RFLP Analysis of Fragments of the 18S rRNA and Cox1 Genes to Identify Sarcocystis cruzi in Water Buffalo (Bubalus bubalis) In Guilan Province, North of Iran.

Background: Sarcocystosis is a zoonotic disease worldwide caused by Sarcocystis spp., some of these species can show clinical and subclinical manifestations, resulting in financial losses. Our study was performed for identifying Sarcocystis sp., in slaughtered buffalo by PCR-RFLP based strategy with sequencing in Guilan, North of Iran. Methods: Overall, 400 fresh muscle samples were prepared via naked-eye observation from 100 buffaloes (esophagus, diaphragm, shoulder, and thigh), followed by the digestion of samples. The PCR was done to amplify partial parts of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I (Cox1) genes. Then, the PCR products were digested by endonuclease SspI, DraI, and FokI. Sequencing of all species was done to confirm the RFLP results. Results: Five macroscopic cysts (1.25%) were visible in the sample by naked-eye examination. Furthermore, 293 samples (73.25%) were found to be Sarcocystis sp. positive through tissue digestion and microscopic observation, whereas 376 samples (94%) were positive by PCR. In addition, the findings of PCR-RFLP and nucleotide sequence samples exhibited the infection of buffaloes with S. cruzi. Conclusion: Based on the data presented herein, Bovine sarcocystosis caused by S. cruzi is very common in buffalo in the Guilan region. Regarding the high prevalence of sarcocystosis, developing disease control and prevention policies for buffaloes is necessary, and a change of attitude in traditional farming is recommended.

Accurate and rapid detection of Fasciola hepatica copro-DNA in sheep using loop-mediated isothermal amplification (LAMP) technique.

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop-mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross-sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens' parasitological examination was performed by using the direct wet mount and formalin-ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica-positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51-0.72 and p < .001). According to the results of chi-square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3- to 4-year-old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.

Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain.

BACKGROUND: There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum. METHODS: Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot. RESULTS: The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein. CONCLUSION: We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.