Recent advances in genome editing of bloodstream forms of Trypanosoma congolense using CRISPR-Cas9 ribonucleoproteins: Proof of concept.

African Animal Trypanosomosis (AAT or Nagana) is a vector-borne disease caused by Trypanosomatidae, genus Trypanosoma. The disease is transmitted by the bite of infected hematophagous insects, mainly tsetse flies but also other blood-sucking insects including stomoxes and tabanids. Although many trypanosome species infect animals, the main agents responsible for this disease with a strong socio-economic and veterinary health impact are Trypanosoma congolense (T. congolense or Tc), Trypanosoma vivax (T.vivax), and to a lesser extent, Trypanosoma brucei brucei (T.brucei brucei or Tbb). These parasites mainly infect livestock, including cattle, in sub-Saharan Africa, with major repercussions in terms of animal productivity and poverty for populations which are often already very poor. As there is currently no vaccine, the fight against the disease is primarily based on diagnosis, treatment and vector control. To develop new tools (particularly therapeutic tools) to fight against the disease, we need to know both the biology and the genes involved in the pathogenicity and virulence of the parasites. To date, unlike for Trypanosoma brucei (T.brucei) or Trypanosoma cruzi (T.cruzi), genome editing tools has been relatively little used to study T. congolense. We present an efficient, reproducible and stable CRISPR-Cas9 genome editing system for use in Tc bloodstream forms (Tc-BSF). This plasmid-free system is based on transient expression of Cas9 protein and the use of a ribonucleoprotein formed by the Cas9 and sgRNA complex. This is the first proof of concept of genome editing using CRISPR-Cas9 ribonucleoproteins on Tc-BSF. This adapted protocol enriches the "toolbox" for the functional study of genes of interest in blood forms of the Trypanosoma congolense. This proof of concept is an important step for the scientific community working on the study of trypanosomes and opens up new perspectives for the control of and fight against animal trypanosomosis.

Experimental evidence that immune trypanolysis using the LiTat 1.3 and LiTat 1.5 variant antigen types is not specific to Trypanosoma brucei gambiense in pigs.

In the context of the human African trypanosomiasis elimination process, reliable and accurate diagnostic tools are crucial for exploring the role of a potential animal reservoir of Trypanosoma brucei gambiense. The immune trypanolysis test (TL) using the variant antigen types (VAT) LiTat 1.3 and LiTat 1.5, described as a specific serological method to detect people infected by T. b. gambiense, seems to be a promising tool. However, its specificity was recently questioned during field animal surveys. The present study evaluates the performance of TL during experimental T. b. brucei infection in pigs. Eight infected pigs and four uninfected pigs were followed up with blood and plasma collection. Blood was used for parasitological investigation. TL was performed on the plasma with the LiTat 1.3, LiTat 1.5 and LiTat 1.6 VATs. All control pigs remained negative to parasitological investigation and TL. Trypanosomes were detected in all the infected pigs and the first detection was between 10 and 14 days post infection (dpi). TL results showed that infected pigs developed antibodies against the three VATs. The first antibody detections by TL occurred between 14 and 21 dpi for antibodies directed against LiTat 1.6, 21 and 168 dpi for antibodies directed against LiTat 1.5 and 70, and 182 dpi for antibodies directed against LiTat 1.3. This study highlights for the first time that TL using LiTat 1.3 and LiTat 1.5 VATs is not specific to T. b. gambiense. Development of specific diagnostic tools for the detection of T. b. gambiense infections in animals, especially in pigs, is still needed.

Title: Évidence expérimentale que la trypanolyse basée sur les types d'antigène variable LiTat 1.3 et LiTat 1.5 n'est pas spécifique de Trypanosoma brucei gambiense. Abstract: Dans le contexte d'élimination de la trypanosomiase humaine Africaine, des outils de diagnostic fiables et précis sont essentiels afin d'explorer le rôle d'un potentiel réservoir animal de Trypanosoma brucei gambiense. La trypanolyse (TL) qui utilise les types d'antigène variable (TAV) LiTat 1.3 et LiTat 1.5, et qui est décrite comme une méthode sérologique spécifique pour détecter les personnes infectées par T. b. gambiense, semble être un outil prometteur. Cependant, sa spécificité a été récemment remise en question lors d'enquêtes sur les animaux. La présente étude évalue la performance de ce test lors d'une infection expérimentale à T. b. brucei chez le porc. Huit porcs infectés et quatre porcs témoins non infectés ont été suivis avec des prélèvements de sang et de plasma. Le sang a été utilisé pour l'examen parasitologique. La TL a été réalisée sur les échantillons de plasma avec les TAV LiTat 1.3, LiTat 1.5 et LiTat 1.6. Tous les porcs témoins ont été négatifs en parasitologie et à la TL. Les trypanosomes ont été détectés sur tous les porcs infectés avec les premières détections entre 10 et 14 jours post-infection (jpi). Les résultats de la TL ont montré que les porcs infectés ont développé des anticorps contre les trois TAV. Les premiers anticorps détectés par la TL étaient dirigés contre le LiTat 1.6 entre 14 et 21 jpi, puis le LiTat 1.5 entre 21 et 168 jpi et enfin le LiTat 1.3 entre 70 et 182 jpi. Cette étude démontre pour la première fois que la TL basée sur les TAV LiTat 1.3 et LiTat 1.5 n'est pas spécifique de T. b. gambiense. Il est donc toujours nécessaire et urgent de développer un outil de diagnostic spécifique pour la détection des infections à T. b. gambiense chez les animaux, notamment chez les porcs.

Diagnosis of animal trypanosomoses: proper use of current tools and future prospects.

Reliable diagnostic tools are needed to choose the appropriate treatment and proper control measures for animal trypanosomoses, some of which are pathogenic. Trypanosoma cruzi, for example, is responsible for Chagas disease in Latin America. Similarly, pathogenic animal trypanosomoses of African origin (ATAO), including a variety of Trypanosoma species and subspecies, are currently found in Africa, Latin America and Asia. ATAO limit global livestock productivity and impact food security and the welfare of domestic animals. This review focusses on implementing previously reviewed diagnostic methods, in a complex epizootiological scenario, by critically assessing diagnostic results at the individual or herd level. In most cases, a single diagnostic method applied at a given time does not unequivocally identify the various parasitological and disease statuses of a host. These include "non-infected", "asymptomatic carrier", "sick infected", "cured/not cured" and/or "multi-infected". The diversity of hosts affected by these animal trypanosomoses and their vectors (or other routes of transmission) is such that integrative, diachronic approaches are needed that combine: (i) parasite detection, (ii) DNA, RNA or antigen detection and (iii) antibody detection, along with epizootiological information. The specificity of antibody detection tests is restricted to the genus or subgenus due to cross-reactivity with other Trypanosoma spp. and Trypanosomatidae, but sensitivity is high. The DNA-based methods implemented over the last three decades have yielded higher specificity and sensitivity for active infection detection in hosts and vectors. However, no single diagnostic method can detect all active infections and/or trypanosome species or subspecies. The proposed integrative approach will improve the prevention, surveillance and monitoring of animal trypanosomoses with the available diagnostic tools. However, further developments are required to address specific gaps in diagnostic methods and the sustainable control or elimination of these diseases.

A review on the diagnosis of animal trypanosomoses.

This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing "Nagana" or animal African trypanosomosis [AAT]), Trypanosoma evansi ("Surra") and Trypanosoma equiperdum ("Dourine"), and Trypanosoma cruzi, a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called "atypical human infections by animal trypanosomes" [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on "One Health," by advancing and preserving animal, human and environmental health.

The COMBAT project: controlling and progressively minimizing the burden of vector-borne animal trypanosomosis in Africa.

Vector-borne diseases affecting livestock have serious impacts in Africa. Trypanosomosis is caused by parasites transmitted by tsetse flies and other blood-sucking Diptera. The animal form of the disease is a scourge for African livestock keepers, is already present in Latin America and Asia, and has the potential to spread further. A human form of the disease also exists, known as human African trypanosomosis or sleeping sickness. Controlling and progressively minimizing the burden of animal trypanosomosis (COMBAT) is a four-year research and innovation project funded by the European Commission, whose ultimate goal is to reduce the burden of animal trypanosomosis (AT) in Africa. The project builds on the progressive control pathway (PCP), a risk-based, step-wise approach to disease reduction or elimination. COMBAT will strengthen AT control and prevention by improving basic knowledge of AT, developing innovative control tools, reinforcing surveillance, rationalizing control strategies, building capacity, and raising awareness. Knowledge gaps on disease epidemiology, vector ecology and competence, and biological aspects of trypanotolerant livestock will be addressed. Environmentally friendly vector control technologies and more effective and adapted diagnostic tools will be developed. Surveillance will be enhanced by developing information systems, strengthening reporting, and mapping and modelling disease risk in Africa and beyond. The socio-economic burden of AT will be assessed at a range of geographical scales. Guidelines for the PCP and harmonized national control strategies and roadmaps will be developed. Gender equality and ethics will be pivotal in all project activities. The COMBAT project benefits from the expertise of African and European research institutions, national veterinary authorities, and international organizations. The project consortium comprises 21 participants, including a geographically balanced representation from 13 African countries, and it will engage a larger number of AT-affected countries through regional initiatives.

Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei.

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.

Modulation of trypanosome establishment in Glossina palpalis palpalis by its microbiome in the Campo sleeping sickness focus, Cameroon.

The purpose of this study was to investigate factors involved in vector competence by analyzing whether the diversity and relative abundance of the different bacterial genera inhabiting the fly's gut could be associated with its trypanosome infection status. This was investigated on 160 randomly selected G. p. palpalis flies - 80 trypanosome-infected, 80 uninfected - collected in 5 villages of the Campo trypanosomiasis focus in South Cameroon. Trypanosome species were identified using specific primers, and the V4 region of the 16S rRNA gene of bacteria was targeted for metabarcoding analysis in order to identify the bacteria and determine microbiome composition. A total of 261 bacterial genera were identified of which only 114 crossed two barriers: a threshold of 0.01% relative abundance and the presence at least in 5 flies. The secondary symbiont Sodalis glossinidius was identified in 50% of the flies but it was not considered since its relative abundance was much lower than the 0.01% relative abundance threshold. The primary symbiont Wigglesworthia displayed 87% relative abundance, the remaining 13% were prominently constituted by the genera Spiroplasma, Tediphilus, Acinetobacter and Pseudomonas. Despite a large diversity in bacterial genera and in their abundance observed in micobiome composition, the statistical analyzes of the 160 tsetse flies showed an association with flies' infection status and the sampling sites. Furthermore, tsetse flies harboring Trypanosoma congolense Savanah type displayed a different composition of bacterial flora compared to uninfected flies. In addition, our study revealed that 36 bacterial genera were present only in uninfected flies, which could therefore suggest a possible involvement in flies' refractoriness; with the exception of Cupriavidus, they were however of low relative abundance. Some genera, including Acinetobacter, Cutibacterium, Pseudomonas and Tepidiphilus, although present both in infected and uninfected flies, were found to be associated with uninfected status of tsetse flies. Hence their effective role deserves to be further evaluated in order to determine whether some of them could become targets for tsetse control of fly vector competence and consequently for the control of the disease. Finally, when comparing the bacterial genera identified in tsetse flies collected during 4 epidemiological surveys, 39 genera were found to be common to flies from at least 2 sampling campaigns.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Molecular detection of 7SL-derived small RNA is a promising alternative for trypanosomosis diagnosis.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.

Mini-review on CRISPR-Cas9 and its potential applications to help controlling neglected tropical diseases caused by Trypanosomatidae.

The CRISPR-Cas system, which was originally identified as a prokaryotic defense mechanism, is increasingly being used for the functional study of genes. This technology, which is simple, inexpensive and efficient, has aroused a lot of enthusiasm in the scientific community since its discovery, and every month many publications emanate from very different communities reporting on the use of CRISPR-Cas9. Currently, there are no vaccines to control neglected tropical diseases (NTDs) caused by Trypanosomatidae, particularly Human African Trypanosomiasis (HAT) and Animal African Trypanosomoses (AAT), and treatments are cumbersome and sometimes not effective enough. CRISPR-Cas9 has the potential to functionally analyze new target molecules that could be used for therapeutic and vaccine purposes. In this review, after briefly describing CRIPSR-Cas9 history and how it works, different applications on diseases, especially on parasitic diseases, are reviewed. We then focus the review on the use of CRISPR-Cas9 editing on Trypanosomatidae parasites, the causative agents of NTDs, which are still a terrible burden for human populations in tropical regions, and their vectors.

Cloning, expression, molecular characterization and preliminary studies on immunomodulating properties of recombinant Trypanosoma congolense calreticulin.

Trypanosomes are bloodstream protozoan parasites, which are pathogens of veterinary and medical importance. Several mammalian species, including humans, can be infected by different species of the genus Trypanosoma (T. congolense, T. evansi, T. brucei, T. vivax) exhibiting more or less virulent and pathogenic phenotypes. A previous screening of the excreted-secreted proteins of T. congolense demonstrated an overexpression of several proteins correlated with the virulence and pathogenicity of the strain. Of these proteins, calreticulin (CRT) has shown differential expression between two T. congolense strains with opposite infectious behavior and has been selected as a target molecule based on its immune potential functions in parasitic diseases. In this study, we set out to determine the role of T. congolense calreticulin as an immune target. Immunization of mice with recombinant T. congolense calreticulin induced antibody production, which was associated with delayed parasitemia and increased survival of the challenged animal. These results strongly suggest that some excreted-secreted proteins of T. congolense are a worthwhile target candidate to interfere with the infectious process.

Tolerance to Trypanosomatids: A Threat, or a Key for Disease Elimination?

So far, research on trypanosomatid infections has been driven by 'disease by disease' approaches, leading to different concepts and control strategies. It is, however, increasingly clear that they share common features such as the ability to generate long-lasting asymptomatic infections in their mammalian hosts. Trypanotolerance, long integrated in animal African trypanosomiasis control, historically refers to the ability of cattle breeds to limit Trypanosoma infection and pathology, but has only recently been recognized in humans. Whilst trypanotolerance is absent from the vocabulary on leishmaniasis and Chagas disease, asymptomatic infections also occur. We review the concept of trypanotolerance across the trypanosomatids and discuss the importance of asymptomatic carriage in the current context of elimination.

A comparison of phenotypic traits related to trypanotolerance in five west african cattle breeds highlights the value of shorthorn taurine breeds.

BACKGROUND: Animal African Trypanosomosis particularly affects cattle and dramatically impairs livestock development in sub-Saharan Africa. African Zebu (AFZ) or European taurine breeds usually die of the disease in the absence of treatment, whereas West African taurine breeds (AFT), considered trypanotolerant, are able to control the pathogenic effects of trypanosomosis. Up to now, only one AFT breed, the longhorn N'Dama (NDA), has been largely studied and is considered as the reference trypanotolerant breed. Shorthorn taurine trypanotolerance has never been properly assessed and compared to NDA and AFZ breeds. METHODOLOGY/PRINCIPAL FINDINGS: This study compared the trypanotolerant/susceptible phenotype of five West African local breeds that differ in their demographic history. Thirty-six individuals belonging to the longhorn taurine NDA breed, two shorthorn taurine Lagune (LAG) and Baoulé (BAO) breeds, the Zebu Fulani (ZFU) and the Borgou (BOR), an admixed breed between AFT and AFZ, were infected by Trypanosoma congolense IL1180. All the cattle were genetically characterized using dense SNP markers, and parameters linked to parasitaemia, anaemia and leukocytes were analysed using synthetic variables and mixed models. We showed that LAG, followed by NDA and BAO, displayed the best control of anaemia. ZFU showed the greatest anaemia and the BOR breed had an intermediate value, as expected from its admixed origin. Large differences in leukocyte counts were also observed, with higher leukocytosis for AFT. Nevertheless, no differences in parasitaemia were found, except a tendency to take longer to display detectable parasites in ZFU. CONCLUSIONS: We demonstrated that LAG and BAO are as trypanotolerant as NDA. This study highlights the value of shorthorn taurine breeds, which display strong local adaptation to trypanosomosis. Thanks to further analyses based on comparisons of the genome or transcriptome of the breeds, these results open up the way for better knowledge of host-pathogen interactions and, furthermore, for identifying key biological pathways.

Adaptive admixture in the West African bovine hybrid zone: insight from the Borgou population.

Understanding the adaptive response to environmental fluctuations represents a central issue in evolutionary biology. Population admixture between divergent ancestries has often been considered as an efficient short-term adaptation strategy. Cattle populations from the West African Bos taurus × Bos indicus hybrid zone represent a valuable resource to characterize the effect of such adaptive admixture at the genome level. We here provide a detailed assessment of the global and local genome ancestries of the Borgou breed, one of the most representative cattle of this hybrid zone. We analysed a large data set consisting of 38,100 SNPs genotyped on 203 Borgou and 591 individuals representative of all the different cattle ancestries. At the global genomic level, we show that Borgou is a stabilized admixed breed whose origin (c. 130 years ago) traces back to the great African rinderpest pandemic, several centuries after the last admixture events, the West African zebus originate from (c. 500 years ago). To identify footprints of adaptive admixture, we combined the identification of signatures of selection and the functional annotation of the underlying genes using systems biology tools. The detection of the SILV coat coloration gene likely under artificial selection may be viewed as a validation of our approach. Overall, our results suggest that the long-term presence of pathogens and the intermediate environmental conditions are the main acting selective pressures. Our analytical framework can be extended to other model or nonmodel species to understand the process that shapes the patterns of genetic variability in hybrid zones.

Population dynamics of Glossina palpalis gambiensis symbionts, Sodalis glossinidius, and Wigglesworthia glossinidia, throughout host-fly development.

The tsetse fly (Diptera: Glossinidae), the vector of trypanosomes causing human and animal trypanosomiasis, harbors symbiotic microorganisms including the primary symbiont Wigglesworthia glossinidia, involved in the fly's nutrition and fertility, and the secondary symbiont Sodalis glossinidius, involved in the trypanosome establishment in the fly's midgut. Both symbionts are maternally transmitted to the intrauterine progeny through the fly's milk gland secretions. In this study, we investigated the population dynamics of these symbionts during fly development. Wigglesworthia and Sodalis densities were estimated using quantitative PCR performed on Glossina palpalis gambiensis at different developmental stages. The results showed that the density of the primary Wigglesworthia symbiont was higher than that of Sodalis for all host developmental stages. Sodalis densities remained constant in pupae, but increased significantly in adult flies. The opposite situation was observed for Wigglesworthia, whose density increased in pupae and remained constant during the female adult stage. Moreover, Wigglesworthia density increased significantly during the transition from the pupal to the teneral stage, while mating had a contradictory effect depending on the age of the fly. Finally, tsetse fly colonization by both symbionts appears as a continuous and adaptive process throughout the insect's development. Last, the study demonstrated both symbionts of G. p. gambiensis, the vector of the chronic form of human African trypanosomiasis, to be permanent inhabitants of the colony flies throughout their life span. This was expected for the primary symbiont, Wigglesworthia, but not necessarily for the secondary symbiont, S. glossinidius, whose permanent presence is not required for the fly's survival. This result is of importance as Sodalis could be involved in the tsetse fly vector competence and may constitute a target in the frame of sleeping sickness fighting strategies.

A quasi-exclusive European ancestry in the Senepol tropical cattle breed highlights the importance of the slick locus in tropical adaptation.

BACKGROUND: The Senepol cattle breed (SEN) was created in the early XX(th) century from a presumed cross between a European (EUT) breed (Red Poll) and a West African taurine (AFT) breed (N'Dama). Well adapted to tropical conditions, it is also believed trypanotolerant according to its putative AFT ancestry. However, such origins needed to be verified to define relevant husbandry practices and the genetic background underlying such adaptation needed to be characterized. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 153 SEN individuals on 47,365 SNPs and combined the resulting data with those available on 18 other populations representative of EUT, AFT and Zebu (ZEB) cattle. We found on average 89% EUT, 10.4% ZEB and 0.6% AFT ancestries in the SEN genome. We further looked for footprints of recent selection using standard tests based on the extent of haplotype homozygosity. We underlined i) three footprints on chromosome (BTA) 01, two of which are within or close to the polled locus underlying the absence of horns and ii) one footprint on BTA20 within the slick hair coat locus, involved in thermotolerance. Annotation of these regions allowed us to propose three candidate genes to explain the observed signals (TIAM1, GRIK1 and RAI14). CONCLUSIONS/SIGNIFICANCE: Our results do not support the accepted concept about the AFT origin of SEN breed. Initial AFT ancestry (if any) might have been counter-selected in early generations due to breeding objectives oriented in particular toward meat production and hornless phenotype. Therefore, SEN animals are likely susceptible to African trypanosomes which questions the importation of SEN within the West African tsetse belt, as promoted by some breeding societies. Besides, our results revealed that SEN breed is predominantly a EUT breed well adapted to tropical conditions and confirmed the importance in thermotolerance of the slick locus.

APOL1 expression is induced by Trypanosoma brucei gambiense infection but is not associated with differential susceptibility to sleeping sickness.

Most African trypanosome species are sensitive to trypanolytic factors (TLFs) present in human serum. Trypanosome lysis was demonstrated to be associated with apolipoprotein L-I (APOL1). Trypanosoma brucei (T. b.) gambiense and Trypanosoma brucei rhodesiense, the two human infective trypanosome species, have both developed distinct resistance mechanisms to APOL1 mediated lysis. Whereas T. b. rhodesiense resistance is linked with the expression of the serum resistance associated (SRA) protein that interacts with APOL1 inside the parasite lysosome, inhibiting its lytic action; T. b. gambiense resistance is rather controlled by a reduced expression of the parasite HpHb receptor, limiting APOL1 absorption by trypanosomes. Based on this last observation we hypothesised that variation in the host APOL1 environment could significantly alter T. b. gambiense growth and thus resistance/susceptibility to sleeping sickness. To test this hypothesis, we have measured blood APOL1 relative expression in HAT patients, uninfected endemic controls and serologically positive subjects (SERO TL(+)) that are suspected to control infection to parasitological levels that are undetectable by the available test used in the field. All RNA samples were obtained from medical surveys led in the HAT mangrove foci of Coastal Guinea. Results indicate that APOL1 expression is a complex trait dependant on a variety of factors that need to be taken into account in the analysis. Nevertheless, multivariate analysis showed that APOL1 expression levels were significantly higher in both HAT and SERO TL(+) subject as compared to endemic controls (p=0.006). This result suggests that APOL1 expression is likely induced by T. b. gambiense, but is not related to resistance/susceptibility in its human host.

Intertwining threshold settings, biological data and database knowledge to optimize the selection of differentially expressed genes from microarray.

BACKGROUND: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed. METHODOLOGY/PRINCIPAL FINDINGS: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes. CONCLUSIONS/SIGNIFICANCE: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.

Development and application of an antibody-ELISA to follow up a Trypanosoma evansi outbreak in a dromedary camel herd in France.

An outbreak of trypanosomosis was observed for the first time in metropolitan France in October 2006, when five camels were proved to be infected by Trypanosoma evansi using parasitological methods. The parasite was isolated and used to produce a soluble antigen for antibody-enzyme linked immunosorbent assay (ELISA) in a protocol derived from a method previously developed for sheep and humans but using protein A conjugate. The animals were treated on three instances, alternatively with melarsomine hydrochloride and quinapyramine and followed up on a monthly basis for 2 years with various diagnostic techniques including parasitological, serological and DNA-based methods. Initially, five animals were detected as being positive using ELISA with 83.3% concordance to parasitological tests. Immediately after the first treatment, parasites and DNA disappeared in all animals; antibody levels decreased regularly until ELISA became negative 3-4 months later. Ten months after the first treatment, parasites and antibodies were detected again in one of the camels previously found to be infected. A retrospective study indicated that the weight of this animal had been underestimated; consequently, it had received underdosages of both trypanocides. However, since hypotheses of re-infection or relapse could not be fully substantiated, it is not known whether the ELISA results for this animal were true- or false-negative over a 7-month period. The study confirmed the value of this ELISA using protein A conjugate to detect antibodies directed against T. evansi in camels and the need to use several diagnostic techniques to optimize detection of infected animals. A warning is raised on surra, a potentially emerging disease in Europe.