Investigation of a set of flavonoid compounds as Helicobacter pylori urease inhibitors: insights from in silico studies.

Helicobacter pylori (H. pylori) is a spiral, microaerophilic gram-negative bacterium, which is associated with the destruction of the lining of the stomach, leads to chronic inflammation of the stomach, which can cause stomach and duodenal ulcers. The problems caused by the treatment with antibiotics have caused researchers to use new approaches to treat infections caused by H. pylori, among them specific treatments with flavonoids. Urease enzyme, as one of the most important pathogenic and antigenic factors of this bacterium, is a suitable target for this purpose. In this study, the inhibitory effect of flavonoid compounds compared to acetohydroxamic acid on H. pylori urease enzyme was evaluated using molecular modeling methods. First, the interaction of flavonoids with urease enzyme compared with acetohydroxamic acid was investigated by molecular docking method to produce efficient docking poses. Then the physicochemical properties and toxicity of the best flavonoid compounds were analyzed using the swissadme server. Also, molecular dynamics calculations were performed to precisely understand the interactions between ligands and protein. The results of this study show that all the investigated flavonoid compounds are capable of inhibiting H. pylori urease. Among these compounds, six compounds chrysin, galangin, kaempferol, luteolin, morin and quercetin showed a greater tendency to bind to urease, compared to the acetohydroxamic acid inhibitor. These compounds are desirable in terms of physicochemical properties. This study also revealed that the flavonoids with their hydroxyl groups (-OH) play an important role during bond formation with amino acids Ala278, Ala169, His314, Asp362 and Asn168. Therefore, flavonoid compounds, due to their suitable location in the active site of the urease, create a more effective inhibition than the chemical drug acetohydroxamic acid and can be suitable candidates for the treatment of Helicobacter pylori under in vitro and in vivo investigations.Communicated by Ramaswamy H. Sarma.

Evaluation of apigenin-based biflavonoid derivatives as potential therapeutic agents against viral protease (3CLpro) of SARS-CoV-2 via molecular docking, molecular dynamics and quantum mechanics studies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic COVID-19 disease that affects human respiratory function. Despite the scientific progression made in the development of the vaccine, there is an urgent need for the discovery of antiviral drugs for better performance at different stages of SARS-CoV-2 reproduction. The main protease (Mpro or 3CLpro) plays a pivotal role in the life cycle of the virus, making it an attractive target for the development of antiviral agents effective against the new strains of coronaviruses (CoVs). In this study, a series of apigenin-based natural biflavonoid derivatives as potential inhibitors of coronaviruses 3CLpro was investigated by in silico approaches. For this purpose, the molecular docking was performed to analyze the interaction of the natural biflavonoids with SARS-Cov-2 main protease and for further investigation, docking to the 3CLpro of SARS-CoV and MERS-CoV. Based on docking scores and comparison with the reference inhibitors (ritonavir and lopinavir), more than half of the biflavonoids had strong interactions with the residues of the binding pocket of the coronaviruses 3CLpro and exhibited better binding affinities toward the main protease than ritonavir and lopinavir. The top biflavonoids were further explored through molecular dynamics simulation, binding free energy calculation and residual energy contributions estimated by the MM-PBSA. Also, drug likeness property investigation by Swiss ADME tools and density functional theory (DFT) calculations were performed. The results confirmed that the 3CLpro-amentoflavone, 3CLpro-bilobetin, 3CLpro-ginkgetin, and 3CLpro-sotetsuflavone complexes possess a large amount of dynamic properties such as high stability, significant binding energy and fewer conformation fluctuations. Also, the pharmacokinetics and drug-likeness studies and HOMO-LUMO and DFT descriptor values indicated a promising result of the selected natural biflavonoids. Overall findings indicate that the apigenin-based biflavonoids may inhibit COVID-19 by significant interactions in the binding pocket and those results can pave the way in drug discovery although the effectiveness of these bioactive compounds should be further validated by in-vitro and in-vivo investigations.Communicated by Ramaswamy H. Sarma.

Environmental Contamination of Different Areas of Isfahan Province of Iran with Toxocara spp. Eggs using Molecular Methods.

Background: Toxocariasis is a parasitic disease caused by the larval stage of Toxocara canis and Toxocara cati. Infective stage of this parasite for human develops on soil. So, in this work contamination of the soil of public environments in five geographical areas of Isfahan province of Iran has been investigated. Materials and Methods: In this descriptive study, 355 soil samples were collected from parks, children's playgrounds, student dormitories, and university environments, and examined by Flotation method. The samples were then inspected using microscopic and molecular methods. Results: From the 355 examined soil samples in 77 (21.69%), and 87 (24.50%) cases Toxocara eggs were detected by microscopic and molecular methods, respectively. In the molecular method, 31 (8.70%) cases of T. cati and 44 (12.39%) cases of T. canis were identified. Conclusion: Toxocara eggs were identified in all areas of Isfahan province, although contamination rate was higher in Fereydun Shahr and Semirum counties.

In silico analysis and identification of antiviral coumarin derivatives against 3-chymotrypsin-like main protease of the novel coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a pandemic viral disease caused by SARS-CoV-2 that generated serious damages for both the human population and the global economy. Therefore, it is currently considered as one of the most important global health problems of human societies and there is an urgent need for potent drugs or vaccines which can effectively combat this virus. The chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays a key role in the viral replication inside the host and thus is a promising drug target to design and develop effective antiviral drugs against SARS and other coronaviruses. This study evaluated some antiviral coumarin phytochemicals as potential inhibitors of coronaviruses 3CLpro by in silico approaches such as molecular docking, ADMET prediction, molecular dynamics simulation, and MM-PBSA binding energy calculation. Natural coumarin derivatives were docked to the 3CLpro of SARS-CoV-2 and for further investigation, docked to the 3CLpro of SARS-CoV and MERS-CoV. The docking scores of these natural compounds were compared with 3CLpro referenced inhibitors (ritonavir and lopinavir) and co-crystal inhibitor N3. Molecular docking studies suggested more than half of the coumarin phytochemicals had favorable interaction at the binding pocket of the coronaviruses 3CLpro and exhibited better binding affinities toward 3CLpro than ritonavir and lopinavir. Most antiviral phytochemicals interact strongly with one or both the catalytic dyad residues (His41 and Cys145) and the other key residues of SARS-CoV-2 main protease. Further, MD simulation and binding free energy calculations using MM-PBSA were carried out for three 3CLpro-coumarin complexes and 3CLpro-N3/lopinavir. The results confirmed that the 3CLpro-glycycoumarin, 3CLpro-oxypeucedanin hydrate, and 3CLpro-inophyllum P complexes were highly stable, experience fewer conformation fluctuations and share a similar degree of compactness. Also, the pharmacokinetics and drug-likeness studies showed good results for the selected coumarin phytochemicals.Therefore, the coumarin phytochemicals could be used as antiviral agents in the treatment of COVID-19 after further studies.

QSAR Modeling, Molecular Docking and Molecular Dynamics Simulations Studies of Lysine-Specific Demethylase 1 (LSD1) Inhibitors as Anticancer Agents.

BACKGROUND: Histone Lysine Demetylases1 (LSD1) is a promising medication to treat cancer, which plays a crucial role in epigenetic modulation of gene expression. Inhibition of LSD1with small molecules has emerged as a vital mechanism to treat cancer. OBJECTIVE: In the present research, molecular modeling investigations, such as CoMFA, CoMFA-RF, CoMSIA and HQSAR, molecular docking and Molecular Dynamics (MD) simulations were carried out on some tranylcypromine derivatives as LSD1 inhibitors. METHODS: The QSAR models were carried out on a series of Tranylcypromine derivatives as data set via the SYBYL-X2.1.1 program. Molecular docking and MD simulations were carried out by the MOE software and the SYBYL program, respectively. The internal and external predictability performances related to the generated models for these LSD1 inhibitors were justified by evaluating cross-validated correlation coefficient (q2), noncross- validated correlation coefficient (r2ncv) and predicted correlation coefficient (r2pred) of the training and test set molecules, respectively. RESULTS: The CoMFA (q2, 0.670; r2ncv, 0.930; r2pred, 0.968), CoMFA-RF (q2, 0.694; r2ncr, 0.926; r2pred, 0.927), CoMSIA (q2, 0.834; r2ncv, 0.956; r2pred, 0.958) and HQSAR models (q2, 0.854; r2ncv, 0.900; r2pred, 0.728) for training as well as the test set of LSD1 inhibition resulted in significant findings. CONCLUSION: These QSAR models were found to be perfect and strong with better predictability. Contour maps of all models were generated and it was proven by molecular docking studies and molecular dynamics simulation that the hydrophobic, electrostatic and hydrogen bonding fields are crucial in these models for improving the binding affinity and determining the structure-activity relationship. These theoretical results are possibly beneficial to design new strong LSD1 inhibitors with enhanced activity to treat cancer.

Molecular Modeling Studies of Anti-Alzheimer Agents by QSAR, Molecular Docking and Molecular Dynamics Simulations Techniques.

BACKGROUND: Acetylcholinesterase (AChE), a serine hydrolase, is an important drug target in the treatment of Alzheimer's disease (AD). Thus, novel AChE inhibitors were designed and developed as potential drug candidates, for significant therapy of AD. OBJECTIVE: In this work, molecular modeling studies, including CoMFA, CoMFA-RF, CoMSIA, HQSAR and molecular docking and molecular dynamics simulations were performed on a series of AChE inhibitors to get more potent anti-Alzheimer drugs. METHODS: 2D/3D-QSAR models including CoMFA, CoMFA-RF, CoMSIA, and HQSAR methods were carried out on 40 pyrimidinylthiourea derivatives as data set by the Sybylx1.2 program. Molecular docking and molecular dynamics simulations were performed using the MOE software and the Sybyl program, respectively. Partial least squares (PLS) model as descriptors was used for QSAR model generation. RESULTS: The CoMFA (q2, 0.629; r2ncv, 0.901; r2pred, 0.773), CoMFA-RF (q2, 0.775; r2ncv, 0.910; r2pred, 0.824), CoMSIA (q2, 0.754; r2ncv, 0.919; r2pred, 0.874) and HQSAR models (q2, 0.823; r2ncv, 0.976; r2pred, 0.854) for training and test set yielded significant statistical results. CONCLUSION: These QSAR models were excellent, robust and had good predictive capability. Contour maps obtained from the QSAR models were validated by molecular dynamics simulationassisted molecular docking study. The resulted QSAR models could be useful for the rational design of novel potent AChE inhibitors in Alzheimer's treatment.

Evaluation of immunogenicity and protective effect of DNA vaccine encoding surface antigen1 (SAG1) of Toxoplasma gondii and TLR-5 ligand as a genetic adjuvant against acute toxoplasmosis in BALB/c mice.

AIMS: Toxoplasma gondii is an obligate intracellular, protozoan that causes a high incidence of serious zoonotic parasitic disease in humans. In the present study the immune-protective efficacy of a DNA vaccine encoding SAG1 in combination with a gene sequence encoding FliC of Salmonella typhimurium (Toll-like receptor 5 agonist) was evaluated against acute T. gondii infection in mice. METHODS AND RESULTS: Ninety-nine female inbred BALB/c mice were divided into nine groups of 11 mice and were immunized intramuscularly three times at three-week intervals (days 0, 21 and 42) and challenged with virulent T. gondii RH strain 4 weeks later. The immunization of pVAX1-SAG1 administered with pVAX1-fliC in mice indicated specific humoral responses, with higher IgG antibody titers and a mixed IgG1/IgG2a response than in other groups (with a predominance of IgG2a over IgG2b and IgG1). Also, the cellular immune response elicited high levels of IFN-γ and IL-12 cytokines and low levels of IL-4 production compared to traditional adjuvants. Furthermore, the mice vaccinated with pVAX1-SAG1+pVAX1-fliC survived for slightly longer after the last immunization and challenge with the T. gondii. CONCLUSION: This investigation indicated that cocktail DNA vaccine encoded SAG1 gene of T. gondii and FliC can protect against acute toxoplasmosis.

Evaluation of cellular immunogenicity of recombinant cytochrome p450 cyp141 protein of Mycobacterium tuberculosis in human and mouse model.

Mycobacterium tuberculosis (Mtb) is still considered one of the unsolved problems for the World Health Organization Identifying and selecting an immunogenic antigen capable of generating specific immune responses is generally the goal of all studies being carried out in to designing new vaccines. Accordingly, the present study was conducted to evaluate the immunogenicity of a M. tuberculosis recombinant protein which exist in the regions of the bacterium genome and may be an immunogenic protein. Immunogenicity of purified proteins was measured by PBMC and mouse spleen lymphocytes culturing methods using ELISA after an appropriate amount of time of incubation with Recombinant cytochrome P450 CYP141 protein. Cellular immune responses were determined and compared by measuring IFN-γ and IL4 in human, and mouse groups. The results revealed a high level of IFN-γ in PPD + individuals and the mice immunized with protein and adjuvant. Recombinant cytochrome P450 CYP141 protein proved capable of generating an immune response in mice and people with a history of previous encounters with Mycobacterium tuberculosis bacteria. It, could be considered a tuberculosis vaccine candidate in order to induce a specific effective immune response in both mice and humans.

Molecular diagnosis of potentially human pathogenic Enterocytozoon bieneusi and Encephalitozoon species in exotic birds in Southwestern Iran.

Microsporidia are obligate intracellular parasites that produce spores. The infections caused by these parasites are mostly considered to be opportunistic in immunodeficient patients. Because of the zoonotic nature of microsporidia as well as the increasing prevalence of immunodeficiency diseases, the aim of this study was to evaluate the molecular diagnosis of Enterocytozoon bieneusi (E. bieneusi) and Encephalitozoon spp. in exotic birds in southwestern Iran. Initially, 816 stool specimens were collected and stained by modified trichrome (Weber) staining. The slides were explored using light microscopy. In the next stage, the extracted DNA was amplified using a multiplex/nested PCR method. RFLP with the Mnl1 restriction enzyme was used to differentiate the Encephalitozoon species in the products of the molecular analysis. Out of 816 samples, 138 and 181 cases were found to be positive by the staining and the multiplex/nested-PCR methods, respectively. Of the 181 samples, 103 and 78 samples were positive for E. bieneusi and Encephalitozoon spp., respectively. The Encephalitozoon species were 17 E. cuniculi, 52 E. intestinalis and 9 E. hellem. Of 103 E. bieneusi samples, 57, 39, 2 and 5 cases were detected as genotypes D, M, E and L, respectively. The results showed a relatively high prevalence of microsporidia in exotic birds, and according to the results of the genotyping, these birds can be an important source of microsporidiosis. It is essential that high-risk individuals, including patients with immunodeficiency diseases, receive accurate and valid information about the risk of direct and indirect contact with infected exotic birds.

Molecular identification of Enterocytozoon bieneusi and Encephalitozoon spp. in immunodeficient patients in Ahvaz, Southwest of Iran.

Microsporidia are often considered as an opportunistic infection in patients with impaired immune systems such as transplant recipients and patients with acquired immune deficiency syndrome (AIDS). Due to the increasing prevalence of parasitic infections and immunodeficiency diseases; the aim of the study is to evaluate molecular identification of Enterocytozoon bieneusi and Encephalitozoon spp. in immunodeficient patients in Ahvaz, southwest of Iran. At first, 310 stool samples were collected from patients with immunodeficiency. The specimens were stained by modified trichrome (weber) and were examined microscopically. The extracted DNA samples were evaluated by multiplex/nested PCR method. The products of multiplex/nested PCR were explored by RFLP method using the restriction enzyme of Mnl1. Of 310, 93 samples were suspected positive for microsporidia by the staining. Also, of 310, 88 samples were positive by the multiplex/nested-PCR test that 62 samples were positive for E. bieneusi as well as 26 were detected as Encephalitozoon species that including 3 E. cuniculi, 19 E. intestinalis and 4 E. hellem. Of 62 E. bieneusi, 45, 16 and 1 were detected as genotype D, M and WL11, respectively. Also, Of 3 E. cuniculi, 1 and 2 cases were identified as genotype I and II, respectively. All E. hellem samples were included genotype 1A. Our findings revealed a relatively high prevalence of microsporidia species in immunodeficient patients. The highest risk of this infection is at individuals with impaired immune systems that it can be life-threatening in people with immune system dysfunction. It is essential that the high-risk people should be receiving the information about the risk of direct contact with infected individuals and animals.

Design, synthesis and biological evaluation of novel coumarin-based benzamides as potent histone deacetylase inhibitors and anticancer agents.

Histone deacetylases (HDACs) are attractive therapeutic targets for the treatment of cancer and other diseases. It has four classes (I-IV), among them especially class I isozyme are involved in promoting tumor cells proliferation, angiogenesis, differentiation, invasion and metastasis and also viable targets for cancer therapeutics. A novel series of coumarin-based benzamides was designed and synthesized as HDAC inhibitors. The cytotoxic activity of the synthesized compounds (8a-u) was evaluated against six human cancer cell lines including HCT116, A2780, MCF7, PC3, HL60 and A549 and a single normal cell line (Huvec). We evaluated their inhibitory activities against pan HDAC and HDAC1 isoform. Four compounds (8f, 8q, 8r and 8u) showed significant cytotoxicity with IC50 in the range of 0.53-57.59 µM on cancer cells and potent pan-HDAC inhibitory activity (consists of HDAC isoenzymes) (IC50 = 0.80-14.81 µM) and HDAC1 inhibitory activity (IC50 = 0.47-0.87 µM and also, had no effect on Huvec (human normal cell line) viability (IC50 > 100 µM). Among them, 8u displayed a higher potency for HDAC1 inhibition with IC50 value of 0.47 ± 0.02 µM near equal to the reference drug Entinostat (IC50 = 0.41 ± 0.06 µM). Molecular docking studies and Molecular dynamics simulation of compound 8a displayed possible mode of interaction between this compound and HDAC1enzyme.

Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells.

BACKGROUND: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. OBJECTIVES: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. MATERIALS AND METHODS: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). RESULTS: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. CONCLUSIONS: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development.

Seroprevalence of Toxoplasma gondii and Neospora spp. Infections in Arab Horses, Southwest of Iran.

BACKGROUND: Because of the economic importance of the Arab race horses and also the role of Toxoplasma gondii and Neospora spp. in abortion and reproductive failure of these animals, we decided to perform this study. OBJECTIVES: We designed this study to investigate the seroprevalence of anti-Toxoplasma gondii and anti-Neospora spp. antibodies in Arab horses from 12 cities of Khuzestan province in southwest of Iran. MATERIALS AND METHODS: From October 2009 to March 2011, a total of 235 blood samples were collected from jugular veins of Arab horses of different ages and genders from 12 cities of Khuzestan province. All the sera were tested for anti-Toxoplasma antibodies using the modified agglutination test (MAT) and the existence of anti-Neospora antibodies were tested using N-MAT for Neospora spp. RESULTS: According to the MAT results, antibodies to T. gondii were found in 114 (48.5%) of 235 sera with titers of 1:20 in 84, 1:40 in 19, 1:80 in four, 1:160 in four, and 1:320 in three horses. According to the N-MAT results, antibodies to Neospora spp. were found in 47 (20%) of 235 sera with titers of 1:40 in 39, 1:80 in five, and 1:160 in three horses. We did not observe any statistically significant differences regarding age groups and genders between seropositive and seronegative horses for Neospora spp. using chi-square (χ(2)) test, but it seemed that anti-Toxoplasma antibodies were more prevalent in older horses (≥ 10 years old). CONCLUSIONS: The results indicated that Arab horses are exposed to these parasites in southwest of Iran. Further research is required to determine the genomic structures of these parasites in Arab horses in southwest of Iran.

Stray cats gastrointestinal parasites and its association with public health in ahvaz city, South Western of iran.

BACKGROUND: Cats are the hosts for some zoonotic parasites such as Toxoplasma gondii and Toxocara spp. which are important in medicine and veterinary. Studies on the prevalence of intestinal parasites of cats have received little attention in south west of Iran. OBJECTIVES: The current study aimed to investigate the prevalence of parasites in stray cats in Ahvaz. MATERIALS AND METHODS: Random sampling was carried out from January to May 2012. One hundred and forty fecal samples from stray cats were examined using sucrose flotation method. RESULTS: Gastrointestinal parasites were found in 121 of the 140 (86.4%) examined samples. The parasites detected in stray cats were Toxocara spp. (45%, 63/140), Isospora spp. (21.4%, 30/140), nematode larvae (21.4%, 30/140), Taenia spp. (18.6%, 26/140), Sarcocystis spp. (17.1%, 24/140), Eimeria spp. (15%, 21/140), Blastocystis spp. (14.3%, 20/140), Giardia spp, (10.7%, 15/140), Physaloptera spp. (7.1%, 10/140), and amoeba cyst (5.7%, 8/140) respectively. The prevalence of infection by Joyexiella spp. and hook worms (4.3%, 6/140), for example, Dipylidium caninum (2.9%, 4/140) was similar; and the prevalence of infection by T. gondii and Dicrocoelium dendriticum was similar (1.4%, 2/140). CONCLUSIONS: Since the prevalence of zoonotic gastrointestinal parasites such as Toxocara spp. in stray cats is high, there is a need to plan adequate programs to control these zoonotic parasites.

Molecular characterization of Toxocara spp. from soil of public areas in Ahvaz southwestern Iran.

In the present study, the microscopy and polymerase chain reaction methods were used for detection and identification of soil contamination by Toxocara eggs in squares, streets, public parks, and rubbish dumps in Ahvaz, southwestern Iran. A total of 210 soil samples were collected from different parts of the city and examined by microscopy and polymerase chain reaction (PCR) methods, following sodium nitrate flotation. Nucleotide sequencing was performed to confirm the results of the PCR method. Toxocara eggs were found in 64 and 71 soil samples using the microscopy and PCR methods, respectively. The highest contamination rate was observed in the central part of Ahvaz (39.5% and 46.5% by the microscopy and PCR methods, respectively). Based on internal transcribed spacer 2 (ITS2) PCR identification, 28% of the samples were diagnosed as Toxocara cati and 5.7% as Toxocara canis; no mixed contamination was observed. DNA sequencing of the ITS2 gene confirmed our findings. Compared to the conventional microscopic detection following by flotation, used as the gold standard, the PCR method appears to be rapid and sensitive as well as allows analysis of Toxocara spp. isolated from soil independent of the stage of egg development. Therefore, the PCR method appears to be a valuable tool for the diagnosis and differentiation of Toxocara spp. from soil samples in epidemiological studies, and will help the local health systems in effective prevention and control of disease.

PCR-based molecular characterization of Toxocara spp. using feces of stray cats: a study from Southwest Iran.

Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%). T. canis eggs were found in 4 (6.34%) of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.

Detection of Toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran.

This study was conducted to determine the presence of Toxoplasma gondii in animal milk samples in Iran. From a total of 395 dairy herds in three provinces of Iran, 66 bovine, 58 ovine, 54 caprine, 33 buffalo, and 30 camel herds were studied, and from these parts of Iran, 200 bovine, 185 ovine, 180 caprine, 164 buffalo, and 160 camel milk samples were collected from various seasons. Samples were tested for Toxoplasma gondii by cell line culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) technique. Only the results of cell line cultivation were confirmed by bioassay in cat. Results indicated that all herds were infected with Toxoplasma gondii. The culture method showed that 51 out of 889 milk samples (5.73%) were positive for Toxoplasma gondii, and all 51 positive culture results were positive with bioassay in cat. The Fars province had the highest prevalence of Toxoplasma gondii (6.84%). The ELISA test showed that 41 milk samples (4.61%) were positive for the presence of Toxoplasma gondii, while the PCR showed that 46 milk samples were positive for Toxoplasma gondii. The results showed higher sensitivity of PCR and higher specificity of ELISA. Caprine had the highest (10%) and camel had the lowest (3.12%) prevalence rate of parasite. The summer season had the highest (76.47%) but winter (3.92) had the lowest incidence of Toxoplasma gondii. This study is the first prevalence report of direct detection of Toxoplasma gondii in animal milk samples in Iran.