Protozoan Parasite Research Articles

Determinants of Anemia in Schoolchildren in the Highland Bolivia

Anemia is a health problem of concern among schoolchildren in underprivileged rural regions, where recurrent parasitic infections are common. A cross-sectional study was conducted in 229 schoolchildren in rural highland Bolivia in the department of La Paz, an area with a high prevalence of protozoan and helminth infections, to determine the types and mechanisms of anemia. A substantial proportion of children (40.2%) were found to be anemic based on hemoglobin measurements. No associations were found between low hemoglobin levels and helminth or protozoan infections when evaluating infectious causes of anemia, nor with Giardia lamblia or Blastocystis hominis, which are associated with iron deficiency and nutrient malabsorption and were highly prevalent in this study. The significant association between anemia and hypochromia suggests iron deficiency, aligned with low hemoglobin levels. A total of 39 out of 150 children (26%) had markers consistent with iron deficiency anemia (IDA), 26 out of 127 children (20%) met the criteria for anemia of inflammation (AI). Furthermore, 12 of the 127 tested children (9.4%) met the criteria for mixed AI with IDA according to the soluble transferrin receptor (sTfR)/log ferritin levels, which increased significantly due to overall infections by Hymenolepis nana and Ascaris lumbricoides helminths. The findings highlight the need for integrated public health interventions to address iron nutrition and parasitic infections to effectively prevent anemia in this vulnerable population.

Berberine ameliorates dextran sulfate sodium -induced colitis through tuft cells and bitter taste signalling

BackgroundInflammatory bowel disease (IBD), a persistent gastrointestinal disease, is featured with impaired gut immunity. Previous studies have demonstrated that tuft cells can regulate the intestinal type 2 immune response by activating downstream ILC2 and Th2 cells and repair gut barrier upon invasion of parasitic helminths, bacteria, protozoans, and enteritis through different chemo-sensing receptors, such as bitter taste receptors. Berberine is a widely used in the treatment of diarrhea in clinic, however the mechanism underlying this effect is not clear. In this study, we aim to explore the relationship between berberine and tuft cells in dextran sulfate sodium (DSS) -induced colitis.ResultsOur data showed that berberine significantly ameliorated DSS-induced colitis and regulating type 2 innate immune lymphocytes (ILC2) and Th2 immune cells via tuft cells in the gut. Furthermore, the effect of berberine on colitis was partially abolished by U73122, a bitter taste receptor inhibitor, suggesting that bitter taste signalling pathway played an important role in the effect of berberine on relieving colitis.ConclusionsBerberine ameliorates dextran sulfate sodium -induced colitis through tuft cells and bitter taste signalling. Our study reveals the unique pharmacological mechanisms of berberine in the context of colitis, laying the foundation for further clinical applications of this compound.

Entamoeba histolytica-induced NETs are highly cytotoxic on hepatic and colonic cells due to serine proteases and myeloperoxidase activities

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica, extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica-induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24 h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite.

Molecular characterization of the permanent outer-inner membrane contact site of the mitochondrial genome segregation complex in trypanosomes

The parasitic protozoan Trypanosoma brucei has a single unit mitochondrial genome linked to the basal body of the flagellum via the tripartite attachment complex (TAC). The TAC is crucial for mitochondrial genome segregation during cytokinesis. At the core of the TAC, the outer membrane protein TAC60 binds to the inner membrane protein p166, forming a permanent contact site between the two membranes. Although contact sites between mitochondrial membranes are common and serve various functions, their molecular architecture remains largely unknown. This study elucidates the interaction interface of the TAC60-p166 contact site. Using in silico, in vitro, and mutational in vivo analyses, we identified minimal binding segments between TAC60 and p166. The p166 binding site in TAC60 consists of a short kinked α-helix that interacts with the C-terminal α-helix of p166. Despite the presence of conserved charged residues in either protein, electrostatic interactions are not necessary for contact site formation. Instead, the TAC60-p166 interaction is driven by the hydrophobic effect, as converting conserved hydrophobic residues in either protein to hydrophilic amino acids disrupts the contact site.

Seroprevalence and Molecular Detection of Toxoplasma gondii in Wild Boars (Sus scrofa) in Khuzestan Province, Iran

ABSTRACTIntroductionToxoplasma gondii is a parasitic protozoan capable of infecting warm‐blooded animals, including humans, and is the causative agent of toxoplasmosis. Wild boars (Sus scrofa) serve as reservoirs for zoonotic pathogens like T. gondii, which can cause disease in humans if transmitted. This study aimed to assess the seroprevalence and molecular prevalence of T. gondii in wild boars in Khuzestan Province, Iran.MethodsSeroprevalence of T. gondii antibodies was assessed using enzyme‐linked immunosorbent assay (ELISA), employing a commercial kit (Calbiotech), whereas molecular detection was carried out through polymerase chain reaction (PCR) targeting the B1 gene of T. gondii on tissue samples (tongue, muscle, diaphragm and heart). Thirty wild boars were sampled from the regions of Shush, Shushtar, Dezful and Abadan, and both blood and tissue samples were analysed.ResultsIn this study, the seroprevalence of T. gondii antibodies was found to be 83.3% (25/30) among wild boar serum samples using ELISA. Molecular detection through PCR identified T. gondii DNA in 46.7% (14/30) of tissue samples, with the highest detection rates in tongue tissues (64%), followed by muscle (21%) and diaphragm (14%). No positive results were found in heart samples, and no animal had multiple tissues testing positive. All PCR‐positive cases corresponded with positive ELISA results, and a statistically significant difference was observed in parasite prevalence across different tissues (p = 0.002).ConclusionAlthough consumption of wild boar meat is banned in Iran, illegal hunting and consumption remain a concern. The high prevalence of T. gondii in wild boars poses a potential risk for transmission through the illegal consumption of undercooked or raw meat. This study highlights the need for public health interventions to control the illegal trade of wild boar meat and reduce the risk of toxoplasmosis transmission. Further research is recommended to investigate T. gondii distribution in other tissues, including the brain, and to better understand the parasite's epidemiology in this region.

Leishmaniasis Diagnosed in the United States Successfully Treated With Miltefosine

Abstract Leishmaniasis is an infectious disease caused by the parasitic protozoa species Leishmania and is most often transmitted to humans through the bite of a sandfly. The most common manifestation is a cutaneous lesion. Leishmaniasis is endemic to many countries throughout the world but rarely diagnosed in the United States. Here, we report a 64-year-old man with initially undisclosed recent travel to Sudan, an endemic country, who returned to the United States with cutaneous lesions on his forehead and dorsal hand. After failing initial therapy, a biopsy showed amastigotes (the tissue stage of the parasite), chronic inflammation with numerous plasma cells, granulomatous features, focal necrobiosis, and neutrophilic debris, consistent with leishmaniasis. The lesions did not significantly improve with fluconazole, but miltefosine (100–200 mg daily) resulted in remission over an 8-week course. The purpose of this case report is to highlight the importance of including rare infectious organisms in the differential and briefly discuss diagnostic and treatment methods.

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.

Structures of Trichomonas vaginalis macrophage migratory inhibitory factor.

The unicellular parasitic protozoan Trichomonas vaginalis causes trichomoniasis, the most prevalent nonviral sexually transmitted disease globally. T.vaginalis evades host immune responses by producing homologs of host proteins, including cytokines such as macrophage migration inhibitory factor. T.vaginalis macrophage migration inhibitory factor (TvMIF) helps to facilitate the survival of T. vaginalis during nutritional stress conditions, increases prostate cell proliferation and invasiveness, and induces inflammation-related cellular pathways, thus mimicking the ability of human MIF to increase inflammation and cell proliferation. The production, crystallization and three structures of N-terminally hexahistidine-tagged TvMIF reveal a prototypical MIF trimer with a topology similar to that of human homologs (hMIF-1 and hMIF-2). The N-terminal tag obscures the expected pyruvate-binding site. The similarity of TvMIF to its human homologs can be exploited for structure-based drug discovery.

“Novel chemo-enzymatic synthesis, structural elucidation and first antiprotozoal activity profiling of the atropoisomeric dimers of trans-8-Hydroxycalamenene”

Leishmaniasis and malaria are two debilitating protozoan diseases affecting millions globally, particularly in tropical and subtropical regions. Current therapeutic options face significant challenges due to emerging drug-resistant strains, necessitating the discovery of novel antiprotozoal agents. This study explores, for the first time, the antiprotozoal potential of calamenenes and their dimers, naturally occurring sesquiterpenes found in essential oils, through a novel chemo-enzymatic synthesis approach. Using the laccase from Trametes versicolor, atropoisomeric dimers of (−)- and (+)-8-trans-hydroxycalamenene were synthesized from commercially available (−)- and (+)-menthol. Structural elucidation was achieved via 2D-NMR spectroscopy, electronic circular dichroism, and DFT calculations. In vitro profiling against Leishmania spp and drug-resistant Plasmodium falciparum revealed that calamenene dimers exhibited significantly higher antiprotozoal activity compared to their monomeric counterparts, highlighting the potential of dimeric terpenoids as promising antiprotozoal agents. This work lays the foundation for developing novel antimalarial drugs based on calamenene scaffolds, encouraging further interactome studies to optimize their pharmacological properties.

The emerging role of photodynamic therapy in the treatment of cutaneous infections.

Cutaneous infections pose ongoing challenges to standard treatments due to resistance and limited efficacy. Photodynamic therapy (PDT) emerges as a promising supplement or an alternative to address complicated cases. In this review, we comprehensively review PDT's safety and efficacy in treating cutaneous infections. A PubMed systematic review with search terms for PDT treatment, hair, skin, and nail infections. There were a total of 43 studies on the use of PDT in cutaneous infections which discussed the treatment of viral (N.=20), bacterial (N.=11), fungal (N.=9), and protozoal (N.=3) infections. There is evidence for using PDT, mostly 5-aminolevulinic acid (5-ALA) and methyl aminolevulinate (MAL), in the treatment of cutaneous infections. Most evidence for viral infections involved treatment with 5-ALA PDT in condyloma acuminatum (CoAc), verruca vulgaris, and molluscum contagiosum. In bacterial infections, 5-ALA and MB PDT have been used to achieve complete remission in refractory Pseudomonas and atypical mycobacteria infections without recurrence. In onychomycosis, MAL PDT achieved a 40.9% cure rate and MB PDT showed a 77.8-100% cure rate with no reports of recurrence. Parasitic infections, such as leishmaniasis have also been successfully treated with both 5-ALA and MAL PDT. PDT is a promising treatment option for cutaneous infections, with growing evidence for its utility in treating cutaneous bacterial, viral, fungal, and parasitic infections, particularly those that fail standard treatments. Side effects were mostly limited to localized pain with good outcomes and low recurrence rates.

Evolutionary genomics of Leishmania braziliensis across the neotropical realm

The Neotropical realm, one of the most biodiverse regions on Earth, houses a broad range of zoonoses that pose serious public health threats. Protozoan parasites of the Leishmania (Viannia) braziliensis clade cause zoonotic leishmaniasis in Latin America with clinical symptoms ranging from simple cutaneous to destructive, disfiguring mucosal lesions. We present the first comprehensive genome-wide continental study including 257 cultivated isolates representing most of the geographical distribution of this major human pathogen. The L. braziliensis clade is genetically highly heterogeneous, consisting of divergent parasite groups that are associated with different environments and vary greatly in diversity. Apart from several small ecologically isolated groups with little diversity, our sampling identifies two major parasite groups, one associated with the Amazon and the other with the Atlantic Forest biomes. These groups show different recombination histories, as suggested by high levels of heterozygosity and effective population sizes in the Amazonian group in contrast to high levels of linkage and clonality in the Atlantic group. We argue that these differences are linked to strong eco-epidemiological differences between the two regions. In contrast to geographically focused studies, our study provides a broad understanding of the molecular epidemiology of zoonotic parasites circulating in tropical America.

Tetramerization of deoxyadenosine kinase meets the demands of a DNA replication substrate challenge in Giardia intestinalis.

The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on salvaging deoxyadenosine and other deoxyribonucleosides from its host environment. Here, we report that G. intestinalis has a deoxyribonucleoside kinase with a 1000-fold higher catalytic efficiency (kcat/KM) for deoxyadenosine than the corresponding mammalian kinases and can thereby provide sufficient deoxyadenosine triphosphatelevels for DNA synthesis despite the lack of de novo synthesis. Several deoxyadenosine analogs were also potent substrates and showed comparable EC50 values on cultured G. intestinalis cells as metronidazole, the current first-line treatment, with the additional advantage of being effective against metronidazole-resistant parasites. Structural analysis using cryo-EM and X-ray crystallography showed that the enzyme is unique within its family of deoxyribonucleoside kinases by forming a tetramer stabilized by extended N- and C-termini in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a markedly reduced affinity for the deoxyribonucleoside substrate. The development of highly efficient deoxyribonucleoside kinases via oligomerization may represent a critical evolutionary adaptation in organisms that rely solely on deoxyribonucleoside salvage.

Occurrence of Leishmania spp. in phlebotomine sand flies and dogs in Guelma region, North-eastern Algeria

Leishmania spp. are sand fly-borne parasitic protozoa of worldwide distribution that may severely affect the health and welfare of dogs as well as of other mammalian species, including humans. Algeria is among the most affected countries, counting several cases of Leishmania infantum infection in humans and dogs. Here, we assessed the occurrence of Leishmania species in both phlebotomine sand fly and dogs in the Guelma region, Northeast of Algeria. Sand flies were collected from July to September 2022, followed by a survey of canine leishmaniasis (CanL) from September 2023 to February 2024. Additionally, to understand the risk of human infection a retrospective data on cases of leishmaniasis recorded in the region from 2012 to 2023 were reported. Sand fly specimens and canine blood samples were tested by quantitative PCR (qPCR) for Leishmania spp., while dog serum samples were processed for anti-L. infantum antibodies detection by immunofluorescent antibody test (IFAT). Of the 1478 sand flies (n = 318 females; n = 1160 males), seven species were identified (i.e., Phlebotomus papatasi, Ph. perfiliewi, Ph. sergenti, Ph. perniciosus, Ph. longicuspis, Ph. ariasi, and Sergentomyia minuta). Leishmania spp. was detected in Ph. perniciosus, Ph. perfiliewi, Ph. papatasi, and Ph. sergenti (i.e., 3.7 %; n = 10). The overall seroprevalence rate was 58.2 %, with 1.6 % of dog blood samples positive for L. infantum at molecular screening. Multivariate analysis of the different risk factors revealed that CanL seropositivity was strongly related to dog age (> 1 year) (OR = 8.35, 95 % CI: 3.43–23.93), the autumn season (OR = 2.95, 95 % CI: 1.33–8.62), and lack of insecticide application (OR = 6.69, 95 % CI: 2.61–22.02). A total of 71 human cases of cutaneous (CL) and visceral (VL) leishmaniasis were recorded. Data presented reveal the occurrence of CanL in Guelma region, advocating for control program measures to be implemented in this part of Algeria.

Prevalence and molecular identification of protozoan intestinal parasitic infections in cancer patients and a control group

BackgroundParasitic infections, especially opportunistic infections, are important issues for immunocompromised patients, including cancer patients. In this study, the prevalence of intestinal protozoan infections was investigated in Iranian cancer patients and a control group using microscopic and molecular methods.MethodsThis cross-sectional study was conducted on a group of 158 individuals with gastrointestinal and non-gastrointestinal cancers from hospitals affiliated with the Iran University of Medical Sciences, alongside 158 healthy individuals included in the control group. Stool samples were collected and examined using direct and concentration methods. The modified acid-fast staining method was used to detect coccidian parasite infections. DNA was extracted from the patients’ samples, and PCR and sequencing were performed.ResultsThe overall prevalence of protozoan infections was higher in the cancer patients (31.6%) than in the control group (12.0%), with the difference between the two groups being statistically significant (P = 0.0001). The study identified Blastocystis hominis as the most common protozoan, with a prevalence of 22.8%, followed by Giardia lamblia, Entamoeba coli, Dientamoeba fragilis, and Chilomastix mesnili, with rates of 2.5%, 1.3%, 1.3%, and 0.6%, respectively, in the cancer patients. Cystoisospora belli and Cryptosporidium sp. were found as opportunistic infections in 2.5% and 0.6% of the cancer patients, respectively. Blastocystis hominis, with a prevalence of 9.5%, followed by Giardia lamblia at 1.3%, were the most common parasitic infections in the control group. A statistical difference was found for Cystoisospora belli (P = 0.044) and Blastocystis hominis (P = 0.0013) between the cancer patients and the control group. Cryptosporidium sp. was confirmed as Cryptosporidium parvum, and Cystoisospora belli was confirmed by PCR sequencing.ConclusionChemotherapy, aside from corticosteroids, increases susceptibility to intestinal Protozoan infections in patients with hematological malignancies, particularly those with lymphoma and leukemia. The results indicated a higher risk of intestinal Protozoan infections, including opportunistic infections, in the cancer patients than in the control group. Cystoisospora belli and Cryptosporidium parvum were found to cause diarrhea in hematological malignancy patients undergoing chemotherapy.

One-Step Multiplex Polymerase Chain Reaction Assay for the Detection of Major Disease-Transmitting Mosquito Vectors in India.

Mosquitoes are important vectors that transmit viral, protozoan, and helminthic diseases across the world. Climate change and unplanned urbanization are accelerating the spread of these diseases. Controlling vector-borne diseases can be performed most effectively through vector control. Inadequate knowledge of vector bionomics is an impediment and can lead to inappropriate vector control efforts. However, the conventional methods of vector identification are based on morphological differences, demand a significant amount of time and specific skills, and are often misleading. An efficient and affordable solution is needed to quickly and accurately identify pooled samples from vast geographical territories. To ensure the correct identification of distorted or pooled samples in India, a set of definitive steps is required, including the construction of unique primers and the standardization of a one-step assay based on the second internal transcribed spacer gene of the ribosomal DNA. We have successfully developed and confirmed a highly efficient one-step multiplex reverse transcriptase polymerase chain reaction assay for the accurate identification of major mosquito vectors, especially in the cases of both the adult and larval forms of Anopheles sp., Aedes sp., and Culex sp. Hence, the specificity, universality, and uniqueness of these primers could serve as a critical tool for the rapid one-step and one-reaction identification of mosquitoes to control mosquito-borne disease outbreaks and public health emergencies.

Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome.

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

Evaluation of an attenuated chicken-origin Histomonas meleagridis vaccine for the prevention of histomonosis in chickens

IntroductionHistomonosis, a protozoan disease caused by Histomonas meleagridis, poses a significant economic burden on domestic poultry in China. To reduce the losses caused by this disease in chickens, an attenuated vaccine was developed by exploiting the diminished virulence of H. meleagridis through successive in vitro passages.MethodsFour experiments were conducted to evaluate the viability of attenuated H. meleagridis as a potential vaccine candidate. Experiment 1 evaluated the route of infection (oral vs. intracloacal) and dose (5 × 104, 1 × 105, and 2 × 105H. meleagridis/chicken) using the virulent strain H. meleagridis JSYZ-D10. Experiment 2 evaluated the attenuated effect of the H. meleagridis JSYZ-D168 strain (infection dose: 2 × 105H. meleagridis/chicken). Experiment 3 evaluated the immunoprotective effect of different immunization doses (5 × 104, 1 × 105, and 2 × 105H. meleagridis/chicken). Experiment 4 evaluated the immunoprotective effect of different immunization schedules (immunization at 3 days of age; immunization at 14 days of age; two immunizations, one at 3 days of age and one at 14 days of age; immunization and infection dose: 2 × 105H. meleagridis/chicken).ResultsThe results showed that the intracloacal route of infection was more effective and stable compared to the oral route. The pathogenicity of the JSYZ-D168 H. meleagridis strain was significantly reduced compared to the original virulent strain. Chickens vaccinated by intracloacal immunization at a dose of 2 × 105H. meleagridis/chicken on day 14 provided effective protection against a virulent strain challenge, significantly resulting in increased body weight and reduced lesions in the cecum and liver within 28 days post-immunization (p < 0.05). Poor immunoprotection was obtained either when the immunization dose was 1 × 105H. meleagridis/chicken or when the immunization program was a single immunization at 3 days of age only.DiscussionIn conclusion, the administration of a vaccine provides a measurable degree of protection against the detrimental effects induced by H. meleagridis, thus warranting its endorsement in clinical settings.

Genome-wide quantification of polycistronic transcription in Leishmania major.

Leishmania major is a human-pathogenic, obligate parasite and the etiological agent of the most prevalent, cutaneous form of leishmaniasis, which is an important neglected, tropical disease with ~1.2 million new infections per year. Leishmania, and the whole order Trypanosomatida, are early eukaryotes with highly diverged gene expression and regulation pathways, setting them apart from their mammalian hosts and from most other eukaryotes. Using precision run-on sequence analysis, we performed a genome-wide mapping and density analysis of RNA polymerases in isolated nuclei of the protozoan parasite Leishmania major. We map transcription initiation sites at divergent strand switch regions and head-tail regions within the chromosomes and correlate them with known sites of chromatin modifications. We confirm continuous, polycistronic RNA synthesis in all RNA polymerase II-dependent gene arrays but find small varying RNA polymerase activities in polycistronic transcription units (PTUs), excluding gene-specific transcription regulation, but not PTU-specific variations. Lastly, we find evidence for transcriptional pausing of all three RNA polymerase classes, hinting at a possible mechanism of transcriptional regulation.IMPORTANCELeishmania spp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression in Leishmania but also in the related Trypanosoma is radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation, and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable resource for researchers in the field of eukaryotic microbiology.

Seroepidemiological study of Neospora caninum in equids using c-ELISA in Erbil Province, Iraq.

Equine neosporosis is an intracellular protozoan disease with a global distribution, affecting a diverse range of warm-blooded animals. Neospora caninum Dubey, Carpenter, Speer, Topper et Uggla, 1988 is associated with foetal loss, neurological disease and abortion in equids. No information was available regarding equine N. caninum infection among equids in Iraq. Thus, the aim of this study was to determine the prevalence rate of N. caninum in equines by using a competitive enzyme-linked immunosorbent assay (c-ELISA). A total of 329 blood samples randomly selected from equines, comprising 268 horses and 61 donkeys were examined. The seroprevalence rate of N. caninum was determined as 46% (28/61) for donkeys and 24% (64/268) for horses. The prevalence of N. caninum indicated a significantly higher risk of infection in donkeys compared to horses (P < 0.001). However, the odds of N. caninum infection in draught equids were 8.2 times greater than other equids with a significant difference (P < 0.001). The current study revealed no significant differences in the prevalence of N. caninum across various genders, breeds, clinical statuses, disease histories and among equids that had contact with dogs. While outdoor feeding and mixed (grazing), showed a significant difference (P = 0.003) and (P = 0.75), respectively, in the presence of antibodies against N. caninum compared to indoor feeding (stable). Moreover, the odds of infection in equids with a history of late abortion were 4.8 times higher than those without such a history of abortion (2.20-10.56) with statistical significance (P < 0.001).

Differential Immune Responses of Th1 Stimulatory Chimeric Antigens of Leishmania donovani in BALB/c Mice.

Visceral leishmaniasis (VL) is the third most severe infectious parasitic disease and is caused by the protozoan parasite Leishmania. To control the spread of the disease in endemic areas where the asymptomatic patients act as reservoirs as well as in nonendemic areas, an effective vaccine is indispensable. In this direction, we have developed three chimeric proteins by the combination of three already known Th1 stimulatory leishmanial antigens, i.e., enolase, aldolase, and triose phosphate isomerase (TPI). The newly developed chimeric proteins, i.e., enolase-aldolase, TPI-enolase, and aldolase-TPI along with BCG as an adjuvant were assessed and compared, examining humoral and cellular adaptive immune responses elicited in BALB/c mice. The three chimeric antigens exhibited differential immune responses shown by differences in Th1 and Th2 cytokine production in ex vivo stimulated splenocytes of immunized mice. It was observed that all three chimeric proteins are more immunogenic than their component proteins. However, while comparing the immune response of the three chimeric proteins, aldolase-TPI exhibited a better immunogenic (Th1-type) response, as evidenced by the highest IFN-γ production, a high IgG2a antibody isotype switching, a high % population of CD8+ and CD4+ T-cells, and a significantly high expression of iNOS2. Thus, the results suggest the potential of these chimeric antigens as strong immunogens that can be harnessed in vaccine development against VL.