Reinfection of farm dogs following praziquantel treatment in an endemic region of cystic echinococcosis in southeastern Iran.

Cystic Echinococcosis (CE) as a prevalent tapeworm infection of human and herbivorous animals worldwide, is caused by accidental ingestion of Echinococcus granulosus eggs excreted from infected dogs. CE is endemic in the Middle East and North Africa, and is considered as an important parasitic zoonosis in Iran. It is transmitted between dogs as the primary definitive host and different livestock species as the intermediate hosts. One of the most important measures for CE control is dog deworming with praziquantel. Due to the frequent reinfection of dogs, intensive deworming campaigns are critical for breaking CE transmission. Dog reinfection rate could be used as an indicator of the intensity of local CE transmission in endemic areas. However, our knowledge on the extent of reinfection in the endemic regions is poor. The purpose of the present study was to determine E. granulosus reinfection rate after praziquantel administration in a population of owned dogs in Kerman, Iran. A cohort of 150 owned dogs was recruited, with stool samples collected before praziquantel administration as a single oral dose of 5 mg/kg. The re-samplings of the owned dogs were performed at 2, 5 and 12 months following initial praziquantel administration. Stool samples were examined microscopically using Willis flotation method. Genomic DNA was extracted, and E. granulosus sensu lato-specific primers were used to PCR-amplify a 133-bp fragment of a repeat unit of the parasite genome. Survival analysis was performed using Kaplan-Meier method to calculate cumulative survival rates, which is used here to capture reinfection dynamics, and monthly incidence of infection, capturing also the spatial distribution of disease risk. Results of survival analysis showed 8, 12 and 17% total reinfection rates in 2, 5 and 12 months following initial praziquantel administration, respectively, indicating that 92, 88 and 83% of the dogs had no detectable infection in that same time periods. The monthly incidence of reinfection in total owned dog population was estimated at 1.5% (95% CI 1.0-2.1). The results showed that the prevalence of echinococcosis in owned dogs, using copro-PCR assay was 42.6%. However, using conventional microscopy, 8% of fecal samples were positive for taeniid eggs. Our results suggest that regular treatment of the dog population with praziquantel every 60 days is ideal, however the frequency of dog dosing faces major logistics and cost challenges, threatening the sustainability of control programs. Understanding the nature and extent of dog reinfection in the endemic areas is essential for successful implementation of control programs and understanding patterns of CE transmission.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus.

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Effect of turmeric extract on bone healing in an experimental model of femoral bone fracture.

Objective: Following bone trauma, several factors participate in making a balance between the activity of osteoblasts and osteoclasts. The receptor activator of nuclear factor kappa B ligand (RANKL), receptor activator of nuclear factor kappa B (RANK), and osteoprotegerin (OPG) molecules play critical roles in the healing process via regulation of osteoclasts function. Turmeric is suggested to have an anti-osteogenic potential; however, its effect on accelerating bone healing has not been adequately studied. Here, we used a rat model of femur fracture to explore the effect of treatment with turmeric extract on the bone repair and the expression of RANK, RANKL, and OPG molecules. Materials and Methods: Eight rats were subjected to surgery, randomly divided into two groups, and treated orally with turmeric (200 mg/kg), or olive oil. Four oil-treated rats without bone fracture were used as control group. After six weeks of treatment, the femurs of animals were examined for radiological, histological, and gene expression analysis. Results: X-ray radiography showed thicker callus and a more obscure fracture line in the turmeric group. Furthermore, higher osteoblast percentages but no osteoclasts were observed in turmeric-treated animals, representing better repair of bone in the fracture site. Also, real-time analyses showed that treatment with turmeric reduced RANK and RANKL expression (p<0.0001) and lowered RANKL/OPG ratio (p=0.01) in femoral bone tissue. Conclusion: Our findings indicated the turmeric ability to facilitate bone hemostasis and optimize the expression of key markers involved in the bone metabolism.

Natural history of Echinococcus granulosus microcyst development in long term in vitro culture and molecular and morphological changes induced by insulin and BMP-4.

Introduction: Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus is a disease of worldwide public health and economic importance. The determinants and underlying cellular mechanisms of CE development and fate in intermediate hosts are largely unknown. Hormones and cytokines such as insulin and BMP-4 are the key players in the development, differentiation, and apoptosis. In this study, we evaluated the long term natural history of E. granulosus microcysts in an vitro setting and the molecular and morphological changes induced by the growth factors, insulin and BMP4 during the development of metacestode stage of E. granulosus. Methods: E. granulosus protoscoleces were cultivated and the parasite development was followed in the long term mono-phasic culture for 105 days and the morphometric, molecular and immunohistochemical changes were evaluated, including the microcysts number and size, microcysts development and deformation rates as well as the markers of calcification (Alizarin Red staining) and apoptosis (BAX, BCL2, Caspase-3, Caspase-8 and TNF-α expression) in the microcysts. Also the biological, histological and molecular consequences of insulin and BMP-4 treatment on the parasite development were evaluated. Results: Insulin and BMP-4 treatment of microcysts resulted in significant increase in microcyst formation, increased size, reduced apoptosis and deformation of the microcysts. Alizarin red staining of the microcysts treated with the insulin and BMP-4 confirmed that calcium deposition is significantly lower than the untreated microcysts. Also Alizarin Red staining and Immunohistochemistry of the microcysts indicates that calcium accumulation in deformed microcysts is higher than the normal ones on day 105. The microcysts began to wrinkle and the germinal layer was partially detached from the laminated layer on day 84. Conclusion: Results of the present study suggest that the degenerative changes in hydatid cysts can be slowed down by insulin and BMP-4, indicating that cellular factors and host hormones could contribute to the longevity of hydatid cysts. Significant evidences are provided suggesting that the microcysts cultivated in vitro can undergo calcification and apoptotic processes similar to what have been observed in the natural hydatid infection in the intermediate hosts.

Comparative Analysis of miRNA Expressions in Different Developmental Stages of Echinococcus granulosus in Mono-Phasic and Di-Phasic Culture Systems.

BACKGROUND: The dog tapeworm, Echinococcus granulosus, is a zoonotic parasite affecting humans and livestock across the globe. Basic research on the molecular biology and genetics of E. granulosus improves our understanding of the biology and potential drug targets in various developmental stages of E. granulosus in both definitive and intermediate hosts. There has been increasing interest in the identification of microRNAs in parasitic organisms. The purpose of the current study was to compare the activity of a selected profile of miRNAs in different developmental stages of E. granulosus. METHODS: Different developmental stages of the parasite were obtained from ex vivo as well as in vitro cultured E. granulosus. MicroRNAs were extracted from the ex vivo germinal layer and invaginated protoscoleces as well as the in vitro generated microcysts, evaginated protoscoleces, and strobilated worms. The expression of the selected miRNAs was evaluated by RT-qPCR for each stage. RESULTS: Four out of five miRNAs were present and active in different developmental stages of E. granulosus. A significant over-expression of miR-61 was observed in the germinal layer and during the protoscolex transformation into the microcysts, however, miR-10 was more expressed in the mature strobilated forms than in the other stages. Let-7 and miR-3489 showed a high expression in the germinal layer. CONCLUSION: Differential expression of four miRNAs among different in vitro and ex vivo developmental stages of E. granulosus was documented in the present study. Further experimental investigations are required to elucidate the probable role of the miRNAs in bi-directional differentiation of protoscoleces either into the strobilated worm or to a secondary hydatid cyst and the potential of these miRNAs as drug targets.

Linguatulosis in small ruminants in southeastern Iran: Epidemiological, histopathological and phylogenetic findings and its public health importance.

Linguatulosis, as a zoonotic disease, can infect most ruminants and cause accidental infections in humans. The objective of this study was to explore the epidemiological, histopathological and phylogenetic profiles of Linguatula serrata infection in sheep and goats and its public health importance during 2015-2018. Mesenteric lymph nodes (MLNs) and liver tissue of goats and sheep were selected randomly in Kerman slaughterhouse. Nymphal samples were used for DNA extraction, amplification and subsequently phylogenetic analysis using 18s rRNA and cytochrome C oxidase subunit 1 (cox1). Overall, of 828 examined livestock, 179 (42.4%) goats and 71 (17.5%) sheep were found to be infected with the nymphal stage of L. serrata. A significant difference was observed between linguatulosis and age. In the histopathological assessment, longitudinal and transverse sections of L. serrata nymphs were observed within the cyst-like spaces surrounded by a wall of fine fibrosis and compact lymphocytes. Moreover, comparing with the L. serrata reference sequences, we found only a single nucleotide change in our goat haplotype in 18s genetic region; while much nucleotide variations were observed in cox1 gene sequences. The results of the present study showed a high infection rate among goats and sheep in southeastern Iran. A better understanding of the disease could be achieved when the parasite species, their molecular characterization and the extent of infection in the area are determined. It is fundamental to select a comprehensive control program in order to take proper preventive and therapeutic measures against the infection.

Linguatula serrata in cattle in southeastern Iran: Epidemiological, histopathological and phylogenetic profile and its zoonotic importance.

This study aimed to determine the prevalence, histopathological observations, and phylogenetic analysis of L. serrata in cattle and its potential zoonotic and public health implications in southeastern Iran.The cross-sectional study was performed in cattle in southeastern Iran. Lymph nodes were collected from each cattle and examined by parasitological and histopathological techniques. A binary logistic regression and chi-square tests were implemented to analyze the data. Genomic DNA was randomly extracted from the nymphal stages of Linguatula isolates. Further characterization and phylogenetic relationships were done using two primers for amplification of partial DNA fragments of 18 s rRNA and cytochrome C oxidase subunit 1 (cox1), respectively.The results showed that 64 cattle of the total 404 were infected with L. serrata. There was no significant difference between linguatulosis infection and gender, while age was significantly different (P < 0.05). The cyst-like spaces containing the longitudinal and transverse sections of the L. serrata nymphs were surrounded by granulomatous reactions. The higher nucleotide variation in the cox1 region was supported by estimating the evolutionary divergence between L. serrata isolates and other Linguatula records of ruminants in Iran. The phylogenetic tree confirmed the close evolutionary relationships among all reported records of L. serrata in Iran.The high prevalence of linguatulosis caused by L. serrata declares the existence of a potential risk of FBPs for humans in southeastern Iran. This condition can advance more serious public health problems and requires a comprehensive control program and treatment strategies to prevent the disease.

Quantifying the load of Echinococcus granulosus eggs in experimental dog infection using probe-based copro-qPCR analysis.

Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no "probe-based" molecular quantification tool has been available for E. granulosus. This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of E. granulosus in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard curve analysis, 93% efficiency and coefficients of correlation (Rsq) > 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of E. granulosus infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs.

Cystic Echinococcosis of Camels: 12S rRNA Gene Variation Revealed Changing Pattern of Genetic Diversity Within Echinococcus granulosus sensu lato in the Middle East and North/Sub-Saharan Africa.

Cystic echinococcosis (CE) is one of the most widespread zoonotic diseases, with considerable public health and economic importance. Camels play a significant role in transmission cycle of Echinococcus granulosus especially, in the Middle East and North Africa (MENA). The present study aimed to identify the genetic variation and haplotype distribution of camel isolates of E. granulosus sensu lato using all existing E. granulosus mitochondrial DNA data from camels in different parts of the world. Sequence data from 1,144 camel isolates of E. granulosus s.l. available in the NCBI GenBank including 57 camel hydatid cysts collected in central Iran were used to analyze the nature of genetic variation within the camel isolates of E. granulosus s.l. in MENA region. Fifty-seven camel isolates were also PCR-sequenced on mitochondrial 12S rRNA gene. Haplotype network analysis revealed seven different haplotypes clustered into four major groups. E. intermedius G6 was identified as the most commonly represented genotype in camels followed by G1. Mitochondrial 12S rRNA gene sequence analysis on 57 camel isolates identified three different genotypes, including E. intermedius/G6 (35/57, 61.4%), E. granulosus sensu stricto/G1-G3 (21/57, 36.8%) as well as one isolate identified as E. ortleppi/G5 (1/57, 1.8%). The number of base substitutions per site over 420 positions of partial 12S rRNA gene sequences were shown as 0.000 and 0.004 for E. intermedius (G6) corresponding to the Middle East and sub-Saharan isolates, respectively. Camel isolates of E. granulosus in the MENA region present moderate genetic diversity (Hd = 0.5540-0.6050). The Middle East isolates demonstrated a more diverse population than the North/sub-Saharan isolates, where six out of seven 12S rRNA haplotypes were identified in the former region. E. intermedius (G6 genotype) was shown to be the most common species in the world camel population. In conclusion, camels showed to be an important intermediate host species in the MENA region with different patterns of genetic variation between the Middle East and Africa.

Intestinal Expression of miR-130b, miR-410b, and miR-98a in Experimental Canine Echinococcosis by Stem-Loop RT-qPCR.

Echinococcus granulosus is a zoonotic cestode dwelling in the small intestine of canid definitive hosts. Intermediate hosts are a wide range of domestic and wild ungulates. Human infection with the larval stage causes cystic echinococcosis. Understanding the nature and extent of molecular mechanisms involved in host-parasite interactions helps to answer some very basic questions in the biology of cestode parasites with significant implications in the management and control of cystic echinococcosis. Little is known on the miRNAs expression in the intestinal tissues of dogs infected with E. granulosus. In the present study, expression of a selected profile of miRNAs was evaluated in experimental canine echinococcosis. MiRNAs were extracted from 20 different parts of small intestinal tract of two sibling 3-months-old mix-breed dogs. Complementary DNA was specifically synthesized using an optimized stem-loop system. Intestinal expression of four miRNAs (cfa-let7g, cfa-miR-98, cfamiR-410, cfa-miR-130b) was evaluated using RT-qPCR. The results of the study indicate a significant difference between test and control dogs in cfamiR-130b, cfa-miR-98, and cfa-miR-410 (P ≤ 0.05); however, there was no significant difference for cfa-let7g. The most upregulated miRNAs were cfamiR-130b and cfa-miR-98. An increasing trend for cfa-let7g and a declining trend for cfa-miR-98, cfa-miR-410, and cfamiR-130b were found toward the distal segments of the small intestine. Our study revealed that cfa-miR-98, cfa-miR-410, and cfamiR-130b are involved in the definitive host response in canine echinococcosis. The study provides new information on the molecular basis of interactions between E. granulosus and dogs in terms of miRNA expression and showed that E. granulosus infection could increase the expression of some pro-inflammatory miRNAs at the cellular level in the definitive host.

Biological and morphological consequences of dsRNA-induced suppression of tetraspanin mRNA in developmental stages of Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis, caused by the cestode Echinococcus granulosus, is a neglected tropical disease with remarkable morbidity in humans and a problem of worldwide economic importance in livestock industry. Understanding the molecular basis of the parasite growth and development is essential for the disease diagnosis, management and control. The tetraspanin (TSP) family of proteins are transmembrane proteins with a role in many physiological processes of eukaryotic organisms. TSPs present in the tegumental surface of platyhelminths play pivotal roles in host-parasite interaction. However, little is known about the role of TSPs in growth and development in the Platyhelminthes. To understand the role of TSP1 in the growth and development of E. granulosus we investigated the effect of EgTSP1-specific long dsRNA in different in vitro stages of the parasite. METHODS: Different stages of E. granulosus, protoscoleces and strobilated worms, were cultivated In vitro in di-phasic media. Using long dsRNA and two delivery methods, i.e. electroporation and electro-soaking, EgTSP1 silencing was performed with an EgTSP1-specific dsRNA. The TSP1 expression profile was assessed as well as the biological and ultrastructural properties of the parasites. RESULTS: After three days of dsRNA treatment, EgTSP1 expression was significantly reduced in both stages of E. granulosus as compared to irrelevant/unrelated dsRNA and untreated controls. Silencing expression of EgTSP1 in different stages of E. granulosus resulted in reduced viability and body contractions, inhibition of protoscoleces evagination and distinctive tegumental changes. Ultrastructural morphology of the strobilated worms treated with EgTSP1-specific dsRNA was indicative of the microtriches impairments and vacuolated tegument compared to the control helminths. CONCLUSIONS: Results of the present study suggest that EgTSP1 plays important structural roles in tegument configuration in E. granulosus. EgTSP1 is proved to be a potential target for the development of vaccines and RNAi-based drugs.

Identification of Candida spp. isolated from oral mucosa in patients with leukemias and lymphomas in Iran.

BACKGROUND AND OBJECTIVES: Oral candidiasis is a serious problem for immunocompromised patients, especially patients with hematological malignancies. After becoming a systemic candidiasis it is difficult to diagnose, control and treat in individuals with hematological malignancies. The aim of this study was to diagnose candidiasis in the oral mucosa of patients with leukemias and lymphomas in a timely manner in order to prevent their progression to systemic candidiasis. MATERIALS AND METHODS: In this cross sectional study, 50 clinical samples were collected from the mouth of patients with hematological malignancies undergoing chemotherapy from the oncology units of teaching hospitals in Kerman, Iran. Patients were from Kerman, Sistan-Baluchestan and Hormozgan in south-eastern Iran. Sampling was restricted to patients with diagnosed acute lymphoid leukemia (ALL); acute myeloid leukemia (AML); chronic lymphoid leukemia (CLL); chronic myeloid leukemia (CML); Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Presumptive species identification of fungi was performed using conventional methods like colony characteristics on CHROMagar Candida medium, germ tube production, and assessing the morphology fungi on corn meal agar. Confirmation of presumptive candida isolates was performed using PCR-RFLP. RESULTS: From a total of 50, 14 patients (28%) had positive oral candidiasis. Candida albicans (57.14%) was the most common species followed by Candida glabrata (14.28%), Candida parapsilosis (14.28%), Candida krusei (7.14%) and Candida kefyr (7.14%). Candida albicans had the highest rate of oral infection in ALL (35.71%) and then NHL (28.57%) patients. CONCLUSION: The results indicate that oral candidiasis is a prevalent fungal infection in the patients with hematologic malignancies with C. albicans being the main etiological agent. However, other species of Candida cause similar infections in these patients.

The effect of albendazole sulfoxide on the expression of miR-61 and let-7 in different in vitro developmental stages of Echinococcus granulosus.

Albendazole, as the main anti-echinococcal benzimidazole, has demonstrated safe and effective therapeutic outcomes in the treatment of echinococcosis. The emergence of resistance or reduced response to albendazole sulfoxide (ABZ_SOX) and other benzimidazoles have been demonstrated in several parasitic helminths of medical and veterinary importance. As the genetic makeup and miRNA profile of helminths affects their response to albendazole sulfoxide, the present study was conducted to investigate the expression of miRNAs in different developmental stages of Echinococcus granulosus exposed to albendazole sulfoxide in vitro. Different developmental stages of the helminth were obtained from in vitro cultured E. granulosus in monophasic and diphasic media. In both ABZ-SOX-treated and control parasites miRNAs were extracted from microcysts, intact protoscoleces and strobilated worms with one and three segments. Expression of two miRNAs, let-7 and miR-61 was evaluated using RT-qPCR for each stage. Results of the present study revealed significant differential expression of both let-7 and miR-61 at different drug concentrations. A significant difference of let-7 expression was observed between the strobilated and metacestode stages of E. granulosus exposed to ABZ-SOX. In the treated protoscoleces, let-7 expression was significantly reduced in the presence of ABZ-SOX at 1000 µg/ml concentration. In contrast higher expression levels were documented in the segmented worms. In the microcysts exposed to different drug concentrations a significant decline of miR-61 expression was demonstrated. Also, a significant increase in expression of miR-61 was observed in one proglottid worms as well as the protoscoleces. Under high drug concentration or long-term exposure of the protoscoleces to ABZ-SOX significantly higher miR-61 expression was observed compared to the controls. Our findings suggested that under in vitro benzimidazole exposure the expression of two E. granulosus miRNAs were significantly affected in the microcyst stage. This study presents the first evidence of the nature of benzimidazole effects on miRNA expression in platyhelminths.

Genetic Profile of Hydatid Cysts in Patients with Multi-Organ Involvement: Mixed Infections by Different Strains.

Our knowledge on the susceptibility of humans to different genotypes of the zoonotic tapeworm Echinococcus granulosus and the genetic make-up of the cysts in multi-organ involvements is limited. This study aimed to identify the genotype profile of E. granulosus in patients undergoing hydatid surgery in an endemic area for cystic echinococcosis (CE) in southeastern Iran. Individuals who underwent hydatid cyst surgery were included in this study. Protoscoleces/germinal layers from each individual isolate were washed and kept in -20°C until use. Genotyping was carried out using PCR-sequencing of two mitochondrial CO1 and ND1 genes. Molecular phylogeny and haplotype network analysis of the human isolates were carried out using sequence data obtained from this study and National Center for Biotechnology Information (NCBI) databases. Forty-two patients (23 women and 19 men) participated in the study; the mean age was 43 years. Eighteen (42.9%) and 24 (57.1%) patients were infected by E. granulosus sensu stricto (G1-G3) and Echinococcus intermedius (G6 genotype), respectively. Molecular study showed mixed infection of G1 (in the liver and right lung) and G6 (in left lung) in a patient. The study showed a significantly high proportion of CE patients infected with the G6 genotype particularly in the southern parts of the province. In the present study a human CE patient infected by two species/genotypes of E. granulosus sensu lato is documented.

Calmodulin-specific small interfering RNA induces consistent expression suppression and morphological changes in Echinococcus granulosus.

Among parasitic helminths, biological features of Echinococcus granulosus have been a focus of particular interest in biology and medicine. The determinants and underlying molecular mechanisms of Echinococcus development in different host settings is largely unknown. The phenomenal bi-directional development of E. granulosus protoscoleces into multi-proglottid and/or microcysts, is a fascinating feature of the parasite cultivation. Calmodulin (CaM) is the major intracellular Ca2+ binding protein in plant and animal organisms. Many Ca2+-related processes in the physiology of eukaryotic organisms are CaM-dependent, however little is known on the role of CaM in platyhelminths growth and development. Small interfering (si) RNA-induced manipulations of the genes involving in the parasite development is an opportunity to explore novel approaches for cystic echinococcosis (CE) prevention and management. Regarding the fundamental role of CaM in cellular function of the parasites, in this study, we investigated the molecular and morphological changes induced by siRNA on CaM in different in vitro stages of E. granulosus. Three developmental stages of the tapeworm, protoscoleces, microcysts and strobilated worms, were cultivated in vitro in mono- and di-phasic media and three delivery methods, i.e. electroporation, soaking and electro-soaking, were used for RNA interference. The level of mRNA suppression as well as the phenotypic changes of the parasites were measured. Following RNA interference, EgCaM mRNA suppressions of 65-99% were recorded in different stages of the tapeworm as compared to untreated/unrelated siRNA controls. Lower viability, growth retardation, morphological abnormalities as well as EgCaM expression suppression were documented in the parasite implying potential of siRNA technology for the prevention and management of CE.

Epidemiological and molecular studies on Echinococcus granulosus from free-roaming dogs in Southeast Iran.

BACKGROUND AND AIM: Cystic echinococcosis (CE), as a major zoonotic helminth infection, imposes remarkable socioeconomic burden on many endemic countries across the world, including Iran. Due to the high importance of free-roaming dogs in the transmission of CE, epidemiological and molecular studies in this type of hosts are required in the endemic regions. This study aimed to investigate the epidemiology and genotyping of Echinococcus granulosus isolated from stray dogs in Kerman, Southeast Iran. MATERIALS AND METHODS: Eighty-four samples were isolated from stray dogs in the city and suburbs of Kerman in coordination with the health authorities and the municipality office for rabies control and dog population management. Dog demographic data, including age and sex were collected. The worm was isolated by necropsy and genomic DNA was extracted and partial cytochrome c oxidase subunit 1 gene was amplified using specific primers. Phylogenetic and Templeton-Crandall-Sing (TCS) network analyses were carried out on the sequence data. RESULTS: The overall prevalence of CE in the surveyed dogs was 10.7% (9/84 cases). Out of 84 stray dogs, 33 (39.3%) and 51 (60.7%) cases were male and female, respectively. There was not a statistically significant difference between the infection and gender of dogs. However, infection is shown more in dogs under one year of age with a statistically significant difference (p<0.05). The results of molecular studies indicated E. granulosus G1 genotype for all isolates. The high presence of free-roaming dogs in urban and peri-urban areas and high frequency of parasite in this animal is a risk factor for humans in the region. Haplotype sequence analysis on the dog isolates revealed a close relationship with other E. granulosus isolates in Kerman. CONCLUSION: The findings of this study provide evidence-based data about the epidemiological and molecular characteristics of CE in dog definitive hosts of Southeast Iran. Further studies are required to understand the prevalence and parasite genotypes in dogs in Iran.

Experimental and field investigation of non-biting flies as potential mechanical vectors of Echinococcus granulosus eggs.

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1-3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.

High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.