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  1. 1. Molecular characterization and phylogenetic analysis of zoonotic cestode Echinococcus granulosus from buffalo, sheep and human isolates Mohammad Omer Faruka*, Mohammad Alamgir Hossaina, Sharmin Chowdhurya, Md. Fazal Karimb, Md. Masuduzzamana, AMAM. Zonaed Siddikia aDepartment of Pathology and Parasitology, Chittagong Veterinary and Animal Sciences University, Zakir Hossain Road, Khulshi, Chittagong 4225, Bangladesh bDepartment of Hepatology, Sir Salimullah Medical College and Mitford Hospital, Dhaka, Bangladesh. *Corresponding email: Materials and methods We are grateful to University Grant Commission of Bangladesh (UGC) to support this study under Higher Education Quality Enhancement Project (HEQEP) CP-3220. The research was conducted at the Molecular Pathology Lab of Chittagong Veterinary and Animal Sciences University (CVASU). Acknowledgements Results 1. Dinkel, A., Njoroge, EM., Zimmerman, A., Walz, M., Zeyhle, E., Almahdi, IE., Mackenstedt, U and Romig, T. 2004. A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa. International Journal for Parasitology 34: 645–653. 2. Nakao, M., Yanagida, T., Okamoto, M., Knapp, J., Nkouawa, A., Sako, Y., Ito, A. 2010. State-of-the-art Echinococcus and Taenia: phylogenetic taxonomy of human–pathogenic tapeworms and its application to molecular diagnosis. Infection, Genetics and Evolution 10 (4), 444–452. 3. Richard, KS., Riley, EM., Taylor, DH. and Morris, D.1988. Studies on the effect of short term high dose praziquantel treatment against protoscolices of ovine and equine Echinococcus granulosus within the cyst and in vitro. Tropical Medicine and Parasitology, 39 (4) :269-272. 4. Singha, bb., Sharmaa, JK., Ghataka, S., Sharmaa, R., Bal, MS., Tuli, A and Gill, JPS.2012. Molecular epidemiology of Echinococcosis from food producing animals in north India. Veterinary Parasitology 186 (2012) 503– 506. References © 2016 Property of Molecular Pathology Lab, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi-4225, Chittagong, Bangladesh. Abstract Echinococcosis is caused by the tapeworm of the genus Echinococcus. Of the 4 known species of Echinococcus, 3 are of zoonotic importance in humans. These are Echinococcus granulosus, causing cystic echinococcosis (CE); Echinococcus multilocularis, causing alveolar echinococcosis (AE); and Echinococcus vogeli. But E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10) [2]. Both cystic and alveolar echinococcosis can be transmitted to man through food. This is through contamination of food with parasite eggs and thus could occur where there is the possibility of contamination of food with dog or fox faeces. Fig.3 Schematic presentation of the structure of Hydatid cyst (Source: Live protoscolex Dead protoscolex Before staining After staining Conclusions and recommendations Fig.4. Agarose electrophoresis of PCR amplified partial mitochondrial 12S rRNA gene. The lanes M indicate marker, Lane-S1-S5 DNA samples from different cyst collected from buffalo, sheep and human. Collection of cysts from abattoirs located at Chittagong Metropolitan Area Separation of cyst and aspiration of cystic fluid Collection and preservation of protoscolex at kreb’s Ringer solution or Normal saline Multiple Cysts in sheep liver Multiple Cysts in bullock liver Determination of viability of protoscolex under light microscope and 0.1% Eosin stain [3] Steps for molecular study Genomic DNA extraction Conventional thermocyclerAgarose gel electrophoresis Sample Protoscolexor germinallayer Fig. 5. Agarose electrophoresis of the PCR-derived 434 bp amplicons of the hydatid cyst of E. granulosus mitochondrial Cytochrome oxidase-1 gene, Lanes: (M) 100 bp DNA ladder, lane2 cattle DNA, lane 3,4- buffalo DNA, Lane 5- cattle DNA and lane 6,7,8- goat DNA. Brood capsule Cyst Cyst Sample collection A total of 24 hydatid cysts of buffalo (n=15) and sheep (n=9) were collected from three different slaughterhouses located at Chittagong metropolitan area and 4 human samples were collected from human patients admitted at Sir Salimullah Medical College, Mitford Hospital, Dhaka, Bangladesh. PCR reaction mix a. Primers b. Gotaq Green master mix® c. Template DNA Species No. of samples Positive cases in PCR assay of 12S rRNA gene Positive cases in PCR assay of 12S rRNA gene Buffalo 15 9 3 Sheep 9 6 0 Human 4 2 0 Fig.6. Phylogenic tree of E. granulosus buffalo and sheep isolates of Chittagong, Bangladesh and reference sequences with complete mitochondrial G1sequence (AF297617). Isolates of this study were grouped into G1 strain Fig.8. Phylogenic tree of E. granulosus buffalo and sheep isolates of Chittagong, Bangladesh and reference sequences with complete mitochondrial G3 sequence (KJ559023). Isolates of this study were grouped into G3 strain. Buffalo originated cyst isolated sequences of this study were almost complete identity with complete mitochondrial G3 strain sequence china (KJ559023) and our neighboring Indian cattle and Buffalo reference sequences.300 200 100 254 bp M S1 S2 S3 S4 S5 M L1 L2 L3 L4 500 300 200 100 434 bp What is echinococcosis? Echinococcus granulosus causes cystic echinococcosis (CE) in humans and many domestic animals all over the world including Bangladesh. The aim of present molecular study was to identify genotypes of E. granulosus isolated from buffalo, sheep and human using Polymerase Chain Reaction (PCR) based tools. Two separate marker genes namely 12S rRNA gene and Cytochrome oxidase 1 (Cox-1) gene were used for differentiation of G1, G3 and G5 strains of E. granulosus. Twenty four hydatid cyst samples collected from 504 cases of buffalo and sheep from different slaughterhouses while 4 human samples were collected from human patients admitted at Sir Salimullah Medical College, Mitford Hospital, Dhaka, Bangladesh. Fertile and viable hydatid cysts were evaluated through microscopy. Germinal membrane and or protoscoleces were used for genomic DNA extraction for further PCR assay. E. granulosus G1 strain and G3 strain were successfully characterized by partial amplification of both the genes. Among buffalo samples, 9 out of 15 cases were diagnosed by amplification of partial 12S rRNA gene while only 3 cases were found positive in COX1 gene. With the samples collected from sheep, 6 out of 9 cases were positively detected by partial amplification of 12S rRNA gene. Among 4 human cyst samples two cysts were successfully amplified with 12S rRNA gene. Three buffalo and one sheep sequences have been submitted to GenBank. These sequences were aligned with reference sequence of NCBI resulting in identification of common sheep strain G1 and buffalo strain G3. Present study documented the presence of common sheep strain G1 and Buffalo strain G3 of E. granulosus in Bangladesh. This report is the first molecular level study of Echinococcus sp. in Bangladesh. Further whole genome analyses of different isolates are ongoing and will shed light on many interesting aspect of this important zoonotic pathogen to reveal their transmission dynamics and distribution patterns among human and other animals. Fig.7. Schematic presentation of the life cycle of E. granulosus which requires dog and other canids as definitive hosts and livestock as intermediate host to complete its life cycle. (Source: Table. 1. A total of 28 cysts isolated from animal and human were used for molecular characterization of Echinococcus granulosus. Two separate genes namely12SrRNA gene and Cytochrome oxidase 1 gene fragments were amplified by PCR using previously reported primer pairs [1,4] Live protoscolex Agarose gel image Buffalo and sheep cyst isolated sequences of this study were aligned with Nepalese woman (AB979277), Brazilian sheep and complete mitochondrial 12SrRNA gene of common sheep strain G1 of E. granulosus. E. ortleppi, human france (KJ624625) and E. vogeli (M84670) were drawn as outer group of this phylogenetic tree.  The DNA were successfully extracted and partial mitochondrial 12sRNA gene were amplified according to procedure described earlier [1] while cytochrome oxidase-1 gene were amplified according to separate protocol mentioned elsewhere [4]  Phylogenetic analysis of buffalo and sheep isolates revealed that E. granulosus common sheep strain G1 and buffalo strain G3 are circulating in Chittagong region of Bangladesh  For identification of Tasmanian sheep strain G2, E. equinus (G4), E. ortleppi (G5) and E. canadenesis (G6-G10) other gene marker likely NAD1, Cytochrome B and ITS1 can be used.  Control measures to prevent cystic echinococcosis should be aimed at preventing common sheep strain G1 due to their massive abundance both in human and livestock in Bangladesh Future planned activities  NCBI-GenBank submission of all DNA sequence of different isolates of Echinococcus spp found in human and animals in Bangladesh for wider public access to the novel datasets  Whole genome sequencing (WGS) of different isolates of Echinococcus spp of animal and human originated sample for comprehensive analyses of comparative genomics and transcriptomics  Development of Real Time PCR assay for rapid differentiation of different E. granulosus strains and their quantitative expression in different host species  Comparative proteomics study through SDS-PAGE using proteins extracted from germinal layers of cyst, scolex and protoscolex to identify the constituent proteins  Development of a database of candidate genes and proteins suitable for further analyses toward increasing our understanding of a possible vaccine development in the long run UV illuminator Fig. 1. Gross pathological study with examination of cyst viability Fig2. PCR assay steps with their instruments photograph