Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.

Blood of African Hedgehog Atelerix albiventris Contains 115-kDa Trypanolytic Protein that Kills Trypanosoma congolense.

INTRODUCTION: Protozoan parasites of the Order Trypanosomatida infect a wide range of multicellular plants and animals, causing devastating and potentially fatal diseases. Trypanosomes are the most relevant members of the order in sub-Saharan Africa because of mortalities and morbidities caused to humans and livestock. PURPOSE: There are growing concerns that trypanosomes are expanding their reservoirs among wild animals, which habours the parasites, withstand the infection, and from which tsetse flies transmit the parasites back to humans and livestock. This study was designed to investigate the potentials of the African hedgehog serving as reservoir for African animal trypanosomes. METHODS: Five adult hedgehogs alongside five laboratory mice were intraperitoneally inoculated with 106 and 104 of Trypanosoma congolense cells, respectively, and monitored for parasitemia and survival. Serum from twenty hedgehogs was subjected to trypanocidal activity-guided fractionation by successive ion-exchange and gel-filtration chromatographies, followed by characterization with Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). RESULTS: Hedgehogs were resistant to the infection as no parasite was detected and none died even after 60 days, while all the mice died within 12 days. Both the serum and plasma prepared from hedgehogs demonstrated trypanocidal activity- rapidly killed trypanosomes even when diluted 1000 times. The trypanolytic factor was identified to be proteinaceous with an estimated molecular weight of 115-kDa. CONCLUSION: For the first time, it is here demonstrated that hedgehog blood has significant trypanolytic activity against T. congolense. The potential application of the hedgehog protein for the breeding of trypanosomosis-resistant livestock in tsetse fly belt is discussed.

Genomic scan of selective sweeps in Djallonké (West African Dwarf) sheep shed light on adaptation to harsh environments.

The Djallonké (West African Dwarf) sheep is a small-sized haired sheep resulting from a costly evolutionary process of natural adaptation to the harsh environment of West Africa including trypanosome challenge. However, genomic studies carried out in this sheep are scant. In this research, genomic data of 184 Djallonké sheep (and 12 Burkina-Sahel sheep as an outgroup) generated using medium-density SNP Chips were analyzed. Three different statistics (iHS, XP-EHH and nSL) were applied to identify candidate selection sweep regions spanning genes putatively associated with adaptation of sheep to the West African environment. A total of 207 candidate selection sweep regions were defined. Gene-annotation enrichment and functional annotation analyses allowed to identify three statistically significant functional clusters involving 12 candidate genes. Genes included in Functional Clusters associated to selection signatures were mainly related to metabolic response to stress, including regulation of oxidative and metabolic stress and thermotolerance. The bovine chromosomal areas carrying QTLs for cattle trypanotolerance were compared with the regions on which the orthologous functional candidate cattle genes were located. The importance of cattle BTA4 for trypanotolerant response might have been conserved between species. The current research provides new insights on the genomic basis for adaptation and highlights the importance of obtaining information from non-cosmopolite livestock populations managed in harsh environments.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

CRIg plays an essential role in intravascular clearance of bloodborne parasites by interacting with complement.

Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy.

Route of inoculation influences Trypanosoma congolense and Trypanosoma brucei brucei virulence in Swiss white mice.

Experiments on infections caused by trypanosomes are widely performed in Swiss white mice through various inoculation routes. To better understand the effect of route of trypanosome inoculation on disease outcomes in this model, we characterised the virulence of two isolates, Trypanosoma brucei KETRI 2710 and T. congolense KETRI 2765 in Swiss white mice. For each of the isolates, five routes of parasite inoculation, namely intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) intradermal (ID) and intravenous (IV) were compared using groups (n = 6) of mice, with each mouse receiving 1x104 trypanosomes. We subsequently assessed impact of the routes on disease indices that included pre-patent period (PP), parasitaemia levels, Packed Cell Volume (PCV), bodyweight changes and survival time. Pre-patent period for IP inoculated mice was a mean ± SE of 3.8 ± 0.2 and 6.5 ± 0.0 for the T brucei and T. congolense isolates respectively; the PP for mice groups inoculated using other routes were not significantly different(p> 0.05) irrespective of route of inoculation and species of trypanosomes. With ID and IP routes, parasitaemia was significantly higher in T. brucei and significantly lower in T. congolense infected mice and the progression to peak parasitaemia routes showed no significant different between the routes of either species of trypanosome. The IM and ID routes in T. congolense inoculations, and IP and IV in T. b. brucei induced the fastest and slowest parasitaemia progressions respectively. There were significant differences in rates of reduction of PCV with time post infection in mice infected by the two species and which was more pronounced in sc and ip injected mice. No significant differences in mice body weight changes and survivorship was observed between the routes of inoculation. Inoculation route therefore appears to be a critical determinant of pathogenicity of Trypanosoma congolense and Trypanosoma brucei brucei in murine mouse model of African trypanosomiasis.

Equine trypanosomosis: enigmas and diagnostic challenges.

Equine trypanosomosis is a complex of infectious diseases called dourine, nagana and surra. It is caused by several species of the genus Trypanosoma that are transmitted cyclically by tsetse flies, mechanically by other haematophagous flies, or sexually. Trypanosoma congolense (subgenus Nannomonas) and T. vivax (subgenus Dutonella) are genetically and morphologically distinct from T. brucei, T. equiperdum and T. evansi (subgenus Trypanozoon). It remains controversial whether the three latter taxa should be considered distinct species. Recent outbreaks of surra and dourine in Europe illustrate the risk and consequences of importation of equine trypanosomosis with infected animals into non-endemic countries. Knowledge on the epidemiological situation is fragmentary since many endemic countries do not report the diseases to the World Organisation for Animal Health, OIE. Other major obstacles to the control of equine trypanosomosis are the lack of vaccines, the inability of drugs to cure the neurological stage of the disease, the inconsistent case definition and the limitations of current diagnostics. Especially in view of the ever-increasing movement of horses around the globe, there is not only the obvious need for reliable curative and prophylactic drugs but also for accurate diagnostic tests and algorithms. Unfortunately, clinical signs are not pathognomonic, parasitological tests are not sufficiently sensitive, serological tests miss sensitivity or specificity, and molecular tests cannot distinguish the taxa within the Trypanozoon subgenus. To address the limitations of the current diagnostics for equine trypanosomosis, we recommend studies into improved molecular and serological tests with the highest possible sensitivity and specificity. We realise that this is an ambitious goal, but it is dictated by needs at the point of care. However, depending on available treatment options, it may not always be necessary to identify which trypanosome taxon is responsible for a given infection.

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.

Variant antigen repertoires in Trypanosoma congolense populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the T. congolense VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

Remarkable richness of trypanosomes in tsetse flies (Glossina morsitans morsitans and Glossina pallidipes) from the Gorongosa National Park and Niassa National Reserve of Mozambique revealed by fluorescent fragment length barcoding (FFLB).

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas.

Interspecies quorum sensing in co-infections can manipulate trypanosome transmission potential.

Quorum sensing (QS) is commonly used in microbial communities and some unicellular parasites to coordinate group behaviours 1,2 . An example is Trypanosoma brucei, which causes human African trypanosomiasis, as well as the livestock disease, nagana. Trypanosomes are spread by tsetse flies, their transmission being enabled by cell-cycle arrested 'stumpy forms' that are generated in a density-dependent manner in mammalian blood. QS is mediated through a small (<500 Da), non-proteinaceous, stable but unidentified 'stumpy induction factor' 3 , whose signal response pathway has been identified. Although QS is characterized in T. brucei, co-infections with other trypanosome species (Trypanosoma congolense and Trypanosoma vivax) are common in animals, generating the potential for interspecies interactions. Here, we show that T. congolense exhibits density-dependent growth control in vivo and conserves QS regulatory genes, of which one can complement a T. brucei QS signal-blind mutant to restore stumpy formation. Thereafter, we demonstrate that T. congolense-conditioned culture medium promotes T. brucei stumpy formation in vitro, which is dependent on the integrity of the QS signalling pathway. Finally, we show that, in vivo, co-infection with T. congolense accelerates differentiation to stumpy forms in T. brucei, which is also QS dependent. These cross-species interactions have important implications for trypanosome virulence, transmission, competition and evolution in the field.

Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains.

African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.). In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6). We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP) periods were 8.4 ± 0.9 (range, 4-11) and 4.5 ± 0.2 (range, 4-6) for T. congolense and T. brucei isolates, respectively (p < 0.01). Despite the longer mean PP, T. congolense-infected mice exhibited a significantly (p < 0.05) shorter survival time than T. brucei-infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection-causing T. congolense EATRO 1829 and chronic infection-causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

Micro RNA expression profiles in peripheral blood cells of rats that were experimentally infected with Trypanosoma congolense and different Trypanosoma brucei subspecies.

To identify miRNAs whose expression are differentially regulated during trypanosome infections a microarray targeting more than 600 rat miRNA was used to analyze the miRNA expression profiles between uninfected rats and animals infected by Trypanosoma congolense and Trypanosoma brucei s.l. The potential targets of dysregulated miRNAs as well as their biological pathways and functions were predicted using several bioinformatics software tools. Irrespective of the infecting trypanosome species, eight miRNAs (seven up- and one down-regulated) were dysregulated during infections. Moreover, other miRNAs were differentially regulated in rats infected by specific trypanosome species. Functional analyses of differentially regulated miRNAs indicated their involvement in diverse biological processes. Among these, transcription repressor activity, gene expression control as well as protein transporter activity were predominant. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of dysregulated miRNAs revealed their involvement in several biological pathways and disease conditions. This suggests possible modulation of such pathways following trypanosome infection; for example, the MAPK signaling pathway which is known to play vital roles in apoptosis, innate immune response and response to viral infections was highly affected. Axon guidance was equally highly impacted and may indicate a cross reactivity between pathogen proteins and guidance molecules representing one pathological mechanism as it has been observed with influenza HA. Furthermore, Ingenuity pathway analyses of dysregulated miRNAs and potential targets indicated strong association with inflammatory responses, cell death and survival as well as infectious diseases. The data generated here provide valuable information to understand the regulatory function of miRNAs during trypanosome infections. They improved our knowledge on host-parasite cross-talks and provide a framework for investigations to understand the development of trypanosomes in their hosts as well as the differences in the clinical and pathological evolutions of the disease.

Chronic Trypanosoma congolense infections in mice cause a sustained disruption of the B-cell homeostasis in the bone marrow and spleen.

Trypanosoma congolense is one of the main species responsible for Animal African Trypanosomosis (AAT). As preventive vaccination strategies for AAT have been unsuccessful so far, investigating the mechanisms underlying vaccine failure has to be prioritized. In T. brucei and T. vivax infections, recent studies revealed a rapid onset of destruction of the host B-cell compartment, resulting in the loss of memory recall capacity. To assess such effect in experimental T. congolense trypanosomosis, we performed infections with both the cloned Tc13 parasite, which is considered as a standard model system for T. congolense rodent infections and the noncloned TRT55 field isolate. These infections differ in their virulence level in the C57BL/6 mouse model for trypanosomosis. We show that early on, an irreversible depletion of all developmental B cells stages occur. Subsequently, in the spleen, a detrimental decrease in immature B cells is followed by a significant and permanent depletion of Marginal zone B cells and Follicular B cells. The severity of these events later on in infection correlated with the virulence level of the parasite stock. In line with this, it was observed that later-stage infection-induced IgGs were largely nonspecific, in particular in the more virulent TRT55 infection model.

Virulence of Trypanosoma congolense strains isolated from cattle and African buffaloes (Syncerus caffer) in KwaZulu-Natal, South Africa.

Trypanosoma congolense and Trypanosoma vivax are major species that infect cattle in north-eastern KwaZulu-Natal (KZN), South Africa. Of the two genetically distinct types of T. congolense, Savannah and Kilifi sub-groups, isolated from cattle and tsetse flies in KZN, the former is more prevalent and thought to be responsible for African animal trypanosomosis outbreaks in cattle. Furthermore, variation in pathogenicity within the Savannah sub-group is ascribed to strain differences and seems to be related to geographical locations. The objective of the present study was to compare the virulence of T. congolense strains isolated from African buffaloes (Syncerus caffer) inside Hluhluwe-iMfolozi Park, and from cattle on farms near wildlife parks (< 5 km), to isolates from cattle kept away (> 10 km) from parks. To obtain T. congolense isolates, blood of known parasitologically positive cattle or cattle symptomatically suspect with trypanosomosis, as well as isolates from buffaloes kept inside Hluhluwe-iMfolozi Park were passaged in inbred BALB/c mice. A total of 26 T. congolense isolates were obtained: 5 from buffaloes, 13 from cattle kept near parks and 8 from cattle distant from parks. Molecular characterisation revealed 80% and 20% of isolates to belong to T. congolense Savannah and Kilifi, respectively. To compare virulence, each isolate was inoculated into a group of six mice. No statistical differences were observed in the mean pre-patent period, maximum parasitaemia or drop in packed cell volume (PCV). Significant differences were found in days after infection for the drop in PCV, the patent period and the survival time. These differences were used to categorise the isolates as being of high, moderate or low virulence. Based on the virulence, 12 of 26 (46%) isolates were classified as highly virulent and 27% each as either of moderate or of low virulence. Whilst 11 of 12 high virulent strains were from buffaloes or cattle near the park, only 1 of 7 low virulent strains was from these animals. All the Kilifi T. congolense types were less virulent than the Savannah types. These results confirmed the higher virulence of T. congolense Savannah type compared to Kilifi type and indicated the prevalence of highly virulent strains to be higher in wildlife parks and in cattle near the parks than on farms further away. The geographical location of these strains in relation to the wildlife parks in the area was discussed.

Regulatory T cells enhance susceptibility to experimental Trypanosoma congolense infection independent of mouse genetic background.

BACKGROUND: BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines. METHODOLOGY/FINDINGS: BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10(3)T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25(+) T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4(+)CD25(+)Foxp3(+) T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25(+) cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25(+) T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines. CONCLUSION: Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice.

Sialidases play a key role in infection and anaemia in Trypanosoma congolense animal trypanosomiasis.

Animal African trypanosomiasis is a major constraint to livestock productivity and has an important impact on millions of people in developing African countries. This parasitic disease, caused mainly by Trypanosoma congolense, results in severe anaemia leading to animal death. In order to characterize potential targets for an anti-disease vaccine, we investigated a multigenic trans-sialidase family (TcoTS) in T. congolense. Sialidase and trans-sialidase activities were quantified for the first time, as well as the tightly regulated TcoTS expression pattern throughout the life cycle. Active enzymes were expressed in bloodstream form parasites and released into the blood during infection. Using genetic tools, we demonstrated a significant correlation between TcoTS silencing and impairment of virulence during experimental infection with T. congolense. Reduced TcoTS expression affected infectivity, parasitaemia and pathogenesis development. Immunization-challenge experiments using recombinant TcoTS highlighted their potential protective use in an anti-disease vaccine.

Virulence in Trypanosoma congolense Savannah subgroup. A comparison between strains and transmission cycles.

Trypanosoma congolense strains have been shown to differ in their virulence both between subgroups and within the Savannah subgroup between strains. This review revisits these findings and complements them with information on the virulence of T. congolense Savannah subgroup strains isolated from cattle (domestic transmission cycle) in different geographical areas and of strains isolated in protected areas where trypanotolerant wildlife species are the reservoir of the trypanosomes (sylvatic transmission cycle). The virulence of a total of 62 T. congolense Savannah subgroup strains (50 domestic and 12 sylvatic), determined using a standard protocol in mice, was compared. Virulence varied substantially between strains with, depending on the strain, the median survival time of infected mice varying from five to more than sixty days. The proportion of highly virulent strains (median survival time <10 days) was significantly (P = 0·005) higher in strains from the sylvatic transmission cycle. The analysis highlights repercussions of the domestication of the trypanosomiasis transmission cycle that may have to be taken in consideration in the development of trypanosomiasis control strategies.

The prevalence of African animal trypanosomoses and tsetse presence in Western Senegal.

In 2005, the Government of Senegal initiated a tsetse eradication campaign in the Niayes and La Petite Côte aiming at the removal of African Animal Trypanosomosis (AAT), which is one of the main constraints to the development of more effective cattle production systems. The target area has particular meteorological and ecological characteristics that provide great potential for animal production, but it is unfortunately still infested by the riverine tsetse species Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae). The tsetse project in Senegal has adopted an area-wide integrated pest management (AW-IPM) approach that targets the entire tsetse population within a delimited area. During the first phase of the programme, a feasibility study was conducted that included the collection of entomological, veterinary, population genetics, environmental and socioeconomic baseline data. This paper presents the parasitological and serological prevalence data of AAT in cattle residing inside and outside the tsetse-infested areas of the target zone prior to the control effort. At the herd level, a mean parasitological prevalence of 2.4% was observed, whereas a serological prevalence of 28.7%, 4.4%, and 0.3% was obtained for Trypanosoma vivax, T. congolense and T. brucei brucei, respectively. The observed infection risk was 3 times higher for T. congolense and T. vivax in the tsetse-infested than in the assumed tsetse-free areas. Moreover, AAT prevalence decreased significantly with distance from the nearest tsetse captured which indicated that cyclical transmission of the parasites by tsetse was predominant over mechanical transmission by numerous other biting flies present. The importance of these results for the development of a control strategy for the planned AW-IPM campaign is discussed.