore top-down herbivore control in intensive crops if the right resources are added. This is important because increased natural control reduces the need for pesticide input in intensive agricultural settings, with cascading positive effects on general biodiversity and the environment. Companion plants thus increase biodiversity both directly, by introducing new habitats and resources for other species, and indirectly by reducing mortality of nontarget species due to pesticides. This study provides a comprehensive assessment of how habitat manipulation affects biocontrol services of a natural enemy community including both parasitoids and generalist predators. The trophic interactions between pests, parasitoids and predators were determined to achieve a better systemic understanding of top-down herbivore control, which can be strengthened when natural enemies complement each other or dampened by intraguild interactions. Our approach to selectively enhance the third trophic level to counteract specific herbivores was successful for both predators and parasitoids. Our results show significant positive effects of companion plants on predation of pest eggs and parasitism of pest larvae. Importantly, our data also suggest that carabids, staphylinids and spiders do not substantially interfere with parasitoid biocontrol as parasitoid DNA was rarely detected in predator guts.