Fate of macrosarcocyst of Sarcocystis gigantea in sheep

This study was conducted to detect the fate of macrosarcocysts of Sarcocystis gigantea in the tongue and eosophagus of naturally infected sheep, via collection of 25 samples, 10 of which showed calcification. The results showed presence of white different size grains on the wall of the pale eosophagus, in addition to presence of nodules containing white chalky materials and on cutting by knife produced grunting sound which indicated calcification. Histopathological results showed presence of granulomatous nodules that contained necrotic centers infiltration by inflammatory cells. Some of which were free from zoites in addition to presence of calcium salt precipitation, which represented dystrophic calcification. Eosinophilic myositis appeared in the tongue was associated with ruptured cyst and released zoites in muscular tissue. Some histological sections revealed ruptured macrocystis with thin wall deposited between muscle bundles. In conclusion, this study showed that the fate of macrocysts included the formation of granulomatous nodules associated with dystrophic calcification and dead zoites in eosophagous more than that in the tongue.

Abattoir surveys have recorded infections by macroscopic cyst of S. gigantea in 94.8% and 100% of ovine examined in Mosul (2,3).Infections by macroscopic cyst have been shown to be transmitted by cat (4).The mature sarcocysts are surrounded by primary cyst wall, which derives from the original membrane of the parasitophorous vacuole and it is covered by host cell material.This layer is called secondary cyst wall, or may be also formed by defense cells of the host and may later induce the calcification of the muscle fiber containing the parasite (5).
The aim of this study was to exam the fate of the sarcocysts of S. gigantea in eosophagus and tongue in naturally infected ovine.

Materials and methods
Twenty five samples of sheep eosophagus and tongue naturally infected with macrosarcocysts of S. gigantea were collected from Mosul abattoir.Ten of them were calcified, their grunting sound was heared after cutting by knife, then fixed in 10% neutral buffered formalin, then after 48-72 hr dehydrated by ethyl alcohol 70-100%, cleaning by xylol and embedded in paraffin wax, to prepare blocks, thus by microtomes cutting section at 4-6 μm thickness, stained with H&E (6) other's staining with Von gossa's and Al-Zarine stain (7).

Results
Samples of infected eosophagus and tongue showed white rice-grain sized nodules, nodules including calcified cheesy content and after cutting by knife grunting sound was heard (Fig. 1) other samples showed pale easophagus with round calcified nodules (Fig. 2).Histopathological sections of the tongue showed presence of different size of elongated sarcocysts between muscle bundles each one surrounded by thin layer of muscle fibers which contain numerous bradyzoites.Some of these macrosarcocysts ruptured and released merozoites (bradyzoites), area of which infilterated by inflammatory cells represented by eosinophils (Fig. 3-5), some other sections revealed necrosis and dystrophic calcification in additions to degeneration of muscle fibers (Fig. 6).In eosophageal sarcosporidiosis, histopathologically, revealed typical granuloma, calcified myofibers were locally surrounded by macrophage and numerous inflammatory cells and multinucleated giant cells (Fig. 7,8).In some lesions massive fibrous septa with hyaline degeneration, sarcoplasma fragmentation and mastolysis were observed (Fig. 9), some other sections showed thin cyst wall and necrotic area with dystrophic calcification in muscle bundles.Some macrocysts showed thin cyst wall present between muscle fibers (Fig. 10, 11) which was more evidence in eosophagus than in tongue.

Discussion
The presence of a Sarcocystis spp.Infection as cysts in muscle termed sarcosporidiosis, is differentiated from the clinical syndrome expressed prior to cyst formation,  referred to a sarcocystosis (8).The S. gigantea is very common in Iraq (9), this high prevalence suggests that either sheep are reinfected continuously or that sarcocysts persist for several years in sheep, reinfection is more likely to be the case because the environment is highly contaminated with sporocyst (10) and the severity of natural field infections may therefore be limited by certain host/parasite interaction.The results of this study showed presence of macrosarcocysts of S. gigantea in different size in muscle fibers of tongue and eosophagus (Fig. 1) (1).These results indicated that infection was in chronic form, in addition to color and size confirmed the chronicity and activity of the cyst.Histopathological changes in ovine  tongue infected with macrosarcocysts of S. gigantean showed presence of macrocysts between muscle bundles some of which were ruptured, released bradyzoite concomitant with eosinophilic response in addition to presence dead bradyzoites associated with dystrophic calcification.The results were observed indicated the presence of heavy and chronic infection with Sarcocystis spp.(11).
The present investigation provides evidence that chronic infection with S. gigantea induce body response in the intermediate host, namely increase the macrosarcocysts in Fig. 10: Histological section of eosophagus infected with macrosarcocysts, of S.gigantea showed thin cyst wall of macrosarcocysts in muscular layer (arrow) (H and E stain 90X).sized (12).Evidence of ruptured cyst was similar to previous study (13) who reported that some sarcocysts probably rupture from time to time and thus the antigenic stimulus for antibodies production smaintained.Sarcocysts and enclosed organism are removed by leukocytes an area occupied by sarcocysts may be mineralized.The muscle cyst are prone to become ruptured due to enlargement, after rupturing of the cyst, some liberated large zoites, may become immobilized after binding with the apical complex associated antibody in the immunized host and change to round form (Fig. 2) (14).Binding of the surface membrane and cytoplasmic granules to the corresponding antibody possibly leads to attracks by the eosinophils and lymphocytes on the exposing aspect with some zoites escaping the killing effect, further binding with this antibody intermixed with inflammatory cell debris (15).
Muscle degeneration, myolysis, necrosis and dystrophic calcification other lesions described in our study are consistent with lesions found previously in tongue, oesophagus and skeletal muscle (16) who reported that muscle degeneration and necrosis are common sequelae to myofiber injury regardless of its causes (chemical, metabolic, traumatic and infections) myofiber degeneration can be reversible, if injury progresses beyond the point of no return degeneration become irreversible and necrosis will follow muscle appear pale (Fig. 2).If the calcification is extensive and severe muscle show glistening, white chalkey foci, that observed as granting sound, easinophilic infilteration, hyaline degeneration of muscle fiber, fiber necrosis, dystrophic calcification and sarcosporidiosis, these parameters were increasly detectable along with growing age of animals (17).Evidence of granulomatous reactions and dystrophic calcification in eosophagus were observed in 10 samples, these lesions were correspond with those found previously (18,19), they reported that sarcocysts in granulomas were open and degenerating, whereas those in normal myofiber were infect and non reactive.Other explanations for granulomas lesions include immunolobic responses, such as type I hypersensitivity, type II (cytotoxic reactions), and type IV (cell mediated immunity).Each of these mechanisms has some or all of the component necessary for development of these lesions.In the present study granulomas, dystrophic calcification and ruptured cyst were observed in (Fig. 11).
Conclusion of these results indicates that the fate of sarcocysts are formation of granulomatous concomitant with dystrophic calcification and dead of trophozoite in eosophagus more than in the tongue.