Molecular Characterization of Spirometra erinaceieuropaei from Jungle Cat (Felis chaus) in North of Iran.

PURPOSE: The aim of this study is to conduct a molecular characterization of Spirometra tapeworm from jungle cat (Felis chaus) in Guilan Province, north of Iran using DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) and 12S rDNA sequences. METHODS: Morphological features of the adult tapeworm of Spirometra were evaluated using specific staining and light microscopy. The molecular characterization was performed using partial Cox1 and 12S rDNA regions. Genetic diversity was calculated and phylogenetic trees of the obtained sequences were constructed. RESULTS: Morphological features were compatible with previous description of adult Spirometra erinaceieuropaei. The Cox1 sequence of the specimen showed 100% similarity with S. erinaceieuropaei sequences in GenBank from Korea, China and Iran. Also, the 12S rDNA sequence revealed 99.7% similarity with S. erinaceieuropaei isolates from China and Japan. Intra-species variation within isolates of S. erinaceieuropaei was 0-1.4% and 0-4.6% for Cox1 and 12S rDNA genes, respectively. CONCLUSION: This is the first report of molecular characterization of S. erinaceieuropaei in jungle cat, F. chaus in Iran. Jungle cat probably plays a major role as reservoir host in maintaining of this parasite in this area with favorable climate condition. Needs for further assessment on the role of appropriate hosts, especially intermediate/paratenic hosts as well as the potential risk of human infectivity with sparganosis is emphasized.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas.

PURPOSE: Fascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3-4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase. METHODS: The unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent. RESULTS: The fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered. CONCLUSION: Reaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

Nanoemulsion of Spiramycin against Tachyzoites of Toxoplasma gondii, RH Strain: Preparation, Toxicology, and Efficacy Studies.

Background: Toxoplasma infection is caused by Toxoplasma gondii, which is an intracellular protozoan parasite. This infection consequently lead various congenital disabilities during pregnancy in patients. Spiramycin (Spi), a macrolide antibiotic, is typically recommended for T. gondii infection in pregnant women. We aimed to prepare the nanoemulsion of spiramycin (NE-Spi) and to evaluate the activity of this formulation in tachyzoites of T. gondii, RH strain. Methods: This study was conducted in 2019-2021 at the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. NE-Spi was prepared by spontaneous emulsification. The effects of this nanoemulsion on the viability of cultured cells were measured using MTT assay. To estimate the effects of NE-Spi on tachyzoites of T. gondii, RH strain, different concentrations of NE-Spi, S-Spi (suspension of spiramycin), and NE (nanoemulsion without any spiramycin) were added to tachyzoites and then stored for 30, 60, 90, 120 min and 24 h in 250 µg/ml concentration at room temperature. Finally, Tachyzoites mortality rates were evaluated by trypan blue staining. Of note, flow cytometry was conducted to confirm the obtained results. Results: The final particle size of NE-Spi was calculated to be 11.3 nm by DLS and TEM. Thereafter, using MTT assay, in 62.5 µg/ml concentration of NE-Spi, the Vero cells viability was obtained as 82%. The highest mortality rates of tachyzoites of T.gondii, RH strain were observed at 250 µg/ml concentration and after 120 min of exposure, but it was not significantly different from 24 h of exposure. Conclusion: NE-Spi has lethal efficacy on T. gondii RH strain in-vitro.

Electrochemical immunosensor based on carbon nanofibers and gold nanoparticles for detecting anti-Toxoplasma gondii IgG antibodies.

An electrochemical immunosensor based on carbon nanofibers (CNFs) and gold nanoparticles (AuNPs) was developed for detecting anti-Toxoplasma gondii antibodies  (anti-T. gondii) IgG in human serum. CNFs were produced using electrospinning and carbonization processes. Screen-printed carbon electrode (SPCE) surface was modified with CNFs and AuNPs which were electrodeposited onto the CNFs. Then, T. gondii antigen was immobilized onto the AuNPs/CNFs/SPCE. Afterward, anti-T. gondii IgG positive serum samples were coated on the modified electrode and assessed via adding anti-human IgG labeled with horseradish peroxidase (HRP) enzyme. The morphology of SPCE, CNFs, and AuNPs/CNFs/SPCE surface was characterized using field emission scanning electron microscopy (FESEM) equipped with energy dispersive spectroscopy (EDS). Characterization of CNFs was evaluated by Raman spectroscopy and X-ray diffraction (XRD). Electrochemical characterization of the immunosensor was verified using cyclic voltammetry (CV), and electrochemical response of modified electrode for anti-T. gondii IgG was detected via differential pulse voltammetry (DPV). This immunosensor was detected in the range 0-200 U mL-1 with a low detection limit (9 × 10-3 U mL-1). In addition, the proposed immunosensor was exhibited with high selectivity, strong stability, and acceptable reproducibility and repeatability. Furthermore, there was a strong correlation between results obtained via the designed immunosensor and enzyme-linked immunosorbent assay (ELISA) as gold standard. In conclusion, the developed immunosensor is a promising route for rapid and accurate clinical diagnosis of toxoplasmosis.

In vitro anti-Toxoplasma gondii activity of Ganoderma lucidum extracts.

OBJECTIVE: Ganoderma extracts have the potential to be used as anti-cancer, anti-inflammatory, immunomodulator, and antimicrobial agents, as evaluated in numerous studies. This study was aimed to determine the lethal and inhibitory effects of aqueous, hydroalcoholic, and alcoholic extracts of Ganoderma lucidum on Toxoplasma gondii RH strain tachyzoites, in vitro. RESULTS: All three types of extracts showed toxoplasmacidal effects. The highest percentage of mortality was related to hydroalcoholic extract. The EC50 of Ganoderma extracts for tachyzoites were 76.32, 3.274, and 40.18 for aqueous, hydroalcoholic and alcoholic extracts, respectively. The selectivity index obtained for hydroalcoholic extract was 71.22, showing the highest activity compared to other extracts. According to our findings, the hydroalcoholic part was the most effective substance among the extracts. This basic study showed obvious anti-toxoplasma effect of Ganoderma lucidum extracts. These extracts can be used as candidates for further in-depth and comprehensive studies especially In vivo experiments to prevent toxoplasmosis.

The Therapeutic Efficacy of Zinc Oxide Nanoparticles on Acute Toxoplasmosis in BALB/c Mice.

Background: Toxoplasma gondii infects nearly one-third of the world's population. Due to the significant side effects of current treatment options, identifying safe and effective therapies seems crucial. Nanoparticles (NPs) are new promising compounds in treating pathogenic organisms. Currently, no research has investigated the effects of zinc oxide NPs (ZnO-NPs) on Toxoplasma parasite. We aimed to investigate the therapeutic efficacy of ZnO-NPs against tachyzoite forms of T. gondii, RH strain in BALB/c mice. Methods: In an experiment with 35 female BALB/c mice infected with T. gondii tachyzoites, colloidal ZnO-NPs at concentrations of 10, 20, and 50 ppm, as well as a 50 ppm ZnO solution and a control group, were orally administered four hours after inoculation and continued daily until the mices' death. Survival rates were calculated and tachyzoite counts were evaluated in the peritoneal fluids of infected mice. Results: The administration of ZnO-NPs resulted in the reduction of tachyzoite counts in infected mice compared to both the ZnO-treated and control group (P<0.001). Intervention with ZnO-NPs significantly increased the survival time compared to the control group (6.2±0.28 days, P-value <0.05), additionally, the highest dose of ZnO-NPs (50 ppm) showed the highest mice survival time (8.7±0.42 days). Conclusion: ZnO-NPs were effective in decreasing the number of tachyzoites and increasing mice survival time in vivo. Moreover, there were no significant differences in survival time between the untreated control group and the group treated with zinc oxide, suggesting that, bulk ZnO is not significantly effective in comparison with ZnONPs.

Seroepidemiological study of toxoplasmosis in women referred to a pre-marriage counseling center in Alborz Province, Iran.

OBJECTIVES: The aim of the current study was to assess prevalence of Toxoplasma infection and its associated risk factors in women of childbearing-age in central Iran. RESULTS: Of 400 serum samples assessed for anti-T. gondii antibodies, 81 (20.25%) samples were positive for anti-T. gondii antibodies, including 74 positive samples (91.3%) for anti-T. gondii IgG and seven positive samples (8.7%) for IgG and IgM. Of seven IgG and IgM positive samples, five and two samples were high and low in IgG avidity, respectively. Based on PCR analysis, Toxoplasma infection was detected in one sample with anti-T. gondii IgM and low IgG avidity. The Chi-square test showed significant correlations of T. gondii seropositivity with history of undercooked meat consumption and contacts with cats (p < 0.05). In the present study, 79.75% of the participants were negative for IgG against T. gondii infection. Furthermore, recently acquired Toxoplasma infection was found using IgG avidity and PCR assays among women of childbearing-age in the study area, which would increase the risk of their fetus becoming infected. Educational program and antenatal screening of childbearing-age women for T. gondii infection may be important primary prevention strategies and help reduce the risk of congenital toxoplasmosis in this population.

Growth comparison of Acanthamoeba genotypes T3 and T4 in several culture media.

Acanthamoeba causes severe diseases such as Granulomatous Amebic Encephalitis (GAE) and Acanthamoeba keratitis (AK). Improving the culture media classically used for this amoeba could help to identify it quickly and facilitate its study as a biological model. The purpose of this study was to compare the growth of two Acanthamoeba genotypes (T3 and T4) in several culture media. Acanthamoeba griffini (T3 genotype) and Acanthamoeba castellanii (T4 genotype) were cultured in PYG, TSY, TYI-S-33, RPMI, and RPMI-FBS medium. The number of amoebas grown in different culture media was counted and compared to each other for 14 days. Findings in this research revealed the highest growth in RPMI-FBS medium. For this reason, we can recommend this culture medium to promote the growth of Acanthamoeba in its biological studies.

Sarcocystis infection in beef and industrial raw beef burgers from butcheries and retail stores: A molecular microscopic study.

Sarcocystis is a genus of eucoccidian parasites, which globally infects humans and various animals. In addition to economic losses in livestock industries, the parasite is a zoonosis that infects humans through contaminated beef and pork with the parasite sarcocysts. Therefore, this study was carried out to assess Sarcocystis contamination in beef and industrial raw beef burger samples from butcheries and retail stores in Tehran, Iran. Overall, 180 samples of 90 beefs and 90 raw industrial beef burgers with at least 80% meat were randomly collected in Tehran, Iran. Samples were studied microscopically after peptic digestion. Furthermore, sample genomic DNAs were used in conventional polymerase chain reaction (PCR) to amplify approximately 900-bp fragments from 18S ribosomal DNA. Of 180 samples, 170 samples (94.4%) were microscopically and 161 samples (89.44%) were molecularly positive for Sarcocystis spp. Eucoccidial DNA fragments were detected in 161 samples (89.4%), including 78 (86.6%) beef and 83 (92.2%) beef burger samples. No significant differences were found between the beef and beef burger infestations by Sarcocystis bradyzoites using statistical analysis (P > 0.05). Statistically significant differences were seen between the sample type and the intensity of parasites in samples (P = 0.003). Furthermore, differences between the conventional PCR results (positive/negative) and the intensity of parasites in samples were statistically significant (P < 0.001). The considerable prevalence of Sarcocystis spp. in beef and beef burger samples reflects high transmission of the parasite in meat producing cattle, which is important due to food hygiene. Although the most prevalent bovine species, S. cruzi, is not a zoonosis, it is highly recommended to follow guidelines on the parasite transmission prevention due to the existence of S. hominis as a zoonotic bovine species.

Serological Survey of Neospora caninum and Toxoplasma gondii Co-Infection in Rodents in Northwestern Iran.

BACKGROUND: Our knowledge of the epidemiology of rodents' parasitic agents in Iran is scarce, although some of these pathogens play an important role in human and veterinary medicine, such as Toxoplasma gondii and Neospora caninum. The purpose of this study was to determine the seroprevalence of Toxoplasma gondii and Neospora caninum in rodents of northwestern Iran between Mar and Dec 2015. METHODS: Overall, 157 serum samples from rodents (101 Meriones persicus, 41 Mus musculus, and 15 Cricetulus migratorius) were assayed by the indirect fluorescence antibody test (IFAT) for antibodies to T. gondii and N. caninum. RESULTS: We found a prevalence of 20.38% (32/157) for N. caninum, 35% (55/157) for T. gondii. Co-presence of antibodies to N. caninum and T. gondii was found in 10 (6.36%) rodents. A significant association was found between the rodents species and seropositivity to N. caninum (P<0.05) but there was no association with rodents species for T. gondii. The overall prevalence of the aforementioned parasites was higher in male versus female rodents. CONCLUSION: The high seroprevalence of toxoplasmosis and neosporosis in rodents in the study area has implications for translocation of these infections across wider geographical regions since these rodents are mostly preyed on by cats or dogs; hence, which can transfer the parasite to other hosts.

Nanosilver Colloid Inhibits Toxoplasma gondii Tachyzoites and Bradyzoites in Vitro.

BACKGROUND: Toxoplasma gondii, the coccidian protozoan parasite with worldwide distribution, is the agent of toxoplasmosis. The disease is life threatening in congenital form and in immunocompromised patients. The present study was carried out in 2016 to evaluate the in vitro effects of nanosilver colloid on tachyzoites and bradyzoites of T. gondii, RH and Tehran strains. METHODS: Different concentrations (5, 10, 20 ppm) of nanosilver colloid were added to tachyzoites of T. gondii, RH strain (type I) and bradyzoites and tissue cysts of T. gondii, Tehran strain (type II) and incubated for 30, 60, 90 and 120 minutes. The mortality rates of tachyzoites and bradyzoites were evaluated by trypan blue dye and MTT assay. Then SEM carried out to show the changes between control and exposed parasites. RESULTS: The greatest mortality rate was seen in 20 ppm concentration and after 120 minutes of exposure. By electron microscopy, the structural changes were seen in tachyzoites of RH and tissue cyst of Tehran strain in comparison with control groups. CONCLUSION: Nanosilver colloid was effective on both tachyzoites and bradyzoites of T. gondii, RH and Tehran strains.

Detection of Toxoplasma gondii in Cord Blood Samples from Neonates in Tehran, Iran.

BACKGROUND: Toxoplasmosis is an infectious disease caused by the coccidian protozoan parasite Toxoplasma gondii. The infection is life-threatening in congenital form because of transmission of the parasite from mother to fetus. In order to investigate the prevalence of congenital toxoplasmosis, the present study was performed for detection of IgG and IgM antibodies in cord blood samples of newborns by ELISA method in Tehran, Iran. METHODS: This cross-sectional survey was carried out on 1000 cord blood samples collected from Shahid Mostafa Khomeini Hospital in Tehran, Iran in 2015. Sera were separated and evaluated for the presence of IgG and IgM antibodies against T. gondii by ELISA method. At the same time, whole cord blood samples were stored at -20 °C for complementary PCR test. RESULTS: From 1000 cord blood serum samples 198 cases (19.8%) were positive for anti T. gondii IgG antibody. IgG positive samples were examined for IgM antibody, among them 1 sample had borderline levels of IgM antibody. PCR was performed for this sample but no positive result was seen. CONCLUSION: Although congenital toxoplasmosis is of importance, no acute form of infection was seen in pregnant women in this study.

In vitro culture of Toxoplasma gondii in HeLa, Vero, RBK and A549 cell lines.

Toxoplasma gondii is a protozoan parasite which can be grown in vivo and in vitro. Various cell lines are used for T. gondii culture in vitro. In this study, four cell lines of HeLa, Vero, RBK and A549 were compared with each other for T. gondii tachyzoites culture. The four cell lines were cultured and infected with 5,000,000 tachyzoites, respectively. The number of tachyzoites and viable host cells and pH of the media were assessed in each culture. The highest tachyzoite yield was seen in HeLa cell culture. The lowest number of viable host cells and the lowest pH were seen in HeLa cell line culture. The lowest tachyzoite yield, the highest viable cell and the highest pH were observed in Vero cell line culture. HeLa and Vero cell lines are thus appropriate for rapid and long-term propagations of T. gondii tachyzoites, respectively.

Occurrence of Acanthamoeba Genotypes in Wastewater Samples in Tehran, Iran.

BACKGROUND: Acanthamoeba spp. is potentially pathogenic free-living amoeba that can exist in various water sources. The presence of this amoeba in water sources could be a health hazard as Acanthamoeba could lead to severe diseases such as Acanthamoeba keratitis and encephalitis. This study aimed to determine the genotypes of isolated Acanthamoeba spp. in raw wastewater samples in Tehran, Iran. METHODS: Overall, 90 raw wastewater samples were collected from water treatment facilities in west and south of Tehran, Iran during 2014-2016. Water samples were filtered and cultured on non-nutrient agar (NNA) medium enriched with Escherichia coli. Morphological and molecular analyses were done on positive strains. The pathogenic ability of the isolated strains was determined using physical assays. RESULTS: Totally, 6 out of 90 (6.7%) samples were positive for Acanthamoeba, according to morphological characteristics of double-walled cysts. Genotyping and sequencing of the positive strains showed Acanthamoeba belonging to T4 (83%) and T11 (17%) genotypes. In vitro pathogenicity tests were revealed that five isolates were classified as non-pathogenic strains and one strain belonging to T4 genotype was classified as the highly pathogenic amoebae. CONCLUSION: The current research reflected a low contamination of wastewater sources to Acanthamoeba. More studies regarding the contamination of wastewaters before and after treatment are required in different places of the country.

Cyst Formation from Virulent RH Strain of Toxoplasma gondii Tachyzoite: In Vitro Cultivation.

BACKGROUND: This study was performed to induce conversion of RH strain tachyzoites of Toxoplasma gondii to bradyzoites by pH changing of the culture medium. METHODS: HeLa cell monolayers were infected at a 1:1 tachyzoite to cell ratio. Four hours after infection, the culture medium was removed and replaced with culture medium and 5% FCS, adjusted to pH 8 with NaOH. The culture was maintained at 37 °C without CO2 until the end of the experiment. Cyst-like structures were collected and stained with periodic acid schiff (PAS) staining method. The soluble antigens of cyst-like structures of RH strain, in addition to RH tachyzoite, bradyzoites of avirulent Tehran strain and uninfected HeLa cells were electrophoresed on 12.5% polyacrylamide gel. The gel was stained by coomassie brilliant blue R-250. RESULTS: Four days after infection of HeLa cells with tachyzoites of T. gondii, RH strain, cyst- like structures were noticed and stained with PAS. In the SDS-PAGE, protein bands of these structures had some differences with tachyzoites of RH strain, but there was quite similarity between protein bands of these structures and tissue cysts (bradyzoites) of Tehran strains. P34 and P36 (bradyzoite-specific proteins) were observed only in T. gondii bradyzoites of RH (cyst like structures) and bradyzoites of Tehran strains. CONCLUSION: Alkalization of culture medium to pH 8 induced expression of bradyzoite- specific proteins and production of RH cysts in cell culture.

Seroepidemiological Study of Toxoplasmosis in Women Referred to Arak Marriage Consulting Center during 2012-2013.

BACKGROUND: Toxoplasmosis is a zoonotic and usually asymptomatic infection. This study was carried out to investigate the seroepidemiology of Toxoplasma infection in women referred to Arak Marriage Consulting Center during 2012-2013. METHODS: In this cross-sectional study, serum samples were collected from 400 women referred to Marriage Consulting Center in Arak City, Markazi Province, central Iran during 2012-2013. Anti-Toxoplasma IgG and IgM antibodies were measured by ELISA using homemade antigen. Results were analyzed by SPSS 18 and the correlation between toxoplasmosis and some affecting factors were estimated. RESULTS: Overall, 97 cases (24.3%) had IgG antibodies against T. gondii and 19 cases (4.8%) were positive for IgM antibodies. A significant correlation was seen between T. gondii infection with clinical symptoms, keeping cat as pet animal, education and handling or eating raw or undercooked meat (P < 0.05). CONCLUSION: The prevalence of (24.3%) for Toxoplasma infection was seen in Arak City. It seems that keeping cat as pet and consumption of undercooked liver and uncooked hamburger are the most important transmission routes for the infection in this city. Since the majority of women are Toxoplasma sero-negative (75.7%) in Arak City, using serological tests and health education prior to marriage or during pregnancy is recommended.