Molecular identification of meat samples
The 14 meat samples (12 for donkeys and 2 for horses) infected with sarcocysts were molecularly identified based on mitochondrial cox1 sequences. The 14 newly obtained cox1 sequences were approximately 1350 bp long and shared 91.3–92.5% identity between donkeys and horses. At this locus, the 12 newly obtained nucleotide sequences from donkey meat and the 2 newly obtained nucleotide sequences from horse meat shared 98.7-99.7% and 98.4-99.9% identity, respectively, with those of E. asinus and E. cabullus previously deposited in GenBank.
Morphological observation of sarcocysts in donkeys
Sarcocysts were found in 12 of 32 (37.5%) donkeys. Using LM, the sarcocysts could be divided into two forms: thin-walled and thick-walled (Fig. 1a, c). The thin-walled sarcocysts were macroscopic, measuring 2350–4856 × 110–320 μm [average = 2787 (± 442) × 210 (±64) μm, (±SD); n = 20 isolated from five donkeys] in size, and exhibited numerous short club-like protrusions with lengths of 2.0 to 2.7 μm [mean = 2.4 ± 0.18 μm; n = 20 measurements from 10 sarcocysts); they were septate and contained bradyzoites measuring 14.5–17.4 × 3.5–5.0 μm [average =15.8 (±1.8) × 4.2 (±0.4) μm, n = 20 measurements from three sarcocysts) in size. The thick-walled sarcocysts were microscopic, measuring 1200–3750 × 45–135 μm [average = 2213 (± 126) × 98 (±21) μm, n = 20 isolated from four donkeys] in size and showed hair-like protrusions with lengths of 3.0 to 5.4 μm [mean = 4.2 (±0.25) μm, n = 20 measurements from 10 sarcocysts]; they were septate and contained bradyzoites measuring 12.1–16.2 × 2.5–4.7 μm [average = 14.5 (± 1.2) × 4.1 (±0.3) μm, n = 20 measurements from five sarcocysts) in size.
The ultrastructures of the two forms of sarcocysts exhibited similar morphological characteristics (Fig. 1 b, d): the primary cyst wall had numerous villus protrusions with bundled microtubules in the core, which penetrated diagonally into the ground substances and sometimes reached the interior border of the ground substance. Minute undulations were present over the entire sarcocyst surface. A layer of ground substance was present beneath the protrusions. Overall, the cyst wall type was similar to type 11 classified by Dubey et al. (2016).
Molecular characterization of sarcocysts in donkeys
The three selected genes (18S rDNA, 28S rDNA and mitochondrial cox1) were successfully amplified from six individual sarcocysts (three thin-walled cysts and three thick-wall cysts) isolated from four donkeys. The three 18S rDNA sequences (accession numbers OM971696–OM971698) of the thin-walled sarcocysts were 1591–1614 bp long and shared 97.7–99.8% identity (average 98.4%). The three 18S rDNA sequences (OM971699–OM971701) of the thick-walled sarcocysts were 1589–1607 bp long and shared 97.7–98.5% identity (average 98.2%). The similarity between the two forms was 97.2–99.5% (average 97.8%). The most similar sequences in GenBank to the newly obtained 18S rDNA sequences were those of Sarcocystis spp. obtained from donkeys and horses in different regions, including S. cf. bertrami (KX545381–KX545396) from Chinese donkeys (93.5–97.5% identity, average 96.6%), S. bertrami (MH025625−MH025628 and KX545397−KX545404) from Chinese horses (94.0–97.5% identity, average 96.3%), S. fayeri (LC171838) from an Italian horse (95.2−97.1%, average 96.3), S. fayeri (AB661437−AB661447) from Japanese horses (90.8−97.4% identity, average 94.7%), S. fayeri (AB972440−AB972443 and LC171831−LC171837) from Canadian horses (90.0−97.1% identity, average 94.1%), and S. fayeri (MF614956) from an Egyptian horse (93.7−93.8%, average 93.8%).
The three 28S rDNA sequences (OM971683−OM971685) obtained from thin-walled sarcocysts were 3441−3450 bp length and shared 97.7−98.5 identity (average 98.1%). Only two 28S rDNA sequences (OM971686 and OM971687) of thick-walled sarcocysts were successfully assembled. They were 3445 and 3446 bp in length and shared 98.8% identity. The similarity between the two forms was 97.8-99.6% (average 98.4%). The most similar sequences were those of S. bertrami (MH025629−MH025630) from Chinese horses (94.7−95.1% identity, average 94.9%), followed by those of S. suihominis (MK867471−MK867473) obtained from domestic pigs (90.0−91.2% identity, average 90.7%).
The three mitochondrial cox1 sequences (OM970235−OM970237) of thin-walled sarcocysts were 1085 bp in length and shared 99.2−99.7 identity (average 99.4%). The three mitochondrial cox1 sequences (OM970238−OM970240) of thick-walled sarcocysts were 1085 bp in length and shared 99.2−99.3% identity (average 99.3%). The identity between the two forms was 99.0−99.9% (average 99.4%). The most similar sequence in GenBank was that of Sarcocystis sp. (KY399759) obtained from a Chinese donkey (98.6−99.4% identity, average 98.8%), followed by those of Sarcocystis spp. obtained from horses and donkeys in different regions, including S. fayeri (LC171840−LC171854) from Canadian horses (82.8−84.5% identity, average 83.9%), S. bertrami (MH025631−MH025633, KY399751−KY399755, KY399758, KY399760−KY399762, and MF152616−MF152619) from Chinese horses (82.6−84.5% identity, average 83.6%), S. fayeri (LC171857) from an Italian horse (83.1-83.8% identity, average 83.4%), S. cf. bertrami (KY399747−KY399750, KY399756, KY399757, MF152613−MF152615) from Chinese donkeys (83.1−83.8% identity with an average of 83.4%), and S. fayeri (LC171855 and LC171856) from Japanese horses (82.7−83.4% identity, average 83.1%).
PCR-RFLP based on mitochondrial cox1 obtained from donkey and horse sarcocysts
The PCR-amplified products of mitochondrial cox1 from donkey and horse sarcocysts were successfully digested by EcorRI and HinifI. This produced three fragments (196, 243 and 641 bp) and two fragments (416 and 644 bp) for donkey and horse, respectively (Fig. 2).
Based on the divergence of the 18S rDNA, 28S rDNA and mitochondrial cox1 sequences of donkey and horse sarcocysts, the Sarcocystis parasites of donkeys are different from those of horses. Hence, the name S. asinus needs to be resurrected for the sarcocysts of donkeys.
Phylogenetic analysis based on the 18S rDNA and mitochondrial cox1 sequences of S. asinus sarocysts confirmed their association with Sarcocystis species. In the tree inferred from 18S rDNA sequences (Fig. 3), S. asinus formed an individual clade within a group comprising Sarcocystis spp. obtained from horses and donkeys originating from different areas, including S. bertrami/fayeri (LC171835, KX545399, MH025625, LC171838) from horses and S. ef. bertrami (KX545386) from a donkey. In the tree inferred form mitochondrial cox1 sequences (Fig. 4), the newly obtained mitochondrial cox1 sequences of S. asinus formed an individual clade with Sarcocystis sp. (KY399759) obtained from a donkey within a group comprising Sarcocystis spp. from both horses and donkeys, including S. bertrami/fayeri (MH02563, LC171856, LC171857, and LC171850) from horses and S. cf. bertrami from a donkey (KY399749).