Morphologic characterization of Sarcocystis spp. histologically detected from the diaphragm and tongue of Rattus norvegicus and Rattus tanezumi

This study focused on the detection of Sarcocystis spp. in two species of rats, Rattus norvegicus and R. tanezumi collected from agricultural area in Dasmarinas, Cavite. This aimed to corroborate the presence of Sarcocystis spp. in different species of rats found in the agricultural area. Further the establishment of different Sarcocystis spp in Rattus spp. based on the parasite's morphologic characterization is also emphasized. Sixty-nine rats (36 R. norvegicus and 33 R. tanezumi) were collected through trapping. Individual rodent autopsy for host identification was performed by determining the morphological differences and external measurements prior to dissection. Tissue samples were examined for the presence of white rice-grain sized nodules. Morphological characteristics of the cysts, particularly the size, shape, and presence of protrusions were noted. Statistical analysis using ANOVA for the significant difference on the number of infected rats per species and across muscle type was done at p≤0.05. Infection in R. tanezumi was higher (48.48%) as compared to R. norvegicus (41.67%), the difference however was not significant. Sarcocysts burden was higher in the diaphragm in both rat species. Generally, higher parasite load was observed in R. norvegicus. In both rat species, tissue cysts ranged from spindle-shaped/fusiform to globular and oval-shaped. The sarcocysts in the diaphragm were more varied. Consistently observed in the tongue were globular–shaped sarcocysts while either fusiformand globular-shaped sarcocysts in the diaphragm. These differences suggest infection with more than one species of Sarcocystis. Hence, the presence of Sarcocystis spp. confirmed the infectivity of the parasite to Rattus spp. Moreover, the different morphologic characteristics observed on Sarcocystis prove the possibility of different species of the parasite harboring the Rattus spp.


Introduction
Sarcocystis is a zoonotic protozoan apicomplexan parasite that causes the disease sarcocystosis whose intermediate-definitive host life cycle is attained through a prey-predator relationship (Jeffery et al., 2013;Gokpinar et al., 2014). The parasite reproduces sexually in the gut of the definitive host producing sporocysts containing infective sporozoites (tachyzoites) that are released with the feces and followed by ingestion of the parasite by the intermediate host to invade muscle tissues, thus completing the cycle (Fayer et al., 2015;Ahmed et al., 2016). Humans may serve as definitive hosts in intestinal sarcocystosis caused by Sarcocystis bovihominis (Sarcocystis hominis) and Sarcocystis suihominis with bovine and pigs as intermediate hosts, respectively (Claveria et al., 2001;Ahmed et al., 2016). Intestinal sarcocystosis is transmitted by ingestion of sarcocystscontaminated meat of intermediate hosts. Likewise, humans can serve as intermediate hosts in muscular sarcocystosis involving various final hosts such as cattle, bovine, bubaline, swine and other herbivores, carrying different species of Sarcocystis (Fayer et al., 2015). In muscular sarcocystosis, the parasite is transmitted by feco-oral ingestion of sporocysts by the definitive hosts. Both the muscular or intestinal infections are rarely serious and more of an opportunistic manifestation. There are diverse animal hosts, including swine, birds, snake, rats, cattle, goats and monkeys, recorded in Southeast Asia commonly infected by more than 130 Sarcocystis spp. (Jeffery et al., 2013;Lau et al., 2014). However, sarcocystosis is an uncommon zoonosis of worldwide distribution. In most cases, they have been detected as an incidental finding at autopsy or necropsy. The presence of the parasite in diverse hosts in Southeast Asia is in congruence with the majority cases of the human and animal sarcocystis infections from this part of the world, particularly Malaysia and Thailand (Nichpanit et al., 2010;Latif and Muslim, 2016). In fact, there has been a report of large human outbreaks of sarcocystis infection in Malaysia (Fayer et al., 2015). In the Philippines, Sarcocystis infections have been reported in buffaloes and bovine species . The preliminary report of Sarcocystis was primarily identified in meat of a water buffalo consistent to this finding is the presence of Sarcocystis in the beef sold as ground meat) and muscles of water buffaloes (Claveria et al., 2001). Other hosts that have been infected in the muscles by these protozoans are livestock like hogs, goats and chicken (Claveria et al., 2004). Higher infection has also been observed in muscle tissues of Rattus spp. , one of the important intermediate hosts of this parasite. Sarcocystis spp. infection in both R. norvegicus and R. rattus muscles, particularly in the tongue and diaphragm was noted with thick walled sarcocysts containing numerous zoites. Rats are pestiferous in nature and may be vital in effecting loss of agricultural profit. Because of their close association in human households, agriculture and commercial places, they become successful species to produce in any human dwellings and anthropogenic activities . They are considered efficient organisms and because of such nature, rats can flourish in mixed lands such as agricultural and residential areas. Thus, they are considered as successful species even with the presence of disturbance and human habitations (Salibay and Luyon, 2008). The most common rat species known to survive and inhabit especially near human dwellings and establishments are R. tanezumi and R. norvegicus. Their presence in these areas accounts for the possible transmission of zoonotic diseases like intestinal and muscular sarcocystosis to humans and other animals. This study investigated R. novegicus and R. tanezumi that are known to be invasive species and have close association to different anthropogenic establishments. This study also provides an update on the existing Sarcocystis data in the Philippines, their biology using rats as hosts, specifically R. norvegicus (the domestic rat) and R. tanezumi (the field rat) and their role in the transmission of Sarcocystis in human and animal meat infections.

Collection and identification of rats
Stray rats were collected from agricultural land area in Burol, the Eastern part of Dasmariñas, 3 kilometers away from the city center at 14.3292° N, 120.9439° E. (Figure 1). Trapping of the rats using 20 spring-door wire traps with food bait was done late afternoon, and trapped rats were collected the following morning. In the absence of a rat, traps were left for 2-3 more days as rats may not approach novel objects in their surroundings at the first encounter (Rubio and Asian J Agric & Biol. 2020;8(3):253-259 Sumalde, 1997). During the handling of the captured rats, leather gloves were worn to minimize the chances of being bitten or scratched. Each site was mapped out into subplots to avoid collection of rats at exactly the same plot.

Figure-1. The Agricultural area where Rattus species were collected
Individual rodent autopsy for host identification was performed by determining the morphological differences and external measurements. Verification of pre-identified species was done at the Mammalogy Section, Zoology Division of the National Museum, Manila Philippines. In total, 69 rats (36 R. norvegicus and 33 R. tanezumi) were identified from the collection sites for a period of 12 weeks. Handling of animals is in accordance to DA AO 40 series of 1999 and approved guidelines were adopted from the Philippine Association for Laboratory Animal Science Code of Practice for the Care and Use of Laboratory Animals in the Philippines (PALAS, 2002).

Collection of the infected tissues
The captured rats were individually-caged at the De La Salle University-Dasmariñas (DLSU-D) Laboratory Research Facility and were scheduled for euthanization to harvest their diaphragms and tongues. The captured rats were restrained by an improvised stabilizer covering only the body and limbs prior to euthanization. The rats were euthanized by 150 mg/kg sodium pentobarbital by intraperitoneal route (DA, 1999;PALAS, 2002;Hau and Van Hoosier, 2003;UP-IACUC Guideline, 2016). Confirmation of death was observed with rapid sedation followed by loss of consciousness to ensue death.

Tissue preparation and examination
The protocols Dubey et al. (2015) and of Bittencourt et al. (2016) were followed in the preparation of the tissues. The dislodged tissues of diaphragm and tongue were individually stored in sealed containers and were processed within 24 hours post-collection. Tissue samples were examined for the presence of white rice-grain sized nodules indicative of sarcocystosis. To check for the presence of sarcocysts, thin muscle slices were mounted on glass slides and then teased lightly with fine needles and add a drop of saline solution. Those tissue samples of the diaphragm and tongue suspected to have sarcocysts were fixed in 10% buffered formalin. Paraffin embedding method was done to prepare tissue paraffin blocks. This was further processed using the standard histologic hematoxylin and eosin (H & E) staining technique. One section for each individual muscular tissue was analyzed. A photomicroscope hooked to a computer monitor was used in the examination of tissue sections at the Biology Laboratory, De La Salle University, Dasmarinas, Dasmarinas City, Cavite.

Statistical analysis
Morphological characteristics of the cysts, particularly the size, shape and presence of protrusions were noted. Statistical analysis using ANOVA for the significant difference on the number of infected rats per species and across muscular tissues and the standard deviation was done at p≤ 0.05. To compare the density of sarcocysts in the muscles, the number of the cyst per field of view of the slides (using LPO) was compared per rat species and per type of muscle.

Ethical standards
The procedure was approved by the Ethics Committee of the De La Salle University-Dasmarinas, Philippines (DLSU-DERC-2015-031A).

Results and Discussion
No macroscopic cysts were observed in the tongue or diaphragm of the Rattus species caught in the agricultural areas of Dasmarinas, Cavite, Philippines. However, microscopic examination of the tissues indicated positive for Sarcocystis spp. Of the 69 rats examined, there were more R. tanezumi (48.48%) infected with sarcocysts relative to R. norvegicus (41.67%) ( Table 1). The infection rate between the rat species did not differ significantly. Asian J Agric & Biol. 2020;8(3):253-259 While sarcocysts were detected in the tongue and diaphragm of both rat species, sarcocysts burden was markedly higher in the diaphragm than in the tongue regardless of the rat species (p>0.391) ( Table 2). Some rats of both species harbor the parasites in both tongue and diaphragm. Generally, higher parasite load was observed in R. norvegicus The shape/form and measurements of sarcocysts from spindle-shaped or fusiform to globular to oval-shaped sarcocysts as well the configuration of the sarcocyst wall were variable (Figs. 2,3), suggesting infection with more than one species of Sarcocystis. In both rat species, tissue cysts ranged from spindleshaped/fusiform ( .Notably, the sarcocysts in the diaphragm were more varied ranging from oval, elongated and globular forms. Those in the tongue, sarcocysts were either globular or oval in shape.

Figure 2. Photomicrographs of sarcocysts in the diaphragm muscle of R. norvegicus (A-F) and R. tanezumi (G-J).
Note fusiform, globular and oval shaped cysts, bananashaped bradyzoites (Br), distinct protrusion ( The sarcocysts also display distinct villar protrusions ( Figure 2C, 2F; Figure 3F) and striations ( Figure 2C; Figure 3F) on their cyst walls. Although no measurement of thickness of the wall was taken to quantitatively distinguish them, visual observations showed varying thickness of the wall. Both the thinwalled and thick-walled sarcocysts observed in the two rat species were divided by septa forming a series of internal compartments and appearing in white lines. These sarcocysts contained the banana-shaped zoites of the parasite. In general bigger tissues cysts in the diaphragm and smaller ones in the tongue were observed in both species. The forms of the sarcocysts in the diaphragm and in the tongue suggest that rats are infected with at two species, one with globular-shaped sarcocysts, and the other fusiform-shaped sarcocysts. Consistently observed in the tongue tissues were globular -shaped sarcocysts while either fusiform-and globular-shaped sarcocysts were observed in the diaphragm Many studies worldwide have detected Sarcocystis in domestic animals, such as dogs and cats (Latif and Muslim, 2016), farm animals (Gokpinar et al., 2014;Bittencourt et al., 2016), wildlife  and humans (Nichpanit et al., 2010;Lau et al., 2014;Fayer et al., 2015). The presence of Sarcocystis spp in different species of rats have been recorded by Munday and Mason (1980) in skeletal muscles of rodents that served as intermediate hosts of this parasite. Among the Rattus spp. that harbor Sarcocystis spp. are the Eastern swamp rat (R. lustreolus); Southern bush rat (R. fuscipes) black rat (R. rattus) Norway rat (R. norvegicus); long-tailed rat (Pseudomys higginsi) and broad-toothed rat (Mastacomys fuscus). The findings in the present study, using R. tanezumi and R. norvegicus, confirmed the infectivity of Sarcocystis spp. in animals in the Philippines. The presence of sarcocysts in rats is indicative of the role of rats in the completion of the life cycle of the parasite. Moreover, the role of the rats as a prey of many predatory animals (mammals, reptiles, birds) pave the way for the Sarcocystis spp. to reach their definitive host where their sexual cycle would be completed (Hu et al., 2012). The observation on Sarcocystis distribution in the muscles of rodents in the current study is consistent with the previous reports elsewhere (Hu et al., 2012;Latif and Muslim, 2016), as well as in the Philippines . The detection of both developing and mature sarcocysts indicates the active parasite transmission in the research site. With regard to the species of Sarcocystis in rats, a related study in Thailand (Jäkel et al., 1997) reported six Sarcocystis spp. in the skeletal muscles of wild rodents. Included were S. singaporensis, S. villivillosi, and S. murinotechis, which showed a striated cyst wall at the light microscopic level, while Sarcocystis cymruensis, S. sulawesiensis, and S. zamani possessed smooth cyst walls. In the present study, the presence and absence of striation in some sarcocysts were also observed. Furthermore, the noted morphological variations (e.g. size, shape, and cyst wall) suggest that there are probably several Sarcocystis species present in one host (Latif and Muslim, 2016). Such morphological variations were in congruence with the characteristics of sarcocysts infecting the diaphragm and tongue muscles of the examined rats in the present study. The detection of sarcocysts in the examined rats underscores the rats' role as intermediate hosts in the completion of the parasite's life cycle (Hu et al., 2012). In addition, the presence of these pestiferous animals within the agricultural areas where many farmers and other rural dwellers habituate reinforce the transmission of the parasite to domestic animals and pets (dogs and cats) and farm animals (cattle, water buffaloes, pigs, chicken). Hence, exposure of humans directly to infected rodents, or indirectly through animals infected by these rodents, pose risks of foodborne and waterborne zoonotic sarcocystosis. In this context, it can be said that the presence of sarcocysts-infected rats can possibly pose public health risk due to their proximity to human inhabited areas (Salibay and Luyon, 2008) where humans are likely to be infected opportunistically by Sarcocystis spp. While the current study proved the presence of Sarcocystis spp. in R. norvegicus and R. tanezumi, the identification of the species of Sarcocystis cannot be done by the use of light microscopy alone. The observed variations in the length and width of the cysts in the present study may be related with the age of the cysts (Bittencourt et al., 2016) and cannot be used solely for the identification of the parasite. In addition, the histologic examination by light microscopy may allow the identification of thick wall and thin wall cysts, but they do not permit the differentiation of the species, even using high magnification which limits to 1000x. And since a full view of the cyst surface, which is an essential feature for the characterization of the cysts morphology (Dubey et al., 1989) was not microscopically obtained, this study did not attempt to identify the species of Sarcocystis. Hence, extensive studies using more powerful microscopic technique and biomolecular analysis are needed to aid possible parasite identification present in Rattus spp. at the species level.

Conclusion
The presence of Sarcocystis spp. confirmed the infectivity of the parasite to Rattus spp. Moreover, the different morphologic characteristics observed on Sarcocystis proved that different species of the parasite harbor the Rattus spp. Hence, the role of rats in prey-predator relationship among organisms is suggestive of the significant spread of Sarcocystis spp. in other host animals, including humans. This poses the risk to transferring Sarcocystis spp infecting man through ingestion of contaminated meat, thereby increasing public health concerns, especially those within the vicinity of the agricultural areas where these rats dominate.