Identification and Genetic Variation of Fasciola Species from Tabriz,North- Western Iran

Background

Fascioliasis is considered as the most important helminthic infection of cattle and sheep. Traditional approaches using morphological and biologic characters cannot cause a certainty in the accurate and precise identification and intra-specific differences of Fasciola spp. In this study, we identified Fasciola species using ITS-1 marker and described genetic variation of each species of the parasite in isolates from Tabriz slaughterhouse in West Azerbaijan Province, north- western Iran.

Methods

Overall, 100 samples (50 from sheep and 50 from cattle) morphologically detected as Fasciola worms were studied for identification of Fasciola species by PCR-RFLP method and intra-species variation of the parasite using RAPD-PCR technique.

Results

A region of approximately 460bp in all samples was successfully amplified. There were no identifiable variations among the size of PCR products. Two and three fragments in samples correspond to F. hepatica and F. gigantica was seen, respectively, through PCR-RFLP method. No difference was seen in digestion pattern according to host (sheep or cattle). Different types of each species of the parasite was observed using RAPD-PCR technique.

Conclusion

We could have an estimate of frequency of F. hepatica and F. gigantic and different genotypes of the parasite in isolates from one locality in north- western of Iran. By extension of such studies in future to other animal hosts (buffalo and goat) and including more regions to sampling, the reliability of the results and their application for control programs in zoonotic diseases will be increased.     

Molecular Differentiation of Fasciola Species and Characterization of Genetic Diversity of F. gigantica Using NADH Dehydrogenase I (ND1) Gene in the Endemic Areas of Iran

Background:

Fasciola hepatica and F. gigantica are the causative agents of fasciolosis in domestic animals and humans. Based on the morphometric criteria, differential diagnosis between them is problematic. In addition, intermediate forms of Fasciola have been found in Iran, which makes the differentiation more difficult. The aim of the present study was to provide molecular evidence for the existence of F. gigantica in Iran using sequencing analysis of ND1 and PCR-RFLP analysis of ITS2 regions and to study the intraspecies variations of F. gigantica based on mitochondrial ND1 gene polymorphism.

Methods:

Forty Fasciola spp. samples collected from four distinct provinces (Fars, Khuzestan, Gilan, Khorasan Razavi) in Iran were collected for morphological and molecular characterization. In molecular method, PCR-RFLP analysis of ITS2 using pagI restriction enzyme was used as a screening approach for F. gigantica differentiation. Then mitochondrial DNA sequence variations in the ND1 gene were used for phylogenetic analysis.

Results:

Based on the morphometric criteria and RFLP analysis, 14 parasitic samples were initially identified to be F. gigantica. Phylogenetic results showed that there are at least 10 different genotypes of F. gigantica in Iran, which are different from those existing in the GenBank. Twenty-six points out of 410 base pairs of sequenced ND1 gene in 10 varieties of F. gigantica were diagnosed to be polymorphic. From 26 points of polymorphism, only eight resulted in the post-translational amino acid changes in ND1 gene product structure.

Conclusion:

Data revealed noticeable genetic diversity (up to 4.63%) between different varieties of F. gigantica in Iran.     

Superoxide Dismutase (SOD) Enzyme Activity Assay in Fasciola spp. Parasites and Liver Tissue Extract

Background

The purpose of this comparative study was to detect superoxide dismutase (SOD) activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level.

Methods

Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues), 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference.

Results

Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass). Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05).

Conclusion

Fasciola species and liver infection are effective causes on SOD enzyme activity level.     

PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855) in the Infected Lymnaea auricularia (Linnaeus, 1785) Snails from Northwestern Iran

Background

Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855) larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785) from northwestern Iran using a molecular approach.

Methods

A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP) patterns specific for the detection of F. gigantica.

Results

Of the total collected snails 496 (7.34 %) were L. auricularia, among which 4.64% (23 out of 496) were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496) of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails). Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites.

Conclusion

Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.     

Bioaccumulation of Some Heavy Metals in the Liver Flukes Fasciola hepatica and F. gigantica

Background

Heavy metals tend to bioaccumulate in living organisms, and their accumulation has been a major concern. As mammals are known to excrete heavy metals via their bile, it seems to be very promising to analyse metal burdens of parasites that infect the biliary tree such as liver flukes of the genus Fasciola. The present study was carried out to evaluate F. hepatica and F. gigantica as bioaccumulators of heavy metals, and to estimate their use as sensitive markers of environmental pollution with heavy metals.

Methods

A total of 36 slaughtered buffaloes (26 infected and 10 controls) collected from the slaughter-house of Tanta City, Egypt were used. Samples of muscle and liver tissues were taken from each buffalo. A total of 44 adult Fasciola flukes were collected from the 26 infected buffaloes. Quantification of some heavy metals (Cd, Cr, Cu, Pb and Zn) in samples was carried out using electrothermal atomic absorption spectrometry.

Results

Results revealed different concentrations of heavy metals in different host tissues. The adult flukes were classified into F. hepatica (n = 25) and F. gigantica (n = 19). The bio-concentration factor (BCF) of Cr was significantly higher in F. hepatica (P = 0.0465) while BCF of Zn was significantly higher in F. gigantica (P = 0.0189). A comparative study between the two species as regards the BCF was never done before.

Conclusion

The obtained results indicate the possibility of use of Fasciola flukes as markers of environmental pollution with some heavy metals.     

Morphological and Molecular Discrimination of Fasciola Species Isolated From Domestic Ruminants of Urmia City,Iran

Background:

The trematodes of the genus Fasciola (the liver flukes) are among the well-known instances of food-borne parasites worldwide. Differentiation of Fasciola species is important because of their different transmission and epidemiological characteristics. The current study was undertaken to discriminate Fasciola species in the domestic ruminants of Urmia city, Iran.

Methods:

Adult flukes were isolated from the naturally infected livers of the slaughtered water buffaloes and sheep. The flukes were initially identified based on morphological and morphometric parameters. A 618-bp-long fragment of the 28SrRNA gene of Fasciola was amplified by polymerase chain reaction (PCR). The amplified fragment was digested by DraII or AvaII enzymes for a restriction fragment length polymorphism (RFLP) analysis and sequenced for the phylogenetic tree construction.

Results:

Based on the morphometric examination, the flukes belonged to F. hepatica, F. gigantica and an intermediate Fasciola form. The PCR-RFLP analysis was able to differentiate F. hepatica from F. gigantica. While the phylogenetic reconstruction justified, to some extent, the morphological diagnosis, it failed to segregate F. hepatica from F. gigantica identified in this and the previous studies.

Conclusion:

To resolve fully the problem of taxonomy and evolution in Fasciola species, employing a broad range of molecular and morphological approaches is necessary. This is crucial for epidemiological surveys and successful clinical management of their infection.     

Detection of Coproantigens by Sandwich ELISA in Rabbits Experimentally Infected with Fasciola gigantica

Background

The study was targeted to report the appearance of coproantigens in feces and circulating antibodies in the serum of Fasciola gigantica experimentally infected rabbits.

Methods

Copro Hyper Immune Serum (HIS) and Excretory-Secretory Hyper Immune Serum (ES HIS) antigens were used in a sandwich ELISA for the detection of F. gigantica antigens in feces of 12 rabbits experimentally infected with different doses of F. gigantica encysted metacercariae (EMC) (10, 25 and 30 EMC). The relation between time of appearance of coproantigens in feces and anti-Fasciola antibodies in serum was evaluated.

Results

The earliest diagnostic coproantigen was recorded at 21^st, 25^th and 28^th day post-infection (p.i.) in groups of rabbits infected with 30, 25 and 10 F. gigantica EMC respectively. Both HIS and ES HIS were able to detect coproantigens in feces of rabbits infected with 30 EMC at day 21 p.i. The appearance of F. gigantica coproantigens in feces of infected rabbits was concurrent to the appearance of anti-Fasciola antibodies in blood (3^rd week p.i.). However, coproantigen has specific ability for direct assessment of active infection with minimal cross-reaction with other heterologous parasitic infections.

Conclusion

The findings hold promise for a more accurate diagnostic technique in the near future for suspected Fasciola infection.     

Carbohydrate Detection and Lectin Isolation from Tegumental Tissue of Fasciola hepatica

Background

Fascioliasis is a chronic hepatic disease and may be resulted from mechanical/molecular parasite adhesion to host liver tissue. The aim of this study was to detect surface carbohydrate and lectin, carbohydrate-binding protein isolation that might be responsible of this molecular binding.

Methods

The present experimental work was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Fasciola hepatica parasites were collected from abattoir (Saman, Tehran, Iran) and surface mannose-carbohydrate was detected by fluorescein isothiocyanate (FITC) conjugated lectin (Lentil). Lectin of tegumental tissue from F. hepatica was isolated by affinity chromatography and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results

Mannose carbohydrate was observed on the surface of tegumental tissue from parasite under fluorescence microscope. Carbohydrate-binding protein or lectin with MW of 50 kDa also was isolated from homogenized tegument of helminth.

Conclusion

These results are important for understanding of molecular pathogenesis of F. hepatica at the chronic phase of fascioliasis     

Sequence Analysis of the Second Internal Transcribed Spacer (ITS2) Region of rDNA for Species Identification of Trichostrongylus Nematodes Isolated From Domestic Livestock in Iran

Background

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification.

Methods

Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species.

Results

A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings.

Conclusion

Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species.     

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 electrophoresis, 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 transcribed spacer 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 by sequencing 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 electrophoresis method and is suggested for verification of Leishmania species.     

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.

Methods

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.     

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods

Background

Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.

Methods

Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.

Results

Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.

Conclusion

The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Iranian isolates which may be related to metronidazole resistance.     

Phylogenetic Study of Haemonchus Species from Iran Based On Morpho-Molecular Characterization

Background:

Haemonchosis has a negative effect on the farming industry throughout the world, especially in the tropic and sub-tropic countries. The present study was carried out to differentiate Haemonchus species from its main hosts in Iran, including sheep, goat and camel.

Methods:

The identification took place based on the morphometrics of the spicules and molecular characters. Two hundred seventy adult male nematodes were collected from the abomasums of different ruminants (90 samples from each animal) at the slaughterhouses from different localities in Iran. Samples were morphologically identified according to the spicules’ morphometric measurements. In the section on molecular study, 10 samples of each Haemonchus isolates were genetically examined. A simple PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the second internal transcribed spacer of ribosomal DNA (ITS2-rDNA) were described to confirm the PCR results.

Results:

PCR-RFLP profile obtained from the restriction enzyme HPa1 in H. contortus and H. longistipes indicated 1 (278 bp) and 2 (113 and 135 bp) different fragments, respectively. The morphological parameters clearly distinguish H. contortus from H. longistipes. Moreover, regarding the ITS2-rDNA, sequences of 295 bp and 314 bp were obtained from H. contortus and H. longistipes, respectively.

Conclusion:

The genotypic results are in agreement with the phenotypic findings of both species.     

Investigation of Double-Band Electrophoretic Pattern of ITS-rDNA Region in Iranian Isolates of Leishmania tropica

Background

Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.

Methods

Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.

Results

Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.

Conclusion

Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.     

Strongyloides stercoralis: The Most Prevalent Parasitic Cause of Eosinophilia in Gilan Province,Northern Iran

Background

Eosinophilia occurs in a wide variety of situations such as parasitic infections, allergic disorders, and malignancies. Most cases of eosinophilia of parasitic origin, especially those with a tissue migration life cycles consists of human infections by helminth parasites. The aim of present study was to determine the parasitic causes of eosinophilia in patients in a major endemic area of human fascioliasis in Gilan Province, northern part of Iran.

Methods

One hundred and fifty patients presenting with an elevated eosinophilia attending infectious disease clinics with or without clinical symptoms, were examined. After clinical history evaluation and physical examination, coprological examinations were performed using the formalin-ether and the Kato-Katz techniques for detection of Fasciola sp. and intestinal parasites.

Results

Forty two percent of patients were infected with S. stercoralis, nine (6%) were found to be infected with Fasciola sp. while only a single patient (0.7%) were infected by Ttrichostrongylus sp.

Conclusion

Local clinicians in Gilan may consider eosinophilia as a suggestive indication for diagnosis of human fascioliasis, especially when microscopic stool and/or serological tests are negative. Based on the results, local physicians should consider S. stercoralis as the potential causes of eosinophilia in patients with elevated eosinophilia.     

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 (BZ^ss 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.     

Identification of Echinococcus granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

Background

The aim of this study was DNA extraction from protoscolecses of Echinococcus granulosus and identification of these strains in West-Azerbaijan Province, north western Iran.

Methods

Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restriction enzymes of Taq 1, Rsa 1 and Alu 1.

Result

Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex). The results of RFLP analysis also were the same for all isolates. PCR-RFLP patterns restriction enzymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approximately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples.

Conclusions

Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex) is dominant genotype in this province.     

Seroprevalence of Neospora caninum Infection in Dairy Cattle in Tabriz,Northwest Iran

Background

The aim of this study was to determine the seroprevalence of antibody to Neospora caninum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran.

Methods

In this cross-sectional study serum samples were collected from 266 healthy and aborted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to detect of antibody against N. caninum using the commercially ELISA kit.

Results

Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%.

Conclusion

Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cattle are recommended.     

Genetic Variability of Antigen B2 of Human,Sheep, Goats,Camel and Cattle Isolates of Echinococcus granulosus in Iran

Background

Antigen B (AgB) is frequently used for immuno-diagnosis of human cystic echinococcosis (CE). Echinococcus granulosus AgBs show a high degree of genetic variability in different hosts or in different CE endemic areas. The present study aimed to evaluate the genetic polymorphisms of encoding antigen B2 gene (AgB2) among different Iranian isolates of E. granulosus.

Methods

A total of 50 CE isolates were collected from human, sheep, cattle, goat and camels, 10 isolates from each intermediate host of E. granulosus. Total genomic DNA from either protoscolices or germinal layer was extracted from each cyst and PCR-RFLP followed by DNA sequencing was used to evaluate sequence variation and polymorphism of AgB2 in the isolates.

Results

After the PCR amplification, using AgB2 primers, an almost 400 bp band was amplified in all of the isolates. The PCR products were digested with Alu1 restriction endonuclease. After restriction enzyme digestion with Alu1‚ sheep and human isolates gave a similar pattern of RFLP with the gene size of approximately 140 and 240bp and camel and goat isolates gave a similar pattern, but different from sheep and human, with the gene size of approximately 150 and 250bp. Sequence analysis showed the most genetic similarity of AgB2 between human and sheep isolates.

Conclusion

Findings of this study revealed the differences in the sequences of AgB2 within and between the Iranian isolates of E. granulosus. These differences may affect the performance of any diagnostic test which uses AgB.     

Molecular Epidemiology of Cryptosporidiosis in Iranian Children,Tehran, Iran

Background

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein (GP60) gene.

Methods

Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly.

Results

Out of 794 collected samples, 19 (2.40%) were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 (89.47%) of the positive isolates were Cryptosporidium parvum and 2 (10.52%) were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa (6/17) and IId (11/17). The most common allele in all 17 isolates belonged to IId A20G1a (41.18%). A22G1 (IF) subtype was detected in two C. hominis isolates of the children.

Conclusion

The predominancy of C. parvum species (specially, IId A20G1a subtype) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.