Sequence variation in mitochondrial cox1 and nad1 genes of ascaridoid nematodes in cats and dogs from Iran

The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0-2.3, 0-1.3 and 0-1.0 for pcox1 and 0-2.0, 0-1.7 and 0-2.6 for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5-16.6 for pcox1 and 11.9-26.7 for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes. © Cambridge University Press 2014

Immunotherapy Using Autoclaved L. major Antigens and M. vaccae with Meglumine Antimoniate, for the Treatment of Experimental Canine Visceral Leishmaniasis

Background: To evaluate immunotherapy against canine visceral leishmaniasis, Leishmania ma­jor antigen and heat-killed Mycobacterium vaccae (SRL172) were used as stimulators of immune de­fense mechanisms and the results were compared with standard chemotherapy meglumine antimoni­ate.Methods: Nineteen mongrel dogs aging 1-3 years old were used in this experiment. Infection was carried out in 15 out of 19 dogs using L. infantum, isolated from a naturally infected poly-symptomatic dog.Results: All the cases showed positive serologic results by direct agglutination test during 30-60 days following inoculation. In the first group, which was under chemotherapy (GlucantimeR), one of the members showed recurrence of the disease despite rapid effect of the therapeutic protocol. Im­munotherapy using SRL172 caused complete cleaning of the parasite in group 2, but the speed was less than Glucantime. Immunotherapy using L. major antigen combined with M. vaccae in group 3 and combine administration of immunotherapy and chemotherapy in group 4 both were with relapsing of one case in each group. Group 5 and 6 were consisted of positive and negative con­trol dogs, respectively.Conclusion: Immunotherapy seems to be an adjuvant in treatment of canine leishmaniasis but it needs more investigation for final confirmation

Induction of apoptosis by alcoholic extract of combination verbascum thapsus and ginger officinale on Iranian isolate of trichomonas vaginalis

Introduction: The genus Malassezia is an important skin resident of human. The present study aimed to analyze in vitro activity of the essential oils of Lavandula stoechas, Cuminum cyminum and Artemisia sieberi against clinical strains of Malassezia species. Methods: A total of 47 Malassezia strains, including Malassezia furfur, Malassezia globosa and Malassezia obtuse, were used in this study. A disk diffusion technique was selected for testing the susceptibility of Malassezia strains to the essential oils. Results: All the essential oils showed in vitro activity against Malassezia strains, with M. furfur and M. obtusa being the highest and lowest susceptible of the strains, respectively. The highest antifungal activity was associated with the essential oil of C. cyminum (mean ± SD: 50.0 ± 0.0 mm), followed by L. stoechas (mean ± SD: 46.8 ± 3.1 mm) and A. sieberi (mean ± SD: 36.9 ± 5.7 mm). The inhibition zone ranges were 12.5 to 15.6 mm (mean ± SD: 14.4 ± 1.6 mm) for ketoconazole and 11.6 to 13.3 mm (mean ± SD: 12.4 ± 0.9 mm) for fluconazole. Although all the antifungal drugs were found to possess good antifungal activities against Malassezia strains, their effects were lower than the activities shown by the essential oils tested (P < 0.05). Conclusion: These results indicated that the essential oils tested, especially the one from C. cyminum, inhibited the growth of clinical strains of Malassezia, implying its potential use in the treatment of Malassezia infections. This indicates that this plant may be useful in preparation of new drugs

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background: Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.Methods: Sixty-three BALB/c mice were injected intra-peritoneal with 5×103 tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were in­jected with phosphate buffer saline as control group. Dot-ELISA was performed for detection of T.gondii antigen in mice sera and capture - ELISA was done as golden standard assay too.Results : Toxoplasma gondii antigen was detected from day 2 in mice sera ; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigene­mia by dot - ELISA, no positive result was detected in control mice by dot- ELISA.Conclusion: Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with cap­ture-ELISA

Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard

Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes. © 2009 Elsevier Inc. All rights reserved

Emergence of a new focus of visceral leishmaniasis due to Leishmania infantum in Golestan Province, north-eastern of Iran

Over the last decade, a few cases of visceral leishmaniasis (VL) have been reported in some districts of the province of Golestan, in north-eastern Iran. The aim of the present study was to investigate the prevalence of Leishmania infantum infection among humans and domestic dogs by using direct agglutination test (DAT) and PCR assays in the eastern zone of the province. Between 2011 and 2012, blood samples were randomly collected from 450 humans and 50 domestic dogs, in the eastern zone of Golestan Province including 7 villages from Marave-tappeh district where new cases of human VL had been recorded there. Each of these samples was tested for anti-Leishmania antibodies, in DAT, and for L. infantum kinetoplast DNA on whole blood, in PCR-based assays. A total of 450 human samples, 6 (1.33 %) were found seropositive and 13 (2.8 %) was found PCR-positive. Of the 50 dog samples, 16 (32 %) were found seropositive and 15 (30 %) were PCR-positive. All PCR-positive dogs were found seropositive except one as well as 6 (46.2 %) PCR-positive humans were also found seropositive. Moreover, the species of L. infantum was detected in all PCR-positive samples. The high prevalence of VL in the study areas offer it has emerged as an endemic focus in the province. Further investigations on the vectors, reservoirs and human population are recommended. © 2013 Indian Society for Parasitology

A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis among Humans and Domestic Dogs in Mazandaran Province, North of Iran

Background: New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmania infantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province. Methods: In this cross-sectional study, blood samples were randomly collected from 402 humans and fortynine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species. Results: None of 402 collected human (402) and dog (49) blood samples showed anti Leishmania infantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1 %) were found PCR-positive corresponding to L.infantum. Conclusion: This study confirms the circulation of L. infantum at least among domestic dogs an

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background: Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies,prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme lectrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribedspacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.Methods: Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed bysequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.Results: ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.Conclusion: ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresismethod and is suggested for verification of Leishmania species