Anti-Toxoplasma gondii activity of rose hip oil-solid lipid nanoparticles.

Toxoplasma gondii is a highly prevalent pathogen, reported from almost all geographical regions of the world. Current anti-T. gondii drugs are not effective enough in immunocompromised patients, encephalitis, chorioretinitis, and congenital toxoplasmosis. Therefore, the prescription of these drugs has been limited. Rose hip oil (RhO) is a natural plant compound, which shows antibacterial, anticancer, and anti-inflammatory activities. In the current study, the anti-T. gondii and cell toxicity effects of solid lipid nanoparticles (SLNs) loaded by RhO (RhO-SLNs) were evaluated. Emulsification sonicated-homogenization method was used to prepare SLNs. RhO-SLNs were characterized, and their anti-T. gondii and cell toxicity effects were evaluated using in vitro analyses. The particle size and the zeta potential of the nanoparticles were 152.09 nm and -15.3 mV nm, respectively. The entrapment efficiency percentage was 79.1%. In the present study, the inhibitory concentration (IC)50 against T. gondii was >1 µg/mL (p-value <.0001). The cell toxicity assay showed cytotoxicity concentration (CC)50 >10 mg/mL (p-value = .017). In addition, at least 75% of T. gondii-infected Vero cells remained alive at concentrations >10 mg/mL. The concentration of 1 mg/mL showed highest anti-Toxoplasma activity and lowest cell toxicity against the Vero cell. Our findings suggest that carrying natural plant compounds with SLNs could be considered an effective option for treatment strategies against T. gondii infections.

Comprehensive analysis of recently discovered lncRNA-associated competing endogenous RNA network in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) arises from the epithelium of the nasopharynx and is characterized by geography-dependent incidence. Despite the high mortality rate, specifically in some ethnic groups, the mechanisms underlying NPC pathogenesis are not thoroughly understood and there is an urgent need to detect the potential and clinically applicable biomarkers to ameliorate the overall survival rate and improve the prognosis of patients. In recent years, research has increasingly focused on the importance of long non-coding RNAs (LncRNAs) in cancer progression. LncRNAs play critical roles in regulating gene expression through mechanisms such as competitively binding to microRNAs (CeRNA). While numerous LncRNAs have been studied in nasopharyngeal carcinoma (NPC), their potential as diagnostic and prognostic biomarkers have not been systematically examined. In the present study, we delve into elucidating the biological functions, molecular mechanisms, and clinical significance of newly identified LncRNAs that serve as sponges for different microRNAs in NPC. We highlight their regulatory mechanisms in promoting cell proliferation, invasion, and metastasis, and discuss their implications in diverse cancer-related signaling pathways. Our overall goal is to emboss the fundamental roles of LncRNA-mediated CeRNA networks in NPC progression, which may open up new avenues for determining the pathogenesis of NPC and developing effective prevention and treatment strategies.

Development of solid lipid nanoparticles-loaded drugs in parasitic diseases.

Parasites cause illnesses with broad spectrum of symptoms from mild to severe, and are responsible for a significant number of outbreaks in the world. Current anti-parasitic drugs are toxic and have significant side effects. Nano-carriers are believed to obviate the limitations of conventional drugs via decreasing side effects and increasing target delivery and drug permeability with a controlled prolonged release of a drug. Solid lipid nanoparticles (SLNs) are lipid nanoparticles (LNPs), which have frequently been practiced. Suitable release rate, stability, and target delivery make SLNs a good alternative for colloidal carriers. SLNs are supposed to have great potential to deliver natural products with anti-parasitic properties. Nanoparticles have employed to improve stability and capacity loading of SLNs, during recent years. This review describes development of SLNs, the methods of preparation, characterization, and loaded drugs into SLNs in parasitic diseases. In addition, we summarize recent development in anti-parasitic SLNs-loaded drugs.

Long intergenic non-protein coding RNA 115 (Linc00115): A notable oncogene in human malignancies.

Long noncoding RNAs (LncRNAs) are RNA transcripts ranging from 200 to 1000 nucleotides that have emerged as critical regulators of gene expression. Growing evidence highlights their involvement in tumor development. In particular, long intergenic non-protein coding RNA115 (Linc00115) has been identified as an oncogene across various human malignancies, with aberrant expression strongly linked to poor clinical outcomes in cancer patients. This review aims to delve into the expression patterns of Linc00115 and elucidate the underlying molecular mechanisms behind its oncogenic properties. Moreover, we discuss the potential utility of Linc00115 as a valuable diagnostic and prognostic biomarker in cancer.

Toward waterborne protozoa detection using sensing technologies.

Drought and limited sufficient water resources will be the main challenges for humankind during the coming years. The lack of water resources for washing, bathing, and drinking increases the use of contaminated water and the risk of waterborne diseases. A considerable number of waterborne outbreaks are due to protozoan parasites that may remain active/alive in harsh environmental conditions. Therefore, a regular monitoring program of water resources using sensitive techniques is needed to decrease the risk of waterborne outbreaks. Wellorganized point-of-care (POC) systems with enough sensitivity and specificity is the holy grail of research for monitoring platforms. In this review, we comprehensively gathered and discussed rapid, selective, and easy-to-use biosensor and nanobiosensor technologies, developed for the early detection of common waterborne protozoa.

Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line.

BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and ß-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed. RESULTS: The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6. CONCLUSION: Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.

Molecular Identification, Subtypes Distribution, and Alleles Discrimination of Blastocystis sp., Isolated from Immunocompromised Subjects in Iran.

Background: Blastocystis sp., is a prevalent protist isolated from humans and animals, which its opportunistic role in immunocompromised patients is still controversial. The current study aimed to evaluate the subtype and alleles distribution of Blastocystis sp., among immunocompromised patients. Methods: Totally, 33 microscopically Blastocystis-positive stool samples, isolated from Guilan province during April 2018 to May 2019 were investigated. Total DNA extraction was performed and the barcoding region of the small subunit ribosomal RNA (SSU rRNA) gene was amplified. Targeted fragments were sequenced to characterize subtypes and relevant alleles. Phylogenetic tree was constructed using Maximum-likelihood and Tamura 3-parameter to illustrate the correlation between subtypes and certain immunodeficiency. Results: Subtype analysis revealed the presence of ST1, ST2, ST3, and ST7 among 13/33 (39.4%), 5 (15.2%), 14/33 (42.4%), and 1/33 (3%), of samples, respectively. ST1 was the major subtype among cancer patients 5/7 (71.42%), while ST3 was the predominant subtype among rheumatoid arthritis (RA) patients 3/6 (50%), internal ward patients 5/10 (50%), and asthma and allergy patients 2/3 (66.66%). ST7 was isolated from a patient hospitalized in internal ward. No significant correlation was seen between the type of immunodeficiency and subtypes (P-value = 0.771). The phylogenetic tree showed no separation regarding the type of immunodeficiency. Conclusion: Among studied immunocompromised patients, ST3 was the most prevalent subtype followed by ST1. There was no specific correlation between subtypes and alleles with type of immunodeficiency. Putative zoonotic alleles were highlighted the probability of zoonotic transmission for Blastocystis sp.

Molecular analysis of internal transcribed spacer 2 of Dicrocoelium dendriticum isolated from cattle, sheep, and goat in Iran.

BACKGROUND: Dicrocoelium dendriticum is a broadly distributed zoonotic helminth, which is mainly reported from domesticated and wild ruminants. There is little data covering the molecular features of this trematode; therefore, current study aimed to molecularly analyze D. dendriticum in livestock. METHODS: Totally, 23 samples of D. dendriticum were collected from cattle, sheep, and goat from Ilam, Lorestan, and Khuzestan, three west and south-west provinces of Iran from February to August 2018. After genomic DNA extraction, the internal transcribed spacer (ITS) 2 fragment was amplified and sequenced in samples. To investigate genetic variations through the ITS 2 fragment of obtained D. dendriticum, phylogenetic tree and network analysis were employed. RESULTS: All 23 samples were successfully amplified and sequenced. Phylogenetic tree showed that our samples were clearly grouped in a clade together with reference sequences. There was no grouping based on either geographical regions or hosts. Network analysis confirmed the phylogenetic findings and showed the presence of nine distinct haplotypes, while our samples together most of sequences, which were previously submitted to the GenBank, were grouped in the Hap1. CONCLUSIONS: Our findings indicated that although ITS 2 fragment discriminate D. dendriticum, this fragment is not suitable to study intra-species genetic variations. Therefore, exploring and describing new genetic markers could be more appropriate to provide new data about the genetic distribution of this trematode.

Free-living amoebae in an oil refinery wastewater treatment facility.

Free Living Amoebae (FLA) are ubiquitous microorganisms reported from harsh environmental conditions. Oil refinery facilities consume vast volumes of water during their processes, generating a large amount of wastewater. The present study aimed to evaluate the wastewater treatment process in an oil refinery wastewater treatment facility (ORWWTF) for the presence of FLA. Water samples were collected from an oil refinery wastewater (ORWW) for nine months. After recording physical-chemical features, samples were cultivated onto non-nutrient agar (NNA). The discriminative fragments of the ribosomal RNA (rRNA) gene were amplified and sequenced to characterize the isolated FLA. Phylogenetic tree, and network analysis were employed to evaluate genetic relationships. The thermo- and osmotolerant tests were performed on the isolated FLA. Twenty-five (32.9%) samples were positive for FLA cultivation. Acanthamoeba spp., Vahlkampfiids, and Vermamoeba spp. were detected, of which Acanthamoeba species were predominant. There was no statistical correlation between pH, NH3, PO4, H2S, and TDS with the presence of FLA. A statistical correlation between the presence of FLA and the type of wastewater treatment plants (WWTPs) was significant (P-value = 0.011). All Acanthamoeba spp. isolates belonged to the genotypes T4 (17/21; 80.95%) and T11 (4/21; 19.05%). Vahlkampfiids were Naegleria spp., (7/10; 70%), Tetramitus aberdonicus (1/10; 10%), Learamoeba spp., (1/10; 10%), and Vahlkampfia spp., (1/10; 10%). All three Vermamoeba spp. were V. vermiformis. The ORWW contains toxic materials, and a few microorganisms can stay active in these environments. This is the first study which isolates FLA from such super harsh conditions. For the first time, T. aberdonicus, and Learamoeba spp., were isolated from oily wastewater. Our findings signify the concern due to the distribution of potentially pathogenic FLA to downstream lands via treated wastewater that may be released after treatment processing.

Formulation of Neem oil-loaded solid lipid nanoparticles and evaluation of its anti-Toxoplasma activity.

BACKGROUND: Toxoplasmosis is caused by an intracellular zoonotic protozoan, Toxoplasma gondii, which could be lethal in immunocompromised patients. This study aimed to synthesize Neem oil-loaded solid lipid nanoparticles (NeO-SLNs) and to evaluate the anti-Toxoplasma activity of this component. METHODS: The NeO-SLNs were constructed using double emulsification method, and their shape and size distribution were evaluated using transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively. An MTT assay was employed to evaluate the cell toxicity of the component. The anti-Toxoplasma activity of NeO-SLNs was investigated using vital (trypan-blue) staining. Anti-intracellular Toxoplasma activity of NeO-SLNs was evaluated in T. gondii-infected Vero cells. RESULTS: The TEM analysis represented round shape NeO-SLNs with clear and stable margins. DLS analysis showed a mean particle size 337.6 nm for SLNs, and most of nanoparticles were in range 30 to 120 nm. The cell toxicity of NeO-SLNs was directly correlated with the concentration of the component (P-value = 0.0013). The concentration of NeO-SLNs, which was toxic for at least 50% of alive T. gondii (cytotoxic concentration (CC50)), was > 10 mg/mL. The ability of NeO-SLNs to kill Toxoplasma was concentration-dependent (P-value < 0.0001), and all concentrations killed at least 70% of alive tachyzoites. Furthermore, the viability of T. gondii- infected Vero cells was inversely correlated with NeO-SLNs concentrations (P-value = 0.0317), and in the concentration 100 µg/mL at least 75% of T. gondii- infected Vero cells remained alive. CONCLUSIONS: Overall, our findings demonstrated that the NeO-SLNs was able to kill T. gondii tachyzoites in concentration 100 µg/mL with a cell toxicity lower than 20%. Such results suggest that employing SLNs as carrier for NeO can effectively kill T. gondii tachyzoites with acceptable cell toxicity. Our findings also showed that SLNs capsulation of the NeO can lead to prolonged release of the extract, suggesting that NeO-SLNs could be also employed to clear cyst stages, which should be further investigated in animal models.

Molecular characterization of Cryptosporidium skunk genotype in raccoons (Procyon lotor) in Iran: concern for zoonotic transmission.

Cryptosporidium spp. are significant zoonotic parasites in humans and animals worldwide. This study aimed to investigate the prevalence of Cryptosporidium infection among raccoon (Procyon lotor) in north of Iran. The fecal samples (n = 30) were collected from raccoons. After DNA extraction, all samples were examined by nested PCR amplification of the 18S ribosomal RNA (rRNA) gene. From 30 raccoon samples, 4 (13.3%) were positive, and the isolates were identified as Cryptosporidium skunk genotype based on sequence analysis. The large distribution of raccoons in northern provinces of Iran and their potency for carrying some human-infecting parasites like Cryptosporidium spp. propose this mammalian as a source for zoonotic parasites.

Soluble total antigen derived from Toxoplasma gondii RH strain prevents apoptosis, but induces anti-apoptosis in human monocyte cell line.

Apoptosis plays crucial role in the pathogenesis of toxoplasmosis, as it limits further development of the disease. The current study aimed to investigate the effects of different concentrations of soluble total antigen (STAg) of Toxoplasma gondii (Nicolle et Manceaux, 1908) on the apoptotic and anti-apoptotic pathways. PMA-activated THP-1 cell line was sensed by T. gondii STAg and the expression patterns of caspase-3, -7, -8, -9, Bax, Bcl-2, and Mcl-1 genes were evaluated. The results showed statistically significant concentration-dependent overexpression of both Bcl-2 (P-value < 0.0001) and Mcl-1 (P-value = 0.0147). The cas-7 showed overexpression in all concentrations (P-value < 0.0001). The cas-3 was suppressed in concentrations 100, 80, and 40 µg, but statistically significant downregulated in concentrations 10 and 20 µg. The Bax was suppressed in concentrations 100 to 20 µg, while it slightly downregulated 1.42 fold (P-value = 0.0029) in concentration 10 µg. The expression of cas-8 and -9 was suppressed in all concentrations. Our results indicated that T. gondii STAg downregulated and suppressed apoptotic and upregulated anti-apoptotic pathways. The upregulation of cas-7 in this study may indicate the role of T. gondii STAg in activation of inflammatory responses.

Evolutionary and phylogenetic analyses of the barcoding region suggest geographical relationships among Blastocystis sp., ST3 in humans.

Blastocystis sp., has 21 distinct subtypes of which ST3 thought to be the most prevalent subtype. This study aims to analyze the global variations of ST3. In total, 496 sequences with more than 400 nucleotides from Asia, Europe, Africa, and America were included in this study. Results show that allele 34 was the most prevalent allele in all continents. The lowest and highest allele diversity were observed in Europe and Africa, respectively. The nucleotide diversity ranged from 0.0077 in Europe to 0.02 in Africa, and haplotype diversity ranged from 0.461 in America to 0.6 in Africa. The haplotype network and Bayesian structure showed at least two major clusters including Asia and Europe-Africa-America. Tajima's D values for all continents were negative and statistically significant, indicating an excess of rare nucleotide variants. Similarly, the Fu's FS test showed negative values for all regions, indicating an excess of rare haplotypes. Pairwise FST exhibited a high genetic differentiation between Asia and other continents. Mismatch analysis for all populations showed a unimodal distribution. Our findings indicate that there are two probable major clusters of Blastocystis sp. ST3, a cluster which is shared between Europe, Africa, and America, and a cluster which is restricted to Asia.

Molecular prevalence and subtype distribution of Blastocystis sp. in Asia and in Australia.

Blastocystis is a prevalent protozoan parasite reported in humans, animals, and environmental samples. Over the past decade, numerous studies have investigated the prevalence and subtype distribution of Blastocystis sp. alongside with its genetic and biochemical features. However, studies on subtype distribution of this protozoan in humans, animals, and environmental samples represent the potential transmission routes. In this review, we evaluated studies performed in Asian countries and in Australia to provide an overview of environmental factors on the prevalence and subtype distribution of Blastocystis sp. among humans, animals, and the environment.

Toxoplasma gondii profilin and tachyzoites RH strain may manipulate autophagy via downregulating Atg5 and Atg12 and upregulating Atg7.

BACKGROUND: Autophagy process is an important defense mechanism against intracellular infection. This process plays a critical role in limiting the development of Toxoplasma gondii. This study aimed to investigate the effects of T. gondii profilin and tachyzoites on the expression of autophagy genes. METHODS AND RESULTS: PMA-activated THP-1 cell line was incubated with T. gondii profilin and tachyzoites for 6 h. After RNA extraction and cDNA synthesis, the expression of Atg5, Atg7, Atg12, and LC3b was evaluated using real-time PCR. The results revealed statistically significant downregulation of Atg5 for 1.43 (P-value = 0.0062) and 4.15 (P-value = 0.0178) folds after treatment with T. gondii profilin and tachyzoites, respectively. Similar to Atg 5, Atg 12 revealed a statistically significant downregulation for profilin (1.41 fold; P-value = 0.0047) and T. gondii tachyzoites (3.25 fold; P-value = 0.011). The expression of Atg7 elevated in both T. gondii profilin (2.083 fold; P-value = 0.0087) and tachyzoites (1.64 fold; P-value = 0.206). T. gondii profilin and tachyzoites downregulated (1.04 fold; P-value = 0.0028) and upregulated (twofold; P-value = 0.091) the expression of LC3b, respectively. CONCLUSIONS: Our findings suggest that T. gondii and profilin may manipulate autophagy via preventing from the formation of Atg5-12-16L complex to facilitate replication of T. gondii and development of toxoplasmosis.

Taxonomy, Population Structure and Genetic Diversity of Iranian Leishmania Strains of Cutaneous and Visceral Leishmaniasis.

BACKGROUND: Despite the broad distribution of leishmaniasis in Iran, there is a little genetic information about the causative agents and epidemiological status of the disease. Genetic diversity of the parasite is suggested to be one of the factors, which influences the clinical manifestations of the disease. In this study, we investigated the genetic variations, population structure, and evolutionary history of Leishmania species from endemic foci of Iran. METHODS: Fifty-two isolates from humans, canines, and rodents from different endemic foci of Iran were used to sequence the N-acetyl glucosamine-1-phosphate transferase (Nagt) gene. Phylogenetic and structure analyses were performed to investigate inter- and intra-species diversity of the Leishmania isolates. RESULTS: In total, 10 haplotypes including L. major (n = 6), L. tropica (n = 2), L. infantum (n = 1) and L. turanica (n = 1) were identified across 52 isolates. Haplotype diversity (Hd) ranged from zero for L. infantum and L. turanica to 0.78 ± 0.136 for L. major. This study identified population structure of Leishmania isolates from different geographical regions of Iran. The results of the phylogenetic tree showed 4 distinct clades for each species of Leishmania. In addition, the highest intraspecies diversity was observed among L. major isolates. No correlation was observed between species and geographic distribution of haplotypes. CONCLUSIONS: Leishmania isolates were identified at the species level using the Nagt gene, low variation within species indicates conservation of this gene in Leishmania. The results provide knowledge into the evolutionary history of Iranian Leishmania isolates.

Molecular characterization and identification of Blastocystis and its subtypes from raccoon (Procyon lotor) in north of Iran.

Blastocystis is a zoonotic protozoan parasite frequently identified in the intestinal tract of humans and a vast variety of animals, worldwide. Here, we assessed the prevalence of Blastocystis and its subtypes in stool samples of raccoons. Stool samples from 30 raccoons were collected. Total DNA was extracted, and the barcoding region of the small subunit ribosomal rRNA (SSU rRNA) gene was amplified and sequenced. Specific fragment for Blastocystis was successfully amplified in five samples (16.66%). Sequencing analysis revealed ST1, ST2, and ST3 among 1, 2, and 2 Blastocystis-positive samples. Our results documented the presence of Blastocystis subtypes 1-3 in raccoons. Subtype 1 showed higher similarity to the human isolates of Blastocystis. However, it seems that raccoons may emerge as reservoirs for Blastocystis and may be linked to zoonotic transmission of the protist.

The First Report and Molecular Analysis of Enterocytozoon bieneusi from Raccoon (Procyon lotor) in North of Iran.

Microsporidia are known opportunistic microorganisms and usually transmitted via the fecal-oral route. However, there is no information about human-infecting microsporidia in wildlife in Iran. This study aimed to investigate and analyze human-infecting microsporidia isolated from raccoons in north of Iran. Totally, 30 fecal samples were collected; then, DNA extraction was performed and specific fragments of the SSU rRNA gene of Enterocytozoon bieneusi and Encephalitozoon species were amplified. After amplification and sequencing the ITS, the results were compared to the GenBank database. Phylogenetic trees and network analysis were employed to explore probable relationships. E. bieneusi was the only detected microsporidia among samples. Genotyping showed the genotypes D, E, and RA in 15/18 (83.33%), 1/18 (5.55%), and 2/18 (11.11%) of samples, respectively. Novel genotypes RA1 and RA2 grouped together and apart from other genotypes. E. bieneusi genotypes D and E clustered with the genotypes previously reported from animals, humans, and environmental samples. Network analysis revealed six distinct sequence types among raccoon's isolates. This study demonstrated that E. bieneusi genotype D was the most prevalent microsporidia among raccoons. It seems that wildlife may play a role in dispersion of microsporidia spores.

Epidemiology of Fascioliasis in Kermanshah Province, Western Iran.

BACKGROUND: We aimed to determine the prevalence of fasciolosis in the definitive hosts (human and livestock) and intermediate (Lymnaea snails) hosts in Kermanshah Province, western Iran from 2014-2016. METHODS: The study on animals was descriptive and retrospective one. All daily records of animals slaughtered in the abattoirs were analyzed. For the study of human fascioliasis, 975 serum samples were collected from different parts of Kermanshah Province and analyzed using ELISA based on excretory-secretory antigens. Moreover, 4400 Lymnaea snails were collected from 25 habitats. The snails were identified and examined for presence of cercariae by shedding method. RESULTS: Fasciolosis was diagnosed in 1.7% of slaughtered animals, which was significantly greater than the other species (P<0.005). There was significant difference (P<0.001) between the prevalence of fasciolosis and seasonal pattern. As for human cases, five cases (0.5%) were positive for fascioliasis. Regarding the seropositivity to fasciolosis, no significant differences were found for age groups, sex, level of education and occupation. No Fasciola infection was seen in snails of the family Lymnaeidae. CONCLUSION: The prevalence of Fasciola parasite was lower compared to other provinces. This is probably due to sequential decline in rainfall and hot climate that makes conditions difficult for the snail intermediate host snails and the larval stages of fasciolid trematodes. The habitual food of people is another important point.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.