VSG Gene Research Articles

Equine Trypanosomiasis: Molecular Detection, Hematological, and Oxidative Stress Profiling.

Surra caused by Trypanosoma evansi (T. evansi) is widely distributed and has significant impact on equine sector and international trades. However, there are no available data about the genetic characterization of this parasite in horses in Egypt. So, the goal of this study was to study the molecular characterization of T. evansi in horses and determine the changes in hematological parameters and oxidative stress associated with T. evansi infection. A total of 12 horses were examined using PCR targeting RoTat 1.2 VSG gene, and we evaluated the changes in hematological and oxidative stress between infected and healthy animals. The results revealed a notable reduction in red blood cell (RBC), hematocrit (HCT), and hemoglobin (Hb) levels in the infected horses, as compared to the control healthy group. In contrast, the infected group showed a substantial increase in mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV). In addition, the infected group exhibited monocytopenia, eosinopenia, and notable lymphocytopenia. Regarding oxidative stress profile, the infected horses showed decreased levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and antioxidant capacity (TAC) compared to the control group. Moreover, the PCR assay targeting RoTat 1.2 VSG gene revealed positive specific band (205 bp) in all examined samples for T. evansi and the phylogenetic analysis demonstrated that strain of this study is closely related to T. evansi isolate in horses from India (MT501210) while showed difference from sequences of other species. The results emphasize the changes in blood composition and the body's response to oxidative stress caused by T. evansi infection in horses.

Molecular and genetic diversity in isolates of Trypanosoma evansi from naturally infected horse and dogs by using RoTat 1.2 VSG gene in Madhya Pradesh, India.

Trypanosoma evansi is a protozoan parasite that can infect a wide range of animals and is widespread around the world. In this study, we analyzed four fatal cases of T. evansi infection using clinical, parasitological, and molecular approaches. We also explored the genetic diversity, demographic history, and population-genetic structure of T. evansi using available Rode Trypanozoon antigenic type (RoTat) 1.2 gene sequences. Clinical findings of infected animals revealed high fever, anemia, weakness, and anorexia. The animals were treated with diminazene aceturate, which was moderately effective, and hematobiochemical parameters showed changes in hemoglobin and glucose levels. The molecular and genetic diversity of T. evansi was analyzed using the RoTat 1.2 VSG gene. Phylogenetic and haplotype analysis revealed two distinct clusters of T. evansi circulating in India. The genetic diversity indices, neutrality tests, gene flow, and genetic differentiation outcomes confirmed the genetic diversity of the T. evansi population, with a lack of uniformity. The identification of two distinct clusters, exhibiting differential demographic histories and evolutionary forces, implies that the clusters may have undergone independent evolutionary trajectories or experienced different environmental pressures. The present findings underlined the need of an early and precise diagnosis in order to treat and control T. evansi infections, and the RoTat 1.2 VSG gene is an important genetic marker for understanding the genetic diversity and evolutionary history of T. evansi. This knowledge can be used to create tailored strategies to control and manage the infection in an endemic region.

Silent Trypanosoma evansi infection in humans from India revealed by serological and molecular surveys, and characterized by variable surface glycoprotein gene sequences

BackgroundThe importance of emerging atypical human trypanosomosis is gaining momentum due to increasing detection and its possible impact on human health. A cross sectional study of atypical human trypanosomosis due to Trypanosoma evansi was carried out in Kolkata and Canning area of West Bengal state of India where previously a death was reported. MethodsIn this study blood and serum samples from 173 individuals were collected during August to December 2014. To check the presence of antibodies against T. evansi, card agglutination test and for the presence of T. evansi specific DNA, PCR were conducted. ResultsT. evansi infection was identified in 5.2% (9/173) human blood samples by CATT serological test (Card agglutination test for trypanosomosis). PCR targeting VSG gene sequences suggested active T. evansi infection in 2.89% (5/173). VSG gene sequences herein determined for five isolates from human cases shared high similarity (89.4–100%). Phylogenetic inference clustered the human isolates with other isolates from different host species from India and other countries, forming a clade exclusive of Indian isolates (84.0 to 100% sequence similarity). ConclusionFirst report of symptomless human T. evansi infection detected by combined serological and PCR assays. First phylogenetic analysis of VSG gene sequences including human isolates of T. evansi in which Indian isolates of T. evansi from human and other hosts clustered in a single clade.

POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.

Trypanosoma brucei causes human African trypanosomiasis and sequentially expresses distinct VSGs, its major surface antigen, to achieve host immune evasion. VSGs are monoallelically expressed from subtelomeric loci, and telomere proteins regulate VSG monoallelic expression and VSG switching. T. brucei telomerase is essential for telomere maintenance, but no regulators of telomerase have been identified. T. brucei appears to lack OB fold-containing telomere-specific ssDNA binding factors that are critical for coordinating telomere G- and C-strand syntheses in higher eukaryotes. We identify POLIE as a telomere protein essential for telomere integrity. POLIE-depleted cells have more frequent VSG gene conversion-mediated VSG switching and an increased amount of telomeric circles (T-circles), indicating that POLIE suppresses DNA recombination at the telomere/subtelomere. POLIE-depletion elongates telomere 3′ overhangs dramatically, indicating that POLIE is essential for coordinating DNA syntheses of the two telomere strands. POLIE depletion increases the level of telomerase-dependent telomere G-strand extension, identifying POLIE as the first T. brucei telomere protein that suppresses telomerase. Furthermore, depletion of POLIE results in an elevated telomeric C-circle level, suggesting that the telomere C-strand experiences replication stress and that POLIE may promote telomere C-strand synthesis. Therefore, T. brucei uses a novel mechanism to coordinate the telomere G- and C-strand DNA syntheses.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle

BackgroundSerious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle.MethodsMolecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia.ResultsA total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83–81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98–100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle.ConclusionWe present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

Molecular identification and phylogenetic analysis of Trypanosoma evansi in dromedaries (Camelus dromedarius) from Iran

Surra is of great concern to countries in the world such as Iran, which have a considerable camel population. The present study was aimed at determining the prevalence of Trypanosoma evansi in the camels of Iran. A total of 167 blood samples from farmed camels were examined for the presence of T. evansi infection using parasitological and molecular methods. Blood smear examination revealed 10 (6%) positive samples, while the PCR method 14 (8.4%) found positive samples. Age, sex, and region were not determined as risk factors for T. evansi infection in this study. The phylogenetic tree inferred from VSG gene sequences of T. evansi clearly separated the sequences of this study into two clades, A and B, which reflects the intrasequence heterogeneity among Iranian isolates. The phylogenetic tree showed that Iranian T. evansi strains are members of the T. brucei clade.

Molecular detection of Trypanosoma evansi in dogs from India and Southeast Asia

Trypanosoma evansi, the causative agent of surra, is a hemoflagellate protozoan mechanically transmitted by hematophagous flies, mainly in tropical and subtropical regions. This protozoan affects several mammalian hosts, including dogs, which are highly susceptible to the infection. To investigate the occurrence of T. evansi in dogs, a total of 672 DNA samples from India (n = 228), Indonesia (n = 57), Malaysia (n = 45), the Philippines (n = 103), Thailand (n = 120), and Vietnam (n = 119) were screened by using species-specific conventional PCR. Of the tested dogs, 10 (1.5%) scored positive to T. evansi. In particular, positive samples were detected in canine blood samples collected from India (n = 4; 1.8%), Indonesia (n = 4; 7%), and Malaysia (n = 2; 4.4%). All tested samples from the Philippines, Thailand and Vietnam were negative. Nucleotide sequence analysis revealed a high variation (i.e. from 0.4% to 6.2%) among the RoTat 1.2 variant surface glycoprotein (vsg) gene. Although the number of sequences included in this analysis is relatively small, this nucleotide variation may indicate the divergence of T. evansi RoTat 1.2 vsg gene among different strains. The high incidence of T. evansi previously reported in cattle and buffaloes in India and Southeast Asia suggests that these animals are the main source of infection to dogs.

Mining the pervasiveness of surra in different animal species of Northeastern states of India: Assam, Mizoram and Tripura.

The online version contains supplementary material available at 10.1007/s12639-021-01392-z.

Associative Genetic Diversity of RoTat 1.2 VSG in Different Trypanosoma evansi Isolates.

Numerous phylogenetic markers have been tested over a period of time for delineating evolutionary history of haemoflagellate-Trypanosoma evansi. To find out the associative genetic diversity, within the various isolates of T. evansi across the globe, based on RoTat 1.2 VSG gene. A total of 5 equine isolates of T. evansi from Northern India were characterized. PCR products were sequenced and sequences were compared with available sequences across India and world. Phylogenetic tree was constructed based on maximum parsimony (MP) method with the tree-bisection-regrafting (TBR) algorithm. Indian isolates formed multiple clades with two haplotypes. The present isolates showed 99.49-100.00% nucleotide homology within themselves. On broader line, Indian isolates were found to be closer to Egyptian isolates than the African counterparts. Few of the Indian isolates showed marked resemblance with a particular Egyptian isolate than with their Indian counter parts. Another remarkable finding is the close association of equine isolates from India with other equine isolates and their clear divergence from isolates of T. evansi affecting other hosts from India and abroad. Vast genetic divergence was seen between the isolates suggesting of multiple distinct lineages of T. evansi amongst the Indian livestock. Interestingly, variations in sequences were seen based on the host range of isolates. The findings are very important from molecular evolutionary point of view.

TRYPANOSOMA EVANSI AND NEOSPORA CANINUM AMONG WATER BUFFALOES (BUBALUS BUBALIS) IN THE PHILIPPINES

The study determined the positivity rate of Trypanosoma evansi and Neospora caninum antibodies in water buffaloes in the province of Nueva Ecija, Philippines using Polymerase Chain Reaction (PCR) for T. evansi and competitive Enzyme-linked Immunosorbent Assay (cELISA) for N. caninum antibodies . A total of 100 whole blood and 100 serum samples were collected to test for T. evansi and N. caninum , respectively. Rotat 1.2 VSG gene was target using PCR for T. evansi detection. Neospora caninum antibody detection was done from the serum samples using cELISA test kit. Results revealed that the positivity rate of T. evansi in Nueva Ecija was 11% (11/100). The positive animals identified were from the municipalities of Munoz (4/16; 25%), Sta. Rosa (3/13; 23.08%) and Talugtug (4/16; 25%). The seropositive rate of Nueva Ecija for N. caninum. was 46% (46/100), seropositive animals were identified in Cabanatuan City, 57.14% (4/7); Science City of Munoz, 43.14% (22/51); Sta. Rosa, 40% (4/10); Sto. Sunday, 50% (6/12); and Talugtug 50% (10/20). The seropositivity rate of N. caninum and the presence of T. evansi in Nueva Ecija may contribute to the cases of abortions in the province and further studies should be employed to confirm the association of these organisms to abortion cases on water buffaloes.

Development of a recombinase polymerase amplification lateral flow assay for the detection of active Trypanosoma evansi infections.

Animal trypanosomosis caused by Trypanosoma evansi is known as "surra" and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings. An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool. Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.

Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription.

In Trypanosoma brucei, genes are arranged in Polycistronic Transcription Units (PTUs), which are demarcated by transcription start and stop sites. Transcription start sites are also binding sites of Origin Recognition Complex 1 (ORC1). This spatial coincidence implies that transcription and replication in trypanosomes must occur in a highly ordered and cooperative manner. Interestingly, a previously published genetic screen identified the T. brucei MCM-BP, which interacts with subunits of MCM helicase, as a protein whose downregulation results in the loss of transcriptional silencing at subtelomeric loci. Here, I show that TbMCM-BP is required for DNA replication and transcription. TbMCM-BP depletion causes a significant reduction of replicating cells in S phase and genome-wide impairments of replication origin activation. Moreover, levels of sense and antisense transcripts increase at boundaries of PTUs in the absence of TbMCM-BP. TbMCM-BP is also important for transcriptional repression of the specialized subtelomeric PTUs, the Bloodstream-form Expression-Sites (BESs), which house the major antigenic determinant (the Variant Surface Glycoprotein, VSG gene) as well as TbORC1 binding sites. Overall, this study reveals that TbMCM-BP, a replication initiation protein, also guides the initiation, termination and directionality of transcription.

Molecular detection of equine trypanosomiasis in the Riyadh Province of Saudi Arabia.

We conducted a cross-sectional study to detect trypanosome infections of horses and donkeys in the Riyadh Province of Saudi Arabia. DNA was extracted from blood samples collected from 368 horses and 142 donkeys, and subjected to universal first ribosomal internal transcribed spacer region (ITS1)-PCR followed by Trypanosoma evansi species-specific RoTat1.2-PCR. The universal ITS1-PCR revealed T. evansi infection in horses ( n = 12; 3.3%) and donkeys ( n = 4; 2.8%). There was no significant effect of sex or age on the prevalence of trypanosomiasis in horses or donkeys. Application of the RoTat1.2-PCR revealed that the RoTat1.2 VSG gene was absent from the positive ITS1-PCR samples of 3 horses and 1 donkey. This discrepancy could be explained by the circulation of T. evansi type B in Saudi Arabia; however, this suspicion requires confirmation.

Evaluation of multiplex PCR assay for detection of Babesia spp, Ehrlichia canis and Trypanosoma evansi in dogs

A multiplex PCR test was evaluated to detect the DNA of three important dog haemoparasites by comparing with singular PCR counterpart on clinical blood samples of dogs in and around Chennai, Tamil Nadu, India. Initial screening of samples was done by microscopic examination of peripheral blood smear and singular PCR and those found exclusively positive for Babesia spp, Ehrlichia canis and Trypanosoma evansi and concurrent infections were used to standardize multiplex PCR. Amplicons of 619 bp, 377 bp and 227 bp corresponding to Babesia spp (18S rRNA gene), E. canis (VirB9 gene), and T.evansi (VSG gene) respectively were amplified, without any non-specific amplification. The laboratory sensitivity (91.7% to 100%) and specificity (100%) of the multiplex PCR were calculated using ‘true positive’ and ‘true negative’ dog blood samples obtained in the initial screening process. Clinical blood samples from 287 dogs were screened using singular PCR and multiplex PCR tests for the presence of genome of Babesia spp, E. canis and T. evansi. The multiplex PCR was found to have high level of diagnostic specificity (97.5%–100%) in the detection of all three dog blood parasites and high level of diagnostic sensitivity (95%) in the detection of T. evansi from field level clinical blood samples compared to the singular PCR. However, the diagnostic sensitivity of the multiplex PCR was found to be low to moderate (40.45%–66.7%) in detection of Babesia spp and E. canis from field level clinical blood samples. The strength of agreement between singular and multiplex PCR assays was ‘moderate’ (0.445), ‘good’ (0.708) and ‘very good’ (0.968) in detection of DNA of Babesia spp, E. canis and T. evansi. The multiplex PCR was found to be 10 fold less sensitive in comparison with the singular PCR counterpart.

MOLECULAR EPIDEMIOLOGICAL STUDIES ON TRYPANOSOMA EVANSI TYPE A AND TYPE B IN CAMELS (CAMELUS DROMEDARIES) FROM FIVE DIFFERENT REGIONS OF SAUDI ARABIA USING THE ITS1 RDNA AND ROTAT 1.2 VSG GENE

Trypanosoma evansi is the most widespread of the pathogenic salivarian trypanosomes and cause a serious disease called (surra) that is affect the domestic animals such camels and horses in Tropical and subtropical countries and often leads to reduced productivity and economic losses. Therefore, the objectives of the present study were to determine the prevalence rates of trypanosomiasis using polymerase chain reaction (PCR) among camels from five different regions of Saudi Arabia and to sequence and characterized the T. evansi from these animals. In the current study, 832 camel blood samples collected from five different regions of Saudi Arabia for detecting T. evansi. A generic ITS1-PCR and RoTat 1.2 VSG gene were applied in this study to analyze camels’ blood samples. Molecular analysis was performed using ITS1-PCRwhich showed that the highest prevalence of trypanosomes was observed in Al-Qaseem province (50.1%) followed Riyadh province (49%), whereas in Hail and the Northern Borders, there were fewer infections with trypanosomes (28.4% &17.6%), respectively. PCR amplification was carried out targeting RoTat 1.2 VSG gene on TS1-positive samples and some of them were negative for RoTat1.2. The test negative in RoTat 1.2 PCR but ITS1 PCR positive could suggest T. evansi type B. Presence of T. evansi type B is interest to the international community, as this has a message to redesign the existing molecular and serological diagnostic markers. However, to our knowledge this the first study demonstrating T. evansi type B out of Africa.

Parasitological and Molecular Detection of Canine Trypanosomiasis From Riyadh Province, Saudi Arabia.

Trypanosoma evansi is the most widespread of the pathogenic salivarian trypanosomes; it causes a serious disease called surra that affects domestic animals such as camels, horses, and dogs, and often leads to reduced productivity and economic losses. Therefore, the objectives of the present study were to determine the prevalence rates of trypanosomiasis using 3 parasitological tests (wet blood film, Giemsa staining, and microhematocrit centrifugation technique) and polymerase chain reaction (PCR) among stray dogs from Riyadh Province, Saudi Arabia. In the current study, 117 dog blood samples collected from certain districts of Riyadh Province showed that 5 of 117 dogs (4.3%) were positive for the genus Trypanosoma. In addition, the findings indicated no effect of dog gender or age on parasite infection. For a more specific diagnosis, PCR amplification of the RoTat 1.2 VSG gene in 5 internal transcribed spacer1-positive samples diagnosed with Trypanosoma indicated that 2 were positive for RoTat 1.2 T. evansi. The absence of the RoTat 1.2 VSG gene in 3 of the 5 T. evansi-positive samples could be explained by the circulation of T. evansi type B in dogs from Saudi Arabia. Thus, this is the first study demonstrating T. evansi type B outside of Africa.

Molecular characterization and sequence phylogenetic analysis of Babesia bigemina cattle isolate from Mathura based on 18S ribosomal DNA gene

VSG is considered to be an important molecule for both molecular and serological based detection of surra. Of late, there are reports of variation and/or absence of RoTat 1.2 evansi VSG from a fewer isolates of murine and wild life origin. Hence, in order to use VSG for routine diagnosis of trypanosomiosis, there is need for selection of that portion of gene, which is conserved in all trypanosomes irrespective of species and geographical distribution. Hence, we identified a 205 bp portion of VSG, which is conserved in Trypanosomes.The present communication deals with molecular cloning and characterization of this Trypanozoan specific VSG gene from T. evansi of horse origin from semi arid region of India and checked its uniqueness by comparing it with various other isolates across the world. The selected truncated showed cent percent homology with all other isolates, thereby, justifying its uniqueness.

Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi

BackgroundThis study was conducted in response to recurring reports from eastern Sudan of camel trypanosomosis that can no longer be treated by currently available trypanocidal drugs. One hundred and eighty-nine blood samples were obtained from camels in different herds and local markets in the western part of Sudan, and a cross-sectional study was carried out between December 2015 and February 2016 to identify the causative agents and possible circulating genotypes.ResultsThe prevalence of trypanosomes detected using the conventional parasitological techniques of Giemsa-stained blood smears, wet blood smears and the microhematocrit centrifugation technique (MHCT) was 7% (13/189), 11% (21/189) and 19% (36/189), respectively. However, a multi-species KIN-PCR targeting the ITS region revealed that the prevalence of Trypanosoma evansi was 37% (70/189), while that of T. vivax was 25% (47/189). Consequently, we used a T. evansi-specific PCR (RoTat1.2 VSG gene) to analyse the KIN-PCR-positive samples and a T. vivax-specific PCR (Cathepsin L-like gene) to analyse all of the samples. The prevalence of T. evansi was 59% (41/70), while the prevalence of T. vivax was 31% (59/189). Mixed infections were detected in 18% (34/189) of the samples. These results were further confirmed by sequencing and a phylogenetic analysis of the complete internal transcribed spacer (ITS) region of T. evansi and the TviCatL gene of T. vivax.ConclusionWe conclude that T. vivax was newly introduced to the camel population and that T. evansi is no longer the single cause of camel trypanosomosis in Sudan. The presence of T. vivax in camels detected in this study is a challenge in the choice of diagnostic approaches, particularly serology, and PCRs. However, an analysis of drug resistance should be performed, and the genotypic variation should be verified. To our knowledge, this is the first molecular study on T. vivax and mixed-infection with T. vivax and T. evansi in Sudanese camels.

Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription.

Seroprevalence of Camel (Camelus dromedarius) Trypanosomiasis, with special Reference to Gene Sequencing of Trypanosoma evansi in Sharkia Governorate

A total of 330 camels ( Camelus dromedarius) of both sexes and with different ages, at different localities in Sharkia Governorate, Egypt, were examined clinically from April 2013 to March 2015. Some adult camels showed emaciation, edema of legs and abdomen which were suspected to be infected by Trypanosoma species. This study aimed to determine the seroprevalence of trypanosomiasis using indirect ELISA test and the results showed that 174 (52.7%) were infected. The infection rate was higher in males (54.8%) than females (42.2%), also, in camels more than 2-years old and in summer and spring seasons. Thirty blood samples from the examined animals by ELISA test (15 positive and 15 negative) were subjected to PCR. The results revealed that 18 samples were positive for T. evansi by PCR. Two positive samples for RoTat1.2 VSG encoding gene were chosen for DNA sequence analysis, one of them was obtained from newly imported camel in a camel farm at Belbais city and the other one belonged to a camel from one of Belbais villages at Sharkia Governorate. Nucleotide sequence alignment of RoTat1.2 VSG gene variants from Egyptian T. evansi showed some heterogeneity with other T. evansi isolates from Egypt. In conclusion, PCR technique is more sensitive and specific than ELISA for the diagnosis of T. evansi infection in camels.