Suppression of E6 Oncogene Induces Apoptosis in CaSki Cervical Cancer Cells.

OBJECTIVE: The most important casuse of cervical cancer incidence and high mortality rate is infection to the human papillomavirus (HPV). The aim of the present study was to investigate the effect of silencing HPV E6 oncogene on cervical cancer cells using specific siRNAs. MATERIALS AND METHODS: CaSki cervical cancer cells, carrying E6 gene, were cultured and then transfected with E6 targeting siRNAs. The cell viability through suppression of E6 expression was explored using MTT assay. Besides, apoptosis induction was investigated by means of flow cytometry using Annexin / PI staining. The changes in the expression of target genes were examined via  Real-Time PCR. RESULTS: E6 gene silencing caused a significant decrease in the survival rate of CaSki cells through remarkable enhancement of apoptosis induction. Moreover, E6 suppression led to significant upregulation of P53, Bax, Caspase-3, and Caspase-9 mRNA expression while downregulated Bcl-2 expression. Interestingly, it was found that suppression of E6 expression could lead to upregulation of  E5 and E7 expression as a compensatory mechanism for E6 deactivation. CONCLUSION: According to the results of this study, suppression of E6 expression using specific siRNAs could be considered as a therapeutic approach for cervical cancer.

Molecular Identification and Genotyping of Babesia canis in Dogs from Meshkin Shahr County, Northwestern Iran.

BACKGROUND: Canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic diseases in dogs. METHODS: A total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of Meshkin Shahr, Ardabil Province, Iran. The presence of Babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18S rRNA gene. RESULTS: Our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with Babesia. All four Babesia-infected dogs were confirmed B. canis by the molecular-based method. Sequence alignments comparison of the B. canis genotypes A and B, it was revealed that all B. canis isolates belonged to genotype B. CONCLUSION: This study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in Iran.

Morphological Description, Phylogenetic and Molecular Analysis of Dirofilaria immitis Isolated from Dogs in the Northwest of Iran.

BACKGROUND: Dirofilariasis is a globally distributed arthropod-borne parasitic disease of mainly canids and felids. We evaluated to extend the knowledge of morpho-molecular characteristics and outer ultrastructure of Dirofilaria immitis isolated from Northwest of Iran. METHODS: Overall, 67 filarial worms including 41 females and 26 males parasites were collected from the cardiovascular system of the 43 stray dogs in Meshkinshar, Ardebil Province, Northwest of Iran in 2017, and subjected to light and scanning electron microscopy (SEM) as well as carmine alum staining for morpho-molecular and identification. Molecular methods were used for confirmation of morphological findings by sequencing of Cyto-chrome c oxidase subunit I (cox1) gene. RESULTS: The partial DNA sequencing of cox1 gene of adult parasites showed considerable homology and close proximity to the previously isolated from Kerman and Meshkinshahr, Iran. The lowest genetic variation and the highest intra-species variability was found in D. immitis and Dirofilaria repens, respectively. No similarity was identified between D. immitis nucleotide sequence and Wolbachia species as its endosymbiont bacteria. CONCLUSION: The SEM technique is an excellent tool for differential recognition of the parasite surface morphology and molecular techniques could differentiate and identify Dirofilaria spp.

Serological Evaluation of Anti-Toxoplasma gondii Antibodies in Patients with Acute Leukemia and Lymphoma through Chemotherapy.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that belongs to the family Coccidae. We aimed to evaluate IgG avidity and the changes of anti-Toxoplasma immunoglobulins M (IgM) and G (IgG) in patients with acute leukemia and lymphoma. METHODS: Ninety eight patients with Acute myeloid leukemia (AML), Acute Lymphoblastic Leukemia (ALL) and lymphoma, selected from patients referring to Imam Reza Hospital of Tabriz (38°04'N 46°18'E), in terms of the presence of anti-Toxoplasma IgM, IgG, IgG avidity antibodies and the major risk factors were evaluated. RESULTS: The results of pre-chemotherapy evaluation showed that of the examined patients, only two cases, one patient with ALL and another patient with lymphoma, had a positive IgM titer. Overall, 46 cases had positive IgG titers, including 20 patients with AML, 15 patients with ALL and 11 patients with lymphoma. Three (3.06%) patients were positive for anti-T. gondii IgM and one of them was with new infection of toxoplasmosis in lymphoma patients. The post-chemotherapy IgG titer evaluation showed 46 [46.9% (95% CI 37.4-56.7)] positive IgG cases that this result was similar to the result of pre-treatment phase. One [1% (95% CI 0.2-5.6)] positive IgG avidity case was detected using ELISA method, in a patient with lymphoma whose IgM was also positive. There was no significant difference between the type of leukemia and the history of contact with cat. CONCLUSION: Performing specialized tests to diagnose toxoplasma infection before starting treatment, in immunodeficiency patients who undergo chemotherapy, is necessary; therefore, these tests should be considered in therapeutic protocols.

Comparative mitochondrial proteomics of Leishmania tropica clinical isolates resistant and sensitive to meglumine antimoniate.

Antimony is an important drug for the treatment of Leishmania parasite infections. In several countries, the emergence of drug-resistant Leishmania species has reduced the effectiveness of this drug. The mechanism of clinical drug resistance is unclear. The aim of this work was to identify mitochondrial proteome alterations associated with resistance against antimonial. A combination of cell fractionation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Label-Free Quantification was used to characterize the mitochondrial protein composition of Leishmania tropica field isolates resistant and sensitive to meglumine antimoniate. LC-MS/MS analysis resulted in the identification of about 1200 proteins of the Leishmania tropica mitochondrial proteome. Various criteria were used to allocate about 40% proteins to mitochondrial proteome. Comparative quantitative proteomic analysis of the sensitive and the resistant strains showed proteins with differential abundance in resistance species are involved in TCA and aerobic respiration enzymes, stress proteins, lipid metabolism enzymes, and translation. These results showed that the mechanism of antimony resistance in Leishmania spp. field isolate may be associated with alteration in enzymes involved in mitochondrial pathways.

An Unusual Location of Hydatid Cyst, Cause of Severe Pollakiuria.

Hydatidosis is an important zoonotic parasite disease in several herbivorous mammals. Human cystic echinococcosis (CE) caused by infection with larval stage of Echinococcus granulosus has been frequently reported from different organs. Here, we report the first case of a urinary echinococcosis in Iran with a pain in the lower left abdominal quadrant and severe frequent urination (pollakiuria). We detected a cyst 120 × 15 mm dimensions with heterogeneous mass contain fluid in the back of the urinary bladder neck between the umbilical region and external urethral sphincter. The patient was candidate for open-abdomen surgery and cysts were resected. The isolated cysts from liver and urinary bladder were referred to pathology laboratory, and the tissue sections were stained with Tri-chrome and Hematoxylin/eosin staining methods. Microscopic examination of prepared tissue sections showed protoscoleces of E. granulosus with specific and thick laminated hyaline layer (non-cellular membrane), with covers the thin activate germinal epithelium, which revealed the diagnosis of a hydatid cyst. This rare case illustrates that CE is the necessity of considering in the differential diagnosis from cysts, abscesses, malignant and benignant tumors, especially is essential in endemic areas of CE.

Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran.

BACKGROUND: The purpose of this study was molecular detection and phylogenetic analysis of Wolbachia species of Dirofilaria immitis. METHODS: Adult filarial nematodes were collected from the cardiovascular and pulmonary arterial systems of naturally infected dogs, which caught in different geographical areas of Meshkin Shahr in Ardabil Province, Iran, during 2017. Dirofilaria immitis genomic DNA were extracted. Phylogenetic analysis for proofing of D. immitis was carried out using cytochrome oxidase I (COI) gene. Afterward, the purified DNA was used to determine the molecular pattern of the Wolbachia surface protein (WSP) gene sequence by PCR. RESULTS: Phylogeny and homology studies showed high consistency of the COI gene with the previously-registered sequences for D. immitis. Comparison of DNA sequences revealed no nucleotide variation between them. PCR showed that all of the collected parasites were infected with W. pipientis. The sequence of the WSP gene in Wolbachia species from D. immitis was significantly different from other species of Dirofilaria as well as other filarial species. The maximum homology was observed with the Wolbachia isolated from D. immitis. The greatest distance between WSP nucleotides of Wolbachia species found between D. immitis and those isolated from Onchocerca lupi. CONCLUSION: PCR could be a simple but suitable method for detection of Wolbachia species. There is a pattern of host specificity between Wolbachia and Dirofilaria that can be related to ancestral evolutions. The results of this phylogenetic analysis and molecular characterization may help us for better identification of Wolbachia species and understanding of their coevolution.

Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran.

BACKGROUND: Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. METHODS: A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. RESULTS: Twenty-eight patients (84.8%) were seropositive, two (6%) were both IgG and IgM positive, while one (3%) showed IgG avidity <40%. Molecular testing confirmed toxoplasmosis in 27 patients (81.8%). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. CONCLUSION: Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis.

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions.

Survey of feline visceral leishmaniasis in Azarshahr area, north west of Iran, 2013.

Leishmania infantum is a causative agent of visceral leishmaniasis or kala-azar, which is endemic in some part of Iran. Azarshahr city located in East Azerbaijan province, North West of Iran, which is endemic for visceral leishmaniasis. This study aimed to investigate the possible reservoir role of cats for visceral leishmaniasis in the Azarshahr area. Totally 65 cats have been trapped alive from villages of Azarshahr county and their serum samples subjected to direct agglutination test (DAT) for L. infantum antibodies. Giemsa stained impression smears have been prepared for parasitological examination of spleen and liver tissue. Also liver and spleen samples of the cats have been cultured in Novy-MacNeal-Nicolle (NNN) medium and also used for PCR. None from 65 samples was positive in NNN culture, PCR and microscopic examination. Fifteen (23.07 %) out of 65 serum samples showed Leishmania specific antibody agglutination at 1:320 dilution or above, but all considered as negative because none of them confirmed by Giemsa stained smears, PCR and NNN culture. According to the findings of the present study, cats are not a reservoir for visceral leishmaniasis in the Azarshahr area.

Seroprevalence of Toxoplasma Gondii Infection among High School Girls in Ajabshir from East Azarbaijan Province, Iran.

INTRODUCTION: Toxoplasmosis is a disease parasite which can infect human and animals. The infection may be serious if is transmitted to the fetus during pregnancy. The aim of this study was to determine the prevalence of specific antibodies and the associated risk factors for toxoplasmosis in students attending high-school in Ajabshir. METHODS: In this descriptive study, 549 blood samples were collected from high school girls. The samples divided into two groups (147 and 402 samples from rural and urban schools respectively). IgG and IgM specific antibodies to Toxoplasma gondii were detected using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of study showed that from 402 urban samples, 50 (12.4) and 34(8.5) cases and from 147 rural samples, 38 (25.9) and 32 (21.8) cases were seropositive for anti-Toxoplasma IgG and IgM antibodies respectively. Of the risk factors studied, the significant association was found between T. gondii-specific antibodies with residency and age. CONCLUSION: Based on data found in our study, 87.6% of young girls from urban areas in Ajabshir did not have antibodies to Toxoplasma and this is a very important issue, because these young women were in fertile age. Therefore required Preventive and control programs especially in these cases in order to reduce the rate of disease.

Cryptosporidium infection in patients with gastroenteritis in sari, iran.

BACKGROUND: Cryptosporidiosis is a common coccidian parasite infection in patients with diarrhea that has worldwide distribution especially in developed countries. Therefore, the aim of this study was to determine the occurrence of Cryptosporidium infection in patients with gastroenteritis admitted to hospitals of Mazandaran University of Medical Sciences by parasitological and molecular methods in Sari, Iran. METHODS: Stool samples were collected from 348 patients with gastroenteritis admitted to the hospitals of Medical University in the Sari and Ghaemshahr cities in Mazandaran Province, Northern Iran in 2010-2011. Oocysts of Cryptosporidium identified using Formalin-Ether concentration method and stained by Aacid-fast staining (AFS) and Auramine phenol fluorescence (APF). Genomic DAN extracted from microscopically positive samples and nested PCR -RFLP by using SSU rRNA that identifies of the species of cryptosporidium. RESULTS: In 348 patients with gastroenteritis, the most clinical symptoms were diarrhea, nausea, vomiting, dehydration, fever and weight loss. 2.3% (8 cases) of diarrheal samples tested by both microscopy and molecular methods were positive for the presence of cryptosporidium. Nested PCR products yielded unique bands of 846 bp, correspond to cryptosporidium. Species diagnosis carried out by digesting the secondary PCR product with SspI restriction enzyme, which noted 3 clearly bands of 449, 254, and 108 bp correspond to Cryptosporidium spp. CONCLUSION: The results of present study on Cryptosporidium spp. in this area can make a background data for control programs and further molecular analyses. Thus, further work needs to determine the origin of Cryptosporidium species in this area.