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.

Fascioliasis associated with chronic cholecystitis in a woman from Sistan and Baluchestan province, a non-endemic region in Southeastern Iran.

BACKGROUND: Fascioliasis, caused by Fasciola hepatica, is a neglected zoonotic food-borne trematodiasis. The Caspian littoral in northern Iran is endemic for the disease, and human fascioliasis is well-known in that region. In the present study, we report the diagnosis, identification, and clinical management of a human case of fascioliasis associated with common bile duct (CBD) obstruction from a non-endemic remote area in southeastern Iran. CASE PRESENTATION: A 42-year-old female was admitted to Afzalipour Medical Center hepatobiliary surgery ward in Kerman with abdominal pain for the past three months. Dilated biliary tract and an ill-defined mass in CBD were reported in abdominal ultrasonography and magnetic resonance cholangiopancreatography, respectively. During distal CBD operation, nine leaf-like motile flatworms were isolated. A morphological study confirmed all the isolates as Fasciola, and further molecular investigations, identified the flukes as F. hepatica using both pepck multiplex PCR and cox1 sequencing. CONCLUSION: Molecular and morphological findings of the study indicated the presence of human fascioliasis in the southeastern province of Sistan and Baluchestan in Iran. Fascioliasis is among the etiologies of chronic cholecystitis, and physicians should consider chronic cholecystitis associated with fascioliasis in the differential diagnosis. In the present report, endoscopic ultrasound was usefully applied for the accurate diagnosis of biliary fasciolosis.

Molecular characterization of ocular dirofilariasis: a case report of Dirofilaria immitis in south-eastern Iran.

BACKGROUND: Dirofilariasis is a zoonotic parasitic infection transmitted from animals to humans by culicid mosquitoes. Although the disease can be caused by Dirofilaria spp. including Dirofilaria immitis and Dirofilaria repens, human ocular dirofilariasis due to D. immitis is relatively rare in the world. This study was aimed to present a case of ocular dirofilariasis caused by D. immitis in southeastern Iran. CASE PRESENTATION: A nematode extracted from the right eye of a 69-year-old man referred with clinical symptoms including itching and redness was examined. After the morphometric analysis, Dirofilaria parasite was detected. Afterwards, a piece of worm body was cut and DNA was extracted and a 680-bp gene fragment amplification and nucleotide sequencing were performed. Phylogenetic analysis revealed a D. immitis roundworm as the causative agent of infection. The patient was treated with antibiotics and corticosteroid and followed up for 1 month. CONCLUSION: The present study provides the second report on ocular dirofilariasis caused by D. immitis isolated from a human in southeast Iran. Based on the available evidence, dirofilariasis in dogs has significantly increased in endemic areas such as Iran. Therefore, physicians should be aware of such zoonotic nematodes so as to take proper and timely action and treatment against the disease.

Genetic and morphometric categorization of Taenia ovis from Sheep in Iran.

Little is known about the genetic and morphological characters of Taenia ovis. The purpose of the present study was to characterize sheep isolates of T. ovis using rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens of Cysticercus ovis were collected from 18 slaughterhouses in Iran. The mean ± s.d. for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4 µm, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3-1.1 and 0.2-1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA between T. ovis haplotypes from present study and eight other Taenia species was found to be 11.3-17.8 and 5.3-16.3%, respectively. Phylogenetic analysis clustered all T. ovis isolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference between T. ovis ovis and T. asiatica was 13.6% that is lesser than the corresponding difference between T. ovis ovis and T. ovis krabbei, warranting the designation of two separate species as T. ovis and T. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

In vitro evaluation on the scolicidal effect of Myrtus communis L. and Tripleurospermum disciforme L. methanolic extracts.

Hydatid disease, a zoonotic disease, is still endemic in many developing countries that is caused by the metacestode of Echinococcus (E.) granulosus. Surgical management is one of the best choices for the treatment of the hydatidosis and using effective scolicidal agents during hydatid surgery is essential to prevent the secondary infection. The aim of the present in vitro study was to evaluate the scolicidal effect of the methanolic extract of Myrtus communis and Tripleurospermum disciforme against protoscoleces of hydatid cyst. Protoscoleces of E. granulosus were aspirated aseptically from infected livers. Various concentrations of M. communis and T. disciforme extracts at different exposure times were examined for their scolicidal activity. Normal saline and silver nitrate were used as negative and positive groups, correspondingly. The viability of protoscoleces was evaluated by 0.1% eosin. The result of the current study indicated that the highest scolicidal effect (100%) of M. communis was obtained at 100 and 50 mg/ml concentrations and LC50 in 10, 20 and 30 min were 11.64 mg/ml, 7.62 mg/ml, and 6.47 mg/ml respectively. The scolicidal activity of T. disciforme was negligible even at high concentration. The findings have shown that the scolicidal activity of M. communis against echinococcosis protoscoleces was strong, while the T. disciforme extract showed fewer effects. However, further studies are required for identification of the active ingredients in the extract and its safety on cells in effective concentrations.

Leishmania tropica isolates from non-healed and healed patients in Iran: A molecular typing and phylogenetic analysis.

The precise identification of the parasite species causing leishmaniasis is essential for selecting proper treatment modality. The present study aims to compare the nucleotide variations of the ITS1, 7SL RNA, and Hsp70 sequences between non-healed and healed anthroponotic cutaneous leishmaniasis (ACL) patients in major foci in Iran. A case-control study was carried out from September 2015 to October 2016 in the cities of Kerman and Bam, in the southeast of Iran. Randomly selected skin-scraping lesions of 40 patients (20 non-healed and 20 healed) were examined and the organisms were grown in a culture medium. Promastigotes were collected by centrifugation and kept for further molecular examinations. The extracted DNA was amplified and sequenced. After global sequence alignment with BioEdit software, maximum likelihood phylogenetic analysis was performed in PhyML for typing of Leishmania isolates. Nucleotide composition of each genetic region was also compared between non-healed and healed patients. Our results showed that all isolates belonged to the Leishmania tropica complex, with their genetic composition in the ITS1 region being different among non-healed and healed patients. 7SL RNA and Hsp70 regions were genetically identical between both groups. Variability in nucleotide patterns observed between both groups in the ITS1 region may serve to encourage future research on the function of these polymorphisms and may improve our understanding of the role of parasite genome properties on patients' response to Leishmania treatment. Our results also do not support future use of 7SL RNA and Hsp70 regions of the parasite for comparative genomic analyses.

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.

Understanding the research and practical needs required to control toxocariasis in Iran.

Human toxocariasis (HT) is a widespread zoonotic infection globally, notably prevalent in tropical areas. Enhancing our understanding of toxocariasis can lead to increased attention towards the socioeconomic impact and control of this neglected zoonosis. We conducted a comprehensive review of all available articles and official documents on toxocariasis in Iran to identify research gaps and critical needs for its control. This review highlights that despite numerous studies exploring various aspects of toxocariasis in definitive and paratenic hosts, as well as humans and environmental contamination, significant data deficiencies and gaps persist across different regions in the country. These gaps involve investigating the worm burden and reinfection rates in definitive hosts, developing more sensitive methods to detect and differentiate of Toxocara species, and understanding the behavior of definitive host animals. Additionally, identifying potential paratenic hosts for HT and exploring the organ-specific affinity and survival duration of Toxocara larvae within these hosts are essential areas for exploration. It's also imperative to comprehend the sylvatic and domestic cycles of the parasite in paratenic hosts. Furthermore, assessing egg density in the environment, exploring potential new sources such as water, and identifying regions with optimal climatic conditions for the survival and development of Toxocara eggs are crucial for the formulation of effective prevention and control strategies. Identifying at-risk groups, developing early diagnosis techniques, employing imaging methods, and identifying long-term complications in humans are also crucial. Community health organizations should prioritize health education for the public and professionals. Furthermore, accurately estimating definitive host populations, monitoring and preventing their movements in public places, implementing regular deworming practices for pets and stray hosts, and recognizing the infection's significance as a health priority are critical. This comprehensive understanding advocates for a holistic "one health" approach to control of HT.

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.

Morphological and molecular characterization of Fasciola isolates from livestock in Golestan province, northern Iran.

BACKGROUND: Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. OBJECTIVE: The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. METHODS: Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019-2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. RESULTS: A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty-one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1-RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. CONCLUSION: The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.

Nasopharyngeal myiasis due to Cephalopina titillator in Southeastern Iran: a prevalence, histopathological, and molecular assessment.

The Cephalopina titillator is one of the most important causative agents of nasal myiasis in camels. This study aimed to explore the prevalence, histopathological effects, and molecular identification of C. titillator infestation in camels of Kerman province, South-Eastern Iran, between 2019 and 2021. The larvae were placed in 10% formalin for histopathological evaluation and species identification. Pieces of larval abdominal segments of C. titillator were selected for extraction of DNA. Partial mitochondrial CO1 genes were sequenced for final analysis. Out of the 870 camels examined, 339 (38.9%) were infested with larval stages of C. titillator. There was a significant difference between age and infection rate (P = 0.001), while no association between males and females (P = 0.074) was found. The infection rate was significantly higher in the winter (P < 0.001) than in the other seasons. In this study, different lesions depending on duration, locations, and the depth of larval adhesion notably degeneration changes, necrosis, and ulceration were observed. Also, in chronic cases, granulation tissue reactions were organized. Cephalopina titillator was confirmed by PCR sequencing analysis using mitochondrial CO1 region. A 582 bp nucleotide sequence was deposited in GenBank under the MW136151 accession number. Phylogenetic analysis of CO1 produced a single uniform sister clade to MZ209004 and MW167083 records from China and Iraq, respectively. The high prevalence of C. titillator in camels in this region and other areas of Iran declares that the country is in an endemic status and displays the existence of the potential risk for camels.

Iranian Hydatid Disease Registry: Establishment and Implementation of a Neglected Tropical Disease Registry.

BACKGROUND: Cystic echinococcosis (CE) or hydatid disease is a global public health concern which imposes considerable economic costs on the communities in endemic regions. CE surveillance data are not adequately reliable. The present study reports the development and outcomes of a CE registry in Iran. METHODS: Hydatid Registry (HydatidReg) was initially established as a single-center registry in 2014 after the ethical approval of KMU. Following a call from MoHME to promote registry of different diseases and health outcomes, a call for participation was announced and all the Iranian Universities of Medical Sciences were requested to contribute to the registry. Subsequently, a nation-wide registry of hydatid disease was established in 2016. With a global perspective, HydatidReg joined the European Register of Cystic Echinococcosis (ERCE). A data collection form based on minimum dataset was designed and standard operating procedures (SOPs) were prepared to ensure standardized patient enrolment in the registry. A biobank system with two-dimensional barcoding was established along with HydatidReg for management and organization of biological specimens. RESULTS: As of March 2021, a total of 690 patients were enrolled in the registry. HydatidReg registered 362 (17.3%) out of the total 2097 patients enrolled in ERCE. Quality control (QC) of the data demonstrated 91.2% completeness and 80% timeliness. In the biobank, 322 biological specimens from 184 CE patients have been deposited including 70 blood, 96 sera and 156 parasite materials. CONCLUSION: High-quality data in the HydatidReg registry provided opportunities for health professionals to improve quality of care and organize meaningful research.

Dynamic modeling of female neutering interventions for free-roaming dog population management in an urban setting of southeastern Iran.

Understanding dynamics of free-roaming dog (FRD) population is critical for planning and implementation of dog population management programs. FRD population size estimation as well as dynamic modeling of dog population under different female dog neutering interventions were investigated in order to determine the most appropriate animal birth control approach. We performed population size estimate of dogs using sight-resight surveys by photography in a randomly selected 25 blocks of the city and all the suburbs of greater Kerman area. Main demographic features were characterized and the dog density distribution was mapped. A dynamic model was developed to predict free-roaming dog population variations after 5 and 10 years. Different scenarios based on 10, 30, 50, 60 and 70% female dog sterilization were considered to predict the effects of animal birth control measures. Free roaming dog population was estimated at 6781 dogs (65.3% males) in Kerman and suburbs with several major population hotspots. Analysis of the dog locations within the city showed that the largest proportion of the dogs were observed in the vacant lots (46.2%). Modeling predictions indicated that, in the absence of management, the free-roaming dog population could increase from a baseline of 6781 to 13,665 dogs (2.02 fold increase) in 5 years and to 19,376 dogs in 10 years (2.86 fold increase). Using a population dynamics model, we simulated five neutering coverages to explore the impact of female neutering on free-roaming dog population size. The 5-year projections of the model have shown that 50% annual female dog sterilization significantly reduced free-roaming dog population by 0.44 comparing to the baseline population. Findings of the present study improve our knowledge on the nature and extent of dog population dynamics in Iran. Effective population control and selection of the most appropriate neutering interventions require a comprehensive knowledge of the characteristics and dynamics of FRD population.

Molecular, Morphological, and Spatial Study of Galba schirazensis (Pulmonata, Lymnaeidae) from Southeastern Iran.

BACKGROUND: Snails of the genus Galba are the intermediate hosts of Fasciola species, the etiological agents of liver fluke disease, fascioliasis. A genetically different but morphologically very similar species in the genus, G. schirazensis, is sympatrically distributed with G. truncatula in some regions of the world. We aimed to investigate the occurrence of G. schirazensis in Kerman province, Iran and to characterize genetically G. schirazensis specimens from southeast Iran. METHODS: Field-collected snails from four localities in Jiroft, Bam and Faryab, Kerman province, southeastern Iran were studied. Hydrological variables including temperature and pH were recorded for each habitat. Each specimen was identified using morphological as well as conchological characteristics. Genetic characterization was performed using PCR-sequencing followed by phylogenetic analyses on nuclear ITS2 as well as mitochondrial cox1 gene fragments. MaxEnt software was used to predict the most appropriate ecological niches for the targeted species. RESULTS: G. schirazensis was found in 4 out of 28 locations. One ITS2 and two cox1 haplotypes were detected among G. schirazensis populations from the four localities. Habitat study showed that G. schirazensis thrives in habitats with alkaline pH. G.schirazensis from South America were clustered with specimens from Bam, Kerman, Iran; however, north Iranian isolates of G. schirazensis were strongly correlated with specimens from Jiroft and Faryab. MaxEnt model for the most appropriate ecological niches of the targeted species predicted environmental suitability for this species in western Africa as well as coastal areas in north and southwestern Africa. CONCLUSION: G. schirazensis is frequently present in southern areas of Kerman Province. At least two genetically different haplotypes are present in southeastern Iran.

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.

Evaluation of the Diagnostic Performance of Recombinant Antigen B1 for Detection of Cystic Echinococcosis Using Lateral Flow Dipstick Test.

BACKGROUND: Human echinococcosis is a neglected zoonotic disease distributed worldwide. It comprises cystic and alveolar forms, the former being the more prevalent disease. Imaging techniques are the first choice for diagnosis of cystic echinococcosis and serology is used as an additional diagnostic technique in doubtful cases or as the sole test in low-resource settings. Rapid diagnostic tests are useful and convenient for immunodiagnosis of cystic echinococcosis in endemic areas, where medical facilities often struggle with limited resources. METHODS: Recently, we have developed Hyd Rapid™, an IgG4 lateral flow dipstick test using recombinant antigen B1 for detection of cystic echinococcosis. This study was performed between 2016 until 2018 at the Institute for Research in Molecular Medicine, Universiti Sains Malaysia. The diagnostic performance of Hyd Rapid™ was tested in-house and at two international laboratories in Switzerland and Iran. RESULTS: The overall diagnostic sensitivity for detection of cystic and alveolar echinococcosis was 95% (56/59). Meanwhile, the diagnostic specificity, with and without exclusion of cysticercosis and fascioliasis, was 100% (n=48) and 88% (63/72), respectively. CONCLUSION: Hyd Rapid™ detected cystic echinococcosis as well as probable cases of alveolar echinococcosis. Therefore, Hyd Rapid™ showed good potential as a serological tool for echinococcosis, and merits further evaluation.

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.