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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
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Short communication
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Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India
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Monika Sharma a, Kangjam Rekha Devi b, Rakesh Sehgal a, Kanwar Narain b, Jagadish Mahanta b, Nancy Malla a,⇑ a b
Department of Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India Regional Medical Research Center (ICMR), Dibrugarh 786 001, Assam, India
a r t i c l e
i n f o
Article history: Received 24 June 2013 Received in revised form 18 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: Taenia solium Cysticerci Sequencing Mitochondrial cox1 gene India
a b s t r a c t Taenia solium taeniasis/cysticercosis is a major public health problem in developing countries. This study reports genotypic analysis of T. solium cysticerci collected from two different endemic areas of North (Chandigarh) and North East India (Didrugarh) by the sequencing of mitochondrial cvtochrome c oxidase subunit 1 (cox1) gene. The variation in cox1 sequences of samples collected from these two different geographical regions located at a distance of 2585 km was minimal. Alignment of the nucleotide sequences with different species of Taenia showed the similarity with Asian genotype of T. solium. Among 50 isolates, 6 variant nucleotide positions (0.37% of total length) were detected. These results suggest that population in these geographical areas are homogenous. Ó 2014 Published by Elsevier B.V.
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1. Introduction
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Taenia solium, a neglected zoonotic parasite is endemic in underdeveloped countries where pig raising and pork consumption are not restricted (Dorny et al., 2009). It is a cause of serious concern due to increase in morbidity and considerable economic loss through wastage of infected organs. Taeniasis is a disease of human intestinal tract caused by the adult tapeworm. The infection is acquired by the ingestion of undercooked pork contaminated with cysticerci. After ingestion, cysticerci migrate to the intestine, where it develops into an adult tapeworm and release proglottids filled with eggs which are passed in the stools. The ingestion of eggs by the intermediate host (pigs and human) results in the development of cysts in the soft tissues which progress to cysticercosis. Intestinal taeniasis has only mild clinical manifestations and may remain asymptomatic. Human cysticercosis is caused when man becomes an accidental intermediate host either by ingestion of T. solium eggs or by autoinfection. The larval stage often localizes in subcutaneous tissue, skeletal muscles, central nervous system (CNS) and eye. Neurocysticercosis (NCC), the most severe form of cysticercosis results from the presence of cysticerci in the central
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⇑ Corresponding author. Tel.: +91 172 27555169; fax: +91 172 2744401. E-mail addresses: [email protected] (M. Sharma), krekha75@yahoo. co.in (K.R. Devi), [email protected] (R. Sehgal), [email protected] (K. Narain), [email protected] (J. Mahanta), [email protected] (N. Malla).
nervous system. It is a major cause of adult acquired epilepsy and other neurological morbidity in many areas of the world (Nash and Garcia, 2011). It is estimated that approximately 50 million Q3 people are infected with taeniasis/cysticercosis and 50,000 deaths occur from cysticercosis annually, worldwide (CDC, 1993; Hoberg, 2002; Eddi et al., 2003). In India, taeniasis has a prevalence of 2–38% and 8.7–50% patients presenting with recent onset of seizure had NCC (Rajshekhar, 2004). In a seroprevalence study in and around Chandigarh, anti-cysticercus antibodies were found in 17.3% subjects with highest prevalence (24%) reported form slum areas, however only 8% of seropositive had history of epilepsy, suggestive of neurocysticercosis (Khurana et al., 2006). A hospital based clinico-serological study from Dibrugarh, Assam showed the seroprevalence of 78.43% in patients with ring enhancing lesion in Computed Tomography (CT) scan of brain suggestive of neurocysticercosis in this area (Kotokey et al., 2006). The prevalence of porcine cysticercosis varies from 7–26% in north and south India (Rajshekhar, 2004). Knowledge of the genetic structure of cestode parasites can be applied to the epidemiology and control of these parasites, because genetic variants may differ in their infectivity and pathogenicity (Thompson and Lymbery, 1995). Mitochondrial DNA analysis of T. solium worldwide revealed that T. solium could be divided into two genotypes, Asian and American genotypes (Nakao et al., 2002). Clinical manifestations in human cysticercosis appear to be well correlated with the genotypes of T. solium (Campbell et al., 2006). In Asian countries, majority of the patients with
1567-1348/$ - see front matter Ó 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.meegid.2013.12.010
Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
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neurocysticercosis are presented with a single enhancement brain lesion and a very few patients have massive infections with multiple cysts (Singhi, 2011). In Latin America, the most common presentation of NCC is multiple cysts without signs of inflammation. Subcutaneous cysticercosis (SCC) is rare in Latin America (Cruz et al., 1994) but very common in Asia (Garcia et al., 2003). A possible reason for the clinical difference in different geographical areas might correlate with the variation in genotypes of T. solium, although other factors such as nutritional status and ethnic difference of patients cannot be ignored. Molecular technique based on Polymerase chain reaction (PCR) has been used for the differential diagnosis of species and strains and to gain knowledge of genetic diversity in the parasitic population (Brouwer et al., 2001; Eom et al., 2002; Kral’ova, 1996; Siles-Lucas et al., 1993). Mitochondrial sequences are widely used in molecular biology as genetic markers for ecological, phylognetic and evolutionary studies. Genetic variations provide the genetic material for natural selection leading to allele fixation within population and speciation. Within cox1, differential nucleotides are dispersed over the entire length and served as diagnostic marker for human taenid cestodes i.e. T. solium, Taenia saginata and Taenia asiatica or differentiation of 2 genotypes of Taenia i.e. Asian genotype and American/African genotype (Yamasaki et al., 2004). Martinez-Hernandez et al., 2009 have analyzed the different mitochondrial gene (cox1, cyt b and nad) and nuclear genome (5.8S+ITS1+18S, LMWA1 and LMWA2) sequences and it was found that most of polymorphic sites were present in cox1 gene. So in the present study, genetic analysis of isolates of T. solium cysticerci from 2 different endemic regions of India i.e. Chandigarh located in North India and Dibrugarh in North East India which are 2585 km apart was carried by comparing sequences of mitochondrial gene: cytochrome c oxidase subunit 1 (cox1).
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2. Materials and methods
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T. solium cysticerci were collected from 50 freshly slaughtered and heavily infected pigs from slaughter houses located in Chandigarh (n = 25) and Dibrugarh (n = 25) respectively. The pigs were raised in widely different geographical areas in North and North East India and thus were considered from different pig breeds. The carcass containing cysts were transported to the department of Parasitology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh and Regional Medical Research Center, Dibrugarh, respectively for collection of cysticerci. The cysticerci were separated, washed with distilled water and examined under the microscope for morphological confirmation. These were fixed in 95% ethanol and stored at 20 °C till further use. Cyst from each animal was considered as an isolate. The cysts collected at Dibrugarh were transported to Chandigarh under refrigerated condition for further processing.
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2.1. Molecular analysis
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DNA extraction: The cyst samples were washed thrice in PBS to remove ethanol and genomic DNA was extracted from each sample by QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. For molecular identification, PCR amplification of cox1 gene was performed by using the primers and PCR conditions as described previously (Nakao et al., 2002) with minor changes. Briefly, PCR was carried out in a 50 ll reaction mixture containing 2 ll DNA, 0.2 mM premixed solution of dNTP, 10 pmol of each primer, 1x PCR buffer, and 1 U of TaqDNA polymerase (Bangalore Genei). Amplification program included an initial denaturation step of 95 °C for 5 min and 30 cycles each of denaturation (95 °C for
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30 s), annealing (56 °C for 30 s), extension (72 °C for 90 s) and final extension of 72 °C for 10 min. After agarose gel electrophoresis (1%), PCR products were purified and sequenced.
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2.2. Phylogenetic analysis
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Previously published sequences of Taenia species retrieved from the National Center for Biotechnology (http://www.ncbi.nlm.nih. gov) were used as the reference sequences (Fig. 1). Nucleotide sequence analysis was performed with BLAST sequence algorithms and sequences were aligned using Clustal W (Thompson et al., 1994). T. saginata and T. asiatica sequences were used as outgroup The genetic distance was calculated by using Kimura two-parameter distance estimates and samples were clustered using the Neighbour joining algorithm using Mega 4 software. Bootstrap analysis was performed using 1000 replicates.
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3. Results
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T. solium cysticerci were identified by the partial sequencing of cox1 gene. The size of amplified PCR product was 1800 bp. For analysis nucleotide sequences of 1620 bp from all these isolates were aligned with reference sequences of other taenid cestodes i.e. T. saginata, T. asiatica and Asian and American/African genotype of T. solium (Fig. 1). In this study, single nucleotide polymorphism was found and no addition or deletions were detected. Among 50 North and North East Indian isolates, 4 haplotypes were found. These haplotypes were differed at 6 variant nucleotide positions (0.37% of total length). Among 25 North Indian isolates, three haplotypes (Accession number KC709806, n = 20; KC709808, n = 2 and KC709809, n = 3) were found and these showed polymorphism at nucleotide positions 52 (G–A), 291 (A–G) and 524 (C–T). The predominant haplotype reported in this study with accession number KC709806 showed 100% homology with cox1 gene sequence of T. solium cysticercus from India, Nepal, Japan, Madagascar (AB066489, AB491986, AB516957, AB781355, respectively), 99.9% homology with Madagascar (AB781357), 99.8% homology with China, Thiland, Korea, Nepal, Madagascar (AB066485/GQ402327, AB066487, DQ089663, AB491985, AB781359, respectively), 99.5% homology with Indonesia: Irian Jaya (AB066488), 99% homology with Tanzania (AB066493) and 98.8% homology with Brazil, Cameroon and Mexico and Madagascar (AB066492, FN995665, FN995660 and AB781361, respectively). Isolates in this haplotype showed 99.8% homology to North East Indian isolates Q4 (KC7098011). The nucleotide sequence of all 25 North East Indian isolates (Accession number KC709811) showed 100% homology with each other and differed from North Indian isolates at nucleotide positions 366(C–T), 1041 (A–G) and 1164 (C–T). Interestingly, these isolates like American and African genotype has ‘T’ at position 1164 and showed 99.8% homology with cox1 gene sequence of T. solium cysticercus from India (India, Nepal, Japan, Madagascar (AB066489, AB491986, AB516957, AB781355, respectively), 99.7% homology with Madagascar (AB781357), 99.6% homology with China, Thiland, Korea, Nepal, Madagascar (AB066485/GQ402327, AB066487, DQ089663, AB491985, AB781359, respectively), 99.3% homology with Indonesia:Irian Jaya (AB066488), 99% homology with Tanzania (AB066493), 98.8% homology with Madagascar (AB781361) 98.9% homology with Mexico (FN995660) and 98.7% homology with Brazil, Cameroon (AB066492, FN995665), respectively. Further in dendrogram analysis, all the isolates were clustered with Asian genotype of T. solium (Fig. 1). All the North Indian
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Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
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Fig. 1. Dendrogram constructed with the cox1 mitochondrial gene sequence obtained from human taeniid cestodes. The sequences were compared with sequence of reference genotypes. Scale in the tree represents similarity indices. All red coloured branches represent the Asian genotype of Taenia solium and Blue coloured branches represent African/American genotype. Pink and Green coloured branches represent the outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 209 210
isolates were clustered in one clade and all the North East Indian isolates clustered together and represented the same clone.
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4. Discussion
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Taeniasis and cysticercosis is endemic in many parts of India and several factors including cultural, socioeconomical, agricultural and environmental contribute to the disease burden (Prasad et al., 2008). In addition to this, lack of education and knowledge about the life cycle of the parasite, as well as the lack of legislation for meat inspection and offal disposal at local abattoirs, contributes to the transmission. Planning of control strategies for the prevention of disease depend on multiple parameters and the genotyping of isolates of T. solium may be one of the parameter in the formulation of control strategies for the prevention of transmission of this parasite. The genetic diversity of T. solium isolates collected from Peru, Colombia, Mexico, India, China, and Philippine has been studied by the sequencing of cox1, ITS1 and diagnostic antigen Ts14. The nucleotide variation was found to be minimal (Hancock et al., 2001). All 15 isolates from Peru had identical cox1 sequences. Furthermore, a clonal structure and local lineages with probable
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events of genetic recombination without genetic flow within them was shown by Random amplified polymorphic DNA (RAPD) in T. solium cysticerci recovered from naturally infected pigs from Mexico, Honduras and Africa (Maravilla et al., 2003; Vega et al., 2003). In the present study, T. solium isolates were collected from 2 distant endemic geographical areas of India to find out the genetic variation by sequencing of cox1 gene. All the isolates were clustered with other Asian genotypes. These results are in agreement with the findings of previous study (Nakao et al., 2002), where the sequencing of cox1 and cyt b gene of 13 pig isolates of T. solium from various regions of Asia (China, India, Irian Jaya and Thailand), Africa (Cameroon, Mozambique and Tanzania) and Latin America (Bolivia, Brazil, Ecuador, Mexico and Peru) have showed that all the T. solium isolates from Asian countries should be classified as Asian genotype and all the isolates from American and African countries should be classified together as American/African genotype. Among 25 North Indian isolates 3 halplotypes differed at positions 52, 291 and 524 were found, whereas in North East Indian isolates no nucleotide variation was observed. Among these two populations 6 variant nucleotide positions (0.37% of total length) were detected but these populations when compared with the reference sequence of Africa and America showed 20 variants
Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
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nucleotide positions. In another study within cox1 gene, 28 variant nucleotide positions (1.7% of total length) were reported among the 13 isolates collected from different region of Asia, America and Africa (Nakao et al., 2002). These results are expected as T. solium is a hermaphrodite organism capable of self fertilization, which is responsible for low genetic variation and increased genetic differentiation among populations (Campbell et al., 2006). In this study some sequences from Indian isolates are found close to Madagascar (Accession number AB781355, AB781357 and AB781359) as % of homology varies from 100% to 99.6% and they were grouped in Asian isolate. However, the other sequence from Madagascar with accession number AB781361 was found to be clustered with American and other African sequences. These results in the present study are in concordant with earlier study from Madagascar, where 11/13 nucleotide sequences of T. solium from Madagascar were found to be clusred with Asian genotype and two were found to be clustered with African/American genotype (Michelet et al., 2010). The findings suggest that the isolates of T. solium from Madagascar have originated from two different geographical areas (Michelet and Dauga, 2012). Interestingly in our study, sequence of T. solium cysticerci isolate obtained after surgical resections from ocular cysticercosis patient from Chandigarh, India with ‘Accession no KC709810’ also showed 100% similarity with other North Indian isolates extracted from muscles of pig (Personal communication). These results suggest the genetic similarity between cysticerci from pig and human in North India. This observation is in concordant to the earlier study from Indonesia and Mexico, in which nucleotide sequence from cysticerci from human eye and brain were almost identical to those obtained from pigs, indicating that the cysticerci of T. solium from pigs and human are genetically very close (Hinojosa-Juarez et al., 2008). To the best of our knowledge, this study is the first report of genotyping of large number of Indian animal isolates of T. solium cysticerci from two distant geographical areas. In conclusion, the result of the present study demonstrated the very low genetic variation in the cox 1 gene of cysticerci of T. solium isolates collected from two distant geographical areas and all the sequences were well clustered with the published sequences of Asian genotypes of T. solium. Information regarding genetic variation in T. solium is limited. This study on large number of samples may have provided preliminary information to design future goals which may have important implications in the planning of control strategies for the prevention of disease.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2013.12. 010.
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Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
No. of Pages 4, Model 5G
3 January 2014 Infection, Genetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
2
Short communication
6 4 7
Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India
5 8 9 10 11 13 12 14 1 2 6 9 17 18 19 20 21 22 23 24 25 26 27 28
Q1
Monika Sharma a, Kangjam Rekha Devi b, Rakesh Sehgal a, Kanwar Narain b, Jagadish Mahanta b, Nancy Malla a,⇑ a b
Department of Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India Regional Medical Research Center (ICMR), Dibrugarh 786 001, Assam, India
a r t i c l e
i n f o
Article history: Received 24 June 2013 Received in revised form 18 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: Taenia solium Cysticerci Sequencing Mitochondrial cox1 gene India
a b s t r a c t Taenia solium taeniasis/cysticercosis is a major public health problem in developing countries. This study reports genotypic analysis of T. solium cysticerci collected from two different endemic areas of North (Chandigarh) and North East India (Didrugarh) by the sequencing of mitochondrial cvtochrome c oxidase subunit 1 (cox1) gene. The variation in cox1 sequences of samples collected from these two different geographical regions located at a distance of 2585 km was minimal. Alignment of the nucleotide sequences with different species of Taenia showed the similarity with Asian genotype of T. solium. Among 50 isolates, 6 variant nucleotide positions (0.37% of total length) were detected. These results suggest that population in these geographical areas are homogenous. Ó 2014 Published by Elsevier B.V.
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1. Introduction
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Taenia solium, a neglected zoonotic parasite is endemic in underdeveloped countries where pig raising and pork consumption are not restricted (Dorny et al., 2009). It is a cause of serious concern due to increase in morbidity and considerable economic loss through wastage of infected organs. Taeniasis is a disease of human intestinal tract caused by the adult tapeworm. The infection is acquired by the ingestion of undercooked pork contaminated with cysticerci. After ingestion, cysticerci migrate to the intestine, where it develops into an adult tapeworm and release proglottids filled with eggs which are passed in the stools. The ingestion of eggs by the intermediate host (pigs and human) results in the development of cysts in the soft tissues which progress to cysticercosis. Intestinal taeniasis has only mild clinical manifestations and may remain asymptomatic. Human cysticercosis is caused when man becomes an accidental intermediate host either by ingestion of T. solium eggs or by autoinfection. The larval stage often localizes in subcutaneous tissue, skeletal muscles, central nervous system (CNS) and eye. Neurocysticercosis (NCC), the most severe form of cysticercosis results from the presence of cysticerci in the central
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⇑ Corresponding author. Tel.: +91 172 27555169; fax: +91 172 2744401. E-mail addresses: [email protected] (M. Sharma), krekha75@yahoo. co.in (K.R. Devi), [email protected] (R. Sehgal), [email protected] (K. Narain), [email protected] (J. Mahanta), [email protected] (N. Malla).
nervous system. It is a major cause of adult acquired epilepsy and other neurological morbidity in many areas of the world (Nash and Garcia, 2011). It is estimated that approximately 50 million Q3 people are infected with taeniasis/cysticercosis and 50,000 deaths occur from cysticercosis annually, worldwide (CDC, 1993; Hoberg, 2002; Eddi et al., 2003). In India, taeniasis has a prevalence of 2–38% and 8.7–50% patients presenting with recent onset of seizure had NCC (Rajshekhar, 2004). In a seroprevalence study in and around Chandigarh, anti-cysticercus antibodies were found in 17.3% subjects with highest prevalence (24%) reported form slum areas, however only 8% of seropositive had history of epilepsy, suggestive of neurocysticercosis (Khurana et al., 2006). A hospital based clinico-serological study from Dibrugarh, Assam showed the seroprevalence of 78.43% in patients with ring enhancing lesion in Computed Tomography (CT) scan of brain suggestive of neurocysticercosis in this area (Kotokey et al., 2006). The prevalence of porcine cysticercosis varies from 7–26% in north and south India (Rajshekhar, 2004). Knowledge of the genetic structure of cestode parasites can be applied to the epidemiology and control of these parasites, because genetic variants may differ in their infectivity and pathogenicity (Thompson and Lymbery, 1995). Mitochondrial DNA analysis of T. solium worldwide revealed that T. solium could be divided into two genotypes, Asian and American genotypes (Nakao et al., 2002). Clinical manifestations in human cysticercosis appear to be well correlated with the genotypes of T. solium (Campbell et al., 2006). In Asian countries, majority of the patients with
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Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
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neurocysticercosis are presented with a single enhancement brain lesion and a very few patients have massive infections with multiple cysts (Singhi, 2011). In Latin America, the most common presentation of NCC is multiple cysts without signs of inflammation. Subcutaneous cysticercosis (SCC) is rare in Latin America (Cruz et al., 1994) but very common in Asia (Garcia et al., 2003). A possible reason for the clinical difference in different geographical areas might correlate with the variation in genotypes of T. solium, although other factors such as nutritional status and ethnic difference of patients cannot be ignored. Molecular technique based on Polymerase chain reaction (PCR) has been used for the differential diagnosis of species and strains and to gain knowledge of genetic diversity in the parasitic population (Brouwer et al., 2001; Eom et al., 2002; Kral’ova, 1996; Siles-Lucas et al., 1993). Mitochondrial sequences are widely used in molecular biology as genetic markers for ecological, phylognetic and evolutionary studies. Genetic variations provide the genetic material for natural selection leading to allele fixation within population and speciation. Within cox1, differential nucleotides are dispersed over the entire length and served as diagnostic marker for human taenid cestodes i.e. T. solium, Taenia saginata and Taenia asiatica or differentiation of 2 genotypes of Taenia i.e. Asian genotype and American/African genotype (Yamasaki et al., 2004). Martinez-Hernandez et al., 2009 have analyzed the different mitochondrial gene (cox1, cyt b and nad) and nuclear genome (5.8S+ITS1+18S, LMWA1 and LMWA2) sequences and it was found that most of polymorphic sites were present in cox1 gene. So in the present study, genetic analysis of isolates of T. solium cysticerci from 2 different endemic regions of India i.e. Chandigarh located in North India and Dibrugarh in North East India which are 2585 km apart was carried by comparing sequences of mitochondrial gene: cytochrome c oxidase subunit 1 (cox1).
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2. Materials and methods
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T. solium cysticerci were collected from 50 freshly slaughtered and heavily infected pigs from slaughter houses located in Chandigarh (n = 25) and Dibrugarh (n = 25) respectively. The pigs were raised in widely different geographical areas in North and North East India and thus were considered from different pig breeds. The carcass containing cysts were transported to the department of Parasitology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh and Regional Medical Research Center, Dibrugarh, respectively for collection of cysticerci. The cysticerci were separated, washed with distilled water and examined under the microscope for morphological confirmation. These were fixed in 95% ethanol and stored at 20 °C till further use. Cyst from each animal was considered as an isolate. The cysts collected at Dibrugarh were transported to Chandigarh under refrigerated condition for further processing.
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2.1. Molecular analysis
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DNA extraction: The cyst samples were washed thrice in PBS to remove ethanol and genomic DNA was extracted from each sample by QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. For molecular identification, PCR amplification of cox1 gene was performed by using the primers and PCR conditions as described previously (Nakao et al., 2002) with minor changes. Briefly, PCR was carried out in a 50 ll reaction mixture containing 2 ll DNA, 0.2 mM premixed solution of dNTP, 10 pmol of each primer, 1x PCR buffer, and 1 U of TaqDNA polymerase (Bangalore Genei). Amplification program included an initial denaturation step of 95 °C for 5 min and 30 cycles each of denaturation (95 °C for
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30 s), annealing (56 °C for 30 s), extension (72 °C for 90 s) and final extension of 72 °C for 10 min. After agarose gel electrophoresis (1%), PCR products were purified and sequenced.
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2.2. Phylogenetic analysis
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Previously published sequences of Taenia species retrieved from the National Center for Biotechnology (http://www.ncbi.nlm.nih. gov) were used as the reference sequences (Fig. 1). Nucleotide sequence analysis was performed with BLAST sequence algorithms and sequences were aligned using Clustal W (Thompson et al., 1994). T. saginata and T. asiatica sequences were used as outgroup The genetic distance was calculated by using Kimura two-parameter distance estimates and samples were clustered using the Neighbour joining algorithm using Mega 4 software. Bootstrap analysis was performed using 1000 replicates.
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3. Results
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T. solium cysticerci were identified by the partial sequencing of cox1 gene. The size of amplified PCR product was 1800 bp. For analysis nucleotide sequences of 1620 bp from all these isolates were aligned with reference sequences of other taenid cestodes i.e. T. saginata, T. asiatica and Asian and American/African genotype of T. solium (Fig. 1). In this study, single nucleotide polymorphism was found and no addition or deletions were detected. Among 50 North and North East Indian isolates, 4 haplotypes were found. These haplotypes were differed at 6 variant nucleotide positions (0.37% of total length). Among 25 North Indian isolates, three haplotypes (Accession number KC709806, n = 20; KC709808, n = 2 and KC709809, n = 3) were found and these showed polymorphism at nucleotide positions 52 (G–A), 291 (A–G) and 524 (C–T). The predominant haplotype reported in this study with accession number KC709806 showed 100% homology with cox1 gene sequence of T. solium cysticercus from India, Nepal, Japan, Madagascar (AB066489, AB491986, AB516957, AB781355, respectively), 99.9% homology with Madagascar (AB781357), 99.8% homology with China, Thiland, Korea, Nepal, Madagascar (AB066485/GQ402327, AB066487, DQ089663, AB491985, AB781359, respectively), 99.5% homology with Indonesia: Irian Jaya (AB066488), 99% homology with Tanzania (AB066493) and 98.8% homology with Brazil, Cameroon and Mexico and Madagascar (AB066492, FN995665, FN995660 and AB781361, respectively). Isolates in this haplotype showed 99.8% homology to North East Indian isolates Q4 (KC7098011). The nucleotide sequence of all 25 North East Indian isolates (Accession number KC709811) showed 100% homology with each other and differed from North Indian isolates at nucleotide positions 366(C–T), 1041 (A–G) and 1164 (C–T). Interestingly, these isolates like American and African genotype has ‘T’ at position 1164 and showed 99.8% homology with cox1 gene sequence of T. solium cysticercus from India (India, Nepal, Japan, Madagascar (AB066489, AB491986, AB516957, AB781355, respectively), 99.7% homology with Madagascar (AB781357), 99.6% homology with China, Thiland, Korea, Nepal, Madagascar (AB066485/GQ402327, AB066487, DQ089663, AB491985, AB781359, respectively), 99.3% homology with Indonesia:Irian Jaya (AB066488), 99% homology with Tanzania (AB066493), 98.8% homology with Madagascar (AB781361) 98.9% homology with Mexico (FN995660) and 98.7% homology with Brazil, Cameroon (AB066492, FN995665), respectively. Further in dendrogram analysis, all the isolates were clustered with Asian genotype of T. solium (Fig. 1). All the North Indian
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Fig. 1. Dendrogram constructed with the cox1 mitochondrial gene sequence obtained from human taeniid cestodes. The sequences were compared with sequence of reference genotypes. Scale in the tree represents similarity indices. All red coloured branches represent the Asian genotype of Taenia solium and Blue coloured branches represent African/American genotype. Pink and Green coloured branches represent the outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 209 210
isolates were clustered in one clade and all the North East Indian isolates clustered together and represented the same clone.
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4. Discussion
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Taeniasis and cysticercosis is endemic in many parts of India and several factors including cultural, socioeconomical, agricultural and environmental contribute to the disease burden (Prasad et al., 2008). In addition to this, lack of education and knowledge about the life cycle of the parasite, as well as the lack of legislation for meat inspection and offal disposal at local abattoirs, contributes to the transmission. Planning of control strategies for the prevention of disease depend on multiple parameters and the genotyping of isolates of T. solium may be one of the parameter in the formulation of control strategies for the prevention of transmission of this parasite. The genetic diversity of T. solium isolates collected from Peru, Colombia, Mexico, India, China, and Philippine has been studied by the sequencing of cox1, ITS1 and diagnostic antigen Ts14. The nucleotide variation was found to be minimal (Hancock et al., 2001). All 15 isolates from Peru had identical cox1 sequences. Furthermore, a clonal structure and local lineages with probable
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events of genetic recombination without genetic flow within them was shown by Random amplified polymorphic DNA (RAPD) in T. solium cysticerci recovered from naturally infected pigs from Mexico, Honduras and Africa (Maravilla et al., 2003; Vega et al., 2003). In the present study, T. solium isolates were collected from 2 distant endemic geographical areas of India to find out the genetic variation by sequencing of cox1 gene. All the isolates were clustered with other Asian genotypes. These results are in agreement with the findings of previous study (Nakao et al., 2002), where the sequencing of cox1 and cyt b gene of 13 pig isolates of T. solium from various regions of Asia (China, India, Irian Jaya and Thailand), Africa (Cameroon, Mozambique and Tanzania) and Latin America (Bolivia, Brazil, Ecuador, Mexico and Peru) have showed that all the T. solium isolates from Asian countries should be classified as Asian genotype and all the isolates from American and African countries should be classified together as American/African genotype. Among 25 North Indian isolates 3 halplotypes differed at positions 52, 291 and 524 were found, whereas in North East Indian isolates no nucleotide variation was observed. Among these two populations 6 variant nucleotide positions (0.37% of total length) were detected but these populations when compared with the reference sequence of Africa and America showed 20 variants
Please cite this article in press as: Sharma, M., et al. Genetic similarity between Taenia solium cysticerci collected from the two distant endemic areas in North and North East India. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2013.12.010
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nucleotide positions. In another study within cox1 gene, 28 variant nucleotide positions (1.7% of total length) were reported among the 13 isolates collected from different region of Asia, America and Africa (Nakao et al., 2002). These results are expected as T. solium is a hermaphrodite organism capable of self fertilization, which is responsible for low genetic variation and increased genetic differentiation among populations (Campbell et al., 2006). In this study some sequences from Indian isolates are found close to Madagascar (Accession number AB781355, AB781357 and AB781359) as % of homology varies from 100% to 99.6% and they were grouped in Asian isolate. However, the other sequence from Madagascar with accession number AB781361 was found to be clustered with American and other African sequences. These results in the present study are in concordant with earlier study from Madagascar, where 11/13 nucleotide sequences of T. solium from Madagascar were found to be clusred with Asian genotype and two were found to be clustered with African/American genotype (Michelet et al., 2010). The findings suggest that the isolates of T. solium from Madagascar have originated from two different geographical areas (Michelet and Dauga, 2012). Interestingly in our study, sequence of T. solium cysticerci isolate obtained after surgical resections from ocular cysticercosis patient from Chandigarh, India with ‘Accession no KC709810’ also showed 100% similarity with other North Indian isolates extracted from muscles of pig (Personal communication). These results suggest the genetic similarity between cysticerci from pig and human in North India. This observation is in concordant to the earlier study from Indonesia and Mexico, in which nucleotide sequence from cysticerci from human eye and brain were almost identical to those obtained from pigs, indicating that the cysticerci of T. solium from pigs and human are genetically very close (Hinojosa-Juarez et al., 2008). To the best of our knowledge, this study is the first report of genotyping of large number of Indian animal isolates of T. solium cysticerci from two distant geographical areas. In conclusion, the result of the present study demonstrated the very low genetic variation in the cox 1 gene of cysticerci of T. solium isolates collected from two distant geographical areas and all the sequences were well clustered with the published sequences of Asian genotypes of T. solium. Information regarding genetic variation in T. solium is limited. This study on large number of samples may have provided preliminary information to design future goals which may have important implications in the planning of control strategies for the prevention of disease.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2013.12. 010.
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