Sharifdini, Meysam
(Department of Parasitology and Mycology, School of Medicine, Guilan University of Medical Sciences)
,
Heidari, Zahra
(Department of Medical Microbiology, School of Medicine, Ardabil University of Medical Sciences)
,
Hesari, Zahra
(Department of Pharmaceutics, School of Pharmacy, Guilan University of Medical Sciences)
,
Vatandoost, Sajad
(Department of Animal Sciences, School of Biology, Faculty of Sciences, Tehran University)
,
Kia, Eshrat Beigom
(Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences)
The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) metho...
The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) method. PCR amplification of ITS2-rDNA region was performed, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 5.0 software. Six out of 7 isolates had high similarity with Trichostrongylus colubriformis, while the other one showed high homology with Trichostrongylus axei registered in GenBank reference sequences. Intra-specific variations within isolates of T. colubriformis and T. axei amounted to 0-1.8% and 0-0.6%, respectively. Trichostrongylus species obtained in the present study were in a cluster with the relevant reference sequences from previous studies. BLAST analysis indicated that there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human, sheep, and goat isolates from Iran and also human isolates from Laos, Thailand, and France. The ITS2 sequence of T. axei exhibited 99.4% homology with the human isolate of T. axei from Thailand, sheep isolates from New Zealand and Iran, and cattle isolate from USA.
The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) method. PCR amplification of ITS2-rDNA region was performed, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 5.0 software. Six out of 7 isolates had high similarity with Trichostrongylus colubriformis, while the other one showed high homology with Trichostrongylus axei registered in GenBank reference sequences. Intra-specific variations within isolates of T. colubriformis and T. axei amounted to 0-1.8% and 0-0.6%, respectively. Trichostrongylus species obtained in the present study were in a cluster with the relevant reference sequences from previous studies. BLAST analysis indicated that there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human, sheep, and goat isolates from Iran and also human isolates from Laos, Thailand, and France. The ITS2 sequence of T. axei exhibited 99.4% homology with the human isolate of T. axei from Thailand, sheep isolates from New Zealand and Iran, and cattle isolate from USA.
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문제 정의
This province has previously been known prevalent for Trichostrongylus species, both in domestic animals [4] and humans [4,15]. In this study, 7 human trichostrongyliasis cases were detected by formalin-ether concentration technique during a study on evaluation of molecular and parasitological methods for the diagnosis of strongyloidiasis in fecal samples [21]. They were 5 males and 2 females with ages ranging between 23 and 57 years old, residing in Mazandaran Province, northern Iran.
제안 방법
5% agarose gel. DNA sequencing was performed using an ABI 3130xl platform (Applied Biosystems, Foster City, California, USA). The sequence results were edited and analyzed by the Geneious software (www.
The PCR reactions were performed in a final reaction volume of 30 µl containing 15 μl of PCR mix which included 1.25 U Taq DNA polymerase, 200 μM of dNTPs, and 1.5 mM MgCl2 (2x Master Mix RED Ampliqon, Copenhagen, Denmark), 10 pmol of each primer, and 4 μl of DNA sample.
대상 데이터
DNA sequencing was performed using an ABI 3130xl platform (Applied Biosystems, Foster City, California, USA). The sequence results were edited and analyzed by the Geneious software (www.geneious.com) and compared with sequences deposited in GenBank by BLAST program (http://www.ncbi.nlm.nih.gov/). A phylogenetic tree was constructed using the maximum likelihood method based on the Tamura 3-parameter model, and pairwise comparisons were determined of the level of sequence differences within and among species using MEGA 5.
In this study, 7 human trichostrongyliasis cases were detected by formalin-ether concentration technique during a study on evaluation of molecular and parasitological methods for the diagnosis of strongyloidiasis in fecal samples [21]. They were 5 males and 2 females with ages ranging between 23 and 57 years old, residing in Mazandaran Province, northern Iran. For extraction of genomic DNA, fecal samples of the patients were kept in 70% ethanol at room temperature.
데이터처리
gov/). A phylogenetic tree was constructed using the maximum likelihood method based on the Tamura 3-parameter model, and pairwise comparisons were determined of the level of sequence differences within and among species using MEGA 5.0 software. Bootstrap analysis was done based on 1,000 replications.
성능/효과
Current phylogenetic analysis clarified the relation of human Trichostrongylus species from an endemic area of trichostrongyliasis in Iran and those of human and animal species of Trichostrongylus registered in GenBank. Based on pairwise comparisons, there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human and herbivores animals. High zoonotic capacity of this species is probably one of the main reasons of current higher occurrence of human trichostrongyliasis in the study area than that of human hookworms which are not zoonotic.
0%. Based on these variations, 21 isolates of T. colubriformis and 5 isolates of T. axei were classified into 5 and 2 haplotypes for ITS-2, respectively (Fig. 3). Trichostrongylus species of the present study were in a cluster with the relevant reference sequences from previous studies (Fig.
All samples were successfully presented amplification of about 328 bp for the ITS2 gene. Comparisons of the sequences from these isolates with other available reference sequences in GenBank, using BLAST system, revealed that 6 isolates had high similarity (more than 95%) with T. colubriformis, and the other one had high homology with T. axei. These sequences were deposited in GenBank database (accession nos.
colubriformis from sheep (EF427624 and KC998728), goat (KF204576), and cattle (KP150536) in other countries. For these sequences, 99.4% similarities were obtained with T. colubriformis isolates from sheep in Ireland (JF680985), sheep in New Zealand (KC998729) and goat in Malaysia (KF204577). T.
In this study, in spite of the availability of few human Trichostrongylus samples (n= 7), using ITS2 sequence analysis, 6 of them were determined as T. colubriformis, and the other one as T. axei. This result is compatible with the result of a recent molecular study on human trichostrongyliasis in Mazandaran Province, in which T.
colubriformis isolates from sheep in Ireland (JF680985), sheep in New Zealand (KC998729) and goat in Malaysia (KF204577). T. colubriformis sequences of this study presented 98.8% homology with the sequences of T. colubriformis from sheep in New Zealand (KC998730).
axei was also found in humans in Thailand [3] and Iran [15] by molecular techniques. The ITS2 sequence of T. axei in this study exhibited 99.4% homology with the human isolate of T. axei from Thailand (KC337066), sheep isolate from New Zealand (KC998727), sheep isolate from Iran (KJ755059), and cattle isolate from USA (KP150521). T.
후속연구
To sum up, T. colubriformis was found to be the most probable dominant human species in the study area, but further investigation with higher sample size is recommended. This species is also the most possible cause of human trichostrongyliasis infection in Laos [14], Thailand [3], and France [7].
참고문헌 (34)
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