The effect of Trypanosoma vivax infection on late pregnancy and postpartum return to cyclicity in Boran cattle

A study was designed to examine the effect of infection with Trypanosoma vivax KETRI 2501 on the maintenance of pregnancy and postpartum return to reproductive function in susceptible Galana (n = 6) and trypano-tolerant Orma Boran (n = 6) heifers during the third trimester of pregnancy. Of the 12 study animals, 3 Galana and 3 Orma Boran heifers served as controls. One of 3 Galana heifers calved prematurely with subsequent perinatal loss. Of the 2 heifers that produced live calves, 1 calf died shortly after birth, while the other survived. Two of 3 Orma heifers calved prematurely and all 3 calves died shortly after birth. The 6 control heifers produced live calves at term, all of which survived. Infection with T. vivax during the third trimester of pregnancy delayed the resumption of ovarian activity after calving, with the Ormas taking a significantly (P < 0.05) shorter time from calving to ovulation. There was no clear evidence that premature birth was associated with pathological changes in reproductive organs. Results from this study demonstrated that infection with pathogenic T. vivax during late pregnancy influenced the outcome of pregnancy in both susceptible Galana and trypano-tolerant Orma Boran heifers, resulting in premature births, perinatal loss, retained placentae, low birth weights and a prolonged period to the onset of postpartum ovarian activity.     

Feeding behaviour of Glossina pallidipes and g. morsitans centralis on Boran cattle infected with trypanosoma congolense or T. vivax under laboratory conditions

In field studies, tsetse flies (Diptera: Glossinidae) feed more successfully on cattle infected with Trypanosoma congolense Broden (Kinetoplastida: Trypanosomatidae) than on cattle infected with T. vivax Ziemann or uninfected cattle. Here we describe the first laboratory investigation of this phenomenon. In the first experiment, caged Glossina pallidipes Austen were fed for 1 and 5 min on a Boran steer infected with T. congolense clone IL 1180 and on an uninfected steer. Feeding success was recorded in this way five times over several weeks. The same protocol was subsequently used in three additional experiments with the following combinations: G. pallidipes and a steer infected with T. vivax stock IL 3913, G. morsitans centralis Machado and a steer infected with T. congolense, and G. morsitans centralis and a steer infected with T. vivax. The four experiments were replicated once, making eight experiments in total. In three experiments there was increased tsetse feeding success, measured at 1 min, after a steer became infected (T. congolense, two experiments and T. vivax, one experiment). Analysis of all data combined found no significant differences in tsetse feeding success on the different groups of cattle prior to infection, but after infection tsetse feeding success was significantly greater on the infected cattle (P< 0.001). Trypanosoma congolense infection led to a greater increase in tsetse feeding success than T. vivax infection. The increase in feeding success was not related to changes in the level of anaemia, skin surface temperature or parasitaemia. A possible explanation is the effects of trypanosome infection on cutaneous vasodilation and/or blood clotting in infected cattle. When allowed to feed for 5 min, nearly all tsetse engorged successfully and effects of cattle infection on feeding success were not found.     

New epidemiological features on animal trypanosomiasis by molecular analysis in the pastoral zone of Sideradougou, Burkina Faso

A multidisciplinary work was undertaken in the agropastoral zone of Sidéradougou, Burkina Faso to try to elucidate the key factors determining the presence of tsetse flies. In this study the PCR was used to characterize trypanosomes infecting the vector ( Glossina tachinoides and Glossina palpalis gambiensis ) and the host, i.e. cattle. A 2-year survey involved dissecting 2211 tsetse of the two Glossina species. A total of 298 parasitologically infected tsetse were analysed by PCR. Trypanosoma vivax was the most frequently identified trypanosome followed by the savannah type of T. congolense and, to a lesser extent, the riverine forest type of T. congolense , and by T. brucei . No cases of T. simiae were found. From the 107 identified infections in cattle, the taxa were the same, but T. congolense savannah type was more frequent, whereas T. vivax and T. congolense riverine forest types were found less frequently. A correlation was found between midgut infection rates of tsetse, nonidentified infections and reptile bloodmeals. These rates were higher in G.p. gambiensis , and in the western part of the study area. T. vivax infections were related to cattle bloodmeals, and were more frequent in G. tachinoides and in the eastern study area. The PCR results combined with bloodmeal analysis helped us to establish the relationships between the vector and the host, to assess the trypanosome challenge in the two parts of the area, to elucidate the differences between the two types of T. congolense , and to suspect that most midgut infections were originating from reptilian trypanosomes.     

Does isometamidium chloride treatment protect tsetse flies from trypanosome infections during SIT campaigns?

African animal trypanosomosis is a major pathological constraint to cattle breeding across 10 million km2 of sub-Saharan West African countries infested by tsetse flies, their cyclic vectors. The release of sterile males (sterile insect technique [SIT]) is a potentially important control technique aimed at eliminating the vectors. Prior to release, tsetse are generally treated with isometamidium chloride, a trypanocide, to prevent them from transmitting parasites. The present study investigated the preventive action of isometamidium chloride (0.5 mg/L) on the subsequent susceptibility of tsetse released into the wild. A total of 1755 Glossina palpalis gambiensis Vanderplank and 744 Glossina tachinoides Westwood were released, of which 50 and 48, respectively, were recaptured 22-43 days after release. Their probosces were analysed by polymerase chain reaction to identify mature infections with three trypanosome species (Trypanosoma vivax, Trypanosoma brucei sensu lato and Trypanosoma congolense savannah type). Two mature infections with T. vivax and four with T. congolense were detected, indicating that the use of this treatment regimen in an SIT campaign would not totally prevent sterile males from transmitting trypanosomes.     

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.     

Effects of Trypanosoma vivax and Trypanosoma congolense infections on the reaction time and semen characteristics of Zebu (Bunaji) x Friesian crossbred bulls

The effect of trypanosomosis on reaction time and semen characteristics of 12 Zebu (Bunaji) x Friesian crossbred bulls aged between 3 and 5 years was studied for a duration of 12 weeks. Four of the bulls were infected with Trypanosoma vivax, another four with Trypanosoma congolense and the remaining four bulls served as controls. Rectal temperatures and haematological parameters were monitored twice weekly. The pre-infection mean value of the rectal temperature was 38.3 degrees C, and this rose to a mean of between 40.5 and 41.1 degrees C in the infected animals. Concurrently, the infected animals exhibited signs of anaemia shown by pale mucous membranes and decreased packed cell volume (PCV), weight loss, lethargy, weakness and dullness.The reaction time (ejaculation time) of semen collection significantly increased from a pre-infection mean value of 20.46-25.14 s to a mean of 290.33-301.15 s within 12 weeks post-infection. Semen characteristics deteriorated progressively within the same period in the infected bulls. There were highly significant and drastic decreases in sperm concentration and volume of semen and increases in sperm morphological defects. By the third week, all the infected bulls were unfit for breeding because of very poor semen characteristics. Deterioration, also characterized by oligospermia at 6 weeks post-infection in all bulls which later culminated in azoospermia in two bulls infected with T. vivax and two bulls infected with T. congolense continued to the end of the investigation. The present results indicate that trypanosomosis due to T. vivax and T. congolense infections is very pathogenic and devastating in its effect on the reaction time (ejaculation time) and semen characteristics which resulted in very poor semen quality. The practical implication is infertility and sterility in Zebu x Friesian crossbred bulls in trypanosome endemic areas.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.     

Genetic engineering of Trypanosoma (Dutonella) vivax and in vitro differentiation under axenic conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.     

Spatio-temporal distribution of tsetse and other biting flies in the Mouhoun River basin, Burkina Faso

In the Mouhoun River basin, Burkina Faso, the main vectors of African animal trypanosomoses are Glossina palpalis gambiensis Vanderplank and Glossina tachinoides Westwood (Diptera: Glossinidae), both of which are riverine tsetse species. The aim of our study was to understand the impact of landscape anthropogenic changes on the seasonal dynamics of vectors and associated trypanosomosis risk. Three sites were selected on the basis of the level of disturbance of tsetse habitats and predominant tsetse species: disturbed (Boromo, for G. tachinoides) and half-disturbed (Douroula for G. tachinoides and Kadomba for G. p. gambiensis). At each of these sites, seasonal variations in the apparent densities of tsetse and mechanical vectors and tsetse infection rates were monitored over 17 months. Tsetse densities differed significantly between sites and seasons. Of 5613 captured tsetse, 1897 were dissected; 34 of these were found to be infected with trypanosomes. The most frequent infection was Trypanosoma vivax (1.4%), followed by Trypanosoma congolense (0.3%) and Trypanosoma brucei (0.05%). The mean physiological age of 703 tsetse females was investigated to better characterize the transmission risk. Despite the environmental changes, it appeared that tsetse lived long enough to transmit trypanosomes, especially in half-disturbed landscapes. A total of 3021 other biting flies from 15 species (mainly Tabanidae and Stomoxyinae) were also caught: their densities also differed significantly among sites and seasons. Their relative importance regarding trypanosome transmission is discussed; the trypanosomosis risk in cattle was similar at all sites despite very low tsetse densities (but high mechanical vector densities) in one of them.     

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.     

Comparing apples and oranges--model-based assessment of different tsetse-transmitted trypanosomosis control strategies

The current control strategies for tsetse-transmitted trypanosomosis in cattle (trypanocidal drugs, tsetse control and trypanotolerant cattle) are briefly reviewed and their adoption rates in different geographic regions of sub-Saharan Africa are presented. The impact of these control strategies and the potential use of vaccines, should they be developed, on trypanosomosis transmission were compared using a mathematical model. The relative trypanosomosis prevalence compared with no control was estimated across a range of control coverages (from none to complete control coverage) by varying the change in specific model parameters influenced by individual control measures. Based on this comparison, the relative rankings of the effect of control strategies on reducing disease prevalence were: vector control, vaccination, and drug use, in that order. In this model, trypanotolerance was assumed to decrease disease prevalence, but not to influence transmission. Differences in the predicted impact of control measures on the transmission of human sleeping sickness are discussed. Finally, the role of transmission model outputs as inputs for economic models to guide investment decisions for trypanosomosis control is emphasised.     

Control of Glossina longipennis (Diptera: Glossinidae) by insecticide-treated targets at Galana ranch, Kenya, and confirmation of the role of G. longipennis as a vector of cattle trypanosomiasis

Glossina longipennis Corti was studied in Galana Ranch, Kenya over a four year period, in two areas (Tank E and Lali) where the species was abundant and other species were absent or scarce. There was active transmission of trypanosomiasis to cattle in both areas, the parasite species being Trypanosoma vivax Ziemann and T. congolense Broden. Mean infection rates of the G. longipennis were 1.1% and 0. 55% for T. vivax and T. congolense respectively at Tank E, and 0.88% and 0.15% at Lali. Experimental transmission studies showed that cattle in fly-proof enclosures challenged with wild G. longipennis collected from Galana became infected with both trypanosome species. A tsetse control operation in one area (Tank E) using targets impregnated with deltamethrin in an oil formulation reduced the population of G. longipennis by 98% over one year, despite evidence of re-invasion. Populations of G. longipennis in the other area (Lali) were relatively stable over the whole study period. The effect of tsetse control on the incidence of cattle trypanosomiasis at Tank E was less clear than that on tsetse numbers, probably due to the lack of a sustained reduction in tsetse numbers. However, a significant relationship was demonstrated between fortnightly incidence measurements and electric net catches of G. longipennis at Tank E. A further significant predictor of incidence was rainfall in the previous four to seven weeks. This study confirms the importance of G. longipennis as a vector of bovine trypanosomiasis in areas where it is the predominant tsetse present.     

Trypanosoma congolense: expression of a heat shock protein 70 and initial evaluation as a diagnostic antigen for bovine trypanosomosis

A 69-kDa immunodominant protein of Trypanosoma congolense was identified as a member of the hsp70 family that is homologous to mammalian BiP. We report here the expression of the gene encoding the T. congolense BiP in a bacterial system. Dot blotting of the truncated recombinant proteins confirmed that BiP antigenicity is mainly located in the C-terminal third of the molecule. A recombinant fragment corresponding to this region was used as an antigen in an indirect ELISA and an initial evaluation of its diagnostic potential for bovine trypanosomosis was performed. The test showed limited sensitivity for detection of primary-infected cattle but was capable of accurately detecting secondary infections. As BiP and its derivatives may be produced at low cost under stable forms allowing standardization of the tests, they warrant further evaluation as antigens fro serodiagnosis of bovine trypanosomosis.     

Trypanosoma vivax, T. congolense "forest type" and T. simiae: prevalence in domestic animals of sleeping sickness foci of Cameroon

In order to better understand the epidemiology of Human and Animal trypanosomiasis that occur together in sleeping sickness foci, a study of prevalences of animal parasites (Trypanosoma vivax, T. congolense "forest type", and T. simiae) infections was conducted on domestic animals to complete the previous work carried on T. brucei gambiense prevalence using the same animal sample. 875 domestic animals, including 307 pigs, 264 goats, 267 sheep and 37 dogs were sampled in the sleeping sickness foci of Bipindi, Campo, Doumé and Fontem in Cameroon. The polymerase chain reaction (PCR) based method was used to identify these trypanosome species. A total of 237 (27.08%) domestic animals were infected by at least one trypanosome species. The prevalence of T. vivax, T. congolense "forest type" and T. simiae were 20.91%, 11.42% and 0.34% respectively. The prevalences of 7 vivax and T. congolense "forest type" differed significantly between the animal species and between the foci (p < 0.0001); however, these two trypanosomes were found in all animal species as well as in all the foci subjected to the study. The high prevalences of 7 vivax and T congolense "forest type" in Bipindi and Fontem-Center indicate their intense transmission in these foci.     

Detection of selection signatures within candidate regions underlying trypanotolerance in outbred cattle populations

Breeding indigenous African taurine cattle tolerant to trypanosomosis is a straightforward approach to control costs generated by this disease. A recent study identified quantitative trait loci (QTL) underlying trypanotolerance traits in experimental crosses between tolerant N'Dama and susceptible Boran zebu cattle. As trypanotolerance is thought to result from local adaptation of indigenous cattle breeds, we propose an alternative and complementary approach to study the genetic architecture of this trait, based on the identification of selection signatures within QTL or candidate genes. A panel of 92 microsatellite markers was genotyped on 509 cattle belonging to four West African trypanotolerant taurine breeds and 10 trypanosusceptible European or African cattle breeds. Some of these markers were located within previously identified QTL regions or candidate genes, while others were chosen in regions assumed to be neutral. A detailed analysis of the genetic structure of these different breeds was carried out to confirm a priori grouping of populations based on previous data. Tests based on the comparison of the observed heterozygosities and variances in microsatellite allelic size among trypanotolerant and trypanosusceptible breeds led to the identification of two significantly less variable microsatellite markers. BM4440, one of these two outlier loci, is located within the confidence interval of a previously described QTL underlying a trypanotolerance-related trait.
Detection of selection signatures appears to be a straightforward approach for unravelling the molecular determinism of trypanosomosis pathogenesis. We expect that a whole genome approach will help confirm these results and achieve a higher resolving power.     

Participatory investigations of bovine trypanosomiasis in Tana River District, Kenya

Participatory research on bovine trypanosomiasis was conducted with Orma pastoralists in Tana River District, Kenya. The use of participatory methods to understand local perceptions of disease signs, disease causes, disease incidence by cattle age group, seasonal patterns of disease and preferences for indigenous and modern control methods are described. Results indicated that local characterization of diseases called gandi and buku by Orma pastoralists was similar to modern veterinary knowledge on chronic trypanosomiasis and haemorrhagic trypanosomiasis (due to Trypanosoma vivax), respectively. The mean incidence of gandi varied from 10.2% in calves to 28.6% in adult cattle. The mean incidence of buku varied from 3.1% in calves to 9.6% in adults. Pearson correlation coefficients for disease incidence by age group were 0.498 (P < 0.01) and 0.396 (P < 0.05) for gandi and buku, respectively. Informants observed cases of trypanosomiasis in 24.1% of cattle (all age groups); these cases accounted for 41.8% of all sick cattle during the preceding 12-month period. Eight indigenous and three modern trypanosomiasis control methods were identified. Results indicated that an integrated approach to trypanosomiasis control based on private, individual action was well established in the assessment area. When presented with four different trypanosomiasis control methods, community representatives selected 'better use of trypanocides' as the most preferred intervention and 'community-based tsetse control' as the least preferred intervention. This finding prompted researchers to modify the original project activities. Constraints facing the sustainability of community-based tsetse control are discussed.     

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 C?te 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.     

Effect of adjuvants on the humoral immune response to congopain in mice and cattle

ABSTRACT: BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme- inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in AdjuphosTM, TiterMaxTM, purified saponin Quil ATM or GERBUTM showed the best response according to the evaluation criteria and these three were chosen for the cattle vaccination study. The animals were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil ATM showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil ATM as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.