Venturicidin A affects the mitochondrial membrane potential and induces kDNA loss in Trypanosoma brucei.

Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces, inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani, but much less so against Trypanosoma evansi. Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%-50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei, and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs.

Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes.

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.

Cytosolic and Mitochondrial Hsp90 in Cytokinesis, Mitochondrial DNA Replication, and Drug Action in Trypanosoma brucei.

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei.

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

Molecular characterization of Leishmania species from stray dogs and human patients in Saudi Arabia.

Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. To assess the frequency and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania infection were examined for CL by a nested polymerase chain reaction (nPCR). Seven (25.9%) out of 27 human patients scored positive for Leishmania spp. (i.e., L. major in five patients from Riyadh and L. tropica in two patients from Al-Qaseem). Out of 311 dogs, five (1.6%) were infected by L. tropica. Data herein presented demonstrate the occurrence of L. tropica in dogs and humans in Saudi Arabia, as well as the occurrence of L. major in humans.

A Fluorinated Phenylbenzothiazole Arrests the Trypanosoma cruzi Cell Cycle and Diminishes the Infection of Mammalian Host Cells.

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

Leishmania cytochrome b gene sequence polymorphisms in southern Iran: relationships with different cutaneous clinical manifestations.

BACKGROUND: Cutaneous leishmaniasis (CL) caused by Leishmania species, is a geographically extensive disease that infects humans and animals. CL is endemic in half of the 31 provinces of Iran, with 29,201 incidence cases reported in Fars province from 2010 to 2015. CL is polymorphic and may result in lesions characterized by different clinical features. Parasite genetic diversity is proposed to be one of the factors affecting the clinical outcome and lesion characteristics in CL patients. However, there is still very limited data regarding the genetic variation of Leishmania spp. based on the sequencing of Cytochrome b (Cyt b) gene. METHODS: All patients originated from endemic regions in Fars province. The amplification of the Cyt b gene from isolates of 100 patients with disparate clinical forms of CL was accomplished using Nested-PCR. Sequence analysis of the amplified Cyt b was used to scrutinize the genetic variations among Leishmania isolates and connect the results with clinical pictures. The clinical demonstrations were basically of two types, typical and atypical lesions. Molecular phylogenetic tree was constructed using the Neighbor-Joining method, with species/strains from this study compared to species/strains from other geographical regions. RESULTS: Leishmania major was identified as the predominant infecting Leishmania spp. (86% of cases), with the remainder of cases being infected by Leishmania tropica. Clinical examination of patients revealed 12 different clinical CL forms. Among Leishmania samples analyzed, five distinct haplotypes were recognized: three in L. major and two in L. tropica. We found a correlation between clinical outcomes and Cyt b sequence variation of Leishmania spp. involved. Moreover, we observed a higher presence of polymorphisms in L. major compared with L. tropica. This difference may be due to the different eco-epidemiologies of both species, with L. tropica being an anthroponosis compared to L. major, which is a zoonosis. CONCLUSIONS: The sequence analysis of Cyt b gene from 25 L. major and L. tropica strains demonstrated genetic variability of L. major and L. tropica causing CL in southern Iran, and a feasible connection amid the genetic heterogeneity of the parasite, geographical source and clinical appearance of the disease in human was detected.

Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load.

The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ±â€¯1 °C for satellite-DNA and 78.1 °C ±â€¯1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10-3 parasite or 240 target copies, and for kDNA, 2 × 10-4 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.

Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression.

Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of Trypanosoma brucei on its mitochondrial genome (kinetoplast DNA [kDNA]), a target of the antitrypanosomatid drug isometamidium. Here, we report that V-ATPase inhibition with bafilomycin A1 (BafA) provides partial resistance to genetic knockdown of mitochondrial gene expression. BafA does not promote long-term survival after kDNA loss, but in its presence, isometamidium causes less damage to kDNA.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Genetic Variability in Leishmaniasis-Causing Leishmania infantum in Humans and Dogs from North-East Spain.

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring.

Genetic diversity of Leishmania donovani causing visceral and cutaneous leishmaniasis in the Western Ghats, India.

The visceral and atypical cutaneous leishmaniasis (VL and CL) caused by Leishmania donovani is an emerging infectious disease in the Western Ghats, Kerala, India. In this study, L. donovani specific kinetoplast minicircle DNA (k-DNA) sequence analysis was conducted to ascertain the genetic variability among the L. donovani isolates from the Western Ghats. Out of 23 CL and 5 VL suspected patient samples, 18 CL and 3 VL tested positive for k-DNA diagnostic PCR. Subsequently, 17 CL and 3 VL samples were found positive for L. donovani specific k-DNA PCR. Although the genetic diversity among the VL and CL isolates was low, there was clear variation from the parasites reported from other countries. The parasites characterized from the current study were more related to those reported from East Africa and India.

Serological and molecular analysis of Leishmania infection in a recent outbreak of visceral leishmaniasis in South Omo Zone, Ethiopia.

BACKGROUND: Ethiopia has a high burden of visceral leishmaniasis. Recently, there was a significant increase in cases in the South Omo Zone. This study aims to assess the prevalence of Leishmania donovani infection and its associated factors. METHODS: A household-based cross-sectional study was carried out in January 2023 in the South Omo Zone in Ethiopia. Dried blood spot samples were collected from 382 randomly selected study participants. Direct agglutination test (DAT) and kinetoplast DNA real-time PCR tests were performed to detect L. donovani infection. Participants' sociodemographic, clinical and risk factors for L. donovani infection data were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyze the data. Febrile cases were checked for malaria with a multiplex PCR assay. RESULTS: Overall prevalence of L. donovani infection among the sampled population was 32.5% (n=124), of which 41.1% (n=51) was detected by PCR, 33.9% (n=42) by DAT and 25.0% (n=31) by both tests. The majority of the positives were from the Logira (28.2%; n=35) and Dilbayne (29.0%; n=36) villages. Participants residing in Logira (adjusted OR [AOR]: 5.80; 95% CI 1.85 to 18.15) and Dilbayne (AOR: 3.38; 95% CI 1.15 to 9.96) villages and owning cows (AOR: 2.31; 95% CI 1.03 to 5.15) showed an association with Leishmania infection. Plasmodium falciparum was detected in 3.4% (n=2) of 59 febrile participants. CONCLUSIONS: The prevalence of L. donovani infection in the South Omo Zone is high. Further research on the role of cows in the transmission cycle is needed to design the best strategy to control Leishmania infection in the South Omo Zone. Such interventions should focus on the Logira and Dilbayne villages, where most of the infections were identified.

Asymptomatic Leishmania infantum infection in dogs and dog owners in an endemic area in southeast France.

The prevalence of asymptomatic leishmaniasis in dogs and their owners in the main endemic areas of France has not been studied to date. The objective of this study was to quantify asymptomatic Leishmania infantum infection in southeast France in healthy people and their dogs using molecular and serological screening techniques. We examined the presence of parasitic DNA using specific PCR targeting kinetoplast DNA (kDNA) and specific antibodies by serology (ELISA for dogs and Western blot for humans) among immunocompetent residents and their dogs in the Alpes-Maritimes. Results from 343 humans and 607 dogs were included. 46.9% (n = 161/343) of humans and 18.3% (n = 111/607) of dogs were PCR positive; 40.2% of humans (n = 138/343) and 9.9% of dogs (n = 60/607) were serology positive. Altogether, 66.2% of humans (n = 227) and 25.7% of dogs (n = 156) had positive serologies and/or positive PCR test results. Short-haired dogs were more frequently infected (71.8%, n = 112) than long-haired dogs (12.2%, n = 19) (p = 0.043). Dogs seemed to be more susceptible to asymptomatic infection according to their breed types (higher infection rates in scenthounds, gun dogs and herding dogs) (p = 0.04). The highest proportion of dogs and human asymptomatic infections was found in the Vence Region, corresponding to 28.2% (n = 20/71) of dogs and 70.5% (n = 31/44) of humans (4.5/100,000 people). In conclusion, the percentage of infections in asymptomatic humans is higher than in asymptomatic dogs in the studied endemic area. It is questionable whether asymptomatic infection in humans constitutes a risk factor for dogs.

Title: Infection asymptomatique à Leishmania infantum chez les chiens et propriétaires de chiens dans une zone endémique du sud-est de la France. Abstract: La prévalence de la leishmaniose asymptomatique chez les chiens et leurs propriétaires dans les principales zones d'endémie françaises n'a pas été étudiée à ce jour. L'objectif de cette étude était de quantifier l'infection asymptomatique à Leishmania infantum dans le sud-est de la France chez des personnes saines et leurs chiens à l'aide de techniques de dépistage moléculaire et sérologique. Nous avons examiné chez des résidents immunocompétents et leurs chiens dans les Alpes-Maritimes la présence d'ADN parasitaire par PCR spécifique ciblant l'ADN du kinétoplaste (ADNk) et d'anticorps spécifiques par sérologie (ELISA pour le chien et Western Blot pour l'homme). Les résultats de 343 humains et 607 chiens ont été inclus; 46,9 % (n = 161/343) des humains et 18,3 % (n = 111/607) des chiens étaient positifs à la PCR et 40,2 % des humains (n = 138/343) et 9,9 % des chiens (n = 60/607) avaient une sérologie positive. Au total, 66,2 % des humains (n = 227) et 25,7 % des chiens (n = 156) avaient des sérologies positives et/ou des résultats de tests PCR positifs. Les chiens à poils courts étaient plus fréquemment infectés (71,8 %, n = 112) que les chiens à poils longs (12,2 %, n = 19) (p = 0,043). Les chiens semblaient plus sensibles à l'infection asymptomatique selon leurs races (taux supérieurs chez les chiens de chasse et chiens de berger) (p = 0,04). La plus forte proportion d'infections asymptomatiques chez les chiens et les humains a été observée dans la Région de Vence, correspondant à 28,2 % (n = 20/71) des chiens et 70,5 % (n = 31/44) des humains (4,5/100 000). personnes). En conclusion, le pourcentage d'infections chez les humains asymptomatiques est plus élevé que chez les chiens asymptomatiques dans la zone d'endémie étudiée. On peut se demander si une infection asymptomatique chez l'homme constitue un facteur de risque pour les chiens.

High-throughput analysis of the Trypanosoma cruzi minicirculome (mcDNA) unveils structural variation and functional diversity.

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Kinetoplast DNA-encoded ribosomal protein S12: a possible functional link between mitochondrial RNA editing and translation in Trypanosoma brucei.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

Sergentomyia species identification and their screening for possible infection to Leishmania spp. in Kaleybar, East-Azerbaijan province, Iran.

Leishmaniasis is a protozoal and vector-borne disease. World health organization has considered the disease as a neglected tropical disease. Phlebotomus and Lutzumyia species (order: Diptera, family: Psychodidae) are human leishmaniasis vectors in new and old worlds. Sergentomyia spp. (Diptera, Psychodidae) are proven vectors of lizard leishmaniasis. Although some studies have identified human Leishmania parasites in Sergentomyia, their role in parasite circulation is unknown yet. Hence, the parasitological and molecular methods were used to study the possible Leishmania infection of Sergentomyia spp., in the human and canine visceral leishmaniasis endemic area in North West of Iran. Even though Sergentomyia specimens were caught in a dominant number compared to Phlebotomus spp., no Leishmania promastigote or DNA was detected in live-caught or sticky trap-caught specimens, respectively. Sergentomyia spp. are proven vectors of sauroleishmaniasis, and despite several global reports of Leishmania infection in Sergentomyia spp., such findings should be carefully interpreted to avoid false vector incriminations.

Molecular Identification of Cutaneous Leishmaniasis Vectors in Alborz Province, North of Iran.

Cutaneous leishmaniasis (CL) is a vector-borne disease widely distributed in tropical and subtropical areas of North and South America, Europe, Asia, and Africa. Considering the increasing number of CL cases in recent years and the fact that no study has been conducted to identify CL fauna and vectors in Alborz province, this study was carried out to identify sand flies and CL vectors in this region. Sand flies were collected from August to October 2021 from plain and mountainous indoor and outdoor areas of the region using sticky paper traps and were detected morphologically. DNA was extracted from the midguts of female sand flies. In this study, 1157 sand flies were collected and identified. The number of sand flies caught from indoor and outdoor places was 367 (31.72%) and 790 (68.28%), respectively. Overall, six species of flies were of the genus Phlebotomus (Raynal, 1937), including Phlebotomus papatasi (P. papatasi, 695 [60.07%]; Scopoli, 1786), P. kandelakii (13 [1.12%]; Shchurenkova, 1926), P. sergenti (232 [20.05%]; Parrot, 1917), P. major (14 [1.21%]; Annandale, 1910), P. caucasicus (4 [0.35%]; Marzinowsky, 1917), P. alexandri (18 [1.56%]; Alexandri Sinton, 1920), and four were of the genus Sergentomyia (Artemiev, 1978), including Sergentomyia tiberiadis (109 [9.42%]; Adler, Theodor & Lourie, 1930), Sergentomyia baghdadis (53 [4.58%]), Sergentomyia sintoni (14 [1.21%]; Sintoni Pringle, 1933), Sergentomyia clydei (5 [0.43%]). P. papatasi spp. were dominant in indoor and outdoor places, with a prevalence of 695 (60.07%). The Leishmania major (L. major) gene was identified in five samples of P. papatasi spp. This suggests that P. papatasi is the potential vector spp. in the study area. Moreover, L. major was confirmed as the aetiological agent of CL cases in Alborz province. The identification of vectors and parasite spp. is very important for the treatment and operational planning of disease vectors.

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.