Helminth parasites of carnivores in northern Iran.

A total of 100 jackals, 35 stray dogs, 13 cats, two badgers, one fox and one wild cat were collected from two provinces in northern Iran. On post mortem examination 41 species of helminths were recovered, at least 12 of which have been reported infecting humans in various countries. Hookworms were the most commonly found parasites, and Echinococcus granulosus showed the highest intensity of infection. Seven helminth species from jackals, three species from dogs, four species from cats and four species from badgers are reported for the first time in Iran.     

Immunogenicity and efficacy of a bivalent DNA vaccine containing LeIF and TSA genes against murine cutaneous leishmaniasis

There is no effective vaccine for the prevention and elimination of leishmaniasis. For this reason, we assessed the protective effects of DNA vaccines containing LeIF, TSA genes alone, or LeIF–TSA fusion against cutaneous leishmaniasis pEGFP‐N1 plasmid (empty vector) and phosphate buffer saline (PBS) were used as control groups. Therefore, cellular and humoral immune responses were evaluated before and after the challenge with Leishmania major. Lesion diameter was also measured 3–12 weeks after challenge. All immunized mice with plasmid DNA encoding Leishmania antigens induced the partial immunity characterized by increased IFN‐γ and IgG2a levels compared with control groups (p < 0.001). Furthermore, the immunized mice showed significant reduction in mean lesion sizes compared with mice in empty vector and PBS groups (p < 0.05). The reduction in lesion diameter was 29.3%, 34.1%, and 46.2% less in groups vaccinated with LeIF, TSA, and LeIF‐TSA, respectively, than in PBS group at 12th week post infection. IFN/IL‐4 and IgG2a/IgG1 ratios indicated that group receiving LeIF–TSA fusion had the highest IFN‐γ and IgG2a levels. In this study, DNA immunization promoted Th1 immune response characterized by higher IFN‐γ and IgG2a levels and also reduction in lesion size. These results showed that a bivalent vaccine containing two distinct antigens may induce more potent immune responses against leishmaniasis.     

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Objective To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran. Methods A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB/c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA‐PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique. Results Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica. In rural areas, the causative agent of CL was mainly L. major (95%L. major vs. 5%L. tropica), in urban areas it was L. tropica (65%L. tropica vs. 35%L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB/c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area. Conclusion Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country.     

Intranasal bioavailability of insulin from carbopol-based gel spray in rabbits

The purpose of this study was to investigate the nasal absorption of insulin from a carbopol-based nasal gel spray in rabbits. An insulin nasal gel was prepared by dispersing carbopol in distilled water, followed by the addition of insulin solution, then neutralization and viscosity adjustment. The nasal absorption of insulin from the gel, in conscious rabbits, was evaluated in comparison with absorption from an insulin solution. The absolute bioavailability of insulin from the nasal gel was studied using blood glucose level in comparison to intravenous injection. The insulin gel formulation produced a significant hypoglycemic response in rabbits, whereas no response was seen following administration of the insulin solution formulation. The bioavailability of insulin from the nasal gel formulation was 20.6% compared with the intravenous injection. The results of the present study suggest that the carbopol gel promotes the nasal absorption of insulin in rabbit model and due to its sprayability with commercially available spray pumps, could be considered as a preferred platform in nasal drug administration.     

Ultrastructural and molecular characterization of Sarcocystis isolated from camel (Camelus dromedarius) in Iran

Sarcocystis cameli was first described in one-humped camels (Camelus dromedarius), and it is the only species which have so far reported in camels. Although more than 150 species of Sarcocystis were described in various animals, only a few data on camel Sarcocystis ultrastructure were published, and this report is the first for molecular information (DNA sequence and RLFP digestion pattern). The main objective of the present work is to characterize Sarcocystis isolated from camels by electron microscopy and PCR-RFLP methods. Muscle samples were taken from the fresh esophagus, diaphragm, skeletal muscles, and heart of one-humped camels (C. dromedarius) slaughtered in abattoirs of Tehran and Ghazvin provinces, Iran. The dissection and trypsin digestion techniques were applied for the detection of the cysts. The infected samples were fixed in glutaraldehyde and/or frozen at −20°C until use for ultrastructural and molecular studies, respectively. The ultrastructural and molecular studies were carried out contemporaneously. The 18S rRNA gene of the parasites was amplified by PCR. The PCR products were cloned into a pTZ57R/T and sequenced. In addition, the PCR products were digested separately with each of the four restriction enzymes for RFLP. Our results indicated that only microcysts were observed in muscle samples. The microcysts were white, elongated, spindled, and a few spiral-shaped, with mean size 260 × 75 μm which are identical with S. cameli. The ultrastructure of microcyst wall had many non-branched finger-like protrusions irregularly folded. There was a 600-bp specific band amplified after PCR with specific primers. The molecular data for camel Sarcocystis is reported for the first time in Iran and the world.     

The short-term effect of mustard gas on the serum immunoglobulin levels

Mustard gas (MG), as a chemical warfare agent was used by the Iraqi army in Iran-Iraq conflict against military men in the battlefield in 1985.The serum levels of IgG, IgA and IgM of patients exposed to MG in the battlefield were measured by single radial immunodiffusion from day 3 up to one month after exposure to MG. The serum levels of IgG in patients showed significant decrease on day 3 after exposure to MG. However, the levels of IgG in the serum samples collected from the patients during 4-18 days after exposure to MG were found to increase. The increase in serum IgG levels in the sera of patients which were collected during 19-31 days after exposure to MG was found to be highly significant, surpassing those from the controls. The levels of serum IgA in patients during one month after exposure to MG showed alterations similar to those of serum IgG, however the serum alterations of the patients IgA, comparing to those of the normal controls were not significant. The serum levels of IgM in patients did not show marked alterations during one month after exposure to MG comparing to those of the normal controls. The initial decrease in serum levels of IgG in patients is discussed in terms of a possible leakage of IgG into the skin blisters and into other severely affected parts of the body such as respiratory system, whereas the subsequent increase in serum IgG is interpreted as due to (auto) antigenic stimulation of the patients' immune systems.     

Molecular Cloning and Expression of EG95 Gene of Iranian Isolates of Echinococcus granulosus

Background

Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein, against E. granulosus, has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector.

Methods

Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces, and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step, the subcloned gene was expressed in CHO as eukaryotic cell.

Results

EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot.

Conclusion

Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.     

Impaired reproductive function of male rats infected with Toxoplasma gondii

Toxoplasmosis is one of the classical conditions known to have an adverse effect on female reproductive functions, but a few investigations into male reproductive parameters have been performed. This work was carried out to study the effects of Toxoplasma gondii on reproductive function in male rats. Male rats were infected with the RH strain of T. gondii tachyzoites, and following every 10 days from 10 to 70 postinfection (PI), the percentage of body weight to testis weight ratio as well as epididymal sperm parameters (number, motility, viability, and morphology rates), serum testosterone (ST), intratesticular testosterone (ITT), serum lactate dehydrogenase (SLDH), intratesticular lactate dehydrogenase and fructose in seminal vesicles and coagulating glands were measured. The results of the study showed sperm motility, viability and concentration rates were significantly decreased temporary after infection up to 70 days. Sperm abnormality was also increased during these days. In addition, temporary alteration in ST, ITT, SLDH, intratesticular LDH and fructose in seminal vesicle and coagulating gland was observed PI. These findings suggest that toxoplasmosis can cause impermanent impairment on the reproductive parameters of male rats.     

Lipoproteins: from physiological roles to drug delivery potentials

Lipoproteins, the endogenous lipid-protein associations responsible for lipid metabolism within the human body, have attracted interest in recent years for their potential as drug delivery carriers owing to, mainly, their lipophilic/amphiphilic nature, which makes them ideal for interacting with highly lipophilic drugs. After lipoprotein particles have been isolated from the blood, drugs can be "loaded" onto them with a variety of methods. Loading can be done either in soluble/suspended form in a liquid medium or as a dry film. Each method has advantages and disadvantages. The drug-loaded lipoproteins can be modified by the attachment of different ligands that target the particles to specific tissue/cell types within the body. A wide variety of drug molecules both from small molecular or macromolecular structures have been tested successfully, mostly in vitro, for their potential for delivery by lipoprotein carriers.     

The effect of betamethasone and IFN-gamma on replication of Toxoplasma gondii (RH strain) and nitric oxide production in Hela cell culture

Toxoplasmosis is a protozoal infection caused by Toxoplasma gondii. Toxoplasmosis produce severe damage in patients who are immunosuppressed. In those who are immunosuppressed, latent infection can be reactivated resulting in acute disseminating disease. Betamethasone is a synthetic glycocorticoid, used as an anti-inflammatory and immunosuppressant in a wide variety of disorders. The aim of this study was evaluation of betamethasone as an immunosuppressor drug on infected cells by Toxoplasma gondii. In this study, at first HeLa cells were grown in 24 well culture plates in culture medium . When confluent monolayer was obtained, we compared 6 groups to evaluate the effect of betamethasone as a corticosteroid drug (two concentrations 4 and 40 micro g/ml) and the effect of IFN-gamma (100 IU/ml ) on growth, replication and Nitric Oxide (NO) production. The results showed, that high number of plaques were seen in group with 40 mug/ml of betamethasone and the lowest number of plaques were seen in group with 100 IU of IFN-gamma. The difference between plaque number in control and groups treated with IFN-gamma and betamethasone was significant (P<0.05). The groups with betamethasone or IFN-gamma without tachyzoites did not show any effect on cell structures. Replication rates in the wells treated with IFN-gamma were decreased significantly 72 h post inoculation in comparison with control group (P<0.05). There was no significant difference among different groups in NO production. The results indicated that betamethasone increase the invasion of tachyzoites to host cells in vitro.