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 diseas

Molecular Study of Cryptosporidium spp. in Dogs from Southwest of Iran

Background: Cryptosporidium is a protozoan parasite that effects rodents, dogs, calves, humans, and cats. Infection with this parasite is known as cryptosporidiosis. Cryptosporidium spp. may induce clinical or subclinical signs in infected hosts. In the life cycle of this parasite infected dogs freely living in urban and rural areas of Khuzestan province are the definitive hosts that should be considered as a real problem in public health for humans. Objectives: This study aimed at determining the frequency of cryptosporidiosis in dogs in southwest of Iran. Methods: Overall, 350 fresh fecal samples were collected from domestic dogs living in 43 villages, from June 2012 to September 2013. All samples were investigated by Sheather’s concentration method and fecal smears were stained with modified Ziehl-Neelsen followed by light microscope examination, and polymerase chain reaction (PCR). Results: The results revealed that frequency of Cryptosporidium infection was 8% and 12.3%, using direct smear and molecular method, respectively. Conclusions: The present findings indicated that domestic dog feces from southwest of Iranmay contain zoonotic parasites such as Cryptosporidium spp. andmay be a potential risk for humans and other animals, especially when they contaminate the environment. The role of dogs as source of human infection should be investigated by further studies

Detection and genotyping of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran

Abstract To detection and genotype of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran. Between August 2011 and May 2012 at different sites located in the area of the Ahvaz city south west Iran. A total of 200 soil samples were taken from different points of the region. Oocysts were recovered using the flotation method. Then, PCR reactions targeting the GRA6 gene were performed for specific T. gondii detection. The positive samples were studied by RFLP (random amplified fragment length polymorphism) using MseI enzymes to confirm the parasite linage. Toxoplasma DNA was found in 18 samples. Among them, 12 samples were successfully genotyped as GRA6 type III and 6 as GRA6 Type II. This is the first investigation detecting and genotyping T. gondii oocyst in environmental soil samples of Ahvaz, South west of Iran. The results of this study indicated that soil contaminated with T. gondii oocysts especially in public park may play a role in the epidemiology of human toxoplasmosis in southwest of Iran

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 animal

Blood Levels of Oxidant/Antioxidant Parameters in Rats Infected with Toxoplasma gondii

Toxoplasmosis is a common parasitic infection in the world. Since increased free radicals and oxidative stress are reported in many parasitic diseases the purpose of the present study was to evaluate the oxidative stress in acute and chronic toxoplasmosis. RH strains of Toxoplasma tachyzoites were used in the present study. Twenty-five female rats were infected with the parasite while 25 other rats were as the control group that received normal saline. Zero-, 5-, 7-, 10-, and 45-day postinfection (DPI) blood samples were taken. Some parameters related to oxidant and antioxidants such as antioxidant enzymes, malondialdehyde, and total antioxidant capacity were measured. On day 7 after infection, GPX activity and GSH level were significantly increased and in the mentioned day the amount of total antioxidant capacity was significantly reduced. In other cases, there were no significant differences between the groups in different days. Overall, based on the results it seems that, on day 7 after infection, in infected rats responses to oxidative stress were triggered and led to decrease of total antioxidant capacity. Furthermore, glutathione was increased to cope with stress. It seems that probably antioxidant defense system entered the infection to the chronic phase and changed the parasites stage

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. keywords: AdjuvantBALB/C miceDNA vaccineFlagellinSurface antigen 1Toll-like receptor 5Toxoplasma gondi