The Sarcocystidae family includes several intracellular coccidian parasites with heteroxenous life cycles such as Toxoplasma gondii, Neospora caninum, Sarcocystis spp., and Hammondia spp. (Carreno et al. 1998; Mugridge et al. 2000). The life cycles of these protozoans are complex, including a definitive host (DH) and one or several intermediate hosts (IH), and involve different parasitic stages (oocysts/sporocysts, tachyzoites, and bradyzoites in tissue cysts). The oocysts or sporocysts are shed in the feces of the DH to the environment, where they can maintain their infective capacity for long periods of time. On the other hand, the tissue cysts containing bradyzoites are present in different organs and tissues of chronically infected IH, where they persist for long periods, even for life. Tissue cysts also represent one of the infectious elements for the DH, and in the case of T. gondii and N. caninum, also for some IH (Dubey 2022; Dubey et al. 2017).

The DH of T. gondii and H. hammondi are cats and other felids, while domestic dogs and other canids are the DH of N. caninum and H. heydorni. On the other hand, the genus Sarcocystis is comprised of more than 200 species, with several using canids and felids as DH, and mainly herbivores as IH (Dubey et al. 2016).

Toxoplasmosis is a worldwide distributed zoonosis, with clinical relevance in pregnant women and in small ruminants, causing fetal lesions and abortions, and is also an important pathogen in zoo animals and immunosuppressed individuals (Dubey 2022). Neosporosis was first described in dogs with neuromuscular disease, and is one of the main causes of abortion in cattle, inflicting important economic losses worldwide (Dubey et al. 2017). Cattle are also described as IH of different Sarcocystis spp.: S. cruzi whose DH are dogs and other canids, S. hominis and S. heydorni whose DH are primates, S. hirsuta and S. bovifelis with felids as HD, and S. rommeli and S. bovini which are also believed to rely on cats as DH to fulfil their life cycle (Gjerde 2016a, b; Hu et al. 2017). Most infections in cattle are chronic, producing thin-walled (S. cruzi and S. heydorni) or thick-walled (S. hominis, S. hirsuta, S. rommeli, S. bovifelis and S. bovini) tissue cysts, but only S. cruzi is mildly pathogenic to cattle and usually not pathogenic for the DH (Dubey et al. 2016; More et al. 2010). Another relevant species of the Sarcocystis genus is S. neurona, which is considered the main cause of equine protozoal myeloencephalitis (EPM), where equines act as aberrant hosts. Definitive hosts of S. neurona are different species of opossums (Didelphis spp.) which are also DH of other species including S. falcatula (Gondim et al. 2021). Sarcocystis spp. sporocysts are morphologically indistinguishable, therefore, molecular diagnosis is recommended for specific diagnosis when oocysts/sporocysts are found in fecal material of a given DH (Dubey et al. 2016).

The genus Hammondia includes H. hammondi, H. heydorni, and H. triffittae. So far, they have not been reported to cause disease in animals or humans. However, the importance of Hammondia spp. lies in its close phylogenetic relationship with T. gondii and N. caninum, and the possibility of cross-reactions when applying serological diagnostic methods (Hu et al. 2017; Riahi et al. 1998; Riahi et al. 2000).

The different parasitic stages of all these Apicomplexan protozoans express different surface and apical complex proteins, related to host-parasite interaction. Studies carried out in cyst-forming protozoa have shown that the events that trigger the transformation from proliferative tachyzoites to cyst-forming bradyzoites and vice versa is associated with the expression or suppression of a wide range of stage-specific proteins, and this could contribute to immune evasion mechanisms (Dubey, 2017; Dubey, 2022). Most of the currently available information has been derived from T. gondii, since the tissue cyst production is relatively easily achieved in vivo in murine models and in vitro in cell culture by applying different treatments that exert physiological stress (Dubey, 2022). In the case of N. caninum, one of the main drawbacks of studying the bradyzoite stage is the difficulty of obtaining sufficiently abundant and homogeneous biological material (Dubey, 2017).

It has been reported that tachyzoites and bradyzoites of T. gondii and N. caninum can be differentiated by detecting stage-specifically expressed antigens or specific immunodominant antigens (IDAs). In 1991, Sibley et al. proposed a nomenclature system for naming important T. gondii genes based on systems already in use (Sibley et al. 1991). The main 30 kDa tachyzoite surface antigen was named TgSAG1, and the corresponding tgsag1 gene is a member of a superfamily of related genes, termed srs (SAG1-related sequences), encoding structurally related surface proteins of T. gondii. For N. caninum, the same nomenclature system was introduced (Howe and Sibley 1999), and homologous N. caninum and T. gondii antigens were proposed to include the prefix Nc or Tg, respectively (for example, TgSAG1 and NcSAG1). In the same way, the SRS-superfamily members in N. caninum were introduced by Wasmuth et al. (2012). Bradyzoite-specific antigens are, for instance, TgBAG1 in T. gondii (Bohne et al. 1995), and NcSAG4 in N. caninum (Fernandez-Garcia et al. 2006).

Little information is available on the antigens expressed in the different stages of Sarcocystis spp., mostly for S. cruzi and S. neurona. Sarcocystis cruzi sporozoite, merozoite, and bradyzoite antigens were identified by immunoblot (IB) using immune sera from infected animals or monoclonal antibodies (MAb). The number of reactive antigen bands increased as the animals progressed through the course of infection and several IDAs were identified (Dubey et al. 2016). Sera from animals infected with S. neurona reacted with antigen bands of 30kDa (SnSAG1) and 16kDa, identified in extracts of merozoites by IB (de Jesus et al. 2019). However, reactions particularly to the 30kDa proteins band (including SnSAG1) have been referred to as potential cross reaction with other Sarcocystis spp infections (de Jesus et al. 2019; Gondim et al. 2021). We do not have available well characterized anti-Sarcocystis spp. antibodies.

Several studies have reported cross-reactions between related apicomplexan parasites, which can generate false positive results when applying serological diagnostic tests (Gondim et al. 2016; Gondim et al. 2017; Liao et al. 2005; Riahi et al. 1998). A previous study reported that sera from animals generated against H. hammondi tachyzoites cross-reacted with the TgSAG1 antigen. Other studies have reported serological cross-reactions between animals infected with T. gondii and H. hammondi, as well as between animals infected with T. gondii and N. caninum (Gondim et al. 2017). Neospora caninum has also shown some degree of serological cross-reactivity with Sarcocystis spp., mainly in infections prevalent in cattle (Dubey et al. 2017). On the other hand, cross-reactivity between N. caninum and H. heydorni has been suggested, but has not been demonstrated so far (Gondim et al. 2017).

Both MAb and polyclonal antibodies (PAb) have been developed against several of these antigens: MAb anti NcSAG1 (Bjorkman and Hemphill 1998), MAb and PAb anti NcSAG4 (Fernandez-Garcia et al. 2006), PAb anti TgBAG1 (McAllister et al. 1996) and MAb anti TgCC2, (Gross et al. 1995), and have been used extensively in various studies (Vonlaufen et al. 2002; Risco-Castillo et al. 2004; Gondim et al. 2016).

The objective of this work was to evaluate Mab (anti NcSAG1, anti NcSAG4 and anti TgCC2) and/or PAb (anti NcSAG4 and anti TgBAG1) to label specific IDAs in different parasitic stages of N. caninum (oocyst, bradyzoite, and tachyzoite), T. gondii (oocyst, cyst, and tachyzoite), H. heydorni (oocyst), S. cruzi (cyst and bradyzoite) and S. falcatula (sporocyst). Most of these antigens were chosen for labeling considering to test widely used antibodies' and availability analytical specificity, and because they are immunodominant and are shared by many apicomplexan parasites, and in some cases could explain cross-reactions observed in serological studies.