The wide distribution and the zoonotic importance of the different Sarcocystis sp. necessitate the development of preventive measures based on the identification and accurate diagnosis (Salehi et al. 2022).
The presented results showed successful detection of Sarcocystis sp. in the different samples from the esophagus, diaphragm, and skeletal muscles of the slaughtered Egyptian buffaloes (Bubalus bubalis). These findings confirmed the obvious distribution of this parasite and were in context with the previous studies that displayed its prevalence either globally (JyothiSree et al. 2017; Salehi et al. 2022; Rogers et al. 2022) or at the local level (El Shanawany et al. 2019c; Nahed et al. 2014). Molecular characterization of Sarcocystis sp. using the 18S rRNA and the ITS genes were extensively used in many previous studies because of their accuracy in differentiating the closely related species adding to the phylogenetic analyses (Hu et al. 2022; Xue et al. 2019). Although it is known that water buffaloes are the intermediate host for four species, including S. fusiformis, S. buffalonis, S. levinei, and S. dubeyi (Dubey et al. 2014a), we could only detect S. fusiformis. This might be due to collection of samples from only one governorate. These results were previously recorded in Egypt as all examined tongue, heart, esophagus, and skeletal muscle samples were only of the same type (S. fusiformis)at the different governorates but with different prevalence rates(Ghaffar et al. 1978; Khalifa et al. 2008). On the other side, detection of multiple types in the same study was also recorded in Brazil (PORTELLA et al. 2021), Thailand, Lao PDR, and Cambodia (Hongchuta et al. 2021). It is worth noting that S. dubeyi species was determined for the first time in Egyptian water buffaloes with a prevalence rate of 30% in 2011(Hilali et al. 2011).
Nucleotide sequence and phylogeny were the most accurate approaches for the identification of Sarcocystis sp. from the same host or different ones (Greenfield et al. 2022; Rogers et al. 2022), especially since morphological confusion might happen due to similarity in some Sarcocystis sp. from cattle and buffalo (Gjerde et al. 2016).
The phylogenetic analysis based on the 18S rRNA and the ITS genes revealed a close relationship between the obtained isolates with S. fusiformis, mainly those previously isolated in Egypt during the last years, and the S. cafferi isolated in South Africa from African buffaloes (Dubey et al. 2014b), and S. cruzi from cattle beef in the USA (Rosenthal et al. 2008). This may be owed to extensive overlapping in intra- and interspecific gene identity. The obtained results agreed with a recent study conducted in India (Sudan et al. 2021). It was recorded that S. fusiformis is the most common species that infects buffaloes across China, Egypt, India, Iran, and Sweden (Dubey et al. 2015). Furthermore, a lower identity percentage was recorded between our obtained isolates and S. buffalonis previously isolated from infected buffaloes in Egypt (Gjerde et al. 2016). It is important to clarify that there are uncertainties concerning the identity and host species specificity of Sarcocystis species of the water buffalo and cattle and the transmission of Sarcocystis species between cattle and buffalo, but steps to preclude exogenous exposures were not reported by Gjerde et al. (2015).
An assumptive diagnosis of sarcocystosis in intermediate hosts can be made by clinical signs, clinical pathology, and biopsies. Serological diagnostic tests are beneficial for early diagnosis and prevalence studies (Metwally et al. 2014). Developing a highly sensitive, specific, and low-cost kit is necessary to diagnose buffalo’s sarcocystosis. ELISA is a common method for the large-scale detection of different parasitic infections (De Silva et al. 2022; El-Shanawany et al. 2019). But, the critical point for the evaluation of a new immunodiagnostic antigen is determining the sensitivity and specificity of this antigen ( )
This study focused on improving the diagnosis of sarcocystosis by using the developed GlcNAc glycoprotein fraction from S. fusiformis whole cyst crude antigen. The analyzed ROC curves and AUC indicated that the developed GlcNAc glycoprotein fraction proved to be a better serodiagnostic antigen than the whole cyst one for ELISA. The diagnostic sensitivity and specificity of ELISA with SF-GlcNAc was 95.56 and 82.5%, respectively, while those with whole cyst antigen were 68.89% and 67.5%, respectively. Previous studies have explained the lower efficiency of whole cyst antigen (Tenter et al. 1991) by showing that sarcocystosis can be diagnosed more efficiently using purified antigens (Panda et al. 2020). This was consistent with our study and those reporting that detecting the soluble crude extract antigen for sarcocystis displayed 76.4% sensitivity and 66.6% specificity (Mamatha et al. 2008). Also, anti-sarcocystis antibodies were detected in 54.3% of naturally infected water buffaloes using crude cyst antigen (Hamidinejat et al. 2005). However, the sensitivity of the sarcocystis bradyzoites 53 KD purified fraction was (95.83%) with no recorded of cross-reactivity (Sabry and Reda 2008). The glycoprotein of mature Sarcocystis plays a functional role as they are branched fibrilles seen on the surface of the Sarcocystis wall, ground substances of Sarcocystis near the cyst wall, septa, and plasmalemma of merozoites (Radchenko 1992). This structural feature was predicted to mediate the interaction between the parasite and host immune system to evoke the humoral and cellular response (Connick et al. 2020; El Shanawany et al. 2019c). Therefore, the presented isolated SF-GlcNAc showed immunogenic efficacy in eliciting the antibody response to S. fusiformis infection in buffaloes. To our knowledge, the study of glycoproteins for the diagnosis of sarcocystosis is very scarce. However, glycoprotein’s potency and accuracy in diagnosing different parasitic diseases were proved. GlcNAc isolated from Echinococcus granuloses showed diagnostic potency in the diagnosis of camel cystic echinococcosis with 97.3% and 54.5% sensitivity and specificity, respectively (El-Shanawany et al. 2019). Also, (El Shanawany et al. 2019b) proved that the GlcNAc fraction isolated from Toxocara vitulorum cuticle antigen proved 79% diagnostic potency of calves’ toxocariasis by indirect ELISA. C. parvum 15-kDa glycoprotein antigens are immunodominant diagnostic antigens of cryptosporidiosis (Strong et al. 2000; Tilley and Upton 1994). Surface antigen glycoproteins extracted from tachyzoites and sporozoites of the T. gondii shed interesting light on vaccine studies and diagnosis of toxoplasmosis (Cong et al. 2013).
The purified SF-GlcNAc antigen showed no cross-reactivity with cryptosporidiosis, coccidiosis, Giardiasis, and blastocistosis infections. However, significant cross-reactivity was reported between sarcocystosis and toxoplasmosis with 80% relative specificity. This proved that there are common antigenic components or epitopes between T. gondii and sarcocystis species (Gondim et al. 2017). Due to the lack of studies regarding the glycoproteins in the Sarcocystis sp., it was not completely clear whether this cross-reactivity is due to the similarity in the structures of purified SF-GlcNAc and T. gondii. A previous study showed that this cross-reactivity might aid in diagnosing human toxoplasmosis using horse T.gondii antigen (Hassan et al. 2012). Thus, a more in-depth study with positive serum samples during both acute and chronic phases of sarcocystosis is required to determine the cross-reactivity with closely related protozoa T. gondii. Sixteen out of the 89 (17.9%) random test buffalo serum samples were positive using the purified S. fusiformis SF-GlcNAc for ELISA. A low prevalence rate was detected in another governorate (8.3%) in the New Valley (Ahmed et al. 2016). Meanwhile, a high percentage (74%) of infection was detected in the Monufia governorate (El Shanawany et al. 2019c). Therefore, the inconsistency between the prevalence rates might be the reason behind the high specificity and low cross-reactivity percentage (12%) of our SF-GlcNAc-based ELISA method than the previously used ELISA based on crude S. fusiform antigen.