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: mofaruk05@yahoo.com
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:
www.southampton.ac.uk)
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: www.cdc.gov)
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