Epidemiological Survey and Phylogenetic Characterization of Cysticercus tenuicollis Isolated from Tibetan Pigs in Tibet, China

Cysticercus tenuicollis, commonly known as “water bell,” is a larva of Taenia hydatigena, which is the most significant parasite of pigs. However, until now very few information is available regarding the prevalence and genetic characterization of the Cysticercus tenuicollis in Tibetan pigs. Therefore, the aim of this study was to investigate the prevalence and phylogenetic analysis of Cysticercus tenuicollis in Tibetan pigs. For this purpose, the COX2 gene of Cysticercus tenuicollis was amplified and sequenced for the first time in Tibetan pigs. The overall prevalence of Cysticercus tenuicollis was 43.93% in Tibetan pigs, with further distribution of 42.86% in 2014 and 45.35% in 2015. In Tibetan male and female pigs, the prevalence of Cysticercus tenuicollis was 43.39% and 44.56%, respectively. The prevalence of Cysticercus tenuicollis in different growing stages (juveniles, subadults, and adults) varied from 30.20% to 63.79%. The phylogenetic analysis of the Cysticercus tenuicollis isolates showed very close resemblance to 16 reference strains, isolates from Gansu, Hunan, and Sichuan provinces of China. To the best of our knowledge, this is the first report on the prevalence and genetic characterization of Cysticercus tenuicollis derived from Tibetan pigs. The data of present study provides baseline information for controlling cysticerci infections in pigs in Tibetan Plateau, China.


Introduction
Cysticercus tenuicollis is a bubble-like metacestode of Taenia hydatigena that invades the abdominal cavity and liver of pigs, dogs, and other carnivores [1][2][3]. This parasite is generally associated with the significant tissue damage in liver, lungs, and other organs of pigs, cattle, sheep, and other types of livestock [4][5][6][7]. The parasite can infect the pigs of any age and can not only shorten the feed intake but also hinder the growth and development of animals. Therefore, considerable tissue damage of liver or other organs by this parasite may perhaps lead to the death or serious economic losses to the pig industry [8].
The prevalence of Cysticercus tenuicollis in domestic pigs has been reported from certain parts of China. Previously, a low prevalence of Cysticercus tenuicollis (1.08%) in pigs was reported from Hunan province of China, whereas the high prevalence rates (42.43% to 35.98%) were observed in Fujian and Yunnan [9][10][11]; additionally, regional distribution of Cysticercus tenuicollis infection in pigs has been also reported from Sichuan and Yunnan [12]. However, no report has been available about the prevalence of Cysticercus tenuicollis infection in Tibetan pigs.
Tibetan pigs are relatively ancient original indigenous breed, a rare plateau type of pigs in the world, and are the only high altitude pasture pigs in China. Tibetan pigs are mainly distributed in the Qinghai-Tibetan Plateau in China (Qinghai, Sichuan, Yunnan, and eastern Tibet region) [13]. With high proteins and rich amino acids, the Tibetan pig meat is an important source of income for Tibetans nomads [13]. Due to outdoor grazing of Tibetan pigs round the year with the main intermediate hosts (domestic dogs and pi-dog) of the Cysticercus tenuicollis, pigs can be exposed to this infection. Furthermore, due to the lack of knowledge at herdsmen level and no special protection, awareness or insect repellent program of the parasitic infection in study area enhances the risk of this infection in ancient original indigenous breed and can lead to significant economic losses.
To date, no report is available about the prevalence and genetic characterization of Cysticercus tenuicollis in Tibetan pigs, China. The current study was aimed at investigating the prevalence and genetic characterization of cytochrome oxidase subunit 2 (COX2) derived from Cysticercus tenuicollis in Tibetan pigs.

Study Site.
This study was conducted in three counties (Nyingchi, Mainling, and Gongbo'gvamda) of Tibet, China, and samples were collected from pigs at different slaughterhouses in these three counties that have an average elevation of 3100 meters with the largest continuous high elevation ecosystem ( Figure 1).

Samples Collection and Processing.
In present study, a total of 112 samples were collected from Tibetan pigs (independent of age, sex, and breed by using simple random technique for sample collection) at different slaughterhouses of Nyingchi, Mainling, and Gongbo'gvamda of Tibet, in 2014. The information regarding each sample was captured on a prescribed performa. Similarly, a total of 86 samples were also collected from these counties in 2015. After autopsy, each positive sample was recorded and collected ( Figure 2). The larval stages were washed extensively in 0.9% sodium chloride solution and identified through morphological examinations. Subsequently, they were fixed in 75% alcohol (V/V) and stored at −20 ∘ C, until subsequent use and further analysis. Total DNA of the larvae of Cysticercus tenuicollis was extracted using the commercial kit (TIANamp Genomic DNA Kit, TianGen, China), according to manufacturer's instructions. The eluted DNA was stored at −20 ∘ C until further use.
A PCR amplification approach was used to amplify a fragment (∼560 bp) of the COX2 gene. For this purpose polymerase chain reaction primer forward GAAGTTGGT-TACTGAAAAG and reverse ATTCCATGATTAACACCA-C were used with the following cycling parameters: 35 cycles at 95 ∘ C for 30 seconds and 72 ∘ C for 1 minutes, annealing at 55 ∘ C for 30 seconds, and final extension at 72 ∘ C for 5 minutes. ddH 2 O was used as blank control group. PCR products were separated on agarose gel (1.0%) along with ethidium bromide (at the rate of 0.5 g/ml) and electrophoresis was performed on 0.5x TBE buffer at 5 V/cm for 60 minutes. The products were purified using a TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Takara Biotechnology Co., Ltd., Dalian, China), according to manufacturer's instructions. The obtained positive products were sequenced by a commercial company (Quintara Biosciences, Wuhan, China).

Phylogenetic and Sequence
Analysis. The nucleotide sequences of the COX2 gene of Cysticercus tenuicollis were compared with previously reported Cysticercus tenuicollis sequences available at NCBI database (appears in Table 2). Multiple alignments and phylogenetic analysis were conducted using Molecular Evolutionary Genetic Analysis (MEGA 6.0) software. Phylogenetic tree was constructed using the neighbor-joining method. The evolutionary distances were estimated using the Kimura two-parameter method. A bootstrapping test was performed with 1000 duplicates and the transversion/transition rate was set at 2.0.

Statistical Analysis.
The prevalence of Cysticercus tenuicollis in Tibetan pigs relating to different genders and growing stages was analyzed statistically by using Chi-square test, while 95% CI and odd ratio were also determined. The value of < 0.05 was considered as statistically significant.    Table 2). The phylogenetic analysis was performed based on the COX2 gene ( Figure 3 illustrated a close homology. The result showed that the Tibet-COX2 strain was very closely related to reference strains isolated from Gansu, Hunan, and Sichuan provinces, which are very close regions to Tibet (Figure 3). In addition, the nucleotide sequences of isolated strains shared 97.7%-100% sequence identify with the reference strains ( Figure 4).

Discussion
Cysticercus tenuicollis is the larvae stage of Taenia hydatigena, which is a common parasite of pigs, cattle, buffalo, yak, sheep, goat, camel, horse, and human [6]. Many countries in the world have reported Cysticercus tenuicollis since its first report [4,6,7]. This disease remains a severe problem in pigs especially under the age of one year, where there are no effective measures that result in very high mortality. China is the largest pigs producing country, while parasitosis has become a major threat to pigs industry in the country, which causes serious illness [13]. As the only plateau and alpine pasture of pig breeds in China, Cysticercus tenuicollis has not yet been reported in Tibetan pigs. The recent research showed that a high infection rate of Cysticercus tenuicollis was found in Tibetan pigs, which is higher than that reported in pigs from Dali (6.06%) and Pingle (1.11%) [13,14]. The prevalence difference of Cysticercus tenuicollis in our study and the previous reports may be due to the differences in freerange feeding pattern, ecological environment, and existence of the wild dogs, as well as the knowledge level of the breeder [15]. During the survey, we found that the local people were not aware of using antiparasitic drugs, secondly improperly or frequently using the antimicrobial agents. So, it may be the possible reason for the high prevalence rate of Cysticercus tenuicollis. Tibetan pigs are mainly fed by the extensive breeding environment combined with the dry lot husbandry [16]. Under the special breeding system, this animal get more chance to expose to the external environment; among them, subadults and adults have the longest time of outdoor activities which increases a higher infection risk of infecting the Cysticercus tenuicollis, compared with the juveniles Tibetan pigs [6].   Currently studies have showed that the mitochondrial genome structure of Tapeworms is similar to other mitochondrial genomes of eukaryotes that include 12 genes encoding proteins [17]. Among them, the mitochondrial gene cytochrome oxidase subunit 2 was the ideal genetic markers, which was used to make phylogenetic analysis [18]. In our study, the isolated parasite was phylogenetically compared to reference parasite, based on the COX2 gene coding sequence (CDS). The phylogenetic analysis demonstrated that the isolates share a close homology with some reference strains isolated in Gansu, Hunan, and Sichuan. In addition, the nucleotide sequences of the isolated strains shared 97.7%-100% sequence identify with the reference strains.
In conclusion, the present survey revealed the high prevalence of Cysticercus tenuicollis infection in Tibetan pigs; herein, we suggest some measures to prevent and control this infection. At first, anthelmintics must be an important practice to prevent the infection and minimize the serious economic losses. Secondly, it is important to improve the management of swine breeding to reduce the spread of disease through direct contact between dogs and Tibetan pigs. Additionally, practice of undercooked animal innards to dogs in study area should be avoided. Finally, it is necessary to change the husbandry of free-range into captivity system in these rural areas.

Conflicts of Interest
The authors declare that they have no conflicts of interest.