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.

Norwegian Scabies in Two Immune-Compromised Patients: A Case Report.

Norwegian scabies (hyperkeratosis scabies) is an acute form of skin disease seen in immune-compromised patients. This study aimed to describe two cases of Norwegian scabies from Ahvaz, southwest of Iran in 2015. Two patients included a 55 year old man with renal transplant history and a 49 yr old man with diabetic mellitus and autoimmune disease, complained of dermatitis lesions and itching with sever hyperkeratosis, several macula and papules on neck and armpits for one-month duration were referred to a Iran Zamin Medical Diagnostic Laboratory in Ahvaz, Southwestern Iran in 2015. Patients were referred for fungal examination. Scraping from the crusted lesions of skin and slide preparation with 20% KOH was done. Microscopic examination presented that huge infestation of Sarcoptes scabiei in all forms of parasite included adult female, nymph stage and eggs. One of the patients spouse was also infested by Sarcoptes and appeared mild clinical symptoms. The disease was diagnosed with Norwegian scabies and the patients were successfully treated with topical 5% permethrin ointment for two weeks continuously. Overall, Norwegian scabies should be considered in immune- compromised patients with contaminated areas.

Prevalence of Toxocara eggs in the soil of public parks of Khorramshahr city, southwest Iran

Human toxocarosis is a chronic zoonotic disease, which creates various syndromes in humans such as visceral larva migrans, ocular larva migrans, and neurological toxocarosis. This disease is commonly contracted through ingestion of eggs from the Toxocara species. The eggs are transmitted to humans through contact with soil that contaminated with feces of infected animals by Toxocara species worm. This study, investigated the soil from various parks and public areas of Khorramshahr city southwest of Iran for Toxocara egg contamination. One-hundred and fifty soil samples were collected from 21 parks and green public areas from April to July 2018 and examined by the modified floatation method using saturated sucrose solution. The Toxocara eggs were isolated in 27 (18%) out of 150 samples collected. Based on Chi-square test, none of the factors: location of sampling (in city and the suburb), the presence of dogs and cats and the size of the sampling location; was not significant difference by soil contamination by Toxocara eggs. The presence of Toxocara eggs in Khorramshahr parks soil and green public areas is one of the main risk factors for catch the toxocarosis. This is important to develop preventive measures in the city and suburb areas to reduce the Environmental contaminations and thus reduce the incidence of disease in humans.

Nasal Myiasis: A Case Report.

Myiasis is caused by invasion of larvae stage of dipterans fly in living tissue of vertebrate host including human and animals. The most important family is Calliphoridae and included Calliphora, Lucilia, Chrysomyia and Cochliomyia genus. A 35-yr-old man with gastric cancer history referred to Golestan Hospital in Ahvaz Southwest of Iran in 2015. He was infected by nasal myiasis from Lucilia spp. in ICU. The genus of third larvae stage was identified by microscopic examination and culture of pupa. The population of flies inducing myiasis should be controlled in hospitals.

Multiple zoonotic helminth infections in domestic dogs in a rural area of Khuzestan Province in Iran.

BACKGROUND: Echinococcosis and toxocarosis caused by the genus of Echinococcus and Toxocara spp. are among important helminthic diseases worldwide. Limited data on the prevalence of these parasites persuaded us to determine the prevalence of E. granulosus, E. multilocularis, and T. canis infections in domestic dogs in rural areas of Ahvaz, southwestern Iran. Fecal samples from 167 domestic dogs were examined using both microscopy and PCR techniques. Multiplex PCR was performed for the presence of Echinococcus, and Taenia spp. and single PCR for detection of T. canis and Toxascaris leonina. RESULTS: The total occurrence of identified parasites was 65 (38.9%). The microscopic examinations showed that 40 (24%), 18 (10.8%), and four (2.4%) of dogs were infected with taeniid-like, ascarid, and both genera eggs, respectively. Echinococcus granulosus was identified in seven (4.2%), Taenia spp. in 29 (17.4%), and mixed infection with both in 11 (6.6%) samples. Sequencing of PCR-positive samples identified E. granulosus s.s. (G1), 18 T. hydatigena (10.8%), five T. multiceps (3%), three T. serialis (1.8%), one T. ovis (0.6%), one Spirometra erinaceieuropaei voucher (0.6%), and two Mesocestoides corti (1.2%). This is the first report of S. erinaceieuropaei voucher and M. corti in dogs in Iran. Nine (5.4%) and 16 (9.6%) dogs showed infection with T. canis and T. leonina, respectively. Two samples showed coinfection with both ascarids. CONCLUSIONS: Several studies have reported echinococcosis and toxocarosis in intermediate hosts from the southwest of Iran; however, this study is the first molecular research on E. granulosus and T. canis in domestic dogs in a rural area of southwestern Iran. Furthermore, issues of soil contamination with dogs' feces and recent dust storms in Khuzestan may have a role in the spreading of these zoonotic infections to other provinces close to it, and neighboring countries such as Iraq.

The role of climate on prevalence or eradication of vesical schistosomiasis in Khuzestan Province of Iran.

Climate is defined as the combination of climate and air elements of a given region which is usually measured for a period of decades. De-marton climate classification has been established based on many factors, including elements such as temperature and rainfall. Vesicle schistosomiasis is a parasitic disease caused by Schistosoma haematobium. This parasite lives in the blood vessels of the bladder. The parasite can cause hematuria in human and if not treated properly can lead to vesicale carcinoma. The parasite is distributed only in certain parts of the province and it is highly dispersed along the rivers of Dez, Karkheh and Karun with high emissions. In 1970, the prevalence of infection in infected foci was 23.8 %. Campaign against the parasite began in 1958 but it did not encompass all centers of infection. Preventive measures include diagnosis and treatment of patients, public health promotion, health education, drying swamps and ponds, improving the environment, cementing the irrigation canals, and the use of moluscocide eventually leads to changing the ecological and conditions of parasite and snail inhabits. Application of preventive measures resulted in the reduction of infection level to 0.7 % in 1979. By continuing struggle and intensifying preventive measures and changing ecological and climatic environment, in 2008, the examination of 3400 urine samples of students in Andimeshk district revealed no cases of the vesical schistosomiasis. It is concluded that S. haematobium and vesical schistosomiasis is eliminated from Khuzestan province southwest Iran, but the disease is still prevalent in neighboring Iran's western border country (Iraq) and due to the special conditions of its facilities and the traffic between the two countries, it is necessary to control and eradicate the disease in Iraq by using the experiences of Iran in eliminating the disease.

Identification of Leishmania Species Isolated from Human Cutaneous Leishmaniasis in Mehran, Western Iran Using Nested PCR.

BACKGROUND: The incidence of cutaneous leishmaniasis in the city of Mehran has risen sharply in recent years because the city borders Iraq, which has allowed entrance of different Leishmania strains. These strains have different shapes, periods of disease, and healing of lesions. The present study identified and determined cutaneous leishmaniasis species in this region. METHODS: This cross-sectional study was carried out by preparing slides from 92 patients with suspected cutaneous leishmaniasis lesions from Mehran during 2012-2013. Parasite genomic DNA was extracted and CSB2XF and CSB1XR primers were used to amplify the Leishmania minicircle kDNA regions. The parasite species were detected by specific 13Z and LIR primers by applying nested PCR technique. RESULTS: All banding patterns were diagnosed as L. major parasite by comparison of standard models with amplified fragments 560 bp in length from bands. The patients were 56.5% male and 43.5% female. The most frequently-infected age group was the 21-30 years group at a rate of 27.2%. About 56.3% of patients had a single lesion and a significant correlation was observed between age and number of lesions (P > 0.05). CONCLUSION: The nested PCR technique was shown to be an effective method with high sensitivity and specificity for identification of human Leishmania parasites. Molecular analysis revealed that parasites isolated from Mehran were identified as L. major and the disease was rural in form.

Comparison of Proteome Profiling of Two Sensitive and Resistant Field Iranian Isolates of Leishmania major to Glucantime® by 2- Dimensional Electrophoresis.

BACKGROUND: In this study, two-dimensional gel electrophoresis (2-DE) method was applied to determine and compare the protein spots expressed in the two field isolates of Leishmania major and recovered from the patients who were clinically sensitive and resistant to Glucantime® treatment. METHODS: Leishmania parasites were isolated from the cutaneous lesions of two CL infected patients in Shiraz, south of Iran. The species of the two isolates were identified as L. major using Nested-PCR. Sensitivity (Sh-214S) and resistance (Sh-120R) of the two isolates to meglumine antimonite were checked by the standard in vitro assays. Both sensitive and resistant L. major isolates were harvested in RPMI 1640 medium. Protein extractions were performed using TCA/Acetone method and the protein spots were determined by a two-dimensional gel electrophoresis (2-DE). The gels were stained with silver nitrate and analyzed by Image Master 2D Melanie-6 software. RESULTS: About 2967 protein spots were detected. Overall, 89 protein spots represented considerable changes of expression in the resistant isolate of L. major compared to the sensitive isolate. Of these, 60 and 29 protein spots were up-and down regulated, respectively. In addition, 11 protein spots present in the resistant isolate were noticed to be absent in the sensitive isolate. CONCLUSION: A number of proteins showed significant changes of expression in the drug-resistant L. major; moreover, the roles of these proteins probably enhanced the parasite resistance to the drug and increased parasite survival in the cells.

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.

Study on the contamination of Abadan public parks soil with Toxocara spp. eggs.

BACKGROUND: Toxocariasis is one of the most important zoonotic diseases caused by Toxocara larva stage in humans. One of the major transmission routes of infection, especially in children is pica. The aim of this topic was study the contamination of Abadan public parks with Toxocara eggs. MATERIALS AND METHODS: Two hundred and ninety one samples of soil were collected from 31 parks. The samples were examined for Toxocara spp. eggs by modified floatation method using saturated sucrose. The results were analyzed using SPSS version 19 and Chi-square test. RESULTS: Eighty five (29.2%) out of 291 samples were infected with Toxocara spp. eggs, means19 (61.2%) of the 31 parks were contaminated. There was no significant difference between the urban and suburb parks contamination (p = 0.208) but there was significant relation between contamination with Toxocara spp. eggs and traces of cats and dogs presence in the parks (p = 0.001). CONCLUSION: As the contamination of Abadan public parks soil with Toxocara spp. eggs is relatively high, the people and specially children might get the contamination during stay in the parks and measures should be taken to control the stray cats and dogs.

In vitro activity of green tea extract against Leishmania major promastigotes.

BACKGROUND: Systemic and topical treatment options against Leishmaniasis are limited to a few drugs with inconsistent efficacy and unacceptable side effects and none of them is suitable for all forms of the disease. OBJECTIVE: The aim of this study was to search the in vitro activity of green tea extract against L. major promastigotes and compare it with glucantime. METHODS: Extract was prepared by percolation method. The extract was dried and dissolved in DMSO 1% solvent. Leishmania major promastigotes treated with 6 concentrations (3, 6, 12, 24, 48, 96 mg/ml) of the extract. As control positive group glucantime 85 mg/ml and additional untreated control group were included in this study. All cultures were performed in triplicate. The promastigotes were also counted and their flagellate's motilities were assessed microscopically. RESULTS: Ethanolic extract of green tea showed significant leishmanicidal activity against L. major promastigotes in different concentrations. Notably there was a concordance in anti-leishmanial effect of the ethanol extract with the increasing of the dosage (3, 6, 12, 24, 48, 96 mg/ml). In comparison with glucantime the mean alive promastigotes in 12 mg/ml concentration of green tea was almost as same as 85 mg/ml glucantime and higher green tea extract concentrations were higher effective than glucantime. CONCLUSION: Our study revealed a novel pharmacological activity against promastigotes of L. major and suggests that green tea extract has the potential of being used in leishmaniasis but more studies are needed to find out its activity against amastigote and appropriate route of application.

Parasitological and molecular study of the furcocercariae from Melanoides tuberculata as a probable agent of cercarial dermatitis.

Cercarial dermatitis is caused by animal schistosomes in many parts of the world including Iran. Various stages of the parasites have been studied in intermediate and definitive hosts in northern and southwestern Iran; however, no molecular investigation for species identification and classification of these agents has been carried out, so far. In the present study, more than 3,800 aquatic snails were collected from water sources of Khuzestan, southwest Iran. The snails were identified as Lymnaea gedrosiana, Radix auricularia, Melanoides tuberculata, Melanopsis sp. and Physa acuta. They were examined for schistosome cercariae. Two specimens of M. tuberculata were infected with ocellate furcocercariae belonging to the family Schistosomatidae. Molecular studies were carried on these schistosomatid samples. Both samples belong to an unknown schistosome species and genus in sister position to Gigantobilharzia-Dendritobilharzia clade. They differ from other species in their ITS sequence region as well as in their intermediate host specificity--This is one of the first reports on schistosome cercariae from M. tuberculata and the first including molecular data. Due to adaptability and invasiveness of this snail species, this new schistosome species, as a potential causative agent of cercarial dermatitis in humans, needs to be studied further.

Cutaneous leishmaniasis of the lid: a report of nine cases.

Leishmaniasis is a parasitic disease caused by Leishmania species and is classified into three forms; cutaneous, mucocutaneous, and visceral. The eyelid is a rare site involved by leishmaniasis and only makes up 2.5% of cases with cutaneous leishmaniasis (CL). Although CL can affect both upper and lower lids on either their outer or inner aspects, the lateral canthus is most often affected. The most common aspect of lid leishmaniasis is chalazion-like lesions but ulcerous, phagedenic, cancer-like forms, and unilateral chronic granulomatous blepharitis may be observed. When the lid is involved, the disease is usually self-limiting; healing usually takes up to one year, hence early diagnosis and treatment are important. The diagnosis is based on a high index of suspicion regarding the endemicity of the disease in the region. Response to treatment in lid CL cases is quite satisfactory. In this article, we report nine cases of lid leishmaniasis with satisfactory responses to intralesional meglumine antimoniate.