Sarcocystis is a common parasitic protozoan with a worldwide distribution found in a variety of mammals and birds, especially in domesticated food animals. Sarcocysts have been diagnosed in donkeys from the former USSR (Gadaev 1978), Austria (Hinaidy and Loupal 1982), Germany (Matuschka 1983), Morocco (Kirmse 1986), Egypt (Hilali and Nasser 1987; Dubey et al. 2016), China (Hu et al. 2001), and Italy (Passantino et al. 2019). In the present study, the prevalence of sarcocysts in the investigated Chinese donkeys was 37.5% (12/32), and it has been reported to be higher than 22.0% (9/41) in Moroccan donkeys (Kirmse 1986) and 28.6% (40/140) in Italian donkeys (Passantino et al. 2019), but lower than 40% (8/20) in the former USSR (Gadaev 1978), 90.0% (18/20) in Egyptian donkeys (Hilali and Nasser 1987) and 92.3% (24/26) in Chinese donkeys surveyed by our group 20 years ago (Hu et al. 2001).
In the present study, two types of sarcocysts (thin-walled cysts and thick-walled cysts) were observed in the muscle tissues of donkeys under LM. The thin-walled sarcocysts were macroscopic (up to 4856 long and 320 µm wide) and had short club-like protrusions (up to 2.7 µm long); the thick-walled sarcocysts were microscopic (up to 3750 µm long and 135 µm wide) and had villar protrusions (up to 5.4 µm long). The two types of sarcocysts were probably associated with the length of development. Fayer et al. (1983) experimentally infected ponies with sporocysts collected from dogs that had been fed horsemeat containing visible sarcocysts. On Day 127 post-infection (PI), the sarocysts were still immature (metrocytes) and measured 12.6–30 × 50–360 µm. some walls had long protrusions (4.5 µm), and others showed short protrusions (1 to 2 µm). However, on Days 157 and 184 PI, all sarcocysts were mature (bradyzoites) and up to 436.6 µm long. They only had short protrusions. Matuschka et al. (1986) performed a similar experimental infection and observed microscopic sarcocysts (< 1 mm long) and macroscopic sarcocysts (up to 2 mm) in ponies on Day 378 PI. However, on Day 1040 PI, only macroscopic sarcocysts were found, with sizes of up to 9 × 0.5 mm.
The ultrastructure of the sarcocyst wall is useful in evaluating the taxonomy of Sarcocystis species in a given host. Dubey et al. (2015) grouped sarcocysts into at least 42 types and several subtypes based on TEM morphological characteristic of sarcocyst wall. In our materials, the ultrastructures of the thin-walled and thick-walled sarcocysts presented characteristics of TEM type 11: the protrusions of the sarcocyst wall contained bundled microtubules in the core of the protrusions penetrated into the ground substance. Based on the inclination of protrusions over the sarcocyst surface, the TEM types of thin-walled and thick-walled sarcocysts could be subdivided into TEM type 11c and 11a. All ultrastructural descriptions of sarcocysts obtained from donkeys provided by different authors to date conform to the characteristics of TEM type 11 (Hilaili and Nasser 1987; Dubey et al. 2016; Passantino et al. 2019). Among them, sarcocysts from Egyptian donkeys are similar to TEM type 11c (Hilaili and Nasser 1987, Dubey et al. 2016), and those from Italian donkeys are similar to TEM type 11a (Passantino et al. 2019). These sarcocysts were named as Sarcocystis sp. (Hilali and Nasser, 1987), or S. bertrami (Dubey et al. 2016, Passantino et al. 2019).
Morphologically similar sarcocysts frequently occur in different hosts, especially in closely related hosts, as observed in the sibling species S. tenella and S. capracanis found in sheep and goats, respectively, which sometimes creates controversy regarding species identification (Formisano et al. 2012; Dubey and Rosenthal 2013). Currently, PCR assays and sequencing procedures are considered much more practical, accurate, and reliable for the delineation and identification of Sarcocystis species than traditional methods based on morphological characteristics (Gjerde 2013). Therefore, a critical comparison of the molecular characteristics of Sarcocystis species in donkeys and horses should be performed to help reach a final conclusion (Dubey et al. 2016).
In the present study, three genetic markers, 18S rDNA, 28S rDNA, and mitochondrial cox1, were sequenced and analyzed in the two types of sarcocysts found in donkeys. The sequences of the three loci in the two types presented high intraspecific similarities of 97.2 − 99.5% (on average 97.8%), 97.8 − 99.6% (on average 98.4%) and 99.0 − 99.9% (on 99.4%), respectively. Therefore, combined with the similar morphological features observed under TEM, the two types of sracocysts observed in donkeys are inferred to represent a same Sarcocystis species. The comparison of the newly obtained 18S rDNA, 28S rDNA and mitochondrial cox1 sequences with those deposited in GenBank showed identities of 90.0 − 97.5% (average 94.7%), 94.7 − 95.1% (average 94.9%) and 82.6 − 84.5% (average 83.4%), respectively, with those of S. bertrami and S. fayeri obtained from horses. Additionally, the difference between the donkey and horse sarcocysts was proven by PCR-RFLP based on the mitochondrial cox1 sequences of the two parasites. Therefore, the sarcocysts of donkeys should not belong to the species S. bertramior or S. fayeri found in horses.
Cross-infection is a criterion for revealing whether different intermediate hosts harbor the same parasite. To date, there has been only one reported attempt to perform the cross-infection of Sarcocystis between donkey and horse (Matuschka 1983). Tissues from 20 horses naturally infected with sarcocysts were fed to a dog, and those of 10 donkeys were fed to another dog. Both dogs excreted sporocysts. Experimental infections were carried out in 4 ponies (#1–4). One pony (#1) fed donkey-derived sporocysts became febrile on Days 10 and 11 and 19 − 21, but no sarcocysts were detected on Days 44 and 59 PI. The same pony was then fed horse-derived sporocysts on Day 117 and killed on Day 138 PI. Only immature sarcocysts were identified in the carcass. The other three ponies (#2 − 4) were fed horse-derived sporocysts and killed on Days 197, 212, and 21 PI, respectively. Mature sarcocysts were detected in ponies #2 and 3, but no sarcocysts were found in pony #4. These results suggest the possible transmission of the parasite in horses and donkeys. However, Fayer and Dubey (1982) found immature sarcocysts (9.2 × 14.4 µm in sizes) on Day 55 PI and mature sarcocysts (35.6 × 81.8 µm in sizes) on Day 77 PI in horses infected with horse-derived sporocysts. Therefore, the experiments performed by Matuschka (1983) are insufficient to support the cross-infection of Sarcocystis between donkey and horse for reasons including the following: (1) transmission was based only on one pony (#1) fed donkey-derived sporocysts; (2) no mature sarcocysts (on Day 138 PI) were detected in the experimental donkey; and (3) immature sarcocysts (on Day 138 PI) were detected in the experimental donkey, however this animal was infected twice, first with donkey-derived sporocysts and second with horse-derived sporocysts (on Day 117 PI). Therefore, the cross-infection of Sarcocystis between donkey and horse should be attempted, and the available molecular evidence also needs to be supplemented in the future.
In summary, two types of sarcocysts were observed in donkeys in China. Based on morphological traits and analysis of the three genetic markers, the two types are attributed to one parasite. Compared the newly obtained sequences with those of S. bertrami and S. fayeri in horses previously deposited in GenBank, the parasite in donkeys are distinct from S. bertrami and S. fayeri in horses. Gadaev (1978) first proposed the name S. asinus for the sarcocysts found in donkeys, but few cyst morphological characteristics and no molecular data provided. Considering the arguments of Sarcocystis spp. in horse and other equids (Dubey et al., 2015), here, Sarcocystis sp. was used to name the sarcocysts found in donkeys to avoid increasing uncertainty.