​Molecular Detection and Phylogenetic Analysis of the Genotype of Larval Cestode Cysticercus cellulosae of Pigs and Taenia solium of Man

Porcine cysticercosis, caused by Cysticercus cellulosae a larval stage of parasite Taenia solium is considered to be an emerging and re-emerging parasitic disease for both developed and developing countries (Craig and Pawlowski 2002). The infection is closely associated with social conditions like food habits as per religion or as per region as well as environmental conditions such as poor hygiene and free roaming of pigs etc. The people of north eastern region of India are mostly non-vegetarian and consumption of pork is very much preferred by the people of this region, although there is a gap between demand and supply of pork in this region. As a result, a large quantity of pigs are imported in this region from the states outside of north eastern region. Where a large numbers of populations are consuming pork and a large quantity of pigs are imported in this region, it is essential that parasites of zoonotic significance like C. cellulosae should not infect pork eater (human) and hence inspection of pork is essential to detect C. cellulosae. But traditional method of detection of C. cellulosae by post mortem examination of pork has disadvantages like need of expert otherwise may be confused with T. hydatigena cysticerci or hydatid cysts (Kakoti et al., 2017) and may not be reliable in case of light infections (over looked). Molecular diagnostics have been considered and  reported to be highly specific and sensitive (Gonzalez et al., 2006, Sato et al. 2006, Chiesa et al., 2010). Keeping in view of the above, the present study on molecular detection of  larval cestode C. cellulosae of pigs was undertaken. Although a study on  molecular detection of  larval cestode C. cellulosae of pigs in nearby state of north eastern region of India, Assam is available (Kakoti et al., 2017) but they have not undertaken any study on human Taenae solium adult parasites and they used different sets of primers. Phylogenetic study on larval cestode C. cellulosae of pigs or human Taenae solium of this regionnot yet done so far. Hence a study on phylogenetic relation of  C. cellulosae of pigs or human Taenae solium of this region was done to know its relation with other parts of the world.

Collection of samples and extraction of DNA
 
From three prime districts of state Arunachal Pradesh i.e. West Kameng, East Kameng and Papum-pare and two  adjoining bordering districts of the State Assam i.e. Sonitpur and Lakhimpur (Fig 1), a total of  654 numbers of pig carcasses were examined  from  17  market places (Fig 2). Overall 1.83% pigs were found infected with C. cellulosae (Biswakarma et al.  2020). The cysticerci samples from the present study were obtained manually from the infected muscles (Fig 3 and Fig 4) and organs of  these pigs were preserved in phosphate buffer saline  until DNA extraction. Genomic DNA extraction from 3-4 numbers of cysticerci was performed using a spin column kit (D Neasy tissue kit: QUIGEN) as recommended by the manufacturer after manually mincing the cysts into small pieces separately.  Stool samples of human patients who attended out- patient department (OPD) of Community health centers, Nursing homes and District hospitals in Seijosa circle East Kameng district; Bhalukpong, Rupa, Bomdila circles in West Kameng and Naharlagun, Itanagar, Doimukh, Balijan and Banderdewa circles in Papum-Pare of Aunachal Pradesh were collected randomly and examined by salt flotation technique for the presence of T. solium eggs. The segments of tapeworm voided by patients (Fig 5) were then identified for species identification. Finding of T. solium eggs and proglottids in faeces were considered as positive infection in human being. A young 32 year old male person stayed at Bhalukpong was found to pass gravid segments of Taenia solium and those segments were collected in normal saline solutions (NSS) after clearing the debris and faecal materials (Biswakarma 2017) until DNA extraction. The DNA from T. solium segments collected from affected human being were also extracted using the same procedure. A total of 10-15 µl of crude DNA were extracted from the cysticerci and proglottids separately. These crude DNAs were then centrifuged at 5000 rpm for 5 minutes before being preserved at -20^oC in PCR tube.











 
Polymerase chain reaction 
 
Oligonucleotide primers
 
The mitochondrial gene cytochrome b oxidase subunit was amplified by PCR. Two sets of published Primers-Cytb/F (5'-ATAAACTGATAGATTGTGGTTC-3') (Forward) and Cytb/R (5'-CATATGACTGTCTAATGAAGA-AAA-3') (Reverse) were taken for the PCR (Nakao et al., 2002).
 
PCR amplification and detection of PCR product 
 
The PCR was carried out in a reaction mixture of 50 µl containing 2 µl of template DNA ( DNA extracted from the scoleces and proglottids), each dNTP at 200 µM, each primer at 0.5 µM,1U of DNA polymerase ( Taq polymerase) and 1x Taqreaction buffer. For PCR amplification, 30 thermal cycles were employed (Initial denaturation at 94^oC for 3 minutes, 94^oC for 30 seconds, 58^oC for 30 seconds, 72^oC for 60 seconds final extension at 72^oC for 10 seconds). A negative control consisting of a reaction mixture without DNA was used.

For visualization of the PCR product, Agarose gel electrophoresis of amplified DNA was done in 1.5% Agarose gel for 1 hour at 5 volts per cm using 1X Tris acetate EDTA (1X TAE)  running buffer. 4 µl of the PCR product mixed with 3 µl of gel loading dye (6X DNA loading dye, Fermentas) was loaded on to the gel with standard markers (1 Kb bp DNA ladder, Fermentas). The gel was than stained with ethidium bromide (0.5 µg/ml) and visualized under gel documentation system (DNR Bio-Imaging system, Mini Lumi).
 
Sequence homology and phylogenetic analysis
 
PCR products were purified using Q1Aquick PCR purification kit as per manufacturer’s protocols. The purified products were sent to Molbiogen (1^st Base DNA) Malaysia at 4^oC for automated sequencing. The sequences obtained were aligned and the DNA sequence of cytb genes of T. solium termed as CVSc isolates,  were compared with previously available sequences from different hosts in NCBI (National center for Biotechnology Informatics) using BLAST system. Multiple alignments were done using clustalW of the MEGA 7.00 software programme. Phylogenetic tree was constructed using the Neighbor-Joining method in the same software. The robustness of the grouping in the Neighbor-Joining analysis was assessed with 1000 bootstrap resampling.

Results were subjected to analysis conducted using the maximum composite likelihood model. Evolutionary analyses were conducted in MEGA7 software.

The PCR amplification of  cytochrome b oxidase genes from C. cellulosae and segments of T. solium showed positive by this conventional PCR. The positive samples showed clear 1068 bp band in PCR (Fig 6). The present finding of PCR with the presence of 1068 bp band in the slab gel showed specific amplification of cytochrome b oxidase genes.



Alignment of cytb  sequences from NCBI GenBank were analysed by MEGA7. Sequences from isolates of India (AB066574); Brazil (AB066577); Mexico (AB066575); Cameroon (AB066579); Mexico (JQ973078); Tanzania, Mbulu district (AY211881); Tanzania (AB066578); Ecuador (AB066576); Mexico (JQ 97303);  Madagascar (FM958327); Madagascar tanambao (FM958327); Vietnam Bac Ninh province (AY280806); Thailand ( AB066572); Vietnam Ho chi Minh city (KC621095); Vietnam Thaibinh province (KC621100); Japan (AB781363); Indonesia (AB066573); Madagascar (FM 958318); China (AB086256); China (AB066571); Nepal (AB81746); Vietnam (AY280805) were included.

The present isolate had 0.0017 distances with Japan (AB781363), Indonesia (AB066573) and Madagascar (FM958318), 0.0035 distances with Madagascar (FM958323), India (AB066574) and Thailand (AB066572) and 0.0052 distances with Madagascar, tanambao province (FM958327), Nepal (AB781746), Vietnam (AY280805), China (AB086256) and China (AB066571) (Table 1).



The range distances of present isolate with African countries like Cameroon (AB066579) is 0.0176 and with Tanzania (AY211881) is 0.0212. The phylogenic tree constructed based on this finding is depicted in Fig 7 .



The present study recorded a PCR amplification of  cytochrome b oxidase genes from C. cellulosae with a definite product size of 1068 bp from DNA extracted from C. Cellulosae and T. solium. The present finding signifies that the same genomic isolate of both the larval cestode and adult parasite of T. solium is prevailing in the study areas as such. This is in agreement to the findings of Singh et al., (2002). The Primer Set Cytb /F forward 5'-ATAAACTGATA GATTGTGGTTC-3' and Cytb /R reverse 5'-CATATGACTG TCTAATGAAGAAAA-3' have been successfully used by Michelet et al., (2010); Palafox-Fonseca et al., (2013). Besides this primer set also referred in a book (Liu, 2010). The detection of T. solium cysticerci from the infected pig carcasses by PCR amplification of  cytochrome b oxidase genes from C. cellulosae with a definite product size of 1068 bp might be helpful for meat inspection by molecular detection of  C. cellulosae infections in pork. Earlier Kakoty et al., (2017) detected  T. solium cysticerci from the infected pig carcasses and suspected carcasses based on amplification of large subunit rRNA gene (TBR) with a product size of 286 bp from the samples collected from  different mark et places of Sivasagar district of Assam, India. But the study  on amplification of DNA extracted from both  C. Cellulosae of pigs and segments of T. solium of human with same primers were not done by them. In that sense the establishment of prevailing of the same genomic isolate of both the larval cestode and adult parasite of T. solium the study areas may be the first report of its kind from north eastern region of India as well as from whole India.

The neighbors-joining phylogenic trees shows close similarity of the present isolates with that prevailing in other South East Asian countries (viz: Thailand, Vietnam, Japan, Nepal, China and Madagascar). It might be assumed from the present finding that the same genotypic isolate of T. solium parasite is prevalent in the whole of South East Asian region which is in agreement with the finding of Nakao et al., (2002). The similarity of the present isolate with the one found in Madagascar shows that some personnel affected with T. solium might have travelled to Madagascar from any of the countries in South East Asian region and spread the infection there ( despite the fact that Madagascar is a Muslim country and have very little pig population).

It can be concluded from the present study that molecular detection of Cysticercus cellulosae can be done by PCR using the specific sets of primers. The same genomic isolate of both the larval cestode and adult parasite of T. solium is prevailing in the study areas as such. The neighbors-joining phylogenic trees shows close similarity of the present isolates with that prevailing in other South East Asian countries (viz.: Thailand, Vietnam, Japan, Nepal, China and Madagascar). It might be assumed from the present finding that the same genotypic isolate of T. solium parasite is prevalent in the whole of South East Asian region.

The authors acknowledge the “Advance Animal Disease Diagnosis and Management Consortium (ADMaC)” team of the Core Lab, College of Veterinary Science, Khanapara, Guwahati, Assam, for associating the work as one component Research under DBT governance towards fulfillment of projected parameter under the able support of Director of Research (Veterinary) and HOD, Department of Parasitology and P.I. of the project. Sincere help and Cooperation rendered by the Dean, FVSc, Khanapara is thankfully acknowledged.

The authors declare that they have no conflict of interest.