In vitro and ex vivo protoscolicidal effects of hydroalcoholic extracts of Eucalyptus microtheca on protoscoleces of Echinococcus granulosus sensu stricto: A light and scanning electron microscopy (SEM) study.

BACKGROUND: Cystic echinococcosis (CE) is one of the most widespread and important global helminth zoonoses. Treatment relies mainly on surgery and, or percutaneous interventions. However, spillage of live protoscoleces (PSCs) leading to recurrence is a problem during surgery. So, the application of protoscolicidal agents before surgery is required. This study aimed to investigate the activity and safety of hydroalcoholic extracts of E. microtheca against PSCs of Echinococcus granulosus sensu stricto (s.s.) both in vitro and also ex vivo, which is a simulation to Puncture, Aspiration, Injection, and Re-aspiration (PAIR) method. METHODS: Considering the effects of heat on the protoscolicidal effecacy of Eucalyptus leaves, hydroalcoholic extraction was performed by both soxhlet extraction at 80 °C and percolation at room temperature. The protoscolicidal action of hydroalcoholic extracts was assessed by in vitro and ex vivo assessments. Infected sheep livers were collected from the slaughterhouse. Then, the genotype of hydatid cysts (HCs) was confirmed by sequencing and, isolates were limited to E. granulosus s.s. In the next step, ultrastructural changes of Eucalyptus-exposed PSCs were studied by scanning electron microscopy (SEM). Finally, a cytotoxicity test was conducted by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the safety of E. microtheca. RESULTS: The prepared extracts by soxhlet extraction and percolation were, successfully exerted strong protoscolicidal effects in both in vitro and ex vivo tests. The results of in vitro assessment indicated that hydroalcoholic extract of E. microtheca prepared by percolation at room temperature (EMP) and hydroalcoholic extract of E. microtheca prepared by soxhlet extraction at 80 °C (EMS) killed all PSCs (100%) at concentrations of 10 and 12.5 mg/mL, respectively. Also, EMP showed 99% protoscolicidal action after 20 min in an ex vivo setting compared to EMS. SEM micrographs confirmed potent protoscolicidal and destructive effects of E. microtheca against PSCs. The cytotoxicity of EMP was tested on the HeLa cell line using MTT assay. The value of 50% cytotoxic concentration (CC50) was calculated at 46.5 µg/mL after 24h. CONCLUSION: Both hydroalcoholic extracts showed potent protoscolicidal activity and, especially EMP produced remarkable protoscolicidal effects compared to the control group.

Application and evaluation of native antigen B from Echinococcus granulosus sensu stricto and E. canadensis alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting.

Cystic echinococcosis (CE) is one of the most important helminthic diseases in the world with different genotypes distribution. The application of specific genotype antigens together with sera from patients with specific cyst genotypes have not been reported, so far. The present study aimed to apply and evaluate native AgB from Echinococcus granulosus sensu stricto (Eg) and Echinococcus canadensis (Ec) alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting. A total of 47 human sera along with 47 human CE cysts were collected. CE genotypes were determined. Native AgB were prepared from E. granulosus s.s and E. canadensis genotypes. ELISA and Western blot were performed on human specific G1-G3 and G6/G7 genotypes sera. Species specific native AgB were used alone or mixed. The sensitivity of ELISA using alone and mixed 1Eg-1Ec, 1Eg-2Ec, and 2Eg-1Ec of native AgB from E. granulosus s.s and E. canadensis genotypes for human G1-G3 sera were 92.10, 89.47, 97.37, 100, and 100%, respectively; while using AgBs, alone and mixed for human G6/G7 sera were 100%. The sensitivity of Western blotting using native AgB of E. granulosus s.s and E. canadensis genotypes alone and mixed 2Eg-1Ec were 78.95% and 100% for human G1-G3 and G6/G7 genotypes sera, respectively. The mixture of AgB from Echinoccus granulosus sensu stricto and Echinococcus canadensis genotypes increased ELISA sensitivity for the diagnosis of human CE. Preparation and application of native AgB from specific and prevalent genotypes of CE in endemic regions is recommended.

Indirect immunofluorescence assay using embryonated eggs of Toxocara in human toxocariasis diagnosis is unreliable due to autofluorescence nature.

Toxocariasis is caused by infection with the nematode species Toxocara canis and Toxocara cati. Serological methods using eggs, larvae and adult worms of Toxocara spp. as antigen have been used for the diagnosis of human toxocariasis. The current study aimed to evaluate indirect immunofluorescence assay (IFA) using embryonated eggs of Toxocara for diagnosis of human toxocariasis. A total of 58 sera including twenty sera from patients with toxocariasis, 20 from healthy persons and 18 from patients with other parasitic infections were collected and used for the study. The embryonated eggs of Toxocara were prepared as antigen. Indirect immunofluorescence assay was performed using the frozen section of uterus containing embryonated T. canis eggs and unembryonated T. cati eggs. All serum samples had a positive reaction using IFA. The eggs of Toxocara as antigen exposed to the serum samples of toxocariasis, other parasitic infections and healthy persons, followed by IFA gave a bright greenish-yellow fluorescence. A number of samples such as eggs of Toxocara, Toxascaris, Trichuris and strongyloides larvae, and adult worm of Ancylostoma exhibited the bright greenish-yellow autofluorescence under fluorescent microscope. IFA using cryocut of embryonated eggs of Toxocara cannot be used for the diagnosis of human toxocariasis due to the existence of autofluorescence of the unembryonated and embryonated eggs, the second stage larva and adult worms of Toxocara spp.

Protoscolicidal Effects of the Garlic Chloroformic Extract on the Protoscolices of Hydatid Cyst at a Short Exposure Time, up to Five Minutes.

BACKGROUND: The treatment of choice for hydatidosis as an important zoonotic disease is surgery. Different agents are injected into the cyst to prevent secondary hydatidosis. To avoid the side effects of such protoscolicidal agents, considering the high protoscolicidal effects of the garlic extract, we conducted the present study on protoscolices in limited applicable times and compared the extract with some chemical agents. METHODS: Sheep's liver and lung cysts were collected. Ninety tubes were selected and divided into 3 sets (for different exposure times), each one comprising 5 groups of 6 tubes. Each tube contained 3000-4000 protoscolices. The groups were 0.5% cetrimide (as positive control), 20% hypertonic sodium chloride, 0.5% silver nitrate, 0.9% normal saline (as negative control), and the garlic chloroformic extract (200 mg/mL). The viability of the protoscolices was assessed using 0.1% eosin. The ANOVA and LSD were used to compare the mean viability of the protoscolices after exposure to the different agents at different times and concentrations. The data were analyzed using SPSS software, version 17. A P<0.05 was considered significant. RESULTS: Our findings showed that the protoscolicidal effects of the garlic extract at 1 (P<0.001) and 2 (P<0.001 and P=0.003) minutes of exposure were higher than those of sodium chloride and silver nitrate. At 5 minutes of exposure, there was no difference between the garlic extract and sodium chloride (P=0.36); however, the difference between these agents and silver nitrate was significant. CONCLUSION: The garlic chloroformic extract in a short exposure time had high protoscolicidal effects and could substitute other agents.

Evaluation of Toxocara cati Excretory-Secretory Larval Antigens in Serodiagnosis of Human Toxocariasis.

BACKGROUND: Toxocariasis is the clinical term that is applied to infection in the human host with Toxocara species larvae. Serological tests are important tools for the diagnosis of toxocariasis. The aim of this study was to evaluate the excretory-secretory (ES) antigens of T. cati larvae using enzyme-linked immunosorbent assay (ELISA) and also Western blotting for serodiagnosis of human toxocariasis. METHOD: The ES antigens were prepared from T. cati third-stage larvae. Serum samples were obtained from 33 confirmed cases of toxocariasis, 35 patients infected with other parasitic diseases, and 30 from healthy individuals tested with ELISA and immunoblotting. RESULTS: The ELISA showed appropriate performance in term of specificity (96.7%) and sensitivity (97.0%). Electrophoretic analysis of T. cati ES antigens revealed a range of 20- to 150-kDa fractions. The highest sensitivity was achieved with 42- and 50-kDa fractions. CONCLUSION: The ELISA analyses using T. cati ES antigens demonstrated good sensitivity and specificity compared to T. canis ES as antigens for diagnosis of human toxocariasis. Accordingly, application of Western blotting, based on 42- and 50-kDa fractions of ES antigens, can be recommended for the accurate diagnosis of toxocariasis.

Molecular characterization of cystic echinococcosis cysts isolated from cattle in Shiraz, Iran.

Cystic echinococcosis (CE), is one of the common parasitic infections of domestic animals and humans caused by the larval stages of the tapeworm Echinococcus granulosus. This research aimed to identify of CE cysts in cattle isolates from Shiraz slaughterhouse using molecular technique. Thirty CE cysts isolates were collected and total DNA was extracted from protoscolices or the germinal layer of each cyst followed by polymerase chain reaction (PCR) using cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit 1 (NADH-1) mitochondrial genes (~ 450 bp) and sequencing. The sequence results were compared with available reference sequences in GenBank by BLAST system. Phylogenetic analysis was performed using MEGA 5.0 software and the maximum likelihood method. The results showed that all 30 samples examined in this study were identified as E. granulosus sensu stricto (G1-G3 genotypes) with G1 dominant.

Therapeutic effect of curcumin nanoemulsion on cystic echinococcosis in BALB/c mice: a computerized tomography (CT) scan and histopathologic study evaluation.

BACKGROUND: This study aimed to determine the therapeutic efficacy of curcumin nanoemulsion (CUR-NE) in mice infected with Echinococcus granulosus sensu stricto protoscoleces. METHODS: Forty-two inbred BALB/c mice were divided into seven groups of six animals each. Six groups were inoculated intra-peritoneally with 1500 viable E. granulosus protoscoleces, followed for six months and used as infected groups. The infected groups were named as: CEI1 to CEI6 accordingly. The 7th group was not inoculated and was named cystic echinococcosis noninfected group (CENI7). CEI1 and CEI2 groups received 40 mg/kg/day and 20 mg/kg/day curcumin nanoemulsion (CUR-NE), respectively. CEI3 received nanoemulsion without curcumin (NE-no CUR), CEI4 received curcumin suspension (CUR-S) 40 mg/kg/day, CEI5 received albendazole 150 mg/kg/day and CEI6 received sterile phosphate-buffered saline (PBS). CENI7 group received CUR-NE 40 mg/kg/day. Drugs administration was started after six months post-inoculations of protoscoleces and continued for 60 days in all groups. The secondary CE cyst area was evaluated by computed tomography (CT) scan for each mouse before treatment and on the days 30 and 60 post-treatment. The CT scan measurement results were compared before and after treatment. After the euthanasia of the mice on the 60th day, the cyst area was also measured after autopsy and, the histopathological changes of the secondary cysts for each group were observed. The therapeutic efficacy of CUR-NE in infected groups was evaluated by two methods: CT scan and autopsied cyst measurements. RESULTS: Septal calcification in three groups of infected mice (CEI1, CEI2, and CEI4) was revealed by CT scan. The therapeutic efficacy of CUR-NE 40 mg/kg/day (CEI1 group) was 24.6 ± 26.89% by CT scan measurement and 55.16 ± 32.37% by autopsied cysts measurements. The extensive destructive effects of CUR-NE 40 mg/kg/day (CEI1 group) on the wall layers of secondary CE cysts were confirmed by histopathology. CONCLUSION: The current study demonstrated a significant therapeutic effect of CUR-NE (40 mg/kg/day) on secondary CE cysts in BALB/c mice. An apparent septal calcification of several cysts revealed by CT scan and the destructive effect on CE cysts observed in histopathology are two critical key factors that suggest curcumin nanoemulsion could be a potential treatment for cystic echinococcosis.

Report of a two-headed protoscolex of Echinococcus granulosus revealed by phase contrast microscopy in Shiraz, Iran.

Echinococcus granulosus sensu lato can produce cystic echinococcosis (CE)/hydatidosis in different hosts. Each CE cyst can produce numerous protoscoleces by its germinal layer in two forms: evaginated and invaginated. Usually, each protoscolex has one head consisting of a rostellum with two rows of hooks and four suckers. During a study of protoscolicidal agents on sheep Echinococcus granulosus protoscoleces, and investigations on their microscopic changes using Phase contrast microscopy, we observed a case of two- headed evaginated protoscolex. Two heads were attached to a unique bigger size of protoscolex body. Morphological observations showed its dimension around two times of usual protoscolex. There was no space between the bodies, hence one fused body was observed which was clearly shown by Phase contrast microscopy. Each head possessed two rows of hooks. Using micrometry all parts of the two-headed protoscolex, especially hooks were measured and photographed showing all aspects of its morphology including tegument, hooks, and two heads. Specific parts of two-headed protoscolex including suckers, hooks, also calcareous corpuscles were measured. The measurements on the two-headed protoscolex showed that the small hook blade length (SBL) and large hook blade length (LBL) were almost langer than one head protoscolex. A total of 120 calcareous corpuscles in two headed-protoscolex is much higher than one head protoscoleces.

Proteomics investigation of human sera for determination of postoperative indicators of pulmonary cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE)/hydatidosis is an important zoonotic parasitic disease caused by the larval stage of Echinococcus granulosus. The disease is a major health problem all over the world. Finding specific and sensitive biomarkers for follow-up of CE in patients after surgery is essential. Using proteomics methods, the present study aimed to evaluate post-surgical treatment by finding probable biomarker/s in the serum of human lungs CE. METHODS: A total of 24 human sera were tested. These sera included eight confirmed lung/s CE patients sera before surgery (BS), eight sera 12 months post-surgery (12MPS) as well as eight control sera from healthy people. Proteomics methods including 2DE and LC-MS/MS were performed on the specimens followed by bioinformatics analysis. Differentially expressed proteins (DEP) were detected and, separately integrated with protein-protein interaction (PPI) data to construct the PPI network. RESULTS: A total of 171 protein spots were detected in three groups including BS, 12MPS, and control groups; of which a total of 106 DEP have been expressed based on fold changes > = 2 and p-value < 0.05. More analysis was performed and a total of 10 protein spots were selected for identification by mass spectrometry showing the following proteins: APOA1, BGN, SPP2, EAF1, ACOXL, MRPL55, MCTP2, SEPTIN1, B4GALNT1, and ZNF843. Based on centrality parameters of the PPI network (degree and betweenness) five Hub-bottlenecks proteins with significant centrality values were found including APOA1, BGN, SPP2, EAF1, and ACOXL. CONCLUSION: This study showed five proteins as hub-bottleneck proteins; of which APOA1 was more prominent. It can be concluded that a change in expression of this protein in patients' sera could be used as an indicator tool for the achievement of lungs CE surgical therapy.

Evaluation of structural changes of Echinococcus granulosus protoscoleces following exposure to different protoscolicidal solutions evaluated by differential interference contrast microscopy.

The present study was aimed to assess the structural changes in protoscoleces of Echinococcus granulosus sensu stricto following exposure to different natural and chemical protoscolicidal agents using differential interference contrast (DIC)/Nomarski microscopy. Protoscoleces of sheep's liver cysts were collected aseptically. Individually, about 1000 protoscoleces were exposed to 0.5% silver nitrate, 20% hypertonic saline solution, 0.5% cetrimide solution and two different concentrations of garlic chloroformic extraction as well as phosphate-buffered saline (PBS). The protoscoleces viability was assessed using 0.1% eosin solution, and structural modifications in the protoscoleces were examined by DIC/Nomarski microscopy. The results revealed the degeneration of the tegument, disorganization of the hooks, and reduction of the size of the protoscoleces exposed to cetrimide, hypertonic sodium chloride, and silver nitrate. Furthermore, calcareous corpuscles became blurred and opaque and their numbers decreased in all the exposed samples except, those in PBS. The exposed protoscoleces to cetrimide and hypertonic sodium chloride solution showed extensive degeneration of the tegument and disorganization of the hooks. In the group exposed to 200 mg/ml chloroformic garlic extract, the protoscoleces' width decreased. The length, width, and number of calcareous corpuscles also decreased significantly in the silver nitrate-exposed protoscoleces. The study concludes that protoscoleces exposed to different solutions; cetrimide 0.5% and hypertonic sodium chloride 20% caused more pronounced structural changes in the exposed protoscoleces. These changes were well demonstrated by DIC microscopy and can be used as a supplementary tool to evaluate the effects of protoscolicidal agents. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-023-01632-4.

Comparison of Isoenzyme Pattern of Echinococcus granulosus sensu stricto (G1-G3) and E. canadensis (G6/G7) Protoscoleces

Background: Different genotypes of Echinococcus granulosus sensu lato (s.l.) infect humans and ungulate animals, causing cystic echinococcosis. Simultaneous isoenzyme, as well as molecular characterizations of this parasite, has not yet been investigated in Iran. The present study aimed to evaluate the isoenzyme pattern of the E. granulosus sensu stricto (s.s.) and E. canadensis genotypes in Iran. Methods: A total of 32 (8 humans and 24 animals) cystic echinococcosis cysts were isolated from Shiraz, Tehran, Ilam, and Birjand from May 2018 to December 2020. The DNAs were extracted and their genotypes were determined by molecular methods. Enzymes were extracted from the cysts and subjected to polyacrylamide gel electrophoresis. The activities of glucose-6-phosphate sehydrogenase (G6PD), malate dehydrogenase (MDH), malic enzyme (ME), nucleoside hydrolyse 1 (NH1), and isocitrate dehydrogenase (ICD) were examined in the cyst samples using isoenzyme method and compared it with the genotyping findings. Results: DNA sequence analysis of the samples showed that the specimens contained 75% E. granulosus s.s. (G1) and 25% E. canadensis (G6) genotypes. The isoenzyme pattern of ICD in both genotypes produced a six-band pattern with different relative factors. The G6PD also produced two bands with different relative migrations in both genotypes. The MDH and NH1 systems revealed a two-band pattern, while only one band was generated in the ME enzyme in the E. granulosus s.s. genotype. In the E. canadensis, the MDH and NH1 enzymes showed one band, and the ME enzyme represented a two-band pattern. Conclusion: Our findings suggest that E. granulosus s.s. and E. canadensis genotypes have entirely different isoenzyme patterns for NH1, G6PD, MDH, and ME.

Protoscolicidal effects of curcumin nanoemulsion against protoscoleces of Echinococcus granulosus.

BACKGROUND: The aim of the present study was to assess in vitro protoscolicidal effects of curcumin nanoemulsion (CUR-NE) against protoscoleces of cystic echinococcosis (CE)/hydatid cysts. METHODS: The CUR-NE was prepared via spontaneous emulsification of soybean as the oil phase, a mixture of Tween 80 and Tween 85 as the surfactant, ethanol as the co-surfactant and distilled water. Various concentrations of CUR-NE (156, 312, 625 and 1250 µg/ml) were exposed to collected protoscoleces of infected sheep liver hydatid cysts for 10, 20, 30, 60 and 120 min. Viability of the protoscoleces were assessed using eosin exclusion test. Morphological changes of the protoscoleces were observed using differential interference contrast (DIC) microscopy. RESULTS: The mean particle size and zeta potential of CUR-NE included 60.4 ± 14.8 nm and - 16.1 ± 1.1 mV, respectively. Results showed that the viability of the protoscoleces decreased significantly with increases in CUR-NE concentrations (p < 0.001). The mortality rates of protoscoleces with exposure to concentrations of 1250 and 625 µg/ml of CUR-NE for 60 min were 94 and 73.33%, respectively. Mortality of the protoscoleces was 100% after 120 min of exposure to 1250 and 625 µg/ml concentrations of CUR-NE. Using NIC microscopy, extensively altered tegumental surface protoscoleces was observed after protoscoleces exposure to CUR-NE. CONCLUSION: The findings of the present study revealed the in vitro protoscolicidal potential of CUR-NE. Therefore, CUR-NEs are addressed as novel protoscolicidal agents, which can be used as an alternative natural medicine to kill the protoscoleces, owing to their low toxicity and significant inhibition potency. However, further studies are necessary to investigate pharmacologic and pharmacokinetics of CUR-NEs.

Comparison of Native Hydatid Cyst Fluid (HCF), Lyophilized HCF, Antigen B (AgB) and Lyophilized AgB (LAgB) Originated from Echinococcus granulosus Sensu Stricto for Sero-Diagnosis of Active, Transitional and Inactive Human Liver Cystic Echinococcosis.

Background: Cystic echinococcosis (CE) is an important zoonotic parasitic disease caused by the larval stage or metacestode of the tapeworm Echinococcus granulosus sensu lato. Due to treatment protocols for different liver cysts, diagnosis of cyst stages is very important. Different antigens have been used for CE diagnosis. However, each one is more sensitive and effective for the diagnosis of specific CE stages is not known well. We aimed to compare Native Hydatid Cyst Fluid (HCF), Lyophilized Hydatid Cyst Fluid (LHCF), antigen B (AgB) and Lyophilized antigen B (LAgB) originated from E. granulosus sensu stricto (G1-G3) genotype, for sero- diagnosis of active, transitional and inactive human liver CE using ELISA technique. Methods: The HCF was collected aseptically from liver CE cysts of sheep slaughtered from 2018 to 2019 in Shiraz slaughterhouse, Southern, Iran. The cysts were characterized by PCR and sequencing for genotype specification. Four types of antigens were used: HCF, LHCF, AgB and LAgB originated from E. granulosus sensu stricto (G1-G3) genotype. Thirty-three serum samples from active, transitional, and inactive human cysts were collected. Overall, 48 samples from other parasitic diseases and 60 samples from healthy subjects as negative controls were checked using four antigens by ELISA method. Results: The best diagnostic sensitivity with 96.97% was observed by anti-LHCF IgG ELISA test. The best specificity with 95.37% was observed in ELISA test using LAgB. Conclusion: Simultaneous test of sera with anti-LHCF IgG ELISA and anti-LAgB IgG ELISA would be the best in the diagnosis of human liver cystic echinococcosis.

Metal uptake in Psettodes erumei and Hysterothylacium spp. larvae in the Persian Gulf: Evaluation of larvae as bio-indicator.

This study aimed to compare the concentrations of heavy metals in Psettodes erumei as host fish and larvae of Hysterothylacium spp. as its parasite. Moreover, to evaluate the larvae as bio-indicators the uptake of heavy metals, the infected and non-infected fish were also compared. Fresh P. erumei species (n = 19) were randomly sampled during four months from Bushehr County, Iran. The digestive tract of each fish was examined for nematode parasites using a stereomicroscopy. The isolated nematodes were identified, and content of Fe, Cu, Zn, Co, Ni, Cr, As, Cd, Hg, and Pb were measured using ICP-OES. The metal concentrations were simultaneously analyzed for the muscles of infected fish and their parasites, as well as non-infected ones. Of the 19 P. erumei examined, 13 (68.4%) P. erumei were infected with Hysterothylacium spp. larvae. The parasites had significantly higher level of Fe, Cu, Zn, and Ni (with mean value of 7.59, 0.572, 1.223, and 4.623 mg/kg, respectively) than the muscles of the host fishes (with mean value of 3.29, 0.0010, 0.586, and 0.277 mg/kg, respectively) (p < 0.05). Infected hosts showed significantly lower amounts of As element in their muscles (0.050 mg/kg) than non-infected hosts (0.113 mg/kg) (p < 0.05). The findings emphasize the potential role of Hysterothylacium spp. larvae as bio-indicators for monitoring heavy metals pollution in marine ecosystems.

Evaluation of the Ability of Antigen B Originated from Echinococcus granulosus Sensu Stricto and E. canadensis for the Diagnosis of Confirmed Human Cystic Echinococcosis Using ELISA.

Background: Cystic echinococcosis (CE) is an important zoonotic parasitic disease worldwide. Application of species-specific antigen for serodiagnosis of human CE has not been utilized, so far. In this regard, AgB originated from Echinococcus granulosus sensu stricto (G1-G3) and E. canadensis (G6/G7) CE cysts, confirmed by molecular biology and sequencing was used for evaluation of their ability in the diagnosis of confirmed human CE. Methods: The hydatid cyst fluid (HCF) of E. granulosus sensu stricto and E. canadensis species were separately, used for preparation of AgB during 2017-2018 in Shiraz and Tehran, Iran. A total of 45 sera samples from confirmed CE patients, 102 sera from healthy people as negative control and 44 sera from other parasitic diseases, were used for measurement of the diagnostic ability of antigen B originated from E. granulosus sensu stricto and E. canadensis species of CE, alone or in 50%:50% mixture using ELISA method. Results: Overall, 38 (84.4%) out of 45 confirmed human CE were positive by ELISA using AgB originated from E. granulosus sensu stricto. This items for AgB originated from E. canadensis was 39 (86.6) out of 45 serum samples. A total of 39 out of 45 samples (86.6%) showed positivity by a mixture of antigen B originating from both species. The specificity of the above tests was calculated as 93.15%, 96.58%, and 93.84%, respectively. Conclusion: Due to the diversity of the cyst species in human population, application of AgB from prevalent species alone or in combination with other species is suggested.

Anisakis spp, DNA detection in paraffin-embedded tissue biopsies recovered from patients with gastritis using real-time PCR in Bushehr, Persian Gulf, Iran.

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time.

Sero-Epidemiological Study of Human Hydatidosis in Semnan and Sorkheh, Semnan Province, Iran.

Background: To determine the seroprevalence of human cystic echinococcosis/hydatidosis which is one of the most important zoonotic diseases by ELISA using native antigen B in Semnan and Sorkheh, Semnan province, Iran, where no significant information about human infection exists. Methods: Overall, 957 human serum samples were randomly prepared from Semnan, Sorkheh, and its 13 surrounding villages in different seasons from 2017 to 2018. Antigen B was prepared from native hydatid cyst fluid of domestic sheep. All serum samples were evaluated by ELISA while the suspected cases were rechecked. The cut-off was calculated as the X ¯ ±2SD. Results: Overall, 48(5%) out of 957 (422 males and 535 females) were positive for hydatidosis. The seropositivity based on sex showed 20(2.1%) out of 422 in males and 28(2.9%) out of 535 in females. The distribution of seropositive samples based on residence area showed 41 (4.3) out of 882 in urban and 7 (0.7) out of 75 in rural areas. The highest seroprevalence cases was among housewives (2.1%) followed by employers (1.5%). Based on education, source of drinking water, and age groups the highest seropositivity was observed in high school and less, in the plumping water consumers, and 50 to 59 yr old age group, respectively. There was a significant difference between seropositivity with occupation, literacy, and age group (P<0.05). Semnan with 4% seropositivity had the highest prevalence followed by Sorkheh, county. Conclusion: High prevalence of the disease in this area emphasizes the importance of increasing people's awareness about hydatidosis.

Dermal microfilariae of dogs, jackals and cats in different regions of Iran.

BACKGROUND: Due to the complexity of retrieving skin-dwelling microfilariae, filarioids of dogs presenting dermal microfilariae (e.g. Cercopithifilaria spp., Onchocerca lupi) are relatively unknown compared to Dirofilaria spp. and Acanthocheilonema spp. whose microfilariae circulate in the blood. Although Cercopithifilaria spp. and O. lupi filarioids are distributed worldwide, there is a paucity of information on their occurrence in Iran. The aim of this study was to investigate these filarioids in a large population of dogs from different regions of Iran. METHODS: From October 2018 to September 2020, skin biopsies were obtained from dogs housed in shelters (n = 557) and privately owned dogs (n = 26) in seven provinces of Iran (Hamedan, Kermanshah, Yazd, Mazandaran, Khuzestan, Lorestan, Esfahan), as well as from three road-killed jackals (Canis aureus) and three cats (Felis catus) in Hamedan province. The skin biopsies were first soaked in saline solution at room temperature overnight, and examined for dermal microfilariae under the microscope. Positive skin specimens and sediments were tested by PCR for a 304-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and amplicons were sequenced. RESULTS: Microfilariae of Cercopithifilaria spp. were found in skin biopsies of 32 of the 583 (5.5%) dogs tested, with infection rates of up to 25% in Kermanshah. No microfilariae were recovered from skin biopsy samples collected from dogs in Khorramabad and Ahvaz, nor from the examined jackals and cats. None of the privately owned dogs were found to be infected. Morphologic and morphometric characteristics of the microfilariae were consistent with C. bainae. Eighteen skin samples were positive for the cox1 gene, of which 15 sequences showed a nucleotide identity of 100% and three of 93.4% with the reference sequence of C. bainae available in GenBank (haplotype I; GenBank accession number: JF461457). CONCLUSIONS: The data from this study broadens current knowledge on the geographical distribution of C. bainae in dogs in Middle Eastern countries. Further studies on different wild canine species in the country (e.g. jackal, fox, wolf) could provide further information on the epidemiology of these filarioids. A particular focus should be put on zoonotic O. lupi given the reports of its presence in human patients from this country.

Evaluation of a simple Dot-ELISA in comparison with countercurrent immunoelectrophoresis for diagnosis of human hydatidosis.

BACKGROUND: Cystic echinococcosis (CE) is caused by the larval stage of cestode parasite Echinococcus granulosus which has a worldwide distribution with variable geographic incidence. The diagnosis of CE is still problematic since the performance of the available serological assays are not reasonably satisfactory. The present study aimed to develop a specific and simple Dot-ELISA system and compare it with currently available countercurrent immunoelectrophoresis (CCIEP) technique for the diagnosis of human CE. METHODS: Serum samples were collected from thirty five pathologically confirmed CE patients, 25 healthy controls, and 12 non-CE patients. The hydatid cyst fluid (HCF) was aseptically obtained from sheep hydatid cysts. Anti-hydatid cyst antibodies in the serum were evaluated by Dot-ELISA and CCIEP. RESULTS: Findings of the study demonstrated a sensitivity of 100% and specificity of 89.1% for the Dot-ELISA system. Antibody was detected in 80% of patients by CCIEP while a few of non-CE patients and healthy controls had a positive reaction in this system. A sensitivity of 80% and specificity of 62% was calculated for this system. CONCLUSIONS: Dot-ELISA was found to be more sensitive and specific in detecting anti-hydatid cyst antibodies in CE patient in comparison with CCIEP.

Parasitological and molecular study of the furcocercariae from Melanoides tuberculata as a probable agent of cercarial dermatitis.

Cercarial dermatitis is caused by animal schistosomes in many parts of the world including Iran. Various stages of the parasites have been studied in intermediate and definitive hosts in northern and southwestern Iran; however, no molecular investigation for species identification and classification of these agents has been carried out, so far. In the present study, more than 3,800 aquatic snails were collected from water sources of Khuzestan, southwest Iran. The snails were identified as Lymnaea gedrosiana, Radix auricularia, Melanoides tuberculata, Melanopsis sp. and Physa acuta. They were examined for schistosome cercariae. Two specimens of M. tuberculata were infected with ocellate furcocercariae belonging to the family Schistosomatidae. Molecular studies were carried on these schistosomatid samples. Both samples belong to an unknown schistosome species and genus in sister position to Gigantobilharzia-Dendritobilharzia clade. They differ from other species in their ITS sequence region as well as in their intermediate host specificity--This is one of the first reports on schistosome cercariae from M. tuberculata and the first including molecular data. Due to adaptability and invasiveness of this snail species, this new schistosome species, as a potential causative agent of cercarial dermatitis in humans, needs to be studied further.