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T. solium taeniasis/cysticercosis is a neglected tropical zoonosis which is endemic in several developing countries. The adult tapeworm infection occurs only in human (Taeniasis), acquired by the ingestion of undercooked pork contaminated with cysticerci. The infection with larval stage is acquired by ingestion of parasite eggs shed in human feces which can infect both human and pigs. After ingestion, cysticerci migrate to the intestine. Taeniasis has only mild clinical manifestations and may remain asymptomatic. Human cysticercosis occurs when cystic larvae lodge in different organs such as the central nervous system (CNS), skeletal muscles, eye and subcutaneous tissue.5,6
It is estimated that 50 million people are infected with the taeniasis/cysticercosis and 50,000 deaths occur from cysticercosis annually, worldwide.(2-4). In India, Human taeniasis has a prevalence of 2-38% and 8.7-50% of patients presenting with recent onset of seizure had NCC. The prevalence of porcine cysticercosis varies from 7-26%. ( Rajshekhar, V. et al., 2004). In Chandigarh and its surrounding areas, seroprevelence rate of 17.3% with highest prevalence of 24% from slum areas has been reported ; however only 8% of the seropostives had previous history of seizure (Khurana et al 2006; Saigal et al 1984).
In Asian countries, majority of the patients with neurocysticercosis presented with a single enhancement brain lesion and a very few have massive infections with multiple cysts. Conversely, in Latin America the most frequent presentation of NCC is multiple cysts without signs of inflammation. Subcutaenous cysticercosis is rare in Latin America but very common in Asia. Whether these differential pathological manifestations are related to different genetic variants of T. solium is not known yet. It is suggested that 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. Analysis of genetic variation between and within populations determined the future evolutionary change, genetic differentiation and speciation . Molecular techniques based on Polymerase chain reaction (PCR) based has been used for the differential diagnosis of taenia species and strains of T. solium and to gain knowledge of genetic diversity in this parasitic population 2-5
In molecular biology, mitochondrial sequences are widely used as genetic markers for ecological, phylognetic and evolutionary studies. The partial sequencing of cox 1 gene of T. solium isolates obtained from different areas in Asia and America reported the presence of two genotypes: Asian genotypes and American genotypes (Okamato et al ). Later, sequencing of mitochondrial genes of African isolates were found to be clustered with American genotype which reveals the presence of Asian genotype and American/African genotype (Nako et al 2002a). In the present study genetic polymorphisms in T. solium from 2 different geographical regions of India were studied by comparing sequences of mitochondrial genes: cytochrome C oxidase subunit 1 (cox1).
Materials and Methods
Taenia solium cysticerci were collected from 2 different geographical areas i.e Chandigarh and Dibrugarh in India which are 2585 km apart. Cyst samples were obtained from 50 freshly slaughtered and heavily infected pigs from slaughter houses located in Chandigarh (n=25) and Dibrugarh (n=25). The carcass containing cysts were transported to the department of Parasitology, PGIMER for further processing. The cysticerci were separated, washed with distilled water and examined under the microscope. They were fixed in 95% ethanol and stored at -20°C till further use. Cyst from each animal was considered as an isolate.
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 the cox1 gene was performed by using primers and PCR conditions as described previously  with minor changes. Briefly, PCR was carried out in a 50 ul reaction mixture containing 2 μl DNA, 0.2 mM premixed solution of dNTP, 10 pmol of each primer, 1x PCR buffer, and 1 U of TaqDNA polymerase. Amplification program included an initial denaturation step of 95ËšC for 5 min and 30 cycles each of denaturation (95ËšC for 30s), annealing (56ËšC for 30s), extension (72ËšC for 90sec) and final extension of 72ËšC for 10 min. After agarose gel electrophoresis (1%), PCR products were purified and sequenced.
Previously published sequences of T. solium isolates retrieved from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov) were used as the reference sequence (Fig 2). Nucleotide sequence analysis was performed with BLAST sequence algorithms and sequences were aligned using ClustalW . 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.
T. solium cysticerci were molecularly identified by the partial sequencing of Cox 1 gene. The sequence of the amplified product were ranged in length from 1630-1700 bp. Nucleotide sequences of all these isolates were aligned with sequences of other taenid cestodes i.e T. solium, T. saginta and T. asiatica. In this study only point mutations were found and no addition or deletions were reported. All the isolates analysed in the present study differed from the standard strains of America, Africa and China at position 994 and 1466 and like China isolate differed from American and African strains at 13 different positions. The nucleotide sequence of North East Indian isolate were differed from North Indian isolates at position 366, 1041. Interestingly these isolates like American and African strain has 'T' at position 1164. The nucleotide sequence of all the North Indian isolates (except Tae 4, 9, 11) showed 100% similarity with sequence deposited in Genbank with accession number (AB066489) and used as reference strain from India in this study. Interestingly isolates which were obtained after surgical resections from ocular cysticecosis patients from Chandigarh also showed 100% similarity with other North Indian isolates from muscles of pig.
Further in dendrogram analysis all the isolates were clustered with Asian genotype of T. solium in 2 different clades. Clade I contain all the isolates from East India whereas all the North Indian isolates were clustered in clade II. In Cox1 gene, 6 variant nucleotide positions (0.37% of total length) were detected among the 50 isolates.
Taeniasis and cysticercosis is endemic in many parts of India and is a cause of serious concern due to increase in morbidity and considerable economic loss. In India several factors including
cultural, socioeconomical, agricultural and environmental contribute to disease burden[Prasad et al.,]. 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. Genotyping of isolates of T. solium play an important role in the formulation of control strategies for the prevention of transmission of this parasite.
Within cox 1gene, differential nucleotide are dispersed over the entire length and served as diagnostic marker for human taenid cestodes or differentiation of 2 genotypes of Taenia Within Cox 1 gene 11 nucleotides at position, , and served as differential marker for 3 species of Tania and Nucleotides at 13 different positions can differentiate the 2 genotypes of T. solium
In this study to study the genetic variation by sequencing of Cox 1 gene isolates of T. solium isolates were collected from 2 distant endemic geographical area of India. All the isolates were clustered with other Asian genotypes. These results are in agreement the findings of Nako et al(2002); based on the sequencing of Cox 1 and Cyt b gene of 13 pig isolates of T. solium from various regions of Asia (China, India, Irian, Jaya and Thailand ), Africa (Bolivia, Brazil, Ecuador, Mexico and Peru)and Latin.America(Cameroon, Mozambique and Tanzania) they proposed that T. solium should be classified into 2 genotypes Asian genotype and American/ African genotype.
In the present study, Cox1 sequences for North Indian T. solium cysticerci samples from human eye and from pigs placed them into the same group had almost identical sequence to a sample of T. solium from India (AB066489), (Fig. 1A and Table 1). These results suggest the genetic similarity between cysticeri from pig and human. These results are concordant with the earlier studies,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.(Hinjosa-Juarez et al).
In North Indian samples 25 isolates were differed at 3 nucleotide positions but in North East Indian isolates no genetic variation was observed. Among these two populations 6 variant nucleotide positions were detect but these population when compared with the standard strain from Africa and America have 20 variants nucleotide positions. In another syudy withIn Cox1 gene, 28 variant nucleotide positions (1.7% of total length) were reporte among the 13isolates collected fron different region of Asia, America and Africa (Nakao et al., 2002). These types of result are expected as T. solium is a hermaphrodite organism capable of self fertilization, which is responsible for diminish genetic variation and increase genetic differentiation among populations.
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 and all the sequences well clustered with the published sequences of Asian genotype of T. solium