Molecular Detection of Animal Trypanosomes in Different Animal Species in the Visayas Region of the Philippines.

PURPOSE: Animal trypanosomosis is one of the most important parasitic diseases significantly affecting the Philippine economy. It is considered by the government to be the second most important disease of livestock after fasciolosis. A PCR-based molecular survey for trypanosomes in different animals in Bohol, Philippines, was performed to assess the prevalence of trypanosomosis in the area during the rainy and dry season. METHODS: A total of 269 blood samples were collected in two batches in rainy and dry season from different animal species in Ubay Stock Farm in Ubay, Bohol, the Philippines, including 151 samples from water buffaloes, 76 samples from cattle, 35 samples from goats, and 7 samples from horses. DNA was subsequently extracted from these blood samples, and two different PCR assays were employed to detect and identify trypanosomes DNA including ITS1 PCR and CatL PCR. RESULTS: Animal trypanosomes, Trypanosoma evansi and Trypanosoma theileri, were detected in water buffalo (37.7%) [95%CI: 30.4 - 45.7], cattle (44.7%) [95%CI: 34.1 - 55.9], and goats (34.3%) [95%CI: 20.8 - 50.8]. Only T. evansi was detected in horses (28.6%) [95% CI: 8.2 - 64.1]. No clinical signs were observed in all positive animals. CONCLUSION: This highlights the importance of domestic animals that can be infected with no signs but may act as reservoir animals and transmit trypanosomosis to susceptible animals. This study supports the importance of regular surveillance to estimate the prevalence of the disease, emphasizing its various dynamics in the affected areas and supporting efficient intervention measures.

Seroprevalence of Toxoplasma gondii and Neospora caninum in camels recently imported to Egypt from Sudan and a global systematic review.

Introduction: Toxoplasma gondii and Neospora caninum are closely related intracellular protozoan parasites of medical and veterinary concern by causing abortions and systemic illness. Limited or ambiguous data on the prevalence of T. gondii and N. caninum in camels triggered us to conduct this study. Methods: Camels (n = 460) recently imported from Sudan and destined mainly for human consumption, were tested for specific antibodies against these protozoans using commercially available ELISAs. From the two only quarantine stations for camels from Sudan, 368 camels were sampled between November 2015 and March 2016 in Shalateen, Red Sea governorate, and 92 samples were collected between September 2018 and March 2021 from Abu Simbel, Aswan governorate. Results & Discussion: Overall, seropositive rates in camels were 25.7%, 3.9% and 0.8% for T. gondii, N. caninum and mixed infection, respectively. However, marked differences were found between the two study sites and/or the two sampling periods: For T. gondii, a higher rate of infection was recorded in the Red Sea samples (31.5%, 116/368; odds ratio 20.7, 5.0-85.6; P<0.0001) than in those collected in Aswan (2.2%, 2/92). The opposite was found for N. caninum with a lower rate of infection in the Red Sea samples (0.82%, 3/368; odds ratio 23.7, 6.7-83.9; P<0.0001) than in the samples from Aswan (16.3%, 15/92). Additionally, our systematic review revealed that the overall published seroprevalence of T. gondii and N. caninum was 28.6% and 14.3% in camels worldwide, respectively. To the best of our knowledge, this study provides the first record of seroprevalence of both T. gondii and N. caninum in recently imported camels kept under quarantine conditions before delivery to other Egyptian cities and regions. In addition, our review provides inclusive data on the prevalence of T. gondii and N. caninum in camel globally. This knowledge provides basic data for the implementation of strategies and control measures against neosporosis and toxoplasmosis.

Therapeutic Efficacy of Orally Administered Nitrofurantoin against Animal African Trypanosomosis Caused by Trypanosoma congolense Infection.

Animal African trypanosomosis (AAT) leads to emaciation and low productivity in infected animals. Only six drugs are commercially available against AAT; they have severe side effects and face parasite resistance. Thus, the development of novel trypanocidal drugs is urgently needed. Nitrofurantoin, an antimicrobial, is used for treating bacterial urinary tract infections. Recently, we reported the trypanocidal effects of nitrofurantoin and its analogs in vitro. Furthermore, a nitrofurantoin analog, nifurtimox, is currently used to treat Chagas disease and chronic human African trypanosomiasis. Thus, this study was aimed at evaluating the in vivo efficacy of nitrofurantoin in treating AAT caused by Trypanosoma congolense. Nitrofurantoin was orally administered for 7 consecutive days from 4 days post-infection in T. congolense-infected mice, and the animals were observed for 28 days. Compared to the control group, the treatment group showed significantly suppressed parasitemia at 6 days post-infection. Furthermore, survival was significantly prolonged in the group treated with at least 10 mg/kg nitrofurantoin. Moreover, 100% survival and cure was achieved with a dose of nitrofurantoin higher than 30 mg/kg. Thus, oral nitrofurantoin administration has potential trypanocidal efficacy against T. congolense-induced AAT. This preliminary data will serve as a benchmark when comparing future nitrofurantoin-related compounds, which can overcome the significant shortcomings of nitrofurantoin that preclude its viable use in livestock.

First molecular survey of animal trypanosomes in Paraguayan horses.

Despite the epidemic situation of animal trypanosomosis caused by Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma vivax in South American countries, there are no reports for the prevalence of animal trypanosomes in Paraguay. In this study, 408 blood samples were obtained from apparently healthy horses from sixteen departments of Paraguay, for routine medical check-up from August to September 2019, and a polymerase chain reaction (PCR)-based cross-sectional study was carried out to identify trypanosome prevalence. The prevalence of Trypanozoon (T. evansi and T. equiperdum) and T. vivax was 7.11% (29/408) and 26.23% (107/408), respectively. Mixed infections were detected in 4.90% (20/408) of the samples. Some of the selected trypanosome positive samples were confirmed as T. vivax and T. evansi Type A by sequence analysis of the internal transcribe spacer region and RoTat1.2 variant surface glycoprotein gene, respectively. In conclusion, we found higher prevalence of T. vivax than Trypanozoon in Paraguayan horses. However, the genotypic variation should be verified in further studies.

Cattle anaplasmosis and babesiosis: Major tick-borne diseases affecting the cattle industry in Khartoum State, Sudan.

Tick-borne hemoparasitic (TBH) infections are a major problem affecting livestock industries worldwide, particularly in tropical and subtropical regions. This study was carried out in response to repeated reports from local veterinarians in Khartoum State, Sudan, where TBH infections are prevalent in dairy farms. This cross-sectional study was undertaken from October 2017 to April 2018 with the objective of assessing the prevalence and potential risk factors associated with cattle anaplasmosis and babesiosis in the localities of Omdurman, Khartoum, and Khartoum North, Khartoum State. A total of 292 cattle blood samples collected from apparently healthy animals were examined for the presence of A. marginale, Babesia bigemina, and B. bovis using PCR. The overall prevalence of A. marginale and B. bigemina was found to be 40.41% and 3.42%, respectively, while B. bovis was not detected. Mixed infections with A. marginale and B. bigemina were detected in four (1.37%) cattle. The prevalence of the two pathogens was found to be significantly higher in Khartoum and Omdurman than in Khartoum North. However, no significant difference was observed for the prevalence based on sex, age, breed, and mean packed cell volume values. Our findings indicated that A. marginale is a highly prevalent parasite in Khartoum State, which may be a primary constraint to the cattle industry. Inclusion of this pathogen in the diagnostic protocols, and consequent treatment and tick control are necessary. Moreover, the role of B. bigemina infection may exacerbate the situation to some extent in this region.

Molecular Characterization of Ticks and Tick-Borne Pathogens in Cattle from Khartoum State and East Darfur State, Sudan.

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick-pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.

First molecular detection and identification of Trypanosoma evansi in goats from Cebu, Philippines using a PCR-based assay.

The increasing number and severity of surra outbreaks in the Philippines led the government to consider it as the second most important disease of livestock in the country. It is one of the most economically important animal parasitic diseases and has been reported in several animal species, including water buffaloes, cattle, and horses in different regions of the Philippines. However, it has not yet been reported in Cebu, the usual gateway of livestock trade in the area that raises 6% of the 3.75 million goats in the country. In the current study, a PCR-based assay was conducted for the molecular detection and characterization of Trypanosoma evansi in goats in Cebu. A total of 251 goats were randomly sampled from four farms. DNA was extracted and ITS1-PCR was applied to detect different trypanosomes in goats. Eighty-five out of the 251 (33.9%) samples tested positive for T. evansi, two of which were also positive for T. theileri-like trypanosome. The detection rate of T. evansi was slightly higher in male goats (38.3%) than in females (32.5%), and in younger goats (34.5%) than in adults (33.5%). The findings, however, did not differ significantly to suggest any association between sex and age with T. evansi infection in goats. The detection of T. evansi and T. theileri-like trypanosome in goats was confirmed by sequence analysis of ITS1 region. To our knowledge, this is the first report on the molecular detection and identification of caprine T. evansi infection in Cebu, Philippines.

Serological and molecular detection of selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan.

In Sudan, donkeys are important animals, providing transportation and income possibilities. However, the prevalence of parasites in donkeys in Sudan has not been thoroughly characterized. Accordingly, in this study, we aimed to detect selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan, wherein people depend mainly on donkeys for their daily life. In total, 198 blood samples collected from donkeys in a local market in West Omdurman, were screened using serological and molecular diagnostic techniques. Serologically, 52 (26.3%), 56 (28.3%), and 19 (9.6%) samples were positive for trypanosomosis using Card Agglutination Test for Trypanosoma evansi, Trypanosoma evansi crude antigen -based enzyme-linked immuno sorbent assay (ELISA) and recombinant Trypanosoma evansi GM6-4r-based ELISA, respectively. ELISA for equine piroplasmosis revealed 156 (78.8%) and 10 (5.1%)Theileria equi- and Babesia caballi-positive samples, respectively. PCR detected Trypanosoma congolense, subgenus Trypanozoon, Theileria equi, and Babesia caballi in 18 (9.1%), 77 (38.9%), 18 (9.1%), and 8 (4%) samples, respectively. Of the 77 Trypanozoon-positive samples, 35 (45.5%) were confirmed as Trypanosoma evansi type A. To our knowledge, this is the first report of detection of Trypanosoma congolense in donkeys outside of tsetse-infested areas in Sudan.

Prevalence of different trypanosomes in livestock in Blue Nile and West Kordofan States, Sudan.

African animal trypanosomosis, transmitted cyclically by tsetse flies or mechanically by other biting flies, causes serious health problems in livestock. Although tsetse infestations have been observed in Blue Nile State in Sudan, tsetse was eradicated in West Kordofan in 1962, and no further studies have been carried out. Accordingly, in this study, we investigated the prevalence of trypanosomosis in cattle, sheep, and goats in Blue Nile and West Kordofan States, Sudan. This cross-sectional study was conducted using 70 cattle, 62 sheep, and 116 goats, and the microhematocrit centrifugation technique was used as a parasitological test. KIN-multispecies polymerase chain reaction (PCR) was used to detect Trypanozoon sp., Trypanosoma congolense, and T. vivax; RoTat 1.2 variable surface glycoprotein-specific PCR was used to detect T. evansi; and TviCatL PCR was used to specifically detect T. vivax. The seroprevalence of trypanosomosis was assessed using card agglutination tests CATT/ T. evansi. The parasitological prevalence rates were 4% (3/70) in cattle, 2% (1/62) in sheep, and 4% (5/116) in goats. The molecular prevalence rates of T. vivax, the most prevalent parasite, were 99% (69/70) in cattle, 98% (61/62) in sheep, and 84% (98/116) in goats. Trypanozoon (T. evansi or T. brucie) rates were 30% (21/70) in cattle, 32% (20/62) in sheep, and 12% (14/116) in goats. Among Trypanozoon-positive isolates, T. evansi was confirmed in 24% (5/21) of cattle, 70% (14/20) of sheep, and 86% (12/14) of goats. Finally, T. congolense was recorded only in cattle in Blue Nile State, with a prevalence of 14% (10/70). The seroprevalence rates of CATT/T. evansi were 46% (32/70) in cattle, 45% (28/62) in sheep, and 14% (16/116) in goats. Thus, we confirmed molecularly, for the first time, the presence of Trypanozoon, particularly T. evansi and T. vivax, in sheep and goats in Sudan. Our results show that sheep and goats could be an important reservoir for trypanosomes, potentially leading to the spread of the disease to the northern parts of the country following the movement of these animals. These findings provide important insights into the epidemiology of the disease and could affect the establishment of control strategies against trypanosomosis in Sudan.

Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan.

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.

Utilization of crude and recombinant ELISAs for serodiagnosis of camel trypanosomosis in Sudan.

This study was carried out to evaluate the application of CATT/T. evansi, crude and recombinant (TeGM6-4r) antigen ELISAs in the diagnosis of camel trypanosomosis caused by two trypanosome species, T. evansi and T. vivax, in Sudan. Concurrently, the current situation of camel trypanosomosis was investigated based on the results of a serological analysis. The recombinant tandem repeat antigen TeGM6-4r is conserved among salivarian trypanosome species and was highly sensitive in the detection Trypanozoon, and T. vivax. It has been validated in the diagnosis of surra in cattle and water buffalo but not in camels. A comparative evaluation of a crude antigen ELISA and a recombinant antigen GM6 (rTeGM6-4r) ELISA was performed using 189 blood samples, which included 148 samples obtained from different camel herds in Eastern Sudan and 41 samples from camels that had been brought from Western Sudan to local markets. The results showed that the rTeGM6-4r ELISA detected the greatest number of positive samples (n = 118, 62%), while CATT/T. evansi and the crude antigen ELISA detected the lowest number of positive samples (n = 73, 39%). The kappa value of rTeGM6-4r as compared to TeCA ELISA was 0.5515, which indicated moderate agreement. We concluded that the rTeGM6-4r ELISA is the test of choice for use in screening camel for trypanosomosis caused by T. evansi and T. vivax in Sudan.

Development and validation of direct dry loop mediated isothermal amplification for diagnosis of Trypanosoma evansi.

Non-tsetse transmitted Trypanosoma evansi infection (Surra) is one of the most important diseases of camels in north and east Africa and of buffalo and cattle in Asia. Early, accurate and feasible diagnosis is a crucial step towards the control of Surra. Dry format of loop-mediated isothermal amplification (LAMP) diagnostics for the detection of T. evansi was developed, where the detection limit was determined as to equivalent to one parasite per reaction. The assay was validated by testing blood from 48 camels clinically diagnosed to have Surra, which all tested negative microscopically and revealed 43 (89.6%) to be positive for T. evansi when tested by the dry-LAMP. Furthermore, DNA extracted from a randomly selected subset of 20 of these blood samples were then subjected to RoTat1.2-PCR (TaKara Ex Taq), with 14 matching results, with six that were positive by dry-LAMP and negative by PCR. The kappa value of dry-LAMP applied to direct blood was 0.4211, indicating moderate agreement to RoTat 1.2-PCR. In addition, 103 genomic DNA extracted from camels' blood were tested by both dry-LAMP and RoTat1.2-PCR revealed 67 matching results and 31 positive by dry-LAMP and negative by PCR and a further five positives by PCR and negative by dry-LAMP. This novel dry-LAMP method is more sensitive than conventional PCR, direct (without DNA extraction step), is user friendly and does not require cold chain or highly trained personnel.

Author Correction: Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host.

Plasmodium was first identified in a goat in Angola in 1923, and only recently characterized by DNA isolation from a goat blood sample in Zambia. Goats were first domesticated in the Fertile Crescent approximately 10,000 years ago, and are now globally distributed. It is not known if the Plasmodium identified in African goats originated from parasites circulating in the local ungulates, or if it co-evolved in the goat before its domestication. To address this question, we performed PCR-based surveillance using a total of 1,299 goat blood samples collected from Sudan and Kenya in Africa, Iran in west Asia, and Myanmar and Thailand in southeast Asia. Plasmodium DNA was detected from all locations, suggesting that the parasite is not limited to Africa, but widely distributed. Whole mitochondrial DNA sequences revealed that there was only one nucleotide substitution between Zambian/Kenyan samples and others, supporting the existence of a goat-specific Plasmodium species, presumably Plasmodium caprae, rather than infection of goats by local ungulate malaria parasites. We also present the first photographic images of P. caprae, from one Kenyan goat sample.

Detection and molecular characterization of tick-borne pathogens infecting sheep and goats in Blue Nile and West Kordofan states in Sudan.

Tick-borne pathogens (TBPs) are common in livestock of sub-Saharan Africa. However, information regarding TBPs in sheep and goats in Sudan is limited. In this study, 178 blood samples of sheep and goats in Blue Nile and West Kordofan states were investigated for TBPs using PCR. Overall, 110 (61.8%) samples were found to be infected with at least one of the following pathogens: Anaplasma ovis, Theileria ovis, and Ehrlichia ruminantium. Babesia ovis and T. lestoquardi were not identified. A. ovis was the most prevalent pathogen (n = 107, 60.1%), followed by T. ovis (n = 23, 12.9%) and E. ruminantium (n = 1, 0.6%). The prevalence rates of A. ovis and T. ovis were significantly higher in sheep than in goats. Phylogenetic analysis of T. ovis 18S rRNA and A. ovis msp4, groEL, and 16S rRNA, revealed that the pathogens identified in this study are clustered together, indicating similar molecular characteristics. Additionally, phylogenetic analysis of E. ruminantium pCS20 revealed that E. ruminantium in this study belong to the West Africa group, and different to E. ruminantium previously identified in ticks from Sudan. We concluded that TBPs are highly prevalent in the study area and continuous monitoring of TBPs in sheep and goats in Sudan is highly required.

The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis.

Trypanosoma equiperdum, which is the etiological agent of dourine, spreads through sexual intercourse in equines. Dourine (T. equiperdum) has been reported in Mongolia, where it is considered an economically important disease of horses. T. evansi has also been reported in Mongolian domestic animals. The objective of this study was to evaluate the potential application of recombinant T. evansi GM6 (rTeGM6-4r)-based diagnostic methods on a farm with an outbreak of non-tsetse transmitted horse trypanosomosis. Ninety-seven percent homology was found between the amino acid sequences of T. equiperdum GM6 and the GM6 of another Trypanozoon, which also shared the same cellular localization. This finding suggests the utility of rTeGM6-4r-based serodiagnostic methods for epidemiological studies and the diagnosis of both surra and dourine in Equidae. Fifty blood samples were examined from a herd of horses. The diagnostic value of an rTeGM6-4r-based ELISA and an rTeGM6-4r-based immunochromatographic test (ICT) were measured in comparison to a T. evansi crude antigen-based ELISA, which is a diagnostic method recommended by the OIE. However, this is not a perfect diagnostic method for trypanosomosis. Positive serum samples were detected in 46%, 42% and 28% of the tested horses using an rTeGM6-4r-based ELISA, crude antigen-based ELISA and rTeGM6-4r-based ICT, respectively. The sensitivity of rTeGM6-based ELISA was 81%, the specificity was 79%, and the agreement was moderate. We conclude that rTeGM6-4r-based ELISA and ICT represent alternative options for baseline epidemiological studies and the on-site diagnosis of horse trypanosomoses in the field, respectively.

Molecular characterization of bovine Cryptosporidium isolated from diarrheic calves in the Sudan.

Cryptosporidiosis is a common protozoan infection causing morbidity and mortality in young cattle and may be zoonotically transmitted to humans. So far, there is no data available on the presence of Cryptosporidium spp. in the Sudan. The aim of this study was to isolate, identify, and genotype Cryptosporidium oocysts sampled from diarrheic calves housed at different farms in three different municipalities in Khartoum State (Khartoum, Khartoum North, Omdurman). A total of 149 fecal samples were evaluated microscopically for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method and 87 (58.3%) samples tested positive. Positive and negative samples were further analyzed by nested PCR targeting the SSU rRNA region. Positive samples were subjected to restriction enzyme analysis of PCR amplicons (PCR-RFLP). Nested PCR identified Cryptosporidium DNA in 53 samples (35.5%); restriction digestion of the PCR products revealed the presence of C. parvum (73.5%), C. ryanae (13.2%), C. andersoni (7.5%), and C. bovis (1.8%). Species distribution was clearly related to age with C. parvum being the predominant species in dysenteric pre-weaned calves. Sequencing of three genes (SSU rRNA, COWP, and GP60) for three C. parvum isolates originating from the three different municipalities showed that all belong to C. parvum subtype family IId. Based on data obtained by GP60, sequencing the two C. parvum isolates from Khartoum and Omdurman represent subtype IIdA18G1, whereas oocysts isolated in Khartoum North belong to subtype IIdA19G1. The observed genotypes are zoonotic and thus C. parvum in calves is potentially a health risk to humans in Khartoum State, Sudan. To the best of our knowledge, this is the first reported attempt to characterize Cryptosporidium isolated from cattle in the Sudan.

The incrimination of three trypanosome species in clinically affected German shepherd dogs in Sudan.

Canine trypanosomosisis (CT) is a common disease caused by tsetse- and non-tsetse-transmitted trypanosomes worldwide. The severity of the disease varies from acute, sub-acute to chronic with non-specific clinical signs. Here, we attempt in a cross-sectional study to assess the current situation of CT and the role of dogs in transmitting trypanosomes to other domesticated animals. The study was carried out during July 2016 on 50 caged German shepherd dogs in Khartoum State to investigate the prevalence of dog trypanosomosis using both serological (CATT/Trypanosoma evansi) and molecular (KIN-PCR, RoTat1.2 VSG-PCR and TviCatL-PCR) tests to detect possible trypanosome infections. CATT/T. evansi detected antibodies against T. evansi in 15 (30%) dogs, while parasite DNA was detected in 17 (34%) dogs by RoTat1.2 PCR. In contrast, a KIN-PCR detected the subgenus Trypanozoon, Trypanosoma congolense savannah, T. congolense Kenya and T. vivax in 36 (72%), 3 (6%), 1 (2%), and 2 (4%) dogs, respectively. However, a species-specific PCR for Trypanosoma vivax was detected 7 (14%) positive cases. We concluded that CT was caused by at least three species of trypanosomes, namely T. evansi, T. vivax and T. congolense. Trypanozoon other than T. evansi could not be ruled out since other tsetse-transmitted trypanosomes have also been detected and species-specific PCRs were not used. This study illustrates that dogs play an important role in the transmission dynamic and the epidemiology of the abovementioned trypanosome species.

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

BACKGROUND: This 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. RESULTS: The 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. CONCLUSION: We 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.