Molecular Survey on Sarcocystis Species in Slaughtered Sheep in Hamedan, Iran

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Introduction
Sarcocystosis is a global parasitic disease of the musculoskeletal system in various animals, such as mammals, birds, reptiles as well as humansˡ. Sarcocystis spp. is an obligatory intracellular heteroxenous parasite, circulating among definitive carnivorous hosts (canids and felids) as predators and herbivorous intermediate hosts, such as cattle, sheep, goat, buffalo, and camel as preyˡ. Sexual reproduction occurs in definitive hosts, shedding sporocysts into the environment versus feces. In contrast, asexual reproduction and schizogony occur in striated and heart muscles of intermediate hosts, leading to Sarcocystis formationˡ. Hence, ingesting food/water contaminated with sporocysts and devouring carcasses infected with sarcocyst are the main routes of transmission in intermediate and definitive hosts, respectively 2 . In this sense, consumption of highly infected meat from food animals is not suitable for humans 2 .
The most widespread Sarcocystis species include Sarcocystis tenella (S. tenella), S. arieticanis, S. medusiformis, and S. gigantea (ovifelis) in sheep as well as S. hominis (bovihominis), S. cruzi (bovicanis) and S. hirsute (bovifelis) in cattle. Sarcocystis infections in the definitive hosts are frequently asymptomatic, while they may inflict mild disease by S. gigantea in sheep, to acute disease by S. cruzi in cattle and S. capracanis in goat 3 . Cellular degeneration, hemorrhage, and inflammatory lesions are the most observed pathologies induced by the parasite 4 . Invasion of the nervous system is rare, except for S. neurona, which develops the nervous disease in some animal species 4 . In pregnant sheep, the acute disease may result in abortion, fetal death, and still birth 5 . The most prominent pathologic traits of the infection develop following merozoite release in the circulation 6 . Generally, the parasite undergoes two merogony cycles. The first-generation meronts are formed 1-2 weeks upon infection in endothelial cells of blood vessels in most body organs, and the second-generation meronts emerge 3-5 weeks post-infection, leading to systemic intravascular coagulation, perivascular inflammation, necrosis, and hemorrhage 7 . Fever directly correlates to the rate of parasitemia, resulting in anemia due to the constant bleeding related to extravascular hemolysis on an immunologic basis. Animal death occurs after ingestion of a huge number of sporocysts, which usually coincides with second-stage merogony 7 . Sarcocystis species involving canid hosts may result in fetal death and abortion in infected, non-immune animals. Nevertheless, no exact underlying mechanism is determined for Sarcocystis-induced abortion, and the parasite has been rarely isolated from the infected fetus and placenta under experimental conditions 8 .
Diagnosis of the infection in livestock depends on several approaches, including abattoir-based macroscopic examination of infected organs in animal carcasses, microscopic observation of tissue cysts using digestion and/or stamp method as well as trichinoscopy, along with immunohistochemical and molecular techniques 9 . Bradyzoites of various Sarcocystis spp. released by the digestion method could not be differentiated due to the morphological similarities in the bradyzoite's shape 9 . Several serological-based methods are available for detecting sarcocystosis in sheep such as enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT), with significant sensitivity in the late phase of infection 10 . However, discrimination between sheep pathogenic and non-pathogenic Sarcocystis spp. is impossible using such methods 10 . With the advent of molecular techniques, species identification of parasitic agents has been facilitated, which provides a deeper understanding of Sarcocystis epidemiology 11 .
In Iran, the prevalence rates of Sarcocystis infection among sheep and cattle populations were reported at 80% and 100%, respectively. With this in mind, the present crosssectional study aimed to investigate Sarcocystis prevalence rate and involved species in sheep of Hamedan, western Iran, using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Muscle sampling and microscopic examination
A total of 60 samples were collected. Upon referral to an abattoir, Hamedan, Iran, from May 2019 to June 2020, a total number of 20 (10 male and 10 females with the age range of 1-3 years) sheep infected with macroscopic cysts were sampled, and 50 grams of each muscle of heart, scapular, esophageal, and diaphragmatic muscles were necropsied. A total number of 40 sheep lacking macroscopic cysts were also randomly sampled in a similar manner and all specimens were tagged and transferred on ice to the laboratory of the Veterinary Medicine Department of Sanandaj Islamic Azad University, Sanandaj, Iran.

Digestion method
For microscopic examination, the digestion method was usedˡ 2 . Briefly, a digestion solution (containing 5.2 g pepsin, 10 ml hydrochloric acid, and 100 ml buffered phosphate, Merck Germany) was applied to each muscle specimen. Next, the yielded digesta was passed through a clean piece of textile and then centrifuged (Memmert, Germany) at 1500 rpm for 10 minutes. After supernatant elimination, smears were prepared from the remaining pellet, dried, and fixed with methanol. Finally, smears were stained with diluted Giemsa solution (20-30 minutes) and observed under a light microscope (Olympus, Japan) using 100x magnification. Observation of the bradyzoite stages of the parasite was reported as a positive sample ( Figure 2).

DNA extraction and PCR-RFLP analysis
For the purpose of DNA extraction, muscle samples kept at -20 o C were initially put at the ambient temperature. Next, the procedure of DNA purification was done using the Cinnagen tissue DNA extraction kit (Cinnagen Company, Iran), according to the manufacturer's instructions. The DNA was pelleted using ethanol and stored at -80 o C for further use 13 . In the following Sar-F1 (5 GCA CTT GAT GAA TTC TGG CA 3) and Sar-F2 (5' CAC CAC CCA TAG AAT CAA G3) primer pairs were used for the amplification of the target gene, in the context of a thermal cycle as 94 o C for 5 minutes (initial denaturation), 30 cycles of 94 o C for 2 minutes (denaturation), 57 o C for 30 seconds (annealing), 72 o C for 2 minutes (extension), along with a final extension step at 72 o C for 5 minutes. The final PCR products were  13 . The PCR reaction (total volume = 30 µl) was prepared by mixing 12 µl distilled water (DW), 3 µl X10 buffer, 2 µl 1.75 mM MgCl2, 0.5 µl of each primer (10 pmol), 10 µl template DNA, and 0.5 µl Taq polymerase enzyme. After amplification, 5 µl of PCR product was electrophoresed on 1% agarose gel containing DNA-safe stain, and likely bands were visualized using a GelDoc apparatus 14 .
The EcoR1, AvaI, HindII, and TaqI restriction enzymes were selected for the RFLP analysis. For this aim, 10 µl of PCR product, 2 µl of 10X enzyme buffer, and volumes of 1, 2, and 3 µl of enzymes (8 -10 units) were mixed together with DW to reach the final volume to the 20 µl. Next, incubation time periods of 4, 10, and 16 hours were considered at 37 o C 15 . Finally, all PCR products, either mixed with restriction enzymes or not, were loaded onto the agarose gel wells and ran for 90 minutes at ambient temperature. At last, bands were visualized and captured using a Gel Doc apparatus 12 .

Results
In the current study, a total number of 40 sheep were sampled from heart, scapular, esophageal and diaphragmatic muscles, among which 20 samples contained macroscopic cysts and 20 specimens were contaminated with microscopic cysts. In other words, 100% of examined muscles using the digestion method were infected with Sarcocystis spp. Following amplification of 18S rRNA of Sarcocystis DNA using specific primers, a 609 bp band was observed (Figure 3).
The  (Table 1). All obtained bands following electrophoresis were visualized using a GelDoc apparatus.

Discussion
Previously, sarcocystosis was considered a prevalent, innocuous disease of livestock, rendering inappropriate marketability of the infected carcasses 9 . Ovine sarcocystosis is reported in many countries across the globe with very high prevalence rates ranging from 9 to 100%ˡˡ. Since the study demonstrated a high prevalence of sarcocystosis infection by microscopic and macroscopic cysts forming species, sheep could be considered alternative intermediate hosts for However, in recent years, sarcocystosis has been the focus of researchers as a well-recognized, frequent parasitic disease, as the causative agent of several health consequences in livestock (cattle, goat, sheep, and pig), such as fever, anemia, abortion, encephalomyelitis, lameness, paralysis, decreased milk production, muscle pains and finally mortality 2,8 . Nowadays, molecular techniques, such as PCR and RFLP are utilized for the definitive diagnosis of Sarcocystis spp. in animals and humans. Based on several studies, the variable regions of the highly conserved 18S ribosomal subunit are useful genetic markers for differentiating the species of Sarcocystis in different hosts, such as sheep, however, according to recently performed studies, cox1 is better for Sarcocystis species identification in sheep.
Among 4 species, S. tenella and S. arieticanis produce microscopic cysts and have pathogenic subsequences as acute disease presented by abortion, fever, anemia, and anorexia in the early period of infection, and then some chronic disorders maybe develop. On the other side, macrocyst forming species, S. gigantea, and S. medusiformis are considered nonpathogenic, but they can affect the meat quality and marketing and cause economic loss. Sheep may be infected with these four species simultaneously. This rate of microscopic infection in sheep in this area also indicates a high exposure rate of these animals to the sporocysts, which are shed by dogs and possibly wild animals that can be served as the definitive hosts. It may lead to nausea, stomachache, and diarrhea after eating undercooked or raw meat in humans. Among these species, S. tenella and S. arieticanis are pathogenic and contribute to clinical symptoms. In contrast, S. gigantea and S. medusiformis are nonpathogenic, while they may cause low-quality meat and economic losses 16 . Moreover, there were not any cross-reactions with T. gondii and N. caninum in the PCR results. In the current study, molecular investigation demonstrated that all detected Sarcocystis belonged to S. tenella, S. medusiformis, S. arieticanis, and S. gigantea. In previous similar studies throughout the world, Sarcocystis tenella was reported with different prevalence rates in Mongolia (96.9%), Ethiopia (93%), Turkey (47.3%-86.5%), and Iran (33.9%) 16 . Based on the results of the limited study, S. tenella and S. gigantea are present among the studied sheep in Isfahan province 16 . In Lorestan and Khuzestan provinces, Iran, it has been reported that all Sarcocystis isolated from slaughtered sheep belonged to S. gigantea 16 .
The findings of the current study revealed a high prevalence of microsarcocysts infection in sheep. The low rate of macrosarcocysts in comparison with the microsarcocysts in the examined animals may be due to the rare contact between sheep and cats in the region. It was revealed that the abundance of macroscopic and microscopic Sarcocystis was 4.10% and 97.00% in investigated Iraqi sheep, respectively 6 . The high prevalence rate of microscopic Sarcocystis in sheep indicates the importance of the infection for the intermediate hosts 12 . The sex of examined sheep had a significant effect on the prevalence. However, in another study, it was reported that ewes had a higher prevalence of infection (7%) than males (93%) 8 . In addition, the prevalence of Sarcocystis infection in sheep increased as the age of the animals increased 12 .
The results of the present study showed that sheep samples were contaminated by S. tenella, S. gigantea, and S, ariteicanis. In a study on sheep in northwest Iran, Sarcocystis gigantea was the only species isolated from sheep 14 .
The results of the present study were similar to the previous study as the macroscopic cysts in Ziyaran slaughterhouse belonged to S. gigantea and the microscopic cysts belonged to S. tenella and Ariti canis 17 .
Based on the current results, all specimens contained bradyzoites in the microscopic method, which is consistent with other investigations. For instance, 34.28% prevalence of Sarcocystis infection in slaughtered sheep in Mashhad abattoir using macroscopic observations, and an 81.9% positive samples in histopathology method; the prevalence and 100% in northeastern Iran has been reported 17 . The authors finally declared a considerably high prevalence of the infection among sheep in northeastern Iran. Moreover, S. tenella was the most frequent species in the area.
The first PCR-RFLP analysis in slaughtered sheep in Qazvin province, Iran, remarked that 100% of samples examined by digestion and stamp smear methods were positive, among which 40 specimens contained macroscopic cysts and 20 samples had microscopic cysts. Furthermore, 20 samples were related to S. tenella and S. arieticanis. Moreover, S. gigantea and S. medusiformis were detected in macroscopic analysis 18 .
The genotype investigation of 10 tissue samples indicated that the macroscopic genotype was related to S. gigantea and the microscopic cyst was related to S. tenella 17 . The results of the current study revealed that microscopic cysts are more common than macroscopic cysts and can cause economic losses and cause many health consequences.

Conclusion
In the present study, 100% of samples were infected with bradyzoites using the microscopic method. Moreover, PCR-RFLP analysis indicated the presence of S. gigantea, S. tenella, and S. arieticanis among examined sheep. Pathogen species should be considered as the main agants of economic loss in livestock animals. Therefore, the implementation of further complementary studies is suggested in order to improve public health and enhance better recognition of sarcocystosis epidemiology.