Identification of Sarcocystis and Trichinella Species in Muscles of Gray Wolf (Canis lupus) from Lithuania

Abstract

Apicomplexan Sarcocystis and Trichinella nematodes are food-borne parasites whose life cycle is carried-out in various wildlife and domestic animals. The gray wolf (Canis lupus) is an apex predator acting as an ecosystem engineer. This study aimed to identify the species of Sarcocystis and Trichinella found in the muscles of gray wolves in Lithuania. During the 2017-2022 period, diaphragm, heart, and hind leg samples of 15 animals were examined. Microscopical analysis showed the presence of two types of Sarcocystis parasites in 26.7% of the analyzed muscle samples. Based on the sequencing of five loci, nuclear 18S rDNA, 28S rDNA, ITS1, mitochondrial cox1, and apicoplast rpoB, S. arctica, and S. svanai were identified. The current work presents the first report of S. svanai in gray wolf. Phylogenetically, S. svanai clustered together with S. lutrae, infecting various carnivorans, and S. arctica was most closely related to S. felis from domestic cats. Trichinella spp. were found in 12 gray wolves (80%). For the first time, Trichinella species were molecularly identified in gray wolves from Lithuania. Trichinella britovi was confirmed in all of the isolated Trichinella larvae using a multiplex PCR. Gray wolves in Lithuania may serve as a major source of zoonotic pathogens due to the presence of these parasites.

Keywords: Sarcocystis; Trichinella; gray wolf; host-specificity; molecular identification; phylogeny.

Figure 1

Sarcocystis spp. and Trichinella sp. in the gray wolf in Lithuania. The filled diamond shape represents positive individuals, and the empty diamond shape represents negative individuals.

Figure 2

Morphology of Sarcocystis svanai from muscle tissue of a gray wolf. Light micrographs. Fresh preparations (a–c). A portion of the ribbon-shaped sarcocyst (shown by arrows) (a), thin cyst wall (arrows) (b), lancet-shaped bradyzoites (c).

Figure 3

Sarcocyst remnants from muscle tissue of the gray wolf (shown by arrows). Light micrographs (a–c). The elongated shape of sarcocyst-like bradyzoite nodules from limb muscle (a), the nodules of bradyzoites from the diaphragm (b), magnified image of lancet-shaped bradyzoites visible in the nodules of bradyzoites (c).

Figure 4

The phylogenetic placement of S. arctica and S. svanai isolated from gray wolves in Lithuania on the basis of 18S rDNA (a), cox1 (b), rpob (c), 28S rDNA (d), and ITS1 (e). Phylogenetic trees were constructed using Maximum Likelihood method, scaled according to branch length and rooted on Toxoplasma gondii. The dashed line points out that its length does not correspond to the evolutionary distance. Figures next to branches display bootstrap support values. Sequences obtained in the present work are highlighted in indigo. Red rectangles show Sarcocystis species using members of order Carnivora as their intermediate hosts. Sarcocystis species employing birds in their life cycle are indicated with purple rectangles and Sarcocystis species cycling between rodents and birds are indicated with green rectangles.

Figure 5

Morphological and molecular examination of Trichinella spp. in gray wolf. (a) Trichinella spp. larvae found by the method of artificial digestion in the diaphragm. (b) Agarose gel electrophoresis (2%) of multiplex PCR of Trichinella britovi in 5 larvae. M—“GeneRuler Low Range DNA Ladder” molecular weight marker, sizes are in base pairs, positive controls of: Ts—T. spiralis, Tna—T. nativa, Tb—T. britovi, Tps—T. pseudospiralis, 1–5—individual larvae from one sample, and N—Negative control.