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Molecular Typing of Eimeria Ahsata and E. Crandallis Isolated from Slaughterhouse Wastewater

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Molecular Typing of Eimeria Ahsata and E. Crandallis Isolated from Slaughterhouse Wastewater Jundishapur J Microbiol. 2016 April; 9(4):e34140. doi: 10.5812/jjm.34140. Published online 2016 April 23. Letter Molecular Typing of Eimeria ahsata and E. crandallis Isolated From Slaughterhouse Wastewater Kareem Hatam Nahavandi,1 Amir Hossein Mahvi,2 Mehdi Mohebali,1,3 Hossein Keshavarz,1 Sasan Rezaei,1 Hamed Mirjalali,4,5 Samira Elikaei,1 and Mostafa Rezaeian1,* 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran 2Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran 3Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, IR Iran 4Gastroenterology and Liver Disease Research Center, Research institute for Gastroentrology and Liver Diseases, Shahid Beheshti University of Medical Sciences (SBUMS), Tehran, IR Iran 5Foodborne and Waterborne Diseases Research Center, Research institute for Gastroentrology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran *Corresponding author: Mostafa Rezaeian, Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran. Tel: +98-2188973901, E-mail: [email protected] Received 2015 October 28; Revised 2016 February 02; Accepted 2016 February 07. Keywords: 18S rRNA Gene, Iran, Wastewater, Eimeria ahsata, Eimeria crandallis Dear Editor, and DNA sequence variations of E. crandallis and E. ahsata compared with other Eimeria that exist in the GenBank The Eimeria species are host-specific protozoan para- database. Therefore, the present study was undertaken to sites that cause the disease known as coccidiosis in a va- identify genetic characteristics of E. ahsata and E. crandallis riety of animals including cattle, sheep, goats, pigs and in slaughterhouse wastewater samples. poultry throughout the world (1). These parasites invade epithelial tissues of the intestine, causing severe damage Twenty-four grab samples of untreated wastewater in the host and result in significant economic losses (2). (5 L each) were collected from the two slaughterhouse At least twelve species of bovine Eimeria, ten species of treatment plants located in the southwest suburban area ovine Eimeria and nine species of caprine Eimeria occur of Tehran, Iran. The animals slaughtered in these two in Iran (3-5). Eimeria ahsata and E. crandallis are two of a slaughterhouses included cattle, sheep and goats. Wa- group of ovine coccidia of what might be called the ar- ter samples were concentrated by the centrifugal (water- loingi type (6). Conventionally, microscopic examination ether) concentration procedure, as previously described of Eimerian oocytes is the only practical method to dis- (12). Oocytes were sporulated in a 2% (w/v) potassium criminate species of Eimeria (2,7,8). However, the partic- dichromate (K2Cr2O7) solution at room temperature (25°C) ular morphology of a given species may vary considerably. for three weeks. The sporulated oocytes were partially Accordingly, microscopic differentiation is not reliable be- identified as E. ahsata and E. crandallis based on the mor- cause several species have confusing features, along with phology of oocytes at × 40 and × 1,000 magnifications the presence of intraspecies variation (7). Therefore, mor- (13). DNA was extracted using an AccuPrep® stool DNA phological observations should not be used as isolated cri- extraction kit (Bioneer Corporation, Daejeon, South Ko- terion for differentiation of species (9). rea) according to the manufacturer’s protocol. An approx- imately 636-bp fragment of DNA extract was PCR amplified Thus, molecular assays have proven useful for the iden- on the basis of the 18S rRNA gene, as previously described tification of Eimeria spp. to overcome the limitations (14). All PCR amplicons were sequenced in both directions of these traditional procedures (7, 10). However, hema- on an automated DNA analyzer (ABI 3730 XL, Bioneer, South tological and biochemical changes in blood serum, and Korea). histopathological lesions of ovine coccidiosis in sheep and lambs naturally infected by E. crandallis and E. ahsata were Unique nucleotide sequences described in this work mentioned in previous studies (11), but no information were deposited in GenBank and accession numbers is available about the genetic characterization and phylo- KP893746, KP893747, KP893748 and KP893749 were pro- genic positions of E. crandallis and E. ahsata. In the present vided for Iranian E. ahsata and E. crandallis sequences of study, the 18S rRNA gene was employed as a molecular 18S rRNA. genetic approach to investigate the phylogenetic analysis Sporulated oocytes of E. ahsata and E. crandallis were Copyright © 2016, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Hatam Nahavandi K et al. identified in all wastewater samples three weeks after their study, the microscopic examination resulted in positive re- storage at 25°C. All samples that were found to be positive sults for all water samples from both slaughterhouse treat- by microscopic evaluation were confirmed to be Eimeria ment plants. The presence of positive samples for E. ahsata by PCR. BLAST search of our 18S rRNA sequences against and E. crandallis was confirmed by PCR assay. those previously published for other Eimeria spp. revealed In the present study, 18S rRNA sequences of E. crandallis the highest similarity (99% - 100% homology) with E. ah- and E. ahsata had considerable homology with E. arloingi, sata and E. crandallis, with differences observed at 2 - 4 nu- E. bovis and E. zuernii (98% - 99%). Similarly, Khodakaram- cleotides. Genetic distances among Eimeria species were Tafti et al. (4) reported a close relationship between E. ar- in the range of 0.002 to 1.264 nucleotide substitutions per loingi and bovine (E. bovis and E. zuernii) and ovine (E. ah- 100 bp. There was a minor difference in the sequence of sata and E. crandallis) coccidia, based on the 18S sequence 18S rRNA gene in E. ahsata and E. crandallis with E. arloingi analyses. It has also been shown that there is a similar- available in GenBank. Phylogenetic analysis showed that ity between these two species (E. crandallis and E. ahsata) one clade contained E. ahsata, E. crandallis and E. arloingi and E. arloingi, based on microscopic evaluation of their (the most pathogenic Eimeria in goats), and E. ahsata and sporulated oocytes. Identically, E. ahsata and E. crandallis in E. crandallis grouped together in one clade (Figure 1). sheep, E. arloingi in goats and E. bovis and E. zuernii in cattle are highly pathogenic and formed a monophyletic group Figure 1. Phylogenetic Tree Based on 18S rRNA Sequences, Constructed According in the position away from other members in spite of many to the NJ Method, Showing the Position of E. ahsata, E. crandallis and Other Eimeria different biological characteristics and the histopatholog- Species Isosporasuis is Used as an Outgroup ical lesions. The phylogram based on the 18S rRNA se- quences showed that one clade contained E. ahsata, E. cran- 64 Eimeria Brunetti |U67116| 68 Eimeria Maxima |U67117| dallis and E. arloingi, and E. ahsata and E. crandallis grouped 44 Eimeria Acervulina |U67115| together in one clade. Similarly, Bush (16) reported a ten- 53 Eimeria Mitis |U67118| Eimeria Tenella |U67121| dency provided by the phylogenetic analysis of avian Eime- 37 99 Eimeria Necatrix |U67119| ria for E. necatrix and E. tenella, the most pathogenic Eimeria Eimeria Meleagrimitis |AF041437| Cyclospora Cayetanensis |AF111183| 15 58 in chicken. 93 Cyclospora Cercopitheci |AF111184| While the 18S rRNA sequence may not eventually prove 69 Cyclospora Papionis |AF111187| 6 Eimeria Pilarensis |AF324215| to be useful to examine in greater detail the magnitude of 71 Eimeria Separata |AF311643| population variation of a single Eimeria species, it can be Eimeria Langebarteli |AF311640| 2 86 Eimeria Nieschulzi |U40263| used to identify Eimerian oocytes in environmental sam- 85 Eimeria Falciformis |AF080614| 15 ples. 61 Eimeria Sevilletensis |AF311644| Eimeria Catronensis |AF324213| 35 Eimeria Alabamensis |AF291427| Eimeria Hessei |KC333453| Acknowledgments 58 Eimeria Dipodomysis |AF339490| 65 Eimeria Zuernii |AB769665| 86 Eimeria Ellipsoidalis |AB769634| The authors thank Fatemeh Tarighi and Tahereh Reza- Eimeria Bovis |U77084| eian for technical assistance. 98 Eimeria Arloingi |KC507792| Eimeria Ahsata |KP893747| 76 Eimeria Crandallis |KP893746| 50 Eimeria Crandallis |KP893748| Footnotes Eimeria Crandallis |KP893749| Eimeria Gruis |AB194382| Authors’ Contribution: The overall implementation of 99 Eimeria Reichenowi |AB194379| Isospora Suis |U97523| this study was the results of efforts of all authors. All au- 0.02 thors read and approved the final manuscript. Funding/Support: This work was supported by the insti- The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. tute for environmental research (IER) and center for re- search of endemic parasites of Iran (CREPI), Tehran Univer- sity of Medical Sciences (TUMS), Iran (grant no. 92-02-160- Prior to this
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