Trichinella spp

Owsiacki, Robin, Buhler, Kayla J., Sharma, Rajnish, Branigan, Marsha, Fenton, Heather, Tomaselli, Matilde, Kafle, Pratap, Lobanov, Vladislav A., Emilie, Bouchard & Jenkins, Emily, 2020, Trichinella nativa and Trichinella T 6 in arctic foxes (Vulpes lagopus) from northern Canada, International Journal for Parasitology: Parasites and Wildlife 13, pp. 269-274 : 271

2.3. Recovery of larvae of Trichinella spp

Larvae of Trichinella spp . were recovered from the forelimb muscle by Pepsin–HCl digestion and sequential larval sedimentation. This enzymatic digestion method is the internationally accepted gold standard and has successfully been used to test for Trichinella spp . in wild and domestic mammalian species ( Forbes and Gajadhar, 1999; Gajadhar and Forbes, 2010). Fat and connective tissue was removed from the muscle, and 5 g (minimum when available; if less, all available muscle sample) was cut into 1 cm × 1 cm pieces ( Gamble et al., 2000). The remaining muscle was minced in a blender using 3–4 bursts of 10 s each. The tissue was digested in a 1% Pepsin-HCl solution for 1.5 h at 37 ◦ C, followed by sequential sedimentation. Finally, 20 mL of the solution was collected in a Petri dish and examined under a dissecting microscope. After digestion, larvae were morphologically characterized as tightly coiled, lightly coiled, or C-shaped ( Fig. 2 ). Larvae were counted and reported as larvae per gram (LPG) of muscle tissue. From each positive fox, 5 individual larvae and a pool of 10 larvae were collected in microcentrifuge tubes containing 1X PCR Buffer (Applied Biosystems 10X PCR buffer [Foster City, United States] diluted with ultrapure H 20; 10X PCR buffer composed of 100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2; 0.01% gelatin) and stored at – 20 ◦ C until DNA extraction.

2.4. DNA extraction and multiplex PCR

DNA was extracted from larvae using the Proteinase K extraction method as per Scandrett et al. (2018). Multiplex PCR was performed as per Zarlenga et al. (1999). Larvae of six recognized species of Trichinella ( T. spiralis , T. nativa , T. britovi , T. pseudospiralis , T. murrelli and Trichinella T6) were provided by the Canadian Food Inspection Agency (Saskatoon, Canada) as positive controls.

2.5. PCR-RFLP

In order to differentiate T. nativa from T. chanchalensis (T13), PCR-RFLP was performed on DNA of larvae that were identified as T. nativa on multiplex PCR as described previously ( Sharma et al., 2019, 2020). On PCR-RFLP, ~920 bp amplified products of positive controls showed distinct band patterns after restriction digestion: three bands of approximately 407, 377 and 130 bp for Trichinella T6; two bands of approximately 537 and 377 bp for T. nativa (T2); two bands of approximately 507 and 407 bp for T. chanchalensis (T13). In each PCR-RFLP run, positive (T2, T13 and T6) and negative controls were included.

Figures

Fig. 2. Possible shapes of Trichinella spp. larvae after digestion showing Cshape (upper left), tightly coiled (middle), and lightly coiled (lower right). Scale bar as shown.