Evaluation of Benzimidazole Resistance in Haemonchus contortus Using Comparative PCR-RFLP Methods

Background: In order to deworm the ruminants especially of sheep in Iran, consumption of benzimi­dazoles has more than 2 decades history and today farmers are using imidazothiazoles, macrocyclic lactones and mostly benzimidazole compounds (BZs) to treat infected farm animals. It has been demonstrated that the most common molecular mechanism leading to BZsresistance in Haemonchus contortus is a single mutation at amino acid 200 (phenylalanine to tyrosine) of the isotype 1 of beta tubulin gene. According to the report of such mutations in Iranian Telador­sagia circumcincta isolates with Restriction Site Created PCR-RFLP, we decided to evaluate the frequency of such mutations in H. contortus in three different geographical areas of Iran.Methodes: A total of 102 collected adult male H. contortus were evaluated with PCR-RFLP (us­ing PSP1406I as restriction enzyme). By means of a second step to compare function of different methods and to increase sensitivity of detection mechanism, a third of samples were examined by another PCR-RFLP method (using TaaI as restriction enzyme) and finally beta tubulin gene of two samples was sequenced.Results: All of samples were detected as BZss homozygote. Finally, beta tubulin gene sequenc­ing of two samples showed no point mutation at codon 200.Conclusion: It seems that BZresistance of H. contortus in Iran is not a serious problem as antici­pated before

Morphological and Genetic Characteristics of the Liver Hydatid Cyst of a Donkey with Iran Origin

Background: No data is available on morphology and genetic characteristics of Echinococcus granulosus derived from donkeys of Iran, despite of its existence in donkeys.In the present study morphometric variations of the rostellar hooks of protoscoleces and genotype characteristics of hydatid cyst of donkey from Iran were determined. Methods: Protoscoleces prepared from hydatid cyst of donkey of Iran were morphometric and genetic analyzed. The genetic analysis was done using Cox 1 gene by comparative sequence analysis. Results: Our morphometric results showed that donkey of Iran shares 6 out of 7 determined parameters with donkeys of Jordan and 4 out of 7 with 4 available data with Switzerland donkeys. Morphological similarities and dissimilarities were observed with sheep-dog (G1) and camel-dog strains (G6) of Iran. The nucleotide sequence alignment showed that the partial sequence of Cox 1 from donkey had 91% homology with query coverage of 99% to the corresponding sequence of E.equinus, 90% homology to the E. felidis, 90% homology to E. ortleppi, 89% homology to the E. shiquinus, 89% homology to the E. vogeli, 89% homology to the E. oligarthrus, 88% homology to the E. canadensis and 83% homology to the Taenia solium.Additionally, the amino acid sequence of this gene has also some differences between this strain and all known strains of E. granulosus even with E. equinus (G4). Conclusion: Despite of common morphological characteristics of Iranian donkey hydatid cyst with those of donkeys of other parts of the world, genetically it has its. own entity

Benzimidazole -Resistance in Haemonchus Contortus: New PCR-RFLP Method for the Detection of Point Mutation at Codon 167 of Isotype 1 Β-Tubulin Gene

Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer), to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu­lin gene of Haemonchus contortus.Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleo­tide polymorphism (SNP) at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer), in which the nucleotide T at the posi­tion 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc­lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp) was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achiev­ing a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respec­tively.Results: All worms had two alleles encoding for phenylalanine (BZss homozygote) for both codons.Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP

Isolation of DNA from A Single Helminth Using New Developed Kit in Iran and Its PCR Analysis

Background: Nematodes are among the most common and important parasites of man and animal. DNA of a single worm can be used for several purposes, such as identification to the species, subspecies, strain and antihelmenthic resistance. DNA extraction from a single small worm using traditional methods such as phenol extraction technique faces serious prob¬lems. Methods: DNA from 20 single Haemonchus contortus was isolated using DNA isolation kit newly designed in Iran by the Re¬search Unit of Molecular Biological System Transfer (MBST) based on the specific binding of DNA to the carrier. The ge¬nomic DNA was amplified using specific primers derived from β-tubulin isotype 1 in PCR. The specificity of the PCR prod¬ucts was determined using semi-nested PCR technique. Specific PCR-product from β-tubulin gene could be amplified with 1 ng, 100 pg and 10 pg DNA. Results: The used DNA extraction method was safe, with high quality and quantity, fast, easy to handle and not costly for ge¬netic analysis of even a single small worm. Conclusion: The Iran produced DNA extraction Kit is grounded on a selective binding of nucleic acids to a silica-based mem¬brane and is recommended for the isolation of DNA from even small amount of biological materials

Identification and determination of effective and immunogenic antigens of Ascaridia galli in poultry reared in Tabriz area

In a trial, in Tabriz area, the ability of an experimental vaccine from the larval homogenate of Ascaridia galli was evaluated during the year 1384. A group of 5 white leghorn hens were immunized twice with 100 micrograms larval homogenate of Ascaridia galli diluted in 1ml PBS and emulsified in 1ml Freund’s adjuvant orally. Booster immunization was also administered orally on day 21 The other group (5 white leghorn hens) received 1 ml PBS emulsified in 1ml of the same adjuvant. On day 33 each hen was administered approximately 10,000 Ascaridia galli infective eggs orally. Blood samples were collected from the animals in a 10 day interval from the first immunization until infective challenge and weekly thereafter until the end of study. Sera were tested by ELISA and Western-blotting. Hens were necropsied 8 weeks post challenge with infective eggs for recovery of the nematodes. Vaccinated hens showed a 76 percent reduction in mean EPG, 74 percent reduction in the average number of male worms, 79 percent reduction in the average number of female worms and 77 percent reduction in the mean number of total worms. Significance in mean optical density of sera in ELISA was noticed (

Determination of Immunogenic Relevant Antigens in the Excretory-Secretory (ES) Products and the Lysates of Ascaridia galli Larvae

Background: Ascaridia galli, the largest nematode of small intestine of birds, especially the native poultry, may give rise to serious illness, pathological defects and economical losses even in modern poultry production systems. Although various measures have been undertaken to vaccinate poultry against A.galli, no satisfactory results were obtained so far. However, there is no report on the efficacy of excretory-secretory (ES) proteins of A.galli larvae in immunization of poultry. Thus, the aim of the present research project was based on the use of the ES products of the larvae, in order to find the protective anti­gens.Methods: Five hundred native poultry were autopsied and adult A.galli was removed form their intestines. The eggs were harvested form the uterus of female worms and cultured at 25 ˚C in water containing 0.1 N sulphuric acid for almost a fort­night. The larvae were then freed mechanically and kept in Earl's salt solution for a few days. The supernatant solution of alive larvae containing the ES products of the larvae, as well as the sonicated alive and dead larvae, was analyzed by SDS-PAGE.Result: Many protein fractions of 15 kDa up to 200 kDa were demonstrated in lysate of these larvae. Using the serum of a hen, infected with a high numbers of A.galli, an immunogenic antigen was identified between 55 kDa to 72 kDa by Western blotting procedure.Conclusion: Finding the protein band between 55 and 72 kDa can be promising for preparation of vaccine, though more investigations are needed to prove the protective ability of this antigen