Abstract:

Objective To assess the feasibility of using specific sequences of cytochrome C oxidase 1 gene(cox1) and internal transcribed spacer 1 (ITS-1) to detect plerocercoid infection in worm-free pathological tissue of mice. Methods Twenty Kunming mice aged 6-8 weeks were randomly divided into the infection group (15 mice) and the uninfected group (5 mice). Mice in the infected group were orally infected with plerocercoids from snake (5 plecerocoids/mouse), while the uninfected mice received no treatment. The mice were sacrificed on day 14 after infection. Subcutaneous muscle tissues containing plerocercoids (worm-containing tissue), worm-stripped tissues (worm-free tissue) and their correspondant of the uninfected group were taken and cut into sections. A portion of these sections were processed for hematoxylin-eosin (HE) staining, while the other portion was used for DNA extraction with a modified proteinase K digestion method. The cox1 (using 2 pairs of primers, resulting in cox1-1 and cox1-2) and ITS-1 (1 pair of primers) were amplified by PCR, and PCR products from DNA of the worm-free tissues in the infection group were cloned, sequenced and aligned with gene sequences of KJ418421, KF990161 and GQ999947 of plerocercoid. The same method was used to prepare muscle tissues from mice infected with cysticercus cellulosae from pigs and the DNA preparation. The species specificity of cox1 and ITS-1 was verified by PCR. Results PCR amplification on DNA of the worm-containing tissue produced specific bands of 822 bp (ITS-1), 414 bp (cox1-1), and 151 bp (cox1-2) after the first round of PCR. No bands were produced from DNA of the worm-free tissue under the same condition but a further round of PCR using the products of the first round resulted in three bands of the same sizes as above. No bands were seen in the uninfected group, either after the first or the second round of PCR. The cox1-1 sequence in the worm-free tissue shared 98.2%, 99.1% and 99.12% homology with KJ418421, KF990161 and GQ999947 of plerocercoid, respectively. No specific bands were amplified from DNA of cysticercus-infected tissue of pigs. Conclusion Repeated PCR amplification using cox1 and ITS-1 gene sequences as the target can identify plerocercoid infection in worm-free pathological tissues of mice.

Key words: Plerocercoid, Worm-free tissue, Gene detection, Cytochrome C oxidase 1, Internal transcribed spacer 1, Spirometra erinacei