Effect of lysate of Sarcocystis gigantea in rats

This study was conducted to examine the effect of lysate isolated from macrosarcocysts of Sarcocystis gigantea after inoculation into albino rats to follow up the pathological effect on heart, lung and skeletal muscle fibers. The results showed the presence of pathological changes, represented by hyperatrophy, congestion, associated with hyaline degeneration in myocardium of heart. In lung tissues severe congestion in each right and left lobes concomitant with emphysema, thickening was observed in wall of branchioles and blood vessels at 3-7 days have been seen. In skeletal muscles the result revealed the presence of multinecrotic areas which infiltrated with inflammatory cells accompanied by hyaline degeneration in muscle fibers at 3-7 days after treatment.


Introduction
The sexual reproduction of the S. gigantea occurs in the intermediate hosts (sheep, goats, cattle or horses), eventually forming cysts in the skeletal muscle (Sarcocysts) that are filled with bradyzoites.Lysates is a bradyzoites extract of a sexual stages of Sarcocystis spp.are toxic when injected into various mammalian hosts such as rabbits and rats (1).The substances imparting toxicity have not been chemically defined and their mechanism of action, a lysate of S. cruzi bradyzoites was shown to possess lectin activity separable from toxin activity (2).The lysate produced a reaction in rabbit similar to that produced by bacterial endotoxin, which have the ability to stimulate macrophages to produce the monokine called cachectin or tumor necrosis factor TNF, the TNF has been implicated as a cause of a profound wasting, loss of body weight, emaciation, this effect results from the action of TNF-α or other cytokines, either directly suppressing insulin-like growth factor 1 (1GF-1) in various tissue or acting indirectly via reduced feed intake or by action on the pituitary gland (3,4).Lysates which has been regarded as the cause of eosinophilic myositis (EM) syndrome, which has been described as a subacute to chronic inflammatory process of skeletal and cardiac muscle particulate and soluble antigen from schizont and merozoites may attract the large number of cells observed perivascularly and interstitially (5).
The present study was to explored the effect of lysate which was isolated from S. gigantea on weight and pathology in albino rats.

Materials and methods
Twenty male 4-5 weeks old albino rat were used, they were divided into two equal groups.Group 1 (G1) is the control group (not inoculated with lysate), Group 2 (G2) was inoculated, intraperitonealy (IP) with lysate of S. gigantea which was prepared according to (2), each rat recieved about 1 ml the lysate contained 1 mg total protein per ml, these animal were revealed in laberatory house of Vet.Medicine.Rats weight obtained before and after inoculation during 3-7 days, rats were sacrified 3-7 days post inoculation to determine the gross pathological changes and speciemens were taken from lung, heart and skeletal muscle and fixed in 10% neutral formaline, processed routinely by ethyl alcohol and xylol and 4-6µm thick sections were prepared and stained with hematoxylin -eosin stain (H&E) (6).

Animal weight
Table (1) showed reduction in weight of rats treated with lysate after 3 days in G2 as compared with control group.This reduction continuously at seven days the mean of weight gains reached 181.2 gm as compared with control rats which reached to 204.2 gm.

Gross pathological finding Macroscopic findings
After 3 days of inoculation of lysate into rats, gross pathological change which occur characterized by: In lung, lesions represented by severe congestion with some areas of gray hepatization in both right and left lobes.These lesions continuously at 7 days after inoculation.Heart, showed cardiomegaly at 3-7 days of inoculation.Skeletal muscles revealed multifocal discrete 2 mm-5 cm in diameter, greenish to yellowish nodules within the affected muscles.

Histopathological findings
Histological sections of lungs rat treated with lysate at 3 days showed multifocal areas of necrosis infiltrated with neutrophils and macrophags and some eosinophils in addition to hemorrhage, thickening in wall of alveoli and emphysema (Fig. 1 and 2).Also at seven days the lesions that mentioned above are seen, in addition to thickening in wall of bronchiol, hyperplasia in lymph node around bronchi as well as accumulation of inflammatory exudate in lumen of bronchi (Fig. 3) pulmonary arteries, revealed hypertrophy of endothelium and thickening in wall due to proliferation in vascular smooth muscle cells in the media (Fig. 4), congestion of blood vessels and atelechtasis also noticed (Fig. 5).Histopathological section of heart revealed congestion of blood vessels, vacuolar degeneration of myocyte associated with infiltration of inflammatory cells after 3 days (Fig. 6), while after 7 days edema and hyaline degeneration have been seen (Fig. 7).Histopathological sections of skeletal muscles showed lesion, characterized by presence of multifocal necrosis each one infiltrated by inflammatory cells represent by eosinophils and neutrophils in addition to edema and hyaline degeneration of myofibriles at 3-7 days post inoculation (Fig. 8 and 9).

Discussion
The significant reduction in weight of rat inoculated with lysate isolated from S. gigantea, was very close to (7,8) who reported that lysate have ability to release TNF-α which is responsible for the occurance of anorexia and cachexia, it has been observed that effect of lysate of the sarcocysts bradyzoite resembles to the acute phase of sarcocystosis in calves (9), they reported that those result from interleukin-1(IL-1) suppression of food intake and reduction of gastric motility (10) as well as reduced gut motility possibly due to elevated somatostatin (SSN) levels (11), elevated levels of plasma glycerol and free fatty acids may also contribute to decreased appetite, 1L-1, through the stimulation of lymphocytes to produce 1L-2, stimulates adipose tissue lipolysis ; TNF-α by suppressing synthesis of lipoprotein lipase, also contributes to adipocyte lipolysis, also it may be correlated with abnormal nitrogen metabolism that appear to result from the malabsorption and internal recycling of nitrogen lysate (acute sarcocystosis) associated with skeletal muscle wasting resulting from muscle protein degradation to produce amino acids needed during the acute phase response.TNF-α also blocks myogenesis, preventing skeletal muscle from competing for amino acids and ultimately inhibiting muscle growth (4).
Pathologically, the result demonstrated the occurance of stages of pneumonia in lung in addition to congestion of blood vessels and thickening in wall, this indicated the effect of lysate to stimulate the release of chemical mediator which lead to proliferation of vascular smooth muscle cell this result agreed with (12,13).
The toxin may effect on host's immune response that lead to changes in the concentration of cytokine signal TNF-α, 1L-6 and 1L-1 that secreted from macrophages (14).In heart the results illustrated cardiomegaly, fatty change in addition to lymphocytic myocarditis, the result indicate that lysate of S. gigantea bradyzoites induced releasing of oxygen free radical which responsible of lipid peroxidation represented by fatty change apperance, and (15) refers to TNF-α during acute phase of sarcocystosis coincided with a reduction in the number of circulating leukocytes and massive infiltration of mononuclear cells, especially macrophages and lymphocytes, into various tissues, and this result agreed with (16,17).
Results of skeletal muscles indicated the presence of eosinophilic myositis associated with necrosis, this results correlated with previous study (18)(19)(20)(21) and also agreed with the results of (1,22) who refered to the increases in the level of alanine amino transferase ALT to ensure the muscle destruction due to ability of lysate to stimulate the chemical mediators to induse this lesion.The pathogenesis of EM varies; a correlation between serum IL-5 levels and disease severity (23) suggests a role for this interleukin species in eosinophil activation.
The pathological consequences resulting from overproduction of cytokines TNF-α leading to a cascade of shock-like symptoms that could effect the survival of the hosts.

Fig. 4 :
Fig. 4: Histomicrograph of rats lung inoculated with lysate isolated from S. gigantea after 7 days, revealed thickening in the wall of pulmonary blood vessels (a) with congestion (b).H&E.400X.

Fig. 7 :
Fig. 7: Histomicrograph of rats heart inoculated with lysate isolated from S. gigantea after 7 days showed infiltration of inflammatory cells (a) and thickening in the wall of blood vessels (b).H&E.400X.

Table 1 :
Effect of lysate on rat weight via three and seven days.