Trypanosoma brucei RRP44 is involved in an early stage of large ribosomal subunit RNA maturation.

Ribosomal RNA precursors undergo a series of structural and chemical modifications to generate matured RNA molecules that will comprise ribosomes. This maturation process involves a large set of accessory proteins as well as ribonucleases, responsible for removal of the external and internal transcribed spacers from the pre-rRNA. Early-diverging eukaryotes belonging to the Kinetoplastida class display several unique characteristics, in particular in terms of RNA synthesis and maturation. These peculiarities include the rRNA biogenesis and the extensive fragmentation of the large ribosomal subunit (LSU) rRNA. The role of specific endo- and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways. Here, we investigated the function of Trypanosoma brucei RRP44 orthologue (TbRRP44) in rRNA processing. Our results revealed that TbRRP44 depletion causes unusual polysome profile and accumulation of the complete LSU rRNA precursor, in addition to 5.8S maturation impairment. We also determined the crystal structure of TbRRP44 endonucleolytic domain. Structural comparison with Saccharomyces cerevisiae Rrp44 revealed differences in the catalytic site and substitutions of surface residues, which could provide molecular bases for the lack of interaction of RRP44 with the exosome complex in T. brucei.

Lack of specific alleles for the bovine chemokine (C-X-C) receptor type 4 (CXCR4) gene in West African cattle questions its role as a candidate for trypanotolerance.

A panel of 81 Asian, African and European cattle (Bos taurus and B. indicus) was analysed for the whole sequence of the CXCR4 gene (3844bp), a strong candidate for cattle trypanotolerance. Thirty-one polymorphic sites identified gave 31 different haplotypes. Neutrality tests rejected the hypothesis of either positive or purifying selection. Bayesian phylogenetic tree showed differentiation of haplotypes into two clades gathering genetic variability predating domestication. Related with clades definition, linkage disequilibrium analyses suggested the existence of one only linkage block on the CXCR4 gene. Two tag SNPs identified on exon 2 captured 50% of variability. Whatever the analysis carried out, no clear separation between cattle groups was identified. Most haplotypes identified in West African taurine cattle were also found in European cattle and in Asian and West African zebu. West African taurine samples did not carry unique variants on the CXCR4 gene sequence. The current analysis failed in identifying a causal mutation on the CXCR4 gene underlying a previously reported QTL for cattle trypanotolerance on BTA2.

Depletion of the RNA-binding protein RBP33 results in increased expression of silenced RNA polymerase II transcripts in Trypanosoma brucei.

We have characterized the RNA-binding protein RBP33 in Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes.

Allosteric activation of trypanosomatid deoxyhypusine synthase by a catalytically dead paralog.

Polyamine biosynthesis is a key drug target in African trypanosomes. The "resurrection drug" eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.

Trypanotolerance in N'Dama x Boran crosses under natural trypanosome challenge: effect of test-year environment, gender, and breed composition.

BACKGROUND: Trypanosomosis, a protozoal disease affecting livestock, transmitted by Glossina (tsetse) flies is a major constraint to agricultural production in Sub-Saharan Africa. It is accepted that utilization of the native trypanotolerance exhibited in some of the African cattle breeds to improve trypanotolerance of more productive but susceptible breeds, will offer a cost effective and sustainable solution to the problem. The success of this approach is based on the premise that quantitative trait loci previously identified under relatively controlled situations confer useful trypanotolerance under natural field situations. As part of a study to authenticate this hypothesis, a population of 192 cattle, consisting of six batches of N'Dama and Kenya-Boran backcross animals [(N'Dama x Kenya-Boran) x Kenya-Boran] born over the period 2002 to 2006 was constructed. Some of the batches also included pure Kenya-Boran cattle, or N'Dama x Kenya- Boran F1 animals. Each batch was exposed as yearlings to natural field trypanosomosis challenge over a period of about one year; the entire challenge period extending from December 2003 to June 2007. Performance of the animals was evaluated by weekly or biweekly measurements of body weight, packed blood cell volume (PCV), parasitemia score, and number of trypanocide treatments. From these basic data, 49 phenotypes were constructed reflecting dynamics of body weight, packed cell volume (PCV) and parasitemia under challenge. RESULTS: Females were distinctly more trypanotolerant than males. F1, backcross and pure Kenya- Boran animals ranked in that order with respect to trypanotolerance. Overall batch effects were highly significant (p<0.001) for most traits, and were generally more significant than the gender or genetic type effects. The superior trypanotolerance of the F1 animals was expressed in all three components of animal defense strategies against pathogens: Avoidance resistance, and tolerance. CONCLUSIONS: The results show that trypanotolerance derived from the N'Dama is expressed under field conditions; and that the trait is primarily additive in nature, being expressed in heterozygous condition and in a three-quarters Boran genetic background. The results further, underscore the complexity of the trait in the field manifesting all three host disease-control strategies, and show the importance of gender and local environmental conditions in determining response to challenge.

Association studies in QTL regions linked to bovine trypanotolerance in a West African crossbred population.

African animal trypanosomosis is a parasitic blood disease transmitted by tsetse flies and is widespread in sub-Saharan Africa. West African taurine breeds have the ability, known as trypanotolerance, to limit parasitaemia and anaemia and remain productive in enzootic areas. Several quantitative trait loci (QTL) underlying traits related to trypanotolerance have been identified in an experimentally infected F(2) population resulting from a cross between taurine and zebu cattle. Although this information is highly valuable, the QTL remain to be confirmed in populations subjected to natural conditions of infection, and the corresponding regions need to be refined. In our study, 360 West African cattle were phenotyped for the packed cell volume control under natural conditions of infection in south-western Burkina Faso. Phenotypes were assessed by analysing data from previous cattle monitored over 2 years in an area enzootic for trypanosomosis. We further genotyped for 64 microsatellite markers mapping within four previously reported QTL on BTA02, BTA04, BTA07 and BTA13. These data enabled us to estimate the heritability of the phenotype using the kinship matrix between individuals computed from genotyping data. Thus, depending on the estimators considered and the method used, the heritability of anaemia control ranged from 0.09 to 0.22. Finally, an analysis of association identified an allele of the MNB42 marker on BTA04 as being strongly associated with anaemia control, and a candidate gene, INHBA, as being close to that marker.

Farmers' perceptions on trypanosomosis and trypanotolerance character of the taurine Sheko.

In the humid and subhumid tropics, trypanosomosis is an economically important zoonotic protozoan disease of the commonly kept farm animal species and their wild relatives. For example, more than 20% of the humid western and southwestern Ethiopia, which is home to more than 14 million heads of cattle, is under varying levels of trypanosomosis risk. Our study was, therefore, initiated to document farmers' perception on trypanosomosis and Sheko's trypanotolerance character. Our findings showed that trypanosomosis was the most frequently reported cattle disease in the Bench Maji Zone. Accordingly, 76.7% of the farmers reported the epidemiological importance of trypanosomosis, and they also noted that trypanosomosis on average accounted for 63.0% of annualized cattle death. The reported signs of trypanosomosis and trypanotolerance indicators were consistent with literature reports. Moreover, 66.7% of the farmers reported Sheko's trypanotolerance character. In the course of time, smallholder farmers have developed ethnoveterinary practices that are mainly used to prevent the landing of vector flies on the animal. Wet and warm seasons of the year, i.e., spring and, to some extent, the beginning of summer and autumn, were reported as peak periods of trypanosomosis risk. Therefore, this showed the need for incorporating farmers' knowledge in trypanosomosis control programs.

Serological characterizations of tandem repeat proteins for detection of African trypanosome infection in cattle.

Serological diagnosis is a useful method to detect African trypanosome infection in livestock animals. Currently available serological tests utilize whole parasites or crude antigens, and recombinant antigens may improve reproducibility/standardization and reduce production costs. With a goal of identifying such recombinant proteins, we computationally identified proteins with tandem repeat (TR) domain from the parasite proteomes and evaluated their potential for serological diagnosis of African trypanosome infections in cattle. Among those tested, Tbg4 demonstrated the best performance with 92% sensitivity, followed by TbbGM6 (85%), TcoGM6 (85%), Tbg2 (65%) and Tbg5 (65%). Although further evaluations such as investigating cross-reactivity to other infections are needed, our data indicate the potential of these antigens for detection of African trypanosome infection in cattle.

Genetic and expression analysis of cattle identifies candidate genes in pathways responding to Trypanosoma congolense infection.

African bovine trypanosomiasis caused by Trypanosoma sp., is a major constraint on cattle productivity in sub-Saharan Africa. Some African Bos taurus breeds are highly tolerant of infection, but the potentially more productive Bos indicus zebu breeds are much more susceptible. Zebu cattle are well adapted for plowing and haulage, and increasing their tolerance of trypanosomiasis could have a major impact on crop cultivation as well as dairy and beef production. We used three strategies to obtain short lists of candidate genes within QTL that were previously shown to regulate response to infection. We analyzed the transcriptomes of trypanotolerant N'Dama and susceptible Boran cattle after infection with Trypanosoma congolense. We sequenced EST libraries from these two breeds to identify polymorphisms that might underlie previously identified quantitative trait loci (QTL), and we assessed QTL regions and candidate loci for evidence of selective sweeps. The scan of the EST sequences identified a previously undescribed polymorphism in ARHGAP15 in the Bta2 trypanotolerance QTL. The polymorphism affects gene function in vitro and could contribute to the observed differences in expression of the MAPK pathway in vivo. The expression data showed that TLR and MAPK pathways responded to infection, and the former contained TICAM1, which is within a QTL on Bta7. Genetic analyses showed that selective sweeps had occurred at TICAM1 and ARHGAP15 loci in African taurine cattle, making them strong candidates for the genes underlying the QTL. Candidate QTL genes were identified in other QTL by their expression profile and the pathways in which they participate.

Trypanosomosis and phenotypic features of four indigenous cattle breeds in an Ethiopian field study.

We conducted a two-part study in the native home areas of four cattle breeds, Abigar, Gurage, Horro and Sheko, in south-western Ethiopia. The first part of the study investigated livestock keeper knowledge about trypanosomosis and trypanotolerance. For each breed 60 livestock keepers were interviewed, resulting in a total of 240 interviews. The second part of the study focused on biological evidence for trypanotolerance. Blood samples of about 100 head of cattle per breed were collected during peak trypanosomosis challenge period and analyzed for packed cell volume (PCV) and parasitaemia. In addition individual body measurements of the sampled animals were taken and the keepers provided some information regarding their animals. Livestock keeper interviews revealed that trypanosomosis was considered a major problem in all areas (95-100%). Almost all Abigar livestock keepers knew how trypanosomosis is transmitted, whereas only 34-52% of the keepers of the other breeds had that knowledge. Most Sheko keepers (75%) knew of trypanotolerance and claimed to have trypanotolerant animals in their own herds. Among the other three breeds the knowledge of trypanotolerance was much less (8-18%). A majority of the keepers were interested in purchasing trypanotolerant animals. PCV was highest among Horro (26.2) and Sheko (25.1) cattle whereas Abigar had the lowest PCV (20.0). Sheko were least infected by trypanosomes (6%) and had the lowest number of trypanocidal treatments per year (1 treatment/animal and year). Abigar cattle were most infected (23%) followed by Gurage (20%) and Horro (17%). Gurage had by far the highest number of treatments per animal and year (24). There were large differences between the number of cattle perceived by the keepers to be infected, and the number detected from blood sampled, among Abigar, Gurage and Horro. Sheko livestock keepers were better at correctly diagnosing trypanosomosis in their animals. It is concluded that Sheko cattle have higher trypanotolerance attributes of the breeds investigated and a better use of this breed could improve cattle health and household welfare in tsetse-infested areas.

Cytokine mRNA profiling of peripheral blood mononuclear cells from trypanotolerant and trypanosusceptible cattle infected with Trypanosoma congolense.

To examine differences in cytokine profiles that may confer tolerance/susceptibility to bovine African trypanosomiasis, N'Dama (trypanotolerant, n = 8) and Boran (trypanosusceptible, n = 8) cattle were experimentally challenged with Trypanosoma congolense. Blood samples were collected over a 34-day period, and RNA was extracted from peripheral blood mononuclear cells. The expression levels of a panel of 14 cytokines were profiled over the time course of infection and between breeds. Messenger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN genes were significantly downregulated across the time course of infection in both breeds. There was an early increase in transcripts for genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and IL12) in N'Dama by 14 days postinfection (dpi) compared with preinfection levels that was not detected in the susceptible Boran breed. By the time of peak parasitemia, a type 2 helper T cells (T(H)2)-like cytokine environment was prevalent that was particularly evident in the Boran. Increases in transcripts for the IL6 (29 and 34 dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were higher in the Boran compared with N'Dama. These findings highlight the implications for using murine models to study the bovine immune response to trypanosomiasis, where in some cases cytokine expression patterns differ. Overall, these data suggest that the trypanotolerant N'Dama are more capable of responding very early in infection with proinflammatory and T(H)1 type cytokines than the trypanosusceptible Boran and may explain why N'Dama control parasitemia more efficiently than Boran during the early stages of infection.

Understanding bovine trypanosomiasis and trypanotolerance: the promise of functional genomics.

African bovine trypanosomiasis, caused by the protozoan parasite Trypanosoma congolense, is endemic throughout sub-Saharan Africa and is a major constraint on livestock production. A promising approach to disease control is to understand and exploit naturally evolved trypanotolerance. We describe the first attempt to investigate the transcriptional response of susceptible Boran (Bos indicus) cattle to trypanosome infection via a functional genomics approach using a bovine total leukocyte (BOTL) cDNA microarray platform. Four male Boran cattle were experimentally infected with T. congolense and peripheral blood mononuclear cells (PBMC) were collected before infection and 13, 17, 23 and 30 days post-infection (dpi). A reference experimental design was employed using a universal bovine reference RNA pool. Data were normalised to the median of a set of invariant genes (GAPDH) and BRB-Array tools was used to search for statistically significant differentially expressed genes between each time-point. Using a set of 20 microarray hybridisations, we have made a significant contribution to understand the temporal transcriptional response of bovine PBMC in vivo to a controlled trypanosome infection. The greatest changes were evident 13 dpi after parasites were first detected in the blood. Significant differences were observed in clusters of protein kinase C subunits and MHC class I/II related molecules.

Serial analysis of gene expression (SAGE) in bovine trypanotolerance: preliminary results.

In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine Bos taurus) breeds, such as the Longhorn (N'Dama) cattle are well known to control trypanosome infections. This genetic ability named "trypanotolerance" results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an N'Dama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes.

Responses of bovine chimaeras combining trypanosomosis resistant and susceptible genotypes to experimental infection with Trypanosoma congolense.

West African N'Dama cattle have developed a genetic capacity to survive, reproduce and remain productive under trypanosomosis risk. The cellular and molecular bases of this so-called trypanotolerance are not known, but the trait is manifested by the N'Dama's greater capacity to control parasitaemia and anaemia development during an infection. In order to examine the role of the haematopoietic system in trypanotolerance, we have exploited the tendency for the placentas of bovine twin embryos to fuse. Placental fusion in cattle results in bone marrow chimaerism in twins. By comparison with the N'Dama, cattle of the East African Boran breed are relatively susceptible. We evaluated the role of the haemopoietic system in trypanotolerance by comparing the performance of five Chimaeric Boran/N'Dama twin calves with that of singletons of the two breeds. Chimaeric Boran/N'Dama pairs of twins were produced in recipient Boran cows by embryo transfer, and the majority of haemopoietic cells in all twinned individuals were of Boran origin. Thus, N'Dama chimaeras differed from N'Dama singletons in that the bulk of their haemopoietic system was derived from their susceptible Boran twins, while Boran chimaeras differed little from Boran control animals. All cattle became parasitaemic and developed anaemia. The N'Dama chimaeras did not manage their anaemia and white blood cell counts effectively. However, they were able to limit parasitaemia development. These results suggest that trypanotolerance is the result of two mechanisms, one that improves parasite control and is independent of the genetic origin of the haemopoietic tissue, and another that is influenced by haemopoietic tissue genotype and which improves control over anaemia. The capacity to maintain growth during infection was similarly dependent on the genetic origin of the haemopoietic tissue.

Review on the molecular tools for the understanding of the epidemiology of animal trypanosomosis in West Africa.

The epidemiology of animal trypanosomosis around Bobo-Dioulasso (Burkina Faso, West Africa) benefited a lot in the last years from the progress of molecular tools. The two most used molecular techniques were the polymerase chain reaction for the diagnosis of the disease in cattle and the characterization of the trypanosomes in the host and the vector on one hand, and the microsatellite DNA polymorphism in tsetse flies to study the intraspecific genetic variability of the vector on the other hand. The results obtained in the Sideradougou area during a recent two year survey with these techniques, associated with many other georeferenced informations concerning vector and cattle distribution, natural environment, landuse, ground occupation, livestock management, were combined in a Geographical Information System. This new approach of a complex pathogenic system led to a better evaluation of the risk of trypanosome transmission.

Susceptibility to trypanosomosis of three Bos indicus cattle breeds in areas of differing tsetse fly challenge.

Studies to assess the differences in susceptibility to trypanosomosis among Bos indicus cattle breeds (Maasai Zebu, Orma Boran and Galana Boran) were conducted under conditions of varying tsetse fly challenge at the Nguruman escarpment in south-western Kenya, for a period of 1 year. It was found that under tsetse challenge quantified as high, Maasai Zebu and Orma Boran were less susceptible than Galana Boran to trypanosome infections, as judged by the significantly lower incidence of infection, development of less severe anaemia, fewer requirements for trypanocidal drug treatments, higher growth rates and fewer mortalities. In the area where tsetse challenge was considered low as a result of a tsetse fly control operation using odour-baited traps, only the Maasai Zebu and Orma Boran were compared. No significant differences in the incidence of infection, degree of anaemia or growth rates were observed between the two breeds, but all were significantly different from their counterparts in the high tsetse challenge area. These results suggest that there is variation in resistance to trypanosomosis among Bos indicus cattle breeds that could be exploited as part of the integrated trypanosomosis control programmes in East Africa.

Zebu-taurine variation in Y chromosomal DNA: a sensitive assay for genetic introgression in west African trypanotolerant cattle populations.

Owing to increasing scientific and agricultural interest in the disease-resistant (trypanotolerant), indigenous cattle breeds of West and Central Africa, there is a need for a rational genetically based description of populations in the region. The greatest threat to the invaluable genetic resource represented by these animals is that of extensive genetic introgression of distantly related zebu cattle from northern populations which do not share their inherited tolerances. Southern blotting with a chromosome Y-specific probe, btDYZ-1 (locus DYZ1) is shown to be a sensitive assay to detect such introgression. Evidence of historical crossbreeding is reported in two important N'Dama populations previously classed as purely taurine.

Immunosuppression in trypanotolerant N'Dama cattle following Trypanosoma congolense infection.

Tsetse-transmitted Trypanosoma congolense infection causes an impairment of in vitro T cell proliferative responses in Boran (Bos indicus) cattle. To assess the importance of this phenomenon as it may relate to the ability of trypanotolerant cattle to control infection with trypanosomes, T cell proliferative responses to mitogenic stimulus with Concanavalin A were measured in N'Dama (Bos taurus) cattle throughout infection. The responses of peripheral blood mononuclear cells from Boran and N'Dama cattle were similar. Depressed proliferative responses were observed with cells of both breeds at 12 days post infection, after which the responses returned to levels similar to those recorded pre-infection. Immunosuppression was also studied in the lymph nodes of a major histocompatibility complex (MHC)-matched pair of N'Dama cattle. Lymph node cells from the infected animal failed to respond to mitogenic stimulus. Co-culture experiments in which the cells from this node were mixed with either lymph node cells or peripheral blood mononuclear cells from the non-infected MHC-compatible animal revealed the presence of suppressor cells, acting in a prostaglandin-independent manner, capable of arresting mitogen-induced T cell proliferation.