Clinico-haematobiochemical and cardiac alterations in Trypanosoma evansi infected buffaloes of Andhra Pradesh, India.

The present research aimed to document the incidence, clinical signs, haematological, and serum biochemical alterations, as well as electrocardiography and echocardiography findings in 62 buffaloes (selected from a total of 240) infected with Trypanosoma evansi. The study spanned one year, from January 2022 to December 2022. Morphological identification of Trypanosoma evansi was done by the presence of a centrally positioned nucleus with a small sub-terminal kinetoplast at the posterior position through microscopic examination of Giemsa stained peripheral blood smears. The incidence of trypanosomosis were determined to be 26% (62/240) using stained blood smear examination and 41% (98/240) through polymerase chain reaction assay. Clinical signs exhibited by buffaloes with trypanosomosis included the lack of rumination (94%; 58/62), anorexia (90%; 56/62), emaciation (87%; 54/62), loss of milk yield (84%; 52/62), ocular discharges (82%; 51/62), depressed demeanour (81%; 50/62), sunken eye balls (61%; 38/62), fever (60%; 37/62), scleral congestion (56%; 35/62) and intermittent fever (42%; 26/62). Cardiovascular clinical findings in affected buffaloes included tachycardia (44%; 27/62), cardiac arrhythmia (24%; 15/62), cardiac murmurs (19%; 12/62) and muffled heart sounds (18%; 11/62). In the present study, buffaloes with trypanosomosis exhibited significant reduction in haemoglobin (p = 0.008), packed cell volume (p = 0.004), total erythrocyte count (p = 0.003), mean corpuscular volume (p = 0.042), total leucocyte count (p = 0.048) and absolute neutrophil count (p = 0.012); a significant increase in absolute eosinophil count (p = 0.011) and absolute monocyte count (p = 0.008) compared to the apparently healthy buffaloes. Additionally significant decrease in albumin (p = 0.001), A/G ratio (p = 0.007), calcium (p = 0.008), glucose (p = 0.007), phosphorous (p = 0.048), sodium (p = 0.008), potassium (p = 0.041) and chloride (p = 0.046) were observed in buffaloes with trypanosomosis compared to healthy ones. Buffaloes with trypanosomosis also showed significant increase in globulin (p = 0.004), aspartate aminotransferase (p = 0.008), bilirubin (p = 0.034), blood urea nitrogen (p = 0.071), creatinine (p = 0.029), cholesterol (p = 0.046), lactate dehydrogenase (p = 0.009), gamma-glutamyl transferase (p = 0.004) and creatine kinase-myoglobin binding levels (p = 0.005). Electrocardiography explorations in buffaloes with trypanosomosis revealed sinus tachycardia, low voltage QRS complex, ST segment elevation, wide QRS complex, sinus arrhythmia, sinus bradycardia, wandering pace maker, first degree atrio ventricular block, biphasic T wave and tall T wave. Echocardiography examination unveiled cardiac chamber dilatation, ventricular wall thickening and indications of pericarditis/cardiac tamponade. Necropsy was carried on the dead buffaloes during the study period disclosed severely congested blood vessels on epicardial surface, endocardial haemorrhages, and presence of pericardial fluid. Histopathological examination of the heart revealed hyaline degeneration, haemorrhages in the cardiac muscles and varying degrees of degenerative changes. Additionally, the pericardium displayed increased thickness due to presence of more elastic fibres, fibroblast cells in the myocardium, discontinuity of muscle layers, vascular congestion, perivascular mono nuclear cell infiltration and augmented thickness of the endocardium with fibroblast cell proliferation. The study's conclusion highlights cardiac alterations as secondary complications in buffaloes infected with Trypanosoma evansi. Further investigations are recommended to elucidate therapeutic modifications and refine the treatment paradigm.

A Survey on Trypanosoma evansi (Kinetoplastida, Trypanosomatidae) Infection in Domestic Animals in a Surra Endemic Area of Southern Algeria.

Background: Trypanosoma (T.) evansi infection is endemic in dromedary camels (Camelus dromedaries) of southern Algeria. Materials and Methods: In order to assess the presence of T. evansi in other domestic animals living together with dromedary camels, a study was conducted in the wilayate of Béchar, El Bayadh, Ouargla and Tamanrasset, between 2015 and 2017. Authorisation to conduct the survey was obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development and Fisheries). A total of 190 animals were sampled, including 42 cattle (Bos taurus), 11 dogs (Canis familiaris), 44 horses (Equus caballus), 3 donkeys (Equus asinus) and 1 mule, 49 goats (Capra hircus) and 40 sheep (Ovis aries). These animals were examined by parasitological (Giemsa stained thin smear, GST), serological (card agglutination test for trypanosomosis (CATT/T. evansi), enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2 [ELISA/VSG RoTat 1.2], immune trypanolysis [TL]) and molecular tests (T. evansi type A specific RoTat 1.2 PCR). Results and Conclusions: The CATT/T. evansi was positive in 10/42 cattle, 0/11 dogs, 2/48 equids, 27/49 goats and 15/40 sheep. On the other hand, 20/38 cattle, 1/9 dogs, 21/42 equids, 17/44 goats and 31/39 sheep were positive in ELISA/VSG RoTat 1.2. However, no single animal was positive in TL. In addition, the T. evansi parasite could not be demonstrated by either GST or RoTat 1.2 PCR in any of the examined animals. This may suggest cross-reactions of CATT/T. evansi and ELISA/VSG RoTat 1.2 with other pathogenic or commensal trypanosome species such as T. vivax or other parasites. Based on these data, in particular taking into account the high specificity of the TL for T. evansi type A, this study does not support the hypothesis that T. evansi circulates in the studied domestic animal species and that they would act as reservoirs for the parasite that causes trypanosomosis in dromedary camels.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

PURPOSE: This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. METHODS: A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. RESULTS: The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. CONCLUSION: It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Assessment of genetic diversity of Trypanosoma evansi in the domestic animal populations through ITS-1 gene sequence analysis.

Trypanosoma evansi infects domestic animals, causing a debilitating and occasionally fatal disease. The disease leads to significant economic losses to farmers and poses a substantial impediment to the growth of livestock production in developing nations, including India. Considering the challenges associated with managing this infection, there is an urgent need to enhance our understanding of the molecular and genetic diversity of T. evansi. Therefore, this study was planned to analyze the genetic diversity of T. evansi using available internal transcribed spacer-1 (ITS-1) gene sequences from India and compare them with sequences from around the globe. Blood samples used in this study were collected from naturally infected animals including dogs, cattle, and buffaloes in the Indian state of Madhya Pradesh. Using the ITS-1 gene, we amplified a 540 base pairs (bp) segment using polymerase chain reaction (PCR), sequenced it, and identified intra-specific variations. Phylogenetic analysis of 90 sequences, including 27 from India, revealed three distinct clusters with high bootstrap support values. A haplotype network analysis identified 34 haplotypes, with H7 being the most prevalent, indicating a complex evolutionary history involving multiple countries. The genetic analysis of the Indian population revealed distinct characteristics. Despite low nucleotide diversity, there was high haplotype diversity in comparison to other populations. Tajima's D, Fu and Li's D, and Fu and Li's F exhibited non-significant negative values, indicating potential stability. Additionally, the slightly positive values in Fu's Fs, Raggedness (r), and Ramos-Onsins and Rozas (R2) statistics suggested a lack of recent significant selective pressures or population expansions. Furthermore, the presence of genetic differentiation and gene flow among T. evansi populations highlighted ongoing evolutionary processes. These findings collectively depicted a complex genetic landscape, suggesting both stability and ongoing evolutionary dynamics within the Indian population of T. evansi. The findings of this study are important for understanding the evolutionary history and population dynamics of T. evansi, and they may help us develop effective control strategies.

Suramin action in African trypanosomes involves a RuvB-like DNA helicase.

Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.

Histopathology and virulence of an in vitro-adapted Trypanosoma evansi TEDC 953 strain (Thailand isolate) in mice.

Background and Aim: Trypanosoma evansi is a blood and tissue protozoan parasite affecting domestic and wild animals. The T. evansi Thai strain, namely, T. evansi from dairy cattle number 953 (TEDC 953) strain, has been successfully isolated from dairy cattle and cultivated in vitro. The in vitro-cultivated parasite is useful for biological studies, evaluation of novel chemotherapeutic agents, and production of antigens for diagnostic tests. This study aimed to observe the histopathology and virulence of an in vitro-adapted T. evansi TEDC 953 strain in vivo. Materials and Methods: The histopathology and virulence of the TEDC 953 strain were clarified in mice. Six mice were infected with 1 × 105 trypomastigotes of TEDC 953 strain intraperitoneally, and four mice were in the negative control. Parasitemia was monitored daily, and the mice were euthanized on 30 days post-infection (DPI). Internal organs were collected for histopathological examination using hematoxylin and eosin staining. Results: Histopathological lesions were found in the liver, lung, heart, kidney, spleen, and brain of the inoculated mice. The main histopathological feature was lymphoplasmacytic inflammation in parenchyma and perivascular areas of multiple organs, and the severity of histopathological changes was related to the presence of trypomastigotes in the regional vessels. Granulomatous inflammation was seen in meninges, pleura, renal capsule, renal pelvis, and spleen of some infected mice. Four mice died at 17, 24, 26, and 27 DPI with an average parasitemia of 4.05 × 1011 trypomastigotes/mL. The average survival time was 23.5 DPI (mice = 4). Conclusion: This study confirmed that the TEDC 953 strain is infectious and pathogenic in mice after the continuously cultivated in vitro. To replace the use of experimental animals, the in vitro-cultivated parasite can be used instead in further studies.

DOES TRYPANOSOMA EVANSI HAVE THE MAXICIRCLE GENE, OR CAN TRYPANOSOMA EQUIPERDUM BE ISOLATED FROM BOVINES?

Identifying a trypanosome isolate is generally based on morphological observations and molecular identification of one of the genes, usually internal transcribed spacer 1 and 2 of ribosomal DNA (ITS1 rDNA, ITS2 rDNA), a variant surface glycoprotein of Rode Trypanozoon antigen type 1.2 (VSG RoTat 1.2), or expression site-associated genes (ESAG). However, this identification is insufficient because these genes cannot distinguish organisms in the subgenus Trypanozoon to the species level. A molecular approach using at least 5 sets of primers is needed, namely, ITS1, ESAG6/7, MINI, RoTat 1.2, and ND5, for stratified selection to obtain more targeted and conclusive results. Using this method to analyze isolates from Indonesia provided unexpected results: 9 isolates previously identified as Trypanozoon were found to have the kDNA maxicircle gene. Nine isolates of Trypanosoma equiperdum were identified for the first time in Indonesia, isolated from bovine (cattle and buffaloes). The identification of T. equiperdum in the 9 isolates was confirmed by analysis of the nucleotide sequence identity of the nad5-kDNA maxicircle gene.

Molecular and hematological investigation of Trypanosoma evansi infection in Iranian one-humped camels (Camelus dromedarius).

Trypanosoma species cause animal trypanosomiasis that infects many animals. Trypanosoma evansi is an organism that infects camels. There are many economic problems associated with this disease, including lower milk and meat yields and abortions. The purpose of the current survey was molecular study of the presence of Trypanosoma in dromedary camel blood in the south of Iran, and its effects on the hematologic, and some acute-phase protein changes. Blood samples were aseptically collected from the jugular vein of dromedary camels (n = 100; aged from 1 to 6 years) originating from Fars Province in EDTA-coated vacutainers. Genomic DNA from 100 µL of the whole blood was extracted and amplified using a PCR assay based on ITS1, 5.8S, and ITS2 ribosomal regions. Also, the PCR products obtained were sequenced. Moreover, the changes in hematological parameters and serum acute-phase proteins (serum amyloid A, alpha-1 acid glycoprotein, and haptoglobin) were measured. Among 100 tested blood, nine samples (9%, 95% CI: 4.2-16.4%) were found positive by the PCR assay. The phylogenetic tree and blast analysis showed four different genotypes closely related to the strains (accession numbers: JN896754 and JN896755) previously reported from dromedary camels in Yazd Province, center Iran. Based on hematological analysis, normocytic and normochromic anemia and lymphocytosis were detected in the PCR-positive cases compared with the negative group. Furthermore, alpha-1 acid glycoprotein was significantly increased in the positive cases. There was a substantial and positive relation between the number of lymphocytes, and the levels of alpha-1 acid glycoprotein and serum amyloid A in the blood (p = 0.045, r = 0.223 and p = 0.036, r = 0.234, respectively). A noticeable frequency of T. evansi infection was reported in dromedary camels in south Iran. This is the first report on the genetic diversity of T. evansi in this region. There was a significant association among Trypanosoma infection, lymphocytosis, and alpha-1 acid glycoprotein. Trypanosoma-positive camels had a significant decrease in hematocrit (HCT), hemoglobin (Hb), and red blood cell (RBC) values compared to the non-infected group. Further experimental studies are needed to elucidate the hematological and acute-phase protein alteration during a different phase of Trypanosoma spp. infection.

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.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Validation of in vitro-produced and freeze-dried whole cell lysate antigens for ELISA Trypanosoma evansi antibody detection in camels.

Trypanosoma evansi is a blood parasite responsible for surra in mammals, with a high impact in camels and horses. The WOAH-recommended reference method for detecting immunoglobulin G directed against T. evansi is ELISA, using whole cell lysate antigens (WCLAs). WCLAs are prepared with T. evansi produced in laboratory rodents, separated from blood cells using DE-cellulose anion exchange chromatography. As parasite lysates are fragile, antigens are preserved frozen pending use. For these reasons and others, T. evansi WCLAs are not commercially available. They are produced in small quantities, in a limited number of specialized laboratories, and they require a reliable and expensive cold chain for their shipment. In this study, we assessed and validated in vitro production of T. evansi and lyophilization of WCLAs in comparison with the reference method using frozen WCLAs prepared with parasites produced in rodents. Using a set of 400 samples monthly collected from 12 naturally infected camels followed-up for 1384 days, and two batches of referenced serum samples (infected, n = 12; non-infected, n = 15), statistical studies on qualitative and semi-quantitative results of the ELISAs did not show any significant difference when comparing the four combinations of parasites produced in vivo or in vitro, and frozen or freeze-dried WCLSAs. A repeatability study (28 repeats in 9 serum samples) was fully satisfying (p-value = 0.055). With the more convenient in vitro-produced freeze-dried WCLAs it was possible to: (i) avoid the ethical concern of in vivo production, (ii) improve the standardization of antigen production, (iii) secure antigen preservation during shipment and (iv) save a considerable amount of money (DE52-cellulose and dry-ice cold chain being avoided). Additional studies with other Trypanosoma spp are required for further extending ELISA to regional laboratories in enzootic areas, especially in view of the current progress in the "Progressive Control Pathway" (PCP) for trypanosomes in Africa.

Intracellular ROS production and apoptotic effect of quinoline and isoquinoline alkaloids on the growth of Trypanosoma evansi.

Trypanosoma evansi, a hemoflagellate poses huge economic threat to the livestock industry of several countries of Asia, Africa, South America and Europe continents of the world. Limited number of available chemical drugs, incidents of growing drug resistance, and related side effects encouraged the use of herbal substitutes. In the present investigation, the impact of six alkaloids of quinoline and isoquinoline group was evaluated on the growth and multiplication of Trypanosoma evansi and their cytotoxic effect was examined on horse peripheral blood mononuclear cells in an in vitro system. Quinine, quinindine, cinchonine, cinchonidine, berbamine and emetine showed potent trypanocidal activities with IC50/24 h values 6.631 ± 0.244, 8.718 ± 0.081, 16.96 ± 0.816, 33.38 ± 0.653, 2.85 ± 0.065, and 3.12 ± 0.367 µM, respectively, which was comparable to the standard anti-trypanosomal drug, quinapyramine sulfate (20 µM). However, in the cytotoxicity assay, all the drugs showed dose dependent cytotoxic effect and quinine, berbamine and emetine showed selectivity index more than 5, based of ration of CC50 to IC50. Among the selected alkaloids, quinidine, berbamine and emetine exhibited higher apoptotic effects in T. evansi. Likewise, drug treated parasites showed a dose-dependent and time-dependent increase in reactive oxygen species (ROS) production. Therefore, increased apoptosis in combination with ROS generation could be responsible for the observed trypanocidal effect which could be further evaluated in T. evansi-infected mice model.

Surveillance and control of Trypanosoma evansi in the canary Islands: A descriptive analysis.

This study examines the occurrence of Surra, a disease caused by Trypanosoma evansi, in camels in the Canary Islands. The 1997 detection of T. evansi in camels in the Canary Islands led to the implementation of an initial control program, resulting in a decrease in prevalence. Following an outbreak in 2014, and due to the impossibility of eradicating it using the conventional measures, a lazaret was set up to separate positive and suspicious animals, in addition to the control measures previously implemented. Stomoxys calcitrans was the only vector captured, and no other animals tested were found to be positive for T. evansi. In November 2019, the last camels that tested serologically positive were detected; however, since February 2018, no camels that tested positive for PCR have been found in the farms were the outbreak was detected, suggesting that the sanitary measures implemented are adequate. The duration of the outbreak control and potential eradication for the disease has yet to be established. This study provides evidence to facilitate the control of African Animal Trypanosomosis in endemic areas of the world, which may contribute to revise the World Organization for Animal Health (WOAH) protocol to implement recommendations of surveillance and control strategies for animal Trypanosomosis in camels.

Morphological, serological, molecular detection, and phylogenetic analysis of Trypanosoma evansi in horses of different regions in Iran.

Trypanosoma evansi, the causative agent of "surra" is enzootic in Iran. The current study aimed to detect T. evansi in horses from different regions of Iran using morphological, serological, and molecular methods. In 2021, 400 blood samples were collected from horses in eight regions. Eighty horses showed clinical signs such as cachexia (n = 64), fever (n = 36), foot edema (n = 40), and abdominal edema (n = 32), and 320 horses appeared healthy. All samples from the studied regions were evaluated for the presence of trypanosomes using direct analysis of blood smears, mercuric chloride, and PCR-based tests. In total, 12% (95% CI: ± 3.1%), 21% (95% CI: ± 3.9%), and 21% (84) of animals were positive for Trypanosoma in microscopic, serologic, and molecular analyses, respectively. All animals positive for SSU rDNA PCR were from Qom, Semnan, and Golestan regions. Further molecular analyses on 84 PCR-positive horses revealed that 29 horses scored positive in PCR using primers of trypanozoon species and 5 scored positive in PCR using primers of Trypanosoma evansi type A. All samples (n = 5) were from Qom region. The 205-bp fragments of T. evansi RoTat 1.2VSG (accession numbers: ON017789-93) analyzed and compared to other isolates sequence from GenBank BLAST search. It has close similarities with isolates from Pakistan, Egypt, Malaysia, Kenya, and India. Data herein demonstrated that horses from Iran were at high risk of T. evansi infection. Comprehensive control programs, such as those based on the application of repellants and traps, and also, compliance with quarantine standards are recommended for minimizing the risk of the infection.

Parasitological, molecular, and epidemiological investigation of Trypanosoma evansi infection among dromedary camels in Balochistan province.

Surra is a zoonotic disease caused by Trypanosoma evansi (T. evansi), which affects a wide variety of animals worldwide. The disease has a severe impact on the productivity, health, and working capacity of camels and causes mortality and extensive economic losses if not diagnosed early. This is the first comprehensive report on the prevalence of T. evansi infection in dromedaries in Balochistan province. In the present study, 393 blood samples (indigenous, n = 240; imported, n=153) were collected from one-humped camels (Camelus dromedarius) and were tested by molecular methods to estimate the prevalence of T. evansi in three districts (Pishin, Nushki, and Lasbella) of Balochistan province. The overall prevalence of T. evansi among examined camel samples was 28.24% (95% confidence interval (CI): 24.02-32.89%). The risk of T. evansi infection in adult camels (> 10 years) is higher than that in young ones (odd-ration (OR) = 2.7; 95% CI: 1.3357-5.3164%). Moreover, male camels were six times more likely to get an infection than female camels. The detection of T. evansi infection in camels sampled in summer and spring was 3.12- and 5.10-fold higher, respectively, than in camels sampled in winter. In conclusion, our findings showed a high rate of T. evansi infection in camels from the three districts. Our study emphasizes the need for a strict surveillance program and risk assessment studies as prerequisites for control measures.

Parasitological and molecular detection of Trypanosoma evansi in a dog from Tocache, San Martin, Peru.

This study presents the first case report of canine trypanosomiasis caused by Trypanosoma evansi in Peru. The case was admitted to a veterinary clinic in the Peruvian Amazon region of San Martin with severe clinical symptomatology which resulted in the dog's death. Microscopy screening showed the presence of trypomastigotes in blood and bone marrow and postmortem histopathology found damage at the cardiac, lung, kidney and spleen levels. Collected specimens were tested by nested-PCR which were positive for Trypanosoma spp., but negative for T. cruzi. High-throughput sequencing determined that the infecting species was closely related to T. equiperdom/evansi and subsequent phylogenetic analysis confirmed that the sample was related to T. evansi. The presence of T. evansi in the area highlights the need for increased surveillance to assess the impact of surra in the region and to develop measures to prevent socioeconomic damage resulting from infections in domestic and farm animals as well as prevent zoonotic transmission.

Detection of Trypanosoma evansi in a naturally infected cat in Indonesia using bioassay and molecular techniques.

Background and Aim: The prevalence of surra in domestic cat is seldom and it is caused by Trypanosoma brucei and Trypanosoma evansi. However, molecular diagnostic approaches are required owing to similarities in their morphology. In Yogyakarta, a domestic cat was diagnosed with trypanosomiasis; however, the causative species was undetermined. Therefore, we aimed to molecularly and biologically identify the isolate. Materials and Methods: Approximately 1 mL of blood from an infected cat was collected into EDTA tube and separated for inoculation into donor mice, blood smear, and DNA isolation. Two donor mice was then used for increasing the number of parasite in order to infect 10 experimental mice. Parasitemia was monitored daily in each experimental mouse by preparing a wet mount and Giemsa-stained thin blood smear. The blood of experimental mice that reached the peak of parasitemia was then collected and used for DNA isolation. Each blood sample, which collected from infected cat and experimental mice, was then isolated and amplified the DNA by polymerase chain reaction using ITS-1. The parasitemia pattern and viability of the animals were observed to determine the biological characteristics of trypanosomatid, while to assess the molecular characteristics, the internal transcribed spacer (ITS)-1 amplification was used. Results: The prepatent period of this trypanosomatid is between 2 and 4 dpi, whereas the life span of mice is approximately 4-10 dpi. Morphologically, the trypomastigote in the cat blood smear had long slender and intermediate shapes. However, only the long slender form was detected. Among the total of 410 nucleotides (NT) of ITS-1 sequences, 25 NT substitutions differed between the cat and mouse isolates. Phylogenetic analysis revealed that both samples had a close genetic relationship with T. evansi. Conclusion: Trypanosoma evansi, a highly virulent trypanosomatid, was isolated from a cat in Yogyakarta.

In vitro and in vivo evaluation of efficacy of berberine chloride: Phyto-alternative approach against Trypanosoma evansi infection.

Current chemotherapy against the Surra organism, Trypanosoma evansi has several limitations in terms of efficacy, toxicity, availability and emerging resistance. These reasons make the search of new chemo-preventive and chemo-therapeutic agent with high potency and low toxicity. Alkaloid phyto-molecules, berberine has shown promising anti-kinetoplastid activity against T. cruzi, T. congolense, T. brucei, Leishmania donovani and L. tropica. However, till date, there is no investigation of therapeutic efficacy of berberine chloride (BC) against T. evansi. The IC50 value of BC for growth inhibition of T. evansi at 24 h of culture was calculated as 12.15 µM. The specific selectivity index (SSI) of BC was calculated as 19.01 and 10.43 against Vero cell line and Equine PBMC's, respectively. Thirteen drug target genes affecting various metabolic pathways were studied to investigate the mode of trypanocidal action of BC. In transcript analysis, the mRNA expression of arginine kinase 1 remained refractory to exposure with BC, which provides metabolic plasticity in adverse environmental conditions. In contrary, rest all the drug target gene were down-regulated, which indicates that drug severely affect DNA replication, cell proliferation, energy homeostasis, redox homeostasis and calcium homeostasis of T. evansi, leading to the death of parasite in low concentrations. It is the first attempt to investigate in vitro anti-trypanosomal activity of BC against T. evansi. These data imply that phytochemicals as alternative strategies can be explored in the future as an alternative treatment for Surra in animal.

Expression kinetics of cytokines and the humoral antibody response concerning short-term protection induced by radiation-attenuated Trypanosoma evansi in bovine calves.

'Surra', an economically important disease of livestock, is caused by the parasitic blood protozoon Trypanosoma evansi. Both innate and adaptive immunity contribute to the protection against this infection. T-helper cells play a crucial role in the antibody-mediated clearance of T. evansi. We present here the data on the kinetics of expression of important Th1, Th2 and Th17 cytokines, vis-a-vis the dynamics of humoral response in bovine calves following immunization with γ-radiation-attenuated live T. evansi and later challenged with homologous virulent T. evansi. Significant upregulation of the pro-inflammatory Th1 and Th17 cytokines was correlated with the IgG2-mediated protection in the immunized bovine calves post-challenge. The calves were immunized with 5 × 106 500 Gy γ-radiation-attenuated live T. evansi (horse isolate) thrice at 15 days intervals through the subcutaneous route and subsequently, challenged with 1 × 103 virulent T. evansi on day 50. Significantly high serum IgG (1:1600) and IgM (1:800) titres were recorded on week 2 PC, whereas the peak serum IgG2 titre (1:800) was recorded on week 6 PC. Significant upregulation of IFN-γ, TNF, IL-1ß, and IL-2 was recorded between days 1 to 3 PC, while the same for IL-17 was recorded on day 14 PC. The immunized calves were free from parasitemia post-challenge and were clinically healthy till the end of the experiment. Significant upregulation of IL-10 and IL-4 transcripts and a corresponding increase of serum IgG1 titre in the placebo group helped patency of the parasite in an anti-inflammatory environment and clinical exacerbation of the disease. The expression of the important Th1 cytokines was crucial for antibody-mediated short-term protection against a lethal challenge of T. evansi in cattle.

Exploring the potential of invariable surface glycoprotein (ISG65) as promising antigen for diagnosis of Trypanosoma evansi infection.

Trypanosoma evansi, a hemoflagellate protozoan, leads to wasting disease, surra in livestock animals causing huge economic losses. Currently, the preferred assay for surra diagnosis is whole cell lysate (WCL) based ELISA, which requires the use of rodents for WCL preparation. To avoid use of laboratory animals, we used recombinant DNA technology to express T. evansi invariable surface glycoprotein (ISG) in E. coli. The potential of recombinant ISG65 (rISG65) as a diagnostic antigen was investigated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The results indicated that rISG65 reacted with horse T. evansi positive serum giving two bands of approximately 48 kDa and 96 kDa. T. evansi-specific antibodies were detected as early as 10 and 14 days post infection using immunoblot and indirect ELISA, respectively using rISG65 antigen. No cross-reactivity was observed in ELISA and immunoblot with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions were observed between 30 and 100 kDa in T. evansi isolate of horse origin indicating the existence of multiple copies of ISG protein in a single trypanosome. The recombinant ISG has proven to be good candidate antigen to be used in ELISA for serodiagnosis of T. evansi infection in different animals.