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.     

Molecular Characterization of Echinococcus granulosus from Hydatid Cysts Isolated from Human and Animals in Golestan Province,North of Iran

Background

The aim of the present study was to determine the molecular characteristics of Echinococcus granulosus from paraffin-embedded tissues of hydatid cysts isolated from human and protoscoleces of hydatid cysts from sheep, cattle and camel isolates using PCR- RFLP of ITS1- rDNA analysis in Golestan Province, northern Iran.

Methods

E. granulosus isolates from human patients infected with hydatid cyst and protoscoleces from hydatid cysts of sheep, cattle and camel isolates were collected from different hospitals and the abattoir throughout the Golestan Province. In all, 60 E. granulosus genomic DNA were extracted and examined by PCR - ITS1 of rDNA and amplified using BD1 / 4S and EGF1 / EGR2 primers, followed by RFLP using Alu1, Msp1 and TaqI restriction enzymes.

Results

The PCR-ITS1 products obtained from sheep, cattle and human isolates were similar to sheep strain (1000 bp and 391 bp). Majority of the camel samples yielded 295 bp DNA bands. RFLP -ITS1 of E. granulosus with Taq1 in human, sheep and cattle isolates showed similar patterns in the number and size of DNA. RFLP methods in camel isolates showed a different genotype, using Taq1, whereas no DNA bands were observed using Alu1 in camel and human isolates. Therefore, two clearly distinguishable banding patterns of E. granulosus were obtained with the three enzymes, which separating human, sheep and cattle isolates from the camel origin.

Conclusion

The results indicate the possible of transmission of the G1 and G6 genotypes of E. granulosus between livestock animals and human in Golestan Province.     

Genotyping of Echinococcus granulosus Isolates from Human Clinical Samples Based on Sequencing of Mitochondrial Genes in Iran,Tehran

Background

The present study was aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nad1 in Iran.

Methods

Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair (bp) for cox1 and nad1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already deposited in GenBank.

Results

Sixteen, (53.3%), 13 (43.3%), and 1 (3.3%) samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% (n=26) and 10% (n=3) were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 (n=9), G3 (n=1) and G6 (n=1).

Conclusion

In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping.     

Genetic Identification of Orientobilharzia turkestanicum from Sheep Isolates in Iran

Background:

Adult worms of Orientobilharzia turkestanicum live in the portal veins, or intestinal veins of cattle, sheep, goat and many other mammals causing orientobilharziasis. Orientobilharziasis causes significant economic losses to livestock industry of Iran. However, there is limited information about genotypes of O. turkestanicum in Iran.

Methods:

In this study, 30 isolates of O. turkestanicum obtained from sheep were characterized by sequencing mitochondrial cytochrome c oxidase subunit 1 (cox1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) gene. The mitochondrial cox1 and nad1 DNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with O. turkestanicum and that of other members of the Schistosomatidae available in Gen-Bank^™.

Results:

Phylogenetic relationships between them were re-constructed using the maximum parsimony method. Phylogenetic analyses done in present study placed O. turkestanicum within the Schistosoma genus, and indicates that O. turkestanicum was phylogenetically closer to the African schistosome group than to the Asian schistosome group.

Conclusion:

Comparison of nad1 and cox1 sequences of O. turkestanicum obtained in this study with corresponding sequences available in Genbank^™ revealed some sequence variations and provided evidence for presence of microvarients in Iran.     

Genetic Diversity of Human Blastocystis Isolates in Khorramabad,Central Iran

Background

There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.

Methods

This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used.

Results

Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.

Conclusion

The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.     

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.     

Still and Moving Image Evidences for Mating of Echinococcus granulosus Reared in Culture Media

Background

Echinococcus granulosus cultivation is very important for improvement of different aspect of medical and veterinary researches. Despite many advances in this case, there is a missing link for in vitro life cycle of adult worms and it is fertilization. Regarding the researchers’ observations, self-fertilization can be done in worms living in dog intestine, but despite all sorts of experimental techniques, this phenomenon has never been observed in reared worms in culture media. Furthermore, cross fertilization has not been observed in vitro and even in parasites with dog intestinal origin; although it theoretically is possible. During a follow-up of cultivated adult worms, evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in our lab which will be presented here.

Methods

Protoscoleces were aseptically removed from sheep hydatid cysts, washed twice with PBS and then cultivated in S.10E.H culture medium. The stages of parasite growth were observed using an inverted microscope for two months and all stages and behaviors were microscopically photographed. Different movies have also been made from these behavioral features.

Results

After around 55 days post cultivation, some evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in some of the mature adult worms. However, fertile eggs in these parasites have never been observed.

Conclusion

Regarding the above observations, these parasites show tendency to unsuccessful self-mating/fertilization (type 2) which failure could be due to anatomical position and physiological maturation. Also lack of suitable conditions for self-fertilization causes the worms try to do unsuccessful cross- mating/fertilization in culture media     

Vaccination of bovines against Echinococcus granulosus (cystic echinococcosis)

Hydatid disease is an important human zoonosis. Humans become infected from carnivores that are infected with the Echinococcus granulosus tapeworm. Carnivores become infected after consuming hydatid cysts from grazing animals, which are generally sheep, goats and cattle. A vaccine, known as EG95, can protect sheep and goats against cystic echinococcosis. This paper describes the adaptation of the EG95 vaccine for use in cattle. The monitoring of results used serology and also infection with E. granulosus eggs, followed by necropsy. Immunisation with living E. granulosus oncospheres showed that cattle could be immunised against E. granulosus. Immunisation of cattle with EG95 plus QuilA was also successful. A dose-response and adjuvant trial showed best results were achieved with 250 μg of antigen and 5mg of the adjuvant QuilA, which was 5 times the recommended sheep dose. After two vaccinations given one month apart, 90% protection was maintained for 12 months. At 12 months a third vaccination boosted protection to 99% which was maintained for a further 11 months.     

Molecular and Parasitological Study of Cryptosporidium Isolates From Cattle in Ilam,West of Iran

Background

Cryptosporidiosis is one of the most important parasitic infections in human and animals. This study was designed for survey on the prevalence of Cryptosporidium infection in farms of Ilam, west of Iran, using parasitology method and genotyping by Nested PCR-RFLP.

Methods

Fecal samples of 217 cattle were collected fresh and directly from the rectum of cattle. All of the samples were examined by microscopic observation after staining with modified Ziehl-Neelsen (MZN). Genomic DNA extracted by using EURx DNA kit. A Nested PCR-RFLP protocol amplifying 825 bp fragment of 18s rRNA gene conducted to differentiate species and genotyping of the isolates using SspI and VspI as restriction enzymes.

Results

The prevalence of Cryptosporidium infection in cattle using both methods is 3.68%. Most of the positive cattle were calves under six months. Species diagnosis carried out by digesting the secondary PCR product with SspI that C. parvum generated 3 visible bands of 448, 247 and 106 bp and digested by VspI restriction enzyme generated 2 visible bands of 628 and 104bp. In this investigation all of the positive samples were Cryptosporidium parvum.

Conclusion

C. parvum (bovine genotype) detected in all positive cattle samples in Ilam, west of Iran. The results of the present study can help for public health care systems to prevention and management of cryptosporidiosis in cattle and the assessment of cattle cryptosporidiosis as a reservoir for the human infection.     

Molecular Cloning and Expression of EG95 Gene of Iranian Isolates of Echinococcus granulosus

Background

Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein, against E. granulosus, has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector.

Methods

Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces, and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step, the subcloned gene was expressed in CHO as eukaryotic cell.

Results

EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot.

Conclusion

Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.     

Evaluation of Immunogenicity of Novel Isoform of EG95 (EG95-5G1) From Echinococcus granulosus in BALB/C Mice

Background

Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purification and evaluation immunogenicity of new construction of EG95 protein.

Methods

The recombinant plasmid pET32-a+ used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni–NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group) were immunized by injecting 20 μg rEG95 protein formulated in Freund’s and alum adjuvant.

Results

Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-γ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellular immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1.

Conclusion

Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses supporting further development of this vaccine candidate.     

Establishment of a Modified in Vitro Cultivation of Protoscoleces to Adult Echinococcus granulosus; an Important Way for New Investigations on Hydatidosis

Background

Echinococcus granulosus, a zoonotic cestode parasite, causative agent of hydatid cyst is endemic in many parts of the world including the Middle East. Study on different aspects of this parasite is very important and valuable. However, working with adult worms which their habitat situated in the small intestine of canids, is dangerous and risky. Achieving such risky situation needs a controlled condition which is cultivation of the organisms in the laboratory. In this regard, cultivation of E. granulosus protoscoleces leading to adult worms was established in the laboratory for the first time in Iran.

Methods

Under aseptic conditions a number of protoscoleces were cultivated in diphasic S.10E.H medium using CO2 incubator to produce adult worms.

Results

Different forms of parasites including pre-segmentation stages (PS1 - PS4) and segmentation stages (S5-S8) and developing stages in segmented worms (S10-S11) were observed and evaluated in these medium. Finally adult worms contained four proglottids with a large and distinct genital pore were observed 50-55 days post cultivation. These parasites do not produce fertile eggs and conclusively do not have risk of hydatid disease transmission to the researchers.

Conclusion

The mentioned method for producing E. granulosus adult worms can open a new window for researches and facilitate working on different aspects of hydatidosis especially for diagnosis, protection and treatment studies.     

Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody

Background:

Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method.

Methods:

Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA.

Results:

Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

Conclusion:

DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.     

Incidence and Genetic Characterization of Gongylonema pulchrum in Cattle Slaughtered in Mazandaran Province,Northern Iran

Background

The gullet worm, Gongylonema pulchrum Molin, 1857, is a thread-like spirurid nematode found in a variety of mammals worldwide. Its incidences in Iranian cattle of different breed or age have not been reported. The aims of the present study are to disclose the infection status of G. pulchrum in cattle slaughtered in northern region of Iran.

Methods

Full-length esophagi of cattle of 97 native dairy breed and 41 Holstein-Friesian breed were collected at four local abattoirs in Mazandaran Province, northern Iran, from March 2006 to August 2007, and were examined parasitologically. Eight overlapping segments of the small- and large-subunits of rDNA were amplified by PCR, and the obtained nucleotide sequences were characterized.

Results

The incidences of G. pulchrum in female and male native dairy breed were 38.9% and 24.0%, respectively, whereas those in female and male Holstein-Friesian breed were 4.2% and 0%, respectively. The first internal transcribed spacer (ITS1) region of G. pulchrum rDNA showed an intra-individual variation in the sequence and length, and the variation was ascribed to some unstable repeats of "A" or "CA".

Conclusion

Distinct incidences of G. pulchrum infection in native dairy breed and Holstein-Friesian breed might be ascribed to different animal husbandry manners for each breed in Iran; the former breed grazes freely in the pasture, but the latter breed is usually held in a pen. The rDNA sequence of Iranian G. pulchrum, obtained for the first time by us, might facilitate a reliable species identification of the parasite with a wide spectrum of morphological variations.     

Molecular Survey of Toxoplasma gondii in Sheep,Cattle and Meat Products in Chaharmahal va Bakhtiari Province,Southwest of Iran

Background

Toxoplasmosis is a worldwide spread disease. The present study examined the prevalence of Toxoplasma gondii infection among animals of edible meat (cattle and sheep) in Chaharmahal va Bakhtiari Province (Southwest of Iran) in 2012. Furthermore, we attempted for the first time to identify this parasite from the meat products in the province.

Methods

The tongue, brain, femur muscle and liver of 50 sheep and 70 cattle as well as 50 samples of meat products were selected and collected to perform molecular survey using Nested-PCR method.

Results

Of the studied sheep, 38% were infected. The infection rate in the age groups under 1 year, 1-2 years, and more than 2 years was 25%, 35.29% and 52.94%, respectively. The infection rate in femur muscle, brain, liver and tongue was 28%, 32%, 30% and 16%, respectively. Of the studied cattle, 8.57% were infected. The infection rate in the age groups 1-2 years, 2-4 years, and more than 4 years was 3.7%, 9.09% and 14.28%, respectively. Sheep was infected 6 times more than cattle (OR = 6.53 CI = 2.374-18.005).The infection rate among samples of meat products was 12% (6 samples out of 50 samples).

Conclusion

Due to the high rate of this parasitic infection among the slaughtered animals as well as meat products in this region, the use of infected material can be one of the main risk factors of transmission of the parasite to humans.     

Genotyping of Toxoplasma gondii Isolates from Soil Samples in Tehran,Iran

Background

The protozoan parasite Toxoplasma gondii can infect any warm blooded nucleated cells. One of the ways for human infection is ingestion of oocysts directly from soil or via infected fruits or vegetables. To survey the potential role of T. gondii oocyst in soil samples, the present study was conducted in Tehran City, Iran.

Methods

A total of 150 soil samples were collected around rubbish dumps, children''s play ground, parks and public places. Oocysts recovery was performed by sodium nitrate flotation method on soil samples. For molecular detection, PCR reaction targeting B1 gene was performed and then, the positive results were confirmed using repetitive 529 bp DNA fragment in other PCR reaction. Finally, the positive samples were genotyped at the SAG2 locus.

Results

Toxoplasma DNA was found in 13 soil samples. After genotyping and RFLP analysis in SAG2 locus, nine positive samples were revealed type III, one positive sample was type I whereas three samples revealed mixed infection (type, I & III).

Conclusion

The predominant genotype in Tehran soil samples is type III.     

Seroprevalence of Toxoplasma gondii in Sheep,Cattle and Horses in Urmia North-West of Iran

Background

Toxoplasma gondii is a zoonotic protozoan parasite found worldwide and responsible for major economic losses in most classes of livestock. This study was aimed to determine the prevalence of T. gondii infection in sheep, cattle and horses in Urmia, north-west of Iran, using MAT.

Methods

Blood samples of 276 livestock and 26 horses were collected from July 2009 till April 2010. The data were analyzed by the Chi-square, Fisher''s Exact and Cochran''s and Mantel-Haenszel Tests. The level of significance was set at P<0.05.

Results

Thirty-three (21.1%) sheep, 2 (1.6%) cattle and 3 (11.5%) horses were seropositive to T. gondii. Analysis showed that sheep were 15 times more likely to be seropositive comparing to cattle also 2 times more likely to be seropositive than horses.

Conclusion

This study showed seroprevalence of equine T. gondii infection with a considerable rate in sheep in Urmia, northwest of Iran. More comprehensive studies on livestock toxoplasmosis are required for further analysis of the parasite reservoir for human infection.     

Morphometric Analysis of Larval Rostellar Hooks in Taenia multiceps of Sheep in Iran and Its Association with Mitochondrial Gene Variability

Background

The purposes of the present study were morphometric characterization of rostellar hooks of Taenia multiceps and to investigate the association of hook length variation and the variability within two mitochondrial genes of sheep isolates of the parasite.

Methods

Up to 4500 sheep brains were examined for the presence of C. cerebralis. Biometric characters based on the larval rostellar hook size were measured for each individual isolate. Representative mitochondrial CO1 and 12S rRNA gene sequences for each of the isolates were obtained from NCBI GenBank. Morphometric and genetic data were analyzed using cluster analysis, Interclass Correlation Coefficient (ICC) and random effects model.

Results

One hundred and fourteen sheep (2.5%) were found infected with the coenuri. The minimum and maximum number of scoleces per cyst was 40 and 550 respectively. Each scolex contained 22–27 hooks arranged in two rows of large and small hooks. The average total length of the large and small hooks was 158.9 and 112.1 μm, respectively. Using ICC, statistically significant clusters of different hook sizes were identified within the isolates. The length of the large and small hooks was significantly associated with the variability in mitochondrial 12S rRNA gene.

Conclusion

Taenia multiceps, is a relatively important zoonotic infection in Iranian sheep with the prevalence rate of 2.5%. Hook length analysis revealed statistically significant difference among individual isolates. Associations between the rostellar hook length and variability in the mitochondrial 12S rRNA was documented.     

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran

Background

Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran.

Methods

Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced.

Results

A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates.

Conclusion

Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.     

Molecular Identification of Giardia duodenalis Isolates from Fars Province,Iran

Background

Giardia duodenalis is one of the most common human intestinal protozoan parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. The present study aimed to identify the prevalence of G. duodenalis isolates and determine the most common of its assemblages in the patients referring to health centers and hospitals in Fars province, Iran that will be subjected to further molecular investigation.

Methods

We collected 1000 human fecal samples from health centers and hospitals in Shiraz, Iran in a one year period from September 2009 to August 2010. Microscopic examination for the presence of G. duodenalis cysts and trophozoites was performed by direct wet mount before and after the concentration techniques. Extraction of DNA was performed by Phenol-Chloroform-Isoamylalcohol (PCI). G. duodenalis-positive specimens were analyzed by PCR. A fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the forward primer RH11 and the reverse primer RH4. Genotyping was performed using sequence analysis of G. duodenalis glutamate dehydrogenase gene using primers GDHeF, GDHiF, and GDHiR.

Results

The prevalence of Giardia infection was 10.7% (107/1000) examined based on microscopic examination. PCR identified 80% (40/50) of the samples as positive for G. duodenalis based on SSU-rDNA amplification on sucrose gradient samples. Besides, genotyping results indicated 32 isolates (80%) as assemblage AII and 8 isolates (20%) as assemblage BIII and BIV based on the DNA sequence analysis of the glutamate dehydrogenase locus of G. duodenalis.

Conclusion

The findings of this study emphasize that Iran (Fars Province) is a favorable area for giardiasis with an anthroponotic infection route.