Bacterial and Fungal Occurrence in Hydatid Cysts from Livestock in Central Iran.

Echinococcus granulosus sensu lato causes Cystic echinococcosis. This study investigated the bacterial and fungal species in the liver and lung hydatid cysts obtained from sheep, goats, cattle, and camels slaughtered in Yazd abattoir, Central Iran. In this study, 84 hydatid cysts were obtained from 20 sheep, 13 goats, 25 cattle, and 26 camels. The fertility and viability rates were assessed using light microscopy and eosin staining, respectively. The aspirated hydatid cysts were cultured to detect the presence of any bacteria and fungi. Bacterial isolates were identified by biochemical tests. DNA was also extracted from germinal layers, and then genotyping was carried out targeting the cox 1 gene. The statistical analysis was performed by SPSS version 16.0. This study showed that 22.62% (19/84) of hydatid cysts had bacterial occurrence, and none of the samples had fungal species. Among the fertile cysts, 52.6% had bacterial occurrence, of which 40% were viable. Most bacteria detected in hydatid cysts included Staphylococcus saprophyticus, Escherichia coli, and S. epidermidis. Hydatid cysts with bacterial occurrence were identified as G1-G3, G5, and G6/G7. The bacterial species occurrence in hydatid cysts had no significant relationship with fertility and viability (P > 0.05), without any significant relation with viability (P > 0.05), animal species (P > 0.05), involved organ in animals (P > 0.05), and hydatid cyst genotypes (P > 0.05). It should also be mentioned that this is the first study to assess the relationship between hydatid cyst genotyping and the occurrence of fungal and bacterial species.

The role of ATP-binding cassette transporter genes expression in treatment failure cutaneous leishmaniasis.

Leishmaniasis is one of the common diseases transmitted by sand flies in tropical and subtropical regions of the world. Currently, antimonial derivatives are the first line of treatment. Some of the members of the ATP-binding cassette (ABC) family of Leishmania are shown to be associated with no response to treatment. In this study, we evaluated ABCI4, ABCG2, ABCC7, ABCB4, and ABCC3 genes expression in Leishmania isolated from patients with non-healing cutaneous leishmaniasis and treatment response isolates. We selected 17 clinical isolates including 8 treatment failure and 9 treatment response samples from September 2020 to March 2021. The isolates were obtained from patients of Health Center Laboratory of Varzaneh, Isfahan, Iran with cutaneous leishmaniasis. The diagnosis was performed using microscopic observation. The samples were directly collected from the lesions. The expression profiling of genes was assessed using SYBR Green real-time PCR that was analyzed with delta-delta Ct. All treatment failure clinical isolates were L. major. Gene expression analysis in treatment failure isolates showed that the ABC transported genes had a different pattern in each isolate. Treatment failure has been reported for cutaneous leishmaniasis worldwide. Knowledge of the molecular mechanisms of treatment failure could solve this problem. ABC transporter genes are considered controversial over the mechanisms of treatment failure outcomes. In this study, we showed that ABC transporter genes could be considered one of the important mechanisms.

Cystic Echinococcosis in Central Iran: G1 and G6 Genotypes in Patients.

Background: Cystic echinococcosis (CE) is caused by Echinococcus granulosus sensu lato. In Central Iran, no molecular information is available on CE in humans. Therefore, in this study, we identified the genotyping of hydatid cysts obtained from patients with CE in central Iran using mitochondrial cytochrome c oxidase subunit I (cox1) gene. Patients and Methods: Hydatid cysts were obtained from 19 patients referred to Shahid Sadoughi, Mojibian, and Mortaz Hospitals, Yazd, Iran from 2018 to 2020. Informed consent was obtained from all included patients. After DNA extraction, amplification was done using cox1 gene. Phylogenetic analysis was performed using MEGA7. Results: Of the 19 patients, 11 (57.9%) were male and eight (42.1%) were female. The mean age of the patients was 35.645 ± 2.55 years old. Regarding cyst location, of eight isolates from lung, six and two belonged to G1 and G6, respectively; and all liver cysts were G1 genotype. The spleen and neck cysts had G1 and G6 genotypes, respectively (p > 0.05). All cysts with a diameter in the range of 5-10 cm (n = 9) and large cysts (>10 cm; n = 5) were identified as G1 (p = 0.002). The maximum likelihood tree topology demonstrated the maximum similarity of G1 among Iran and worldwide (99%-100% likelihood). Conclusions: Based on our results, it seems that the sheep-dog cycle in the infection of humans by Echinococcus granulosus in this study area has the most important role compared with the other cycles such as the camel-dog one.

Occurrence and genetic characterization of Echinococcus granulosus sensu lato from domestic animals in Central Iran.

BACKGROUND: The species complex of Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis distributed worldwide. There is no genotype information from hydatid cysts in the intermediate hosts in Central Iran. Therefore, in this study, we analyzed the hydatid cysts in livestock slaughtered in an abattoir in this region. Six hundred fifty-seven hydatid cysts were isolated from 97 animals, including sheep, cattle, camels, and goats slaughtered in Yazd abattoir from September 2018 to January 2020. The demographic data was collected as well as cyst location, fertility, and viability. Out of 657 samples, 164 samples were genotyped. Then, phylogenetic analysis was performed using MEGAX. Statistical analyses were done using SPSS version 16.0 by chi-square with a significant difference of less than 0.05. RESULTS: Out of 164 samples, the G1-G3 complex genotype had the most frequency in samples, with 135 cases recognized. The G6/G7 was observed in 19 isolates and G5 was reported in nine samples. One sample was detected as Taenia hydatigena. CONCLUSIONS: This study showed that G1-G3 and G6/G7 genotypes were presented in all animals, but G5 was reported only in cattle, goats, and camels. It is the first molecular identification of cystic echinococcosis in Central Iran. Hence, reporting G5 in livestock in this area should be considered due to transmission to humans.

Relationship between Serum Tumor-Related Markers and Genetically Modified Rice Expressing Cry1Ab Protein in Sprague-Dawley Rats.

Genetically Modified (GM) foods are among the most important achievements of biotechnology. Given the safety importance of transgenic rice, this study was conducted to investigate the effect of GM rice consumption on serum concentrations of tumor markers in rats. In this experimental intervention, we used the blood serum samples from the Biobank taken from 60 males and 60 female Sprague-Dawley (SD) rats fed on three different diets, including rat's standard food, non-GM rice, and GM rice after 90 day. Tumor markers including Carcinogenic embryonic antigen (CEA), Alpha-Fito protein (AFP), Cancer Antigen 19-9 (CA19-9), Cancer Antigen 125 (CA125), and Cancer Antigen15-3 (CA15-3) were assessed by enzyme-linked immune sorbent assay (ELISA) method. Statistical analysis was conducted via SPSS software. The results show that the concentrations of tumor markers were within the normal range in the SD rats treated with diet, and since the concentration of tumor markers was lower than the acceptable index determined, according to the kit standard in both groups, no obvious carcinogenic effect was found. However, these findings are not enough to make a final decision regarding the safety assessment of GM rice consumption.

Molecular characteristic of treatment failure clinical isolates of Leishmania major.

BACKGROUND: Leishmaniasis is a prevalent tropical disease caused by more than 20 Leishmania species (Protozoa, Kinetoplastida and Trypanosomatidae). Among different clinical forms of the disease, cutaneous leishmaniasis is the most common form, with an annual 0.6-1 million new cases reported worldwide. This disease's standard treatment is pentavalent antimonial (SbV) that have been used successfully since the first half of the 20th century as a first-line drug. However, treatment failure is an increasing problem that is persistently reported from endemic areas. It is important to define and standardize tests for drug resistance in cutaneous leishmaniasis. SbV must be reduced to its trivalent active form (SbIII). This reduction occurs within the host macrophage, and the resultant SbIIIenters amastigotes via the aquaglyceroporin1 (AQP1) membrane carrier. Overexpression of AQP1 results in hypersensitivity of the parasites to SbIII, but resistant phenotypes accompany reduced expression, inactivation mutations, or deletion of AQP1. Hence, in this study, a phylogenetic analysis using barcode gene COXII and kDNA minicircle and expression analysis of AQP1 were performed in treatment failure isolates to assess the isolates' molecular characteristics and to verify possible association with drug response. METHODS: Samples in this study were collected from patients with cutaneous leishmaniasis referred to the Diagnosis Laboratory Center in Isfahan Province, Iran, from October 2017 to December 2019. Among them, five isolates (code numbers 1-5) were categorized as treatment failures. The PCR amplification of barcode gene COXII and kDNA minicircle were done and subsequently analyzed using MEGA (10.0.5) to perform phylogenetics analysis of Treatment failures (TF) and Treatment response (TR) samples. Relative quantification of the AQP1 gene expression of TF and TR samples was assessed by real-time PCR. RESULTS: All samples were classified as L. major. No amplification failure was observed in the cases of barcode gene COXII and kDNA minicircle amplification. Having excluded the sequences with complete homology using maximum parsimony with the Bootstrap 500 method, four major groups were detected to perform phylogenetic analysis using COXII. The phylogenetic analysis using the barcode target of minicircle showed that all five treatment failure isolates were grouped in a separate sub-clade. CONCLUSIONS: We concluded that the barcode gene COXII and the minicircle kDNA were suitable for identification, differentiation and phylogenetic analysis in treatment failure clinical isolates of Leishmania major. Also, AQP1 gene expression analyses showed that treatment failure isolates had less expression than TR isolates. The isolate with TF and overexpression of the AQP1 gene of other molecular mechanisms such as overexpression of ATP-binding cassette may be involved in the TR, such as overexpression of ATP-binding cassette which requires further research.

Safety assessment of genetically modified rice expressing Cry1Ab protein in Sprague-Dawley rats.

Rice is considered one of the most important staple food crops. Genetically modified (GM) Bt rice, harbored cry1Ab gene expressing the insect-resistance protein has been developed to resistance to the insects. In this study, we assessed the safety of the GM Bt rice on Sprague-Dawley rats for 90 days. Totally, 120 rats in both sexes were used for three different diets, including 50% GM Bt rice, feeding with 50% rice, and standard feeding. Each 40 SD rats including 20 males and 20 females were considered as each diet. The clinical variables such as body weight and food consumption were measured and a range of clinical tests was examined, including hematology, serum chemistry parameters, urinalysis profile, thyroid, and sex hormone levels. Pathological assessments were also done. The results showed that the mean weekly feed utilization (%) had no significant difference among the studied groups. Also, blood biochemistry, hematological parameters, urine analysis, and hormonal levels had no significant differences among the groups. However, alanine aminotransferase was less in males versus female feeding with GM Bt rice. No histopathological changes were observed among the groups. In conclusion, this study demonstrated that GM Bt rice had no obvious adverse effects on rats' health.

Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major.

OBJECTIVE: Leishmaniasis is caused by different Leishmania spp. Treatment failure (TF) of cutaneous leishmaniasis (CL) is a serious issue that may be due to various reasons, previous studies suggested Leishmania RNA virus (LRV) as a potential cause of TF. Two variant groups of LRV1 and LRV2 are reported. In this study, the presence of LRV1/LRV2 was compared in TF with treatment response (TR) isolates of L. major. Clinical isolates of 15 TF and 15 TR were collected from CL patients referred to the Health Centers of Isfahan. Genomic DNA was extracted to identify Leishmania spp. using ITS1-PCR-RFLP. Identification of LRV1/LRV2 was performed using SYBR Green Real-Time PCR. The statistical analysis to test relationship between the treatment response with Glucantime and the presence of LRV were performed using SPSS 16.0 with Fisher's Exact test. P value of less than 0.05 was considered significant. RESULTS: ITS1-PCR-RFLP results showed that every isolate was identified as L. major. The results showed no LRV1 in any of the samples but 7 TR isolates and 2 TF isolates showed positive for LRV2. Statistical analysis showed no significant difference between the presence of LRV2 and response to Glucantime (p-value = 0.1086). Therefore, other mechanisms might be responsible for TF.

Molecular Analysis of Aquaglyceroporin 1 Gene in Non-Healing Clinical Isolates Obtained from Patients with Cutaneous Leishmaniasis from Central of Iran.

BACKGROUND: Regarding the antimonial-resistant of Leishmania spp., understanding of related mechanism is necessary. One of the most important involved molecules is aquaglyceropin1 (AQP1). The aim of this study was molecular analysis of AQP1 gene from antimonial-resistant clinical isolates and its expression. METHODS: Overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of Yazd and Varzaneh,, located 105km southeast of Isfahan and 240km away from Yazd, were assessed from Jun 2015 to Dec 2017. After sampling, staining was done and evaluated for Leishman by microscope. Samples were collected in RNAlater solution for gene expression analysis in non-healing isolates. DNA extraction was performed from each slide with Leishman body. All patients with L. major isolates detected by ITS1-PCR-RFLP were followed for finding the resistant isolates, consequence of molecular characterization of AQP1 using PCR-RFLP. Gene expression of AQP1 from all resistant isolates was assessed in comparison with the one in a sensitive isolate. Statistical analysis was done using SPSS. The significance level was considered ≤0.05. RESULTS: Five isolates were detected as antimonial resistant. Molecular detection and identification were appeared that all were L. major. The molecular characterization of AQP1 showed G562A mutation. Gene expression of AQP1 in resistant isolates showed 1.67 fold higher than the sensitive isolate. CONCLUSION: We reported a new point mutation of G562A in AQP1 gene involved in molecular mechanism in resistant isolates.

Radioactivity levels in the mostly local foodstuff consumed by residents of the high level natural radiation areas of Ramsar, Iran.

Among High Level Natural Radiation Areas (HLNRAs) all over the world, the northern coastal city of Ramsar has been considered enormously important. Many studies have measured environmental radioactivity in Ramsar, however, no survey has been undertaken to measure concentrations in the diets of residents. This study determined the 226Ra activity concentration in the daily diet of people of Ramsar. The samples were chosen from both normal and high level natural radiation areas and based on the daily consumption patterns of residents. About 150 different samples, which all are local and have the highest consumption, were collected during the four seasons. In these samples, after washing and drying and pretreatment, the radionuclide was determined by α-spectrometry. The mean radioactivity concentration of 226Ra ranged between 5 ± 1 mBq kg-1 wet weight (chino and meat) to 725 ± 480 mBq kg-1 for tea dry leaves. The 226Ra activity concentrations compared with the reference values of UNSCEAR appear to be higher in leafy vegetables, milk and meat product. Of the total daily dietary 226Ra exposure for adults in Ramsar, the largest percentage was from eggs. The residents consuming eggs from household chickens may receive an elevated dose in the diet.