Flagellated Protozoan Parasite Research Articles

Epidemiology and clinical description of Chagas disease in Valencia, Spain, from 2010 to 2020

Chagas disease (CD), caused by the flagellate protozoan parasite Trypanosoma cruzi, is endemic in 21 American countries. It is estimated that about 6-8 million people are infected. Nevertheless, CD should be now considered a worldwide-distributed disease due to migratory movements from endemic to non-endemic regions where the infection risk is related to transplacental transmission to newborns from infected mothers or through blood or organ donations from infected individuals. Spain is the non-endemic country with the highest burden of CD in Europe. This study aims to assess the prevalence and perform a descriptive analysis of patients with CD at Hospital Universitari i Politécnic (HUiP) in Valencia, Spain, from 2010 to 2020, to raise awareness about the importance of the disease.We compared data collected from official sources (National Institute of Statistics and Microbiological Surveillance Network of the Comunitat Valenciana) as well as data from the Health Department-Valencia La Fe, Valencia (Spain). A total of 3713 subjects were analyzed, 3159 women and 554 men, of which 433 were positive, prevalence of 11.7%. Pediatric population accounted for 106 (24.5%) while adult population for 327 (75.5%). Most migrants were from Bolivia (298, 91.1%). Regarding women, 200 (64%) were of childbearing age and 90 (45%) of these were pregnant. DNA detection by PCR was positive only in 4 newborns (3.8%). Treatment with benznidazole was implemented in 204 patients and 107 developed adverse drug reactions (ADRs). In conclusion, the prevalence of CD in our health department is not negligible and poses a challenge for the health system; worrisomely, many patients remain undiagnosed and untreated.

Cytotoxic effects of ivermectin on Giardia lamblia: induction of apoptosis and cell cycle arrest.

Giardia lamblia is a flagellated protozoan parasite causing giardiasis, a common intestinal infection characterized by diarrhea, abdominal cramps, and nausea. Treatments employed to combat this parasitic infection have remained unchanged for the past 40 years, leading to the emergence of resistant strains and prompting the search for new therapeutic agents. This study investigated the cytotoxic effects of ivermectin (IVM) on G. lamblia trophozoites. We conducted dose-response experiments to assess IVM-induced cytotoxicity. We utilized various biochemical and ultrastructural analyses to explore the underlying mechanisms of cell death, including reactive oxygen species (ROS) production, DNA fragmentation, cell cycle arrest, and apoptosis markers. Our findings demonstrate that IVM induces dose-dependent cytotoxicity and triggers cell death pathways. We found that IVM treatment generates elevated levels of reactive oxygen species (ROS), DNA fragmentation, and arrests of trophozoites in the cell cycle's S phase. Additionally, ultrastructural analysis reveals morphological alterations consistent with apoptosis, such as cytoplasmic vacuolization, chromatin condensation, and tubulin distribution. The insights gained from this study may contribute to developing new therapeutic strategies against giardiasis, addressing the challenge posed by drug-resistant strains.

In vitro assessment of Dracocephalum lindbergii for growth inhibition, apoptosis induction, and cytokine modulation against Leishmaniamajor

Leishmaniasis is a vector-borne disease caused by several species of flagellate protozoan parasites belonging to the genus Leishmania, which are part of the Trypanosomatidae family. This study aims to evaluate the in vitro anti-Leishmania activity of Dracocephalum lindbergii Rech.f (D. lindbergii) on the growth, apoptosis induction, and proliferation of Leishmania major. To conduct this study, the aerial parts of D. lindbergii were collected during the flowering stage in May 2022 from North Khorasan province, Iran. The plant material was extracted using various solvents, starting with those of lower polarity and progressing to those of higher polarity. To assess the impact of the D. lindbergii fraction on L. major promastigotes, amastigotes, and macrophages (THP-1), cell viability was determined using the MTT assay. Additionally, flow cytometry with the Annexin V-PE apoptosis detection kit was employed to distinguish between viable, necrotic, and apoptotic promastigotes in response to treatment with the 100 % methanolic fraction of D. lindbergii (D). The expression levels of TNF-α, IFN-γ, IL-12, IL-10, TGF-β, IL4, iNOS, and GAPDH were quantified using real-time PCR (qPCR) on the macrophage cell line. Each treatment approach exhibited marked anti-leishmanial effects across different concentrations over 24, 48, and 72 h of incubation, showing statistically significant differences compared to the untreated control group (P < 0.001). Different concentrations of D, MAT, and AmpB, both individually and in combination, significantly reduced the total number of intramacrophage amastigotes compared to the untreated control groups at 24, 48, and 72 h. The results also showed time-dependent variations in the anti-Leishmania activity of the fraction. In terms of cellular morphology, treated cells exhibited changes such as shrinkage, cytoplasmic condensation, and reduced mobility, particularly noticeable after 24 h of treatment. Additionally, fraction D demonstrated significant antioxidant properties. This study highlights the potential of D. lindbergii as an anti-Leishmania agent, with the 100 % methanolic fraction emerging as a promising candidate for the development of novel treatments for leishmaniasis.

Extreme mitochondrial reduction in a novel group of free-living metamonads

Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered ‘mitochondrion-related organelles’ (MROs) with reduced functions, however the degree of reduction varies. Here, we generate high-quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses placed these organisms in a group we name the ‘BaSk’ (Barthelonids+Skoliomonads) clade, a deeply branching sister group to the Fornicata, a phylum that includes parasitic and free-living flagellates. Bioinformatic analyses of gene models shows that these organisms are predicted to have extremely reduced MRO proteomes in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system Fe/S cluster pathway by lateral gene transfer. One of the isolates, Skoliomonas litria, appears to have lost all other known MRO pathways. No proteins were confidently assigned to the predicted MRO proteome of this organism suggesting that the organelle has been lost. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade demonstrates that mitochondrial functions may be completely lost even in free-living organisms.

CysLT receptor-mediated NOX2 activation is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products.

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B4 (LTB4) and various cysteinyl leukotrienes (CysLTs) which included LTC4, LTD4, and LTE4. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.

Cochlosoma anatis in Poultry and its Industry Impact.

Cochlosoma anatis is a flagellated protozoan parasite classified in the Trichomonadidae family and is the causative agent of cochlosomiasis, an enteric disease of turkeys, waterfowl, and other wild birds. Cochlosomiasis symptoms largely consist of watery diarrhea, lethargic birds, depressed weight gain, and widespread flock morbidity causing flock nonuniformity. The known distribution of C. anatis is centered around areas of turkey production farms in the southeast United States, e.g., North Carolina, Missouri and Arkansas, but has been reported in other states and some other countries. Diagnosis is confirmed through examination of enteric mucosal scrapings using light microscopy. Following the withdrawal of approval of effective antiprotozoal medications for use in commercial animal production, cochlosomiasis has become a greater concern for commercial turkey industry professionals. Transmission of C. anatis occurs via the fecal-oral route, but the organism is fragile outside the host, suggesting the implication of a vector in the introduction of disease to susceptible farms. Research regarding C. anatis pathogenicity, transmission, and environmental involvement has been limited, creating a gap in cochlosomiasis knowledge. Future research is needed to further explore ways to prevent and treat cochlosomiasis, with needs centered on disease pathogenesis, transmission patterns, and prophylaxis and treatment methods.

Heterologous Expression of Human IFNγ and Anti-IL17 Antibody in Leishmania tarentolae Promastigote.

Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function. In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH-hIFNγ-antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47pg/mL. Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH-hIFNγ-antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite. The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania.

Trichomoniasis and Associated Socio-Demographic Factors among Women in Port Harcourt, Nigeria

Human trichomoniasis is a sexually transmitted infection (STI) caused by an anaerobic flagellated protozoan parasite called Trichomonas vaginalis that infects the human urogenital tract. The study is aimed that assessing the association between trichomoniasis and demographic parameters in women attending Rivers State University Teaching Hospital (RSUTH). The cross-sectional study was made up of 650 randomly selected women, 450 of them were tested for trichomoniasis using urine sample while the other 200 were tested using high vagina swab (HVS). With the aid of a well-structured questionnaire, consenting participants provided their socio-demographic data. The results showed that there was an association between educational status and prevalence of trichomoniasis (p&lt;0.05) in both cases of samples. Also, there was association between age and prevalence of trichomoniasis in HVS tested sample (p&lt;0.05). There was no association between trichomoniasis and occupational and marital statuses (p&gt;0.05). This study has revealed that education and age can impact on the dynamics of trichomonial infection among women.

Trichomonas vaginalis and Associated Factors among Pregnant Women Attending Antenatal Care at Bule Hora University Teaching Hospital, Oromia Region, Southern Ethiopia.

Trichomoniasis is caused by a flagellated protozoan parasite called Trichomonas vaginalis. It is one of the most common, curable nonsexually transmitted infections globally. In Ethiopia, complications associated with genital infection in pregnant women are a common problem. Despite the burden of the disease, epidemiological data related to this disease is currently rare in Africa, particularly in Ethiopia. Objective. This research is aimed at assessing the prevalence of Trichomonas vaginalis and associated factors among pregnant women attending antenatal care at Bule Hora University Teaching Hospital. Methods. An institutional-based cross-sectional study was conducted among 196 pregnant women attending ANC at Bule Hora University Teaching Hospital. Structured questionnaires were used to collect sociodemographic and associated factor data. The consecutive sampling technique was used to include study participants. The two vaginal swabs were collected by brushing the vagina with a sterile cotton swab and tested by using direct wet mount and the Giemsa staining. The data were analyzed using SPSS version 26 for logistic regression analysis. A p value < 0.05 with 95% CI was used to declare it statistically significant. Result. An overall prevalence of T. vaginalis among pregnant women was 7.7% (95% with confidence interval (CI), 0.043-0.123). The highest prevalence was observed among the 35-39-year-old age group with 18.2% and among widowed women with 25%. This study revealed that the number of sexual partners (AOR: 3.215, 95% CI: 1.062-9.731) was a significant associated factor of T. vaginalis.Conclusion. The prevalence of T. vaginalis was considerably high among pregnant women in this study. This finding emphasizes the need for routine screening and treatment of pregnant women in the first antenatal care and enhances the need for regular health education for pregnant women at antenatal clinics to make them aware of their health, and avoidance of the risk of trichomoniasis is advised.

Prevalence of Trichomonas gallinae in Birds of Iran and its Pathological Findings: A Review study

Trichomonas gallinae is a flagellated protozoan parasite that affects numerous avian species worldwide, causing a range of diseases collectively termed trichomonosis. This review study aimed to present a comprehensive analysis of the prevalence of T. gallinae in birds of Iran, along with an exploration of its associated pathological findings. Through an extensive search of published studies, scientific databases, and relevant literature, we identified several studies conducted in Iran that focused on the prevalence of T. gallinae in different bird populations as well as their pathological effect. These studies utilized a variety of techniques, including direct microscopic examination, culture methods, and molecular tools to detect and identify the presence of this parasite. This review study indicates that T. gallinae infections are prevalent among various bird species in Iran, including Domestic pigeons, Zebra Finches and Turkey. The prevalence rates reported showed significant differences among various regions and bird species, ranging from as low as 2.5% in zebra finches to as high as 70% in pigeons. The clinical signs of infected birds varied from no clinical signs to visible yellowish-white masses of caseous necrotic material, hyperplasia of mucous cells and significant congestion of lung. These findings underline the importance of T. gallinae as a significant pathogen affecting avian populations in Iran. Using at least two techniques such as culture or PCR in addition to direct smear is recommended for better diagnosis of infection. The study highlights the need for further epidemiological studies to better understand the risk factors associated with T. gallinae transmission and to develop effective prevention and control strategies.

Prevalence and Risk Factors for Giardia Species in Livestock Animals of Iraq

Giardiasis is an infection caused by the protozoan flagellate parasite Giardia spp. in the intestine. G duodenalis, a species complex of diverse genotypes that tend to demonstrate host specificity, is responsible for most veterinary health-related infections; production animals, companion animals, and wildlife can all be infected. Abdominal pain and diarrhea, typically accompanied by steatorrhea, are the most common symptoms of Giardiasis. Cysts or antigens in feces are commonly used to diagnose the disease. Treatment regimens vary and are dependent on the indication. Control methods must include hygiene measures. Despite direct evidence of Giardia sp transmission to humans via polluted water supplies, our analysis will focus on some recent research and transition techniques for Giardia sp in goats. Both animals and people are at risk from this parasite. Keywords: Giardia Species, Goats, Prevalence, Risk Factors, Giardiasis

RNA viruses of Crithidia bombi, a parasite of bumblebees

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi – a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

Genetic diversity and molecular diagnosis of Giardia.

Giardia is a genus of flagellated protozoan parasites that infect the small intestine of humans and animals, causing the diarrheal illness known as giardiasis. Giardia exhibits significant genetic diversity among its isolates, which can have important implications for disease transmission and clinical presentation. This diversity is influenced by the coevolution of Giardia with its host, resulting in the development of unique genetic assemblages with distinct phenotypic characteristics. Although panmixia has not been observed, some assemblages appear to have a broader host range and exhibit higher transmission rates. Molecular diagnostic methods enable researchers to examine the genetic diversity of Giardia populations, enhancing our understanding of the genetic diversity, population structure, and transmission patterns of this pathogen and providing insights into clinical presentations of giardiasis.

On the emergence of eukaryotes and other enigmas

The origin of eukaryotes is one of the most fundamental problems in the entire history of life. How did eukaryotes arise? Previous attempts to solve the problem are very far from an answer, at best they propose a solution to one of the various innovations that ended up culminating in eukaryotes. Based on a hypothetical-deductive methodology, as usual in evolutionary issues, I propose that eukaryotes emerged from the endosymbiotic association between a flagellate parasite and its host, of which the sperm is the main vestige. The hypothesis unifies the solution to the vast array of acquisitions shared by eukaryotes that differentiate them from other beings, remarkably cell nucleus, mitosis, meiosis and sexual reproduction. The solution has a deep impact on understanding the origin and functioning of all complex life forms.

FAKTOR-FAKTOR YANG MEMPENGARUHI PATOGENITAS TRICHOMONAS VAGINALIS

Trichomonas vaginalis is a flagellated protozoan parasite that causes trichomoniasis. Trichomoniasis is a non-viral sexually transmitted infection and is found in the human urogenital tract. The interaction between T. vaginalis with the host is very complex and many factors influence the pathogenicity of T. vaginalis. To determine the factors that influence the pathogenicity of Trichomonas vaginalis. Literature review using the Google Scholar database. The search results for articles matched the criteria with the keyword "pathogenicity of Trichomonas vaginalis". Pathogenicity factors include changes in the trophozoite Trichomonas vaginalis to an ameboid form, cellular pathogenicity (attachment of Trichomonas vaginalis to host cells, cytotoxic and hemolytic activity, phagocytosis), interactions with vaginal flora, symbiont contribution with Trichomonas vaginalis, recognition by the host immune system, and evasion host immune system. Pathogenicity factors include changes in the trophozoite Trichomonas vaginalis to an ameboid form, cellular pathogenicity (attachment of Trichomonas vaginalis to host cells, cytotoxic and hemolytic activity, phagocytosis), interactions with vaginal flora, symbiont contribution with Trichomonas vaginalis, recognition by the host immune system, and evasion host immune system.

Trichomonas tenax: A Neglected Protozoan Infection in the Oral Cavities of Humans and Dogs-A Scoping Review.

Trichomonas tenax is a flagellated protozoan parasite found in the oral cavities of humans and animals and has been associated with periodontal disease, the most prevalent inflammatory disease affecting them all. Studies have shown that T. tenax can cause damage to mammalian cells and secretes virulent proteins, such as cysteine. It is presently considered zoonotic. Despite the few studies that have been done, the pathogenicity of this oral protozoan is still not fully understood. A database search was performed in July 2022 using PubMed and Google Scholar to retrieve data eligible for this study. PRISMA-ScR guidelines were followed to conduct this scoping review. A total of 321 articles were found with 87 included in this review after applying the exclusion criteria. Due to its increasing prevalence worldwide in both humans and dogs, detecting and elucidating the pathogenicity of this parasite is paramount for effective global control and prevention of periodontal disease. However, there is a paucity in the literature on this neglected zoonotic trichomonad, which is in large contrast to the closely related human pathogen T. vaginalis. Here, we comprehensively review the history, morphology and reproduction, host, prevalence, diagnosis, pathogenicity, control, and prevention of T. tenax. Hopefully, this article will call attention to both medical and veterinary professionals as well as epidemiologists on this most neglected and zoonotic protozoan. More epidemiological and clinical studies need to be conducted on T. tenax to gain a better understanding of its pathogenicity, to increase the chances of developing effective drugs to aid in the control of this oral parasite, and reduce the spread of periodontal disease worldwide.

Incidence of Giardiasis Among Population at Wasit Province

Giardia lamblia, an intestinal flagellate parasite is widely prevalent in humans worldwide. A total of 100 stool samples from diarrhoeal patients during three months ( October, November , and December, 2011) were examined by direct smear and lugol’s iodine techniques to investigate Giardia lamblia parasite . The total human patients involved in this study were divided into five age groups . The overall prevalence of G.lamblia infection was 22%. The highest infection appeared the in age group (1-10) years old, and the lowest was in the age group (11-20) years old. Males were highly infected 26 % (13/50) than females 16% (9/50) in patient groups.

Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030.

Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.

A fish kill at the Aposelemis dam (Crete, Greece) caused by heavy parasitism by Ichthyobodo sp.

A fish kill was recorded at the Aposelemis Dam, which is the main reservoir of drinking water for the island of Crete in Greece. Hundreds of goldfish were found dead at a side stream which provides water to the reservoir. The affected fish had been entrapped in a small pond at the side of the stream with practically zero water renewal as the event occurred in August which is a dry season for the island of Crete. The event was alarming for the local community since anthropogenic pollution was initially suspected which could pose a significant human health threat. Following examination of the fish, the mortality was attributed to heavy infection by the parasitic flagellate, Ichthyobodo sp., whilst no pollutants were detected. The parasite was studied through light and scanning electron microscopy and was identified molecularly.

Isolation and in vitro cultivation of Trypanosoma evansi Thai strains

Trypanosoma evansi is a flagellate protozoan parasite responsible for “surra”. To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15–16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.