Molecular Detection and Characterization of Goat Isolate of Taenia hydatigena in Turkey

The aim of this study was to provide molecular detection and characterization of the goat isolate of Taenia hydatigena from Ankara province of Turkey. For this purpose, PCR amplification of small subunit ribosomal RNA (rrnS) and partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) genes were performed in a one-month-old dead goat. According to rrnS-PCR results, parasites were identified as Taenia spp., and partial sequence of mt-CO1 gene was corresponding to T. hydatigena. At the end of the study, we concluded that molecular tools can be used to define species of parasites in cases where the key morphologic features cannot be detected. Nucleotide sequence data of Turkish goat isolate of T. hydatigena was submitted to GenBank for other researchers interested in this subject. By this study, molecular detection and characterization of T. hydatigena was done for the first time in Turkey.


Introduction
Cysticercosis caused by the metacestode of the taeniid cestodes is a global infection, which has veterinary, medical, and economic importance. The adult stages of the parasites infect canids, while the metacestodes develop in several species of domestic and wild intermediate mammalian hosts where they develop as fluid-filled larvae in tissues [1][2][3].
Cysticercus tenuicollis is the metacestode of canine tapeworm Taenia hydatigena, which has been reported in domestic and wild ruminants, pigs, squirrels, and monkeys [1,2]. Metacestodes are found attached to the omentum, mesentery, and occasionally on the liver surface; however, unusual locations of C. tenuicollis have been described as lungs, kidneys, brain, ovaries, uterine tubes, uterus, cervix, and vagina. An aberrant location of C. tenuicollis vesicle inside the chorioallantoic membrane of a goat foetus was reported [4]. Pathogenicity of adult parasites is not high for definitive hosts. However, large numbers of developing cysticerci migrate contemporaneously in the liver of intermediate hosts, producing "hepatitis cysticercosa", a condition whose gross pathology resembles acute fasciolosis and which is often fatal [1,2,5].
The aim of this study was to provide molecular detection and characterization of the goat isolate of T. hydatigena by PCR amplification of small subunit ribosomal RNA (rrnS) and partial sequencing of mt-CO1 gene in Ankara province of Turkey.  Figure 1: Liver of one-month-old dead goat with haemorrhagic tracts (a) and ten recovered cystic samples (b).

Materials and Methods
Liver of a one-month-old dead goat was sent to our laboratory for parasitological examination. For this, liver was cut into one cm 3 pieces and left into warm water for 30 minutes. Then, liver pieces were removed by squeezing, and sediment was examined [1]. 46 cystic samples were collected from the sediment. While ten of them were examined under light microscope, remaining parasites were taken into phosphate-buffered saline (PBS).
PCR conditions were the same for two primer pairs.   The 446 bp region of mt-CO1 gene of one cystic sample was sequenced by a commercial company (Refgen, Ankara, Turkey). The obtained sequences were edited and aligned, using the Bioedit software [12], and then, sequence analysis was undertaken by BLAST algorithms and databases from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov/).

Results
After the macroscopic examination, we detected hemorrhagic tracts and migrating larvae under the liver capsula ( Figure 1). In the microscopic examination, we could not detect scoleces, rostellar hooks, or invagination. The rrnS-PCR with the Cest3 and Cest5 primers yielded 267 bp of amplification product (Figure 2). According to this, parasites were identified as Taenia spp., and mt-CO1-PCR yielded a 446 bp-sized fragment (Figure 3). Partial sequence of mt-CO1 (GenBank accession number: JN827307) was corresponding with T. hydatigena. The alignment of the nucleotide sequences with published sequence results (DQ995656, HQ204206) for T. hydatigena is presented in Figure 4. According to the alignment results, there was 99% identity in nucleotide sequences for T. hydatigena.
In general, it is easy to define the species of Cysticercus when the parasite has a certain size and is in specific predilection site; however, in cases of unusual or aberrant localizations, degenerations, and calcifications, species identification and differentiation from cysts of another etiology may be difficult [3]. In Turkey, studies on C. tenuicollis focus on the prevalence and postmortem morphologic diagnosis of the disease [13][14][15][16][17]. In this study, we could not detect key morphologic features of larvae such as scoleces, rostellar hooks, or invagination in the microscopic examination. Therefore, we tried to detect C. tenuicollis by genus-specific PCR and partial sequencing of mt-CO1 gene. Cest3 and Cest5 primer pair is specific to Taenia spp., but not to Echinococcus spp. [10]. Thus, this primer pair can be used to differentiate C. tenuicollis from the hydatid cysts and other cystic structures. Moreover, partial sequencing of the 446 bp amplicon of JB3 and JB4.5 primer pair enabled us to detect T. hydatigena at species level. At the end of the study, we submitted nucleotide sequence data (JN827307) of the Turkish goat isolate of T. hydatigena to GenBank