Cell Adhesion in Trypanosoma: In Vitro Studies of the Interaction of Trypanosoma vivax with Immobilized Organic Dyes1

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex? Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.     

High Trypanosoma vivax infection rates in water buffalo and cattle in the Brazilian Lower Amazon

Highly sensitive and accurate molecular diagnostic methods have not yet been employed for livestock trypanosomosis in the Brazilian Lower Amazon although the first reports of Trypanosoma vivax and Trypanosoma evansi in Brazil were in water buffalo (Bubalus bubalis) in this region. The present study assessed trypanosomosis in buffalo and cattle raised in communal and seasonally flooding pastures in the state of Pará using the fluorescent fragment length barcoding (FFLB) method. T. evansi was not detected, but high infection rates of T. vivax and T. theileri were revealed by a simplified FFLB standardized in the present study that discriminates all trypanosome species infective to livestock in South America. T. vivax infection rates detected by TviCATL-PCR were 24.6% for cattle (n = 61) and 28.1% for buffalo (n = 89). Using the FFLB method, overall T. vivax infection rates increased to 59.6% and 44.3% for buffalo and cattle, respectively. Furthermore, the predominance of a single microsatellite-based genotype of T. vivax was reinforced in the Lower Amazon. Relevant T. vivax infection rates detected in clinically healthy buffalo and cattle through the sampled years (2008–2017) highlight the need for systematic studies to demonstrate the endemic steady state of T. vivax in this region. Our findings provide baseline information for livestock management, including control of T. vivax dispersal, and the introduction of naïve animals. The growing international trade of live livestock from this very important livestock breeding region represents a serious risk for T. vivax spreading outside Amazonia and Brazil.     

Genome and Phylogenetic Analyses of Trypanosoma evansi Reveal Extensive Similarity to T. brucei and Multiple Independent Origins for Dyskinetoplasty

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F₁F_O-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.     

Trypanosoma vivax displays a clonal population structure

African animal trypanosomiasis, or Nagana, is a debilitating and economically costly disease with a major impact on animal health in sub-Saharan Africa. Trypanosoma vivax, one of the principal trypanosome species responsible for the disease, infects a wide host range including cattle, goats, horses and donkeys and is transmitted both cyclically by tsetse flies and mechanically by other biting flies, resulting in a distribution covering large swathes of South America and much of sub-Saharan Africa. While there is evidence for mating in some of the related trypanosome species, Trypanosoma brucei, Trypanosoma congolense and Trypanosoma cruzi, very little work has been carried out to examine this question in T. vivax. Understanding whether mating occurs in T. vivax will provide insight into the dynamics of trait inheritance, for example the spread of drug resistance, as well as examining the origins of meiosis in the order Kinetoplastida. With this in mind we have identified orthologues of eight core meiotic genes within the genome, the presence of which imply that the potential for mating exists in this species. In order to address whether mating occurs, we have investigated a sympatric field population of T. vivax collected from livestock in The Gambia, using microsatellite markers developed for this species. Our analysis has identified a clonal population structure showing significant linkage disequilibrium, homozygote deficits and disagreement with Hardy-Weinberg predictions at six microsatellite loci, indicative of a lack of mating in this population of T. vivax.     

Non-Invasive In Vivo Study of the Trypanosoma vivax Infectious Process Consolidates the Brain Commitment in Late Infections

Trypanosoma vivax, one of the leading parasites responsible for Animal African Trypanosomosis (Nagana), is generally cyclically transmitted by Glossina spp. but in areas devoid of the tsetse flies in Africa or in Latin American countries is mechanically transmitted across vertebrate hosts by other haematophagous insects, including tabanids. We followed on from our recent studies on the maintenance of this parasite in vivo and in vitro, and its genetic manipulation, by constructing a West African IL1392 T. vivax strain that stably expresses firefly luciferase and is fully virulent for immunocompetent mice. We report here on a study where murine infection with this strain was monitored in vivo using a non-invasive method. Study findings fully support the use of this strain in the assessment of parasite dynamics in vivo since a strong correlation was found between whole body light emission measured over the course of the infection and parasitemia determined microscopically. In addition, parasitemia and survival rates were very similar for mice infected by the intraperitoneal and sub-cutaneous routes, except for a longer prepatent period following sub-cutaneous inoculation with the parasite. Our results clearly show that when administered by the subcutaneous route, the parasite is retained few days in the skin close to the inoculation site where it multiplies before passing into the bloodstream. Ex vivo bioluminescence analyses of organs isolated from infected mice corroborated our previous histopathological observations with parasite infiltration into spleen, liver and lungs. Finally, our study reinforces previous observations on the presence of the parasite in the central nervous system and consequently the brain commitment in the very late phases of the experimental infection.     

Tsetse blood-meal sources,endosymbionts and trypanosome-associations in the Maasai Mara National Reserve,a wildlife-human-livestock interface

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.     

Trypanosoma vivax: sequence of antigenic variants in mice and goats

Three populations composed of different variable antigen types were derived from a clone of the recently isolated mouse-infective Trypanosoma vivax stock Zaria Y486. The antigenic composition of the trypanosome populations which appeared following infection of mice and goats with these populations was compared using the immune lysis test. Significant differences were observed in the antigenic composition of both the initial and relapse populations obtained from goats and mice. These observations suggest that there may be selective growth of different variable antigen types in different hosts with the result that the antigenic composition of a goat-derived population may be greatly altered following subinoculation into normal mice. This can be somewhat reduced however if goat derived populations are subinoculated into lethally irradiated mice. Two antigenically different T. vivax populations were also cyclically transmitted to individual goats in a preliminary study of the effect of tsetse transmission on the antigenic composition of the ingested populations. It was observed that the composition of the first detectable populations in the infected goats was similar but different from that ingested by the tsetse flies.     

Focus: Zoonotic Disease: Molecular Detection of Zoonotic Pathogens in the Blood and Tissues of Camels (Camelus dromedarius) in Central Desert of Iran

Dromedary camels (Camelus dromedarius) play a major economic role in many countries in Africa and Asia. Although they are resistant to harsh environmental conditions, they are susceptible to a wide range of zoonotic agents. This study aimed to provide an overview on the prevalence of selected zoonotic pathogens in blood and tissues of camels in central Iran. Blood, liver, portal lymph node, and brain were collected from 100 apparently healthy camels at a slaughterhouse in Qom city to assess the presence of DNA of Brucella spp., Trypanosoma spp., Coxiella burnetii, and Bartonella spp. PCR products were sequenced bidirectionally and phylogenetic analyses were performed. Eleven percent of camels tested positive for Brucella abortus (3%) and Trypanosoma evansi (8%). Coxiella burnetii and Bartonella spp. DNA was not detected. Our data demonstrate that camels from Iran contribute to the epidemiology of some zoonotic pathogens. Performing proper control strategies, such as vaccination of camels and humans in contact with them, test-and-slaughter policy, and education of the general population is necessary for minimizing the risk of zoonotic infection.     

Microscopic and molecular detection of Deraiophoronema evansi (Lewis, 1882) in domestic Bactrian camels (Camelus bactrianus) of Mongolia

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5–10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi–COI gene sequences were subjected to phylogenetic analyses. The D. evansi–COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.     

A Proline Racemase Based PCR for Identification of Trypanosoma vivax in Cattle Blood

A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764–783 bp and 983–1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.     

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

Background

Trypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia.

Methodology/principal findings

T. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions.

Conclusions/significance

This study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA.     

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels.     

First survey of ticks,tick-borne pathogens (Theileria,Babesia, Anaplasma and Ehrlichia) and Trypanosoma evansi in protected areas for threatened wild ruminants in Tunisia

The aim of the study was to identify ticks present in the environment and wild Tunisian ruminants and to detect tick-borne pathogens and Trypanosoma evansi DNA in these specimens. Sampling was done throughout each season from the environment in three protected areas around Tunisia: El Feidja, Haddaj and Oued Dekouk. Ticks were collected also, from one fawn of Barbary red deer and eight naturally deceased wild ruminants (one Barbary red deer, five Scimitar-horned oryx, one Addax antelope and one Dorcas gazelle), all of which lived in various protected areas. PCR and nested PCRs were performed to detect the presence of Theileria spp., Babesia spp., Trypanosoma evansi, Ehrlichia spp., Anaplasma spp., Anaplasma bovis and Anaplasma phagocytophilum DNA in these tick specimens. A total of 352 ticks were collected, belonging to six different species: Hyalomma excavatum (80.6%), Hyalomma dromedarii (10.2%), Hyalomma marginatum (0.5%), Rhipicephalus bursa (0.5%), Rhipicephalus sanguineus sensu lato (5.1%) and Ixodes ricinus (2.8%). Pathogens have been detected in 25% of H. dromedarii, 9.1% of H. excavatum and 5% of R. sanguineus sensu lato. The percentage of detection of T. evansi was 0.2%. Ehrlichia spp.-Anaplasma spp. were detected in 10.1% of ticks. Anaplasma spp. and A. bovis were detected in 7.6%, and 0.8% of examined ticks, respectively. None of the Theileria spp., Babesia spp., or A. phagocytophilum DNA was detected in the tested ticks. To our knowledge, the present study represents the first identification of these six tick species and the first detection of rickettsial pathogens and T. evansi in North African wild ruminants' species. These results extend the knowledge about the diversity of ticks and tick-borne pathogens in wildlife and justify further investigations of the possible role of R. sanguineus sensu lato in the transmission of T. evansi.     

Trypanosoma brucei s.l.: Microsatellite markers revealed high level of multiple genotypes in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus of Cameroon

To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.     

Trypanosoma vivax: mechanical transmission in cattle by one of the most common African tabanids,Atylotus agrestis

The role of mechanical vectors in the transmission of African livestock trypanosomes has always been controversial relative to tsetse flies, their cyclical vectors. An experiment was carried out in Burkina Faso to demonstrate mechanical transmission of Trypanosoma vivax by one of the most common tabanids in Africa: Atylotus agrestis. Eight heifers (crossbred zebuxBaoulé), free of trypanosome infection, were kept in a corral covered by a mosquito net, together with two heifers infected experimentally with a local stock of T. vivax. On average, 324 A. agrestis, freshly captured with Nzi traps, were introduced daily over 20 days. Parasitological, PCR and serological examinations were carried out regularly to assess infections and levels of parasitaemia. Microscopic examination of buffy-coats indicated that five of the eight receiver-heifers were infected on days 8, 13, 32, 41, and 48. PCR results indicated that these five heifers were already infected by day 13. Mechanical transmission of T. vivax by A. agrestis was demonstrated unequivocally, at a high rate (63% in 13-20 days). Conditions of transmission in this experiment are discussed in terms of natural rates of challenge. The importance of tabanids as mechanical vectors of T. vivax should be re-considered, in light of these results. Creation of tsetse free zones in Africa will generally lead to the disappearance of T. congolense, T. brucei, and most often T. vivax as well; however, in areas where T. vivax can be mechanically transmitted, clearance of tsetse may not be sufficient to eradicate livestock trypanosomosis.     

Trypanosoma vivax: Adaptation of Two East African Stocks to Laboratory Rodents1

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10⁹–10¹⁰ trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

Background

Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km² of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases.

Methodology/Principle Findings

the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4).

Conclusion/Significance

this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.     

Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice

Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse’s productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of highly parasitaemic mice that was detectable by PCR was 0.001 ng, using the ESAG6/7 and TBR1/2 primer, (b) using TBR1/2 primer for parasites purified could detect 0.000001 ng and (c) in the prepatent period PCR detect the presence of parasites earlier than parasitological techniques. Nevertheless, the percentage of detection for PCR varies depending on primer employed with 60% and 66% for ITS1 and 21/22-mer, and 80% for ESAG6/7 and TBR1/2. Consequently, TBR1/2 and ESAG6/7 were the best primers to monitor T. evansi infections in mice. For epidemiological application, such comparative evaluation should be made for detection of T. evansi in livestock such as horses.