Molecular identification of different trypanosome species and subspecies in tsetse flies of northern Nigeria

Background: Animal African Trypanosomiasis (AAT) is caused by several species of trypanosomes including Trypanosoma congolense, T. vivax, T. godfreyi, T. simiae and T. brucei. Two of the subspecies of T. brucei also cause Human African Trypanosomiasis. Although some of them can be mechanically transmitted by biting flies; these trypanosomes are all transmitted by tsetse flies which are the cyclical vectors of Trypanosoma congolense, T. godfreyi, T. simiae and T. brucei. We present here the first report assessing the prevalence of trypanosomes in tsetse flies in Nigeria using molecular tools. Methods: 488 tsetse flies of three species, Glossina palpalis palpalis, G. tachinoides and G. morsitans submorsitans were collected from Wuya, Niger State and Yankari National Park, Bauchi State in 2012. Trypanosomes were detected and identified using an ITS1 PCR assay on DNA purified from the ‘head plus proboscis’ (H + P) and abdomen (ABD) parts of each fly. Results: T. vivax and T. congolense Savannah were the major parasites detected. Trypanosomes prevalence was 7.1 % in G. p. palpalis, 11.9 % in G. tachinoides and 13.5 % in G. m. submorsitans. Prevalences of T. congolense Savannah ranged from 2.5 to 6.7 % and of T. vivax were approximately 4.5 %. Trypanosoma congolense Forest, T. godfreyi and T. simiae were also detected in the site of Yankari. The main biological and ecological determinants of trypanosome prevalence were the fly sex, with more trypanosomes found in females than males, and the site, with T. congolense subspp. being more abundant in Yankari than in Wuya. As expected, the trypanosome species diversity was higher in Yankari National Park than in the more agricultural site of Wuya where vertebrate host species diversity is lower. Conclusions: Our results show that T. congolense Savannah and T. vivax are the main species of parasite potentially causing AAT in the two study sites and that Yankari National Park is a potential reservoir of trypanosomes both in terms of parasite abundance and species diversity

MIF-mediated hemodilution promotes pathogenic anemia in experimental African trypanosomosis

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells ( RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF ( macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis

African Trypanosomes undermine humoral responses and vaccine development : link with inflammatory responses?

African trypanosomosis is a debilitating disease of great medical and socioeconomical importance. It is caused by strictly extracellular protozoan parasites capable of infecting all vertebrate classes including human, livestock, and game animals. To survive within their mammalian host, trypanosomes have evolved efficient immune escape mechanisms and manipulate the entire host immune response, including the humoral response. This report provides an overview of how trypanosomes initially trigger and subsequently undermine the development of an effective host antibody response. Indeed, results available to date obtained in both natural and experimental infection models show that trypanosomes impair homeostatic B-cell lymphopoiesis, B-cell maturation and survival and B-cell memory development. Data on B-cell dysfunctioning in correlation with parasite virulence and trypanosome-mediated inflammation will be discussed, as well as the impact of trypanosomosis on heterologous vaccine efficacy and diagnosis. Therefore, new strategies aiming at enhancing vaccination efficacy could benefit from a combination of (i) early parasite diagnosis, (ii) anti-trypanosome (drugs) treatment, and (iii) anti-inflammatory treatment that collectively might allow B-cell recovery and improve vaccination

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection

Infections with extracellular trypanosomes require control by efficient innate immune mechanisms and can result in the destruction of the mammalian humoral immune system

Salivarian trypanosomes are extracellular parasites that affect humans, livestock, and game animals around the world. Through co-evolution with the mammalian immune system, trypanosomes have developed defense mechanisms that allow them to thrive in blood, lymphoid vessels, and tissue environments such as the brain, the fat tissue, and testes. Trypanosomes have developed ways to circumvent antibody-mediated killing and block the activation of the lytic arm of the complement pathway. Hence, this makes the innate immune control of the infection a crucial part of the host-parasite interaction, determining infection susceptibility, and parasitemia control. Indeed, trypanosomes use a combination of several independent mechanisms to avoid clearance by the humoral immune system. First, perpetuated antigenic variation of the surface coat allows to escape antibody-mediated elimination. Secondly, when antibodies bind to the coat, they are efficiently transported toward the endocytosis pathway, where they are removed from the coat proteins. Finally, trypanosomes engage in the active destruction of the mammalian humoral immune response. This provides them with a rescue solution in case antigenic variation does not confer total immunological invisibility. Both antigenic variation and B cell destruction pose significant hurdles for the development of anti-trypanosome vaccine strategies. However, developing total immune escape capacity and unlimited growth capabilities within a mammalian host is not beneficial for any parasite, as it will result in the accelerated death of the host itself. Hence, trypanosomes have acquired a system of quorum sensing that results in density-dependent population growth arrest in order to prevent overpopulating the host. The same system could possibly sense the infection-associated host tissue damage resulting from inflammatory innate immune responses, in which case the quorum sensing serves to prevent excessive immunopathology and as such also promotes host survival. In order to put these concepts together, this review summarizes current knowledge on the interaction between trypanosomes and the mammalian innate immune system, the mechanisms involved in population growth regulation, antigenic variation and the immuno-destructive effect of trypanosomes on the humoral immune system. Vaccine trials and a discussion on the role of innate immune modulation in these trials are discussed at the end

A systematic review and meta-analysis of trypanosome prevalence in tsetse flies

Background: The optimisation of trypanosomosis control programs warrants a good knowledge of the main vector of animal and human trypanosomes in sub-Saharan Africa, the tsetse fly. An important aspect of the tsetse fly population is its trypanosome infection prevalence, as it determines the intensity of the transmission of the parasite by the vector. We therefore conducted a systematic review of published studies documenting trypanosome infection prevalence from field surveys or from laboratory experiments under controlled conditions. Publications were screened in the Web of Science, PubMed and Google Scholar databases. Using the four-stage (identification, screening, eligibility and inclusion) process in the PRISMA statement the initial screened total of 605 studies were reduced to 72 studies. The microscopic examination of dissected flies (dissection method) remains the most used method to detect trypanosomes and thus constituted the main focus of this analysis. Meta-regression was performed to identify factors responsible for high trypanosome prevalence in the vectors and a random effects meta-analysis was used to report the sensitivity of molecular and serological tests using the dissection method as gold standard. Results: The overall pooled prevalence was 10.3% (95% confidence interval [CI] = 8.1%, 12.4%) and 31.0% (95% CI = 20. 0%, 42.0%) for the field survey and laboratory experiment data respectively. The country and the year of publication were found to be significantly factors associated with the prevalence of trypanosome infection in tsetse flies. The alternative diagnostic tools applied to dissection positive samples were characterised by low sensitivity, and no information on the specificity was available at all. Conclusion: Both temporal and spatial variation in trypanosome infection prevalence of field collected tsetse flies exists, but further investigation on real risk factors is needed how this variation can be explained. Improving the sensitivity and determining the specificity of these alternative diagnostic tools should be a priority and will allow to estimate the prevalence of trypanosome infection in tsetse flies in high-throughput

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-gamma, 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. Author summary Bovine African Trypanosomosis is a parasitic disease of veterinary importance that adversely affects the public health and economic development of sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature and major cause of death. Using murine models, it was shown that the disease is characterized by a well-timed and balanced production of pro-inflammatory cytokine promoting factors followed by an anti-inflammatory response, involving IL-10. The latter is required to attenuate infection-associated pathogenicity and to prevent early host death from uncontrolled hyper-inflammation mediated immunopathologies. However, the cellular source of IL-10 in vivo and the window within which these cells exert their function during the course of African trypanosomiasis remain poorly understood, which hampers the design of effective therapeutic strategies. Using a T. congolense infection mouse model, relevant for bovine trypanosomosis, we demonstrate that during the chronic stage of infection hepatocyte-derived IL-10, but not myeloid cell-derived IL-10, regulates the main infection-associated immunopathologies and ultimately mediates host survival. Hence, strategies that tilt the balance of hepatocyte cytokine production in favor of IL-10 could majorly impact the wellbeing and survival of T. congolense-infected animals. Given the unmet medical need for this parasite infection, our findings offer promise for improved treatment protocols in the field

Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake

It has long been established that the Trypanosoma brucei TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest Trypanosoma congolense paralogue, TcoAT1, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [3H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [3H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [3H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in T. congolense

Discovery of mating in the major African livestock pathogen Trypanosoma congolense

The protozoan parasite, Trypanosoma congolense, is one of the most economically important pathogens of livestock in Africa and, through its impact on cattle health and productivity, has a significant effect on human health and well being. Despite the importance of this parasite our knowledge of some of the fundamental biological processes is limited. For example, it is unknown whether mating takes place. In this paper we have taken a population genetics based approach to address this question. The availability of genome sequence of the parasite allowed us to identify polymorphic microsatellite markers, which were used to genotype T. congolense isolates from livestock in a discrete geographical area of The Gambia. The data showed a high level of diversity with a large number of distinct genotypes, but a deficit in heterozygotes. Further analysis identified cryptic genetic subdivision into four sub-populations. In one of these, parasite genotypic diversity could only be explained by the occurrence of frequent mating in T. congolense. These data are completely inconsistent with previous suggestions that the parasite expands asexually in the absence of mating. The discovery of mating in this species of trypanosome has significant consequences for the spread of critical traits, such as drug resistance, as well as for fundamental aspects of the biology and epidemiology of this neglected but economically important pathogen