Reactivity against Sarcocystis neurona and Sarcocystis falcatula-like in horses from Southeastern and Midwestern Brazil

Abstract Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona. Immunofluorescence antibody tests (IFATs) have been widely used to identify exposure of horses to S. neurona in Brazil. Here we used IFAT to search for IgG antibodies against Sarcocystis falcatula-like (Dal-CG23) and S. neurona (SN138) in sera from 342 horses sampled in Campo Grande, Mato Grosso do Sul state (Midwestern), and São Paulo, São Paulo state (Southeastern), Brazil. The 1:25 cutoff value was chosen to maximize sensitivity of the test. IgG antibodies against S. neurona were detected in 239 horses (69.88%), whereas IgG antibodies against S. falcatula-like were detected in 177 horses (51.75%). Sera from 132 horses (38.59%) reacted against both isolates. Absence of reactivity was evidenced in 58/342 horses (16.95%). The lower cutoff used, and the presence of opossums infected with S. falcatula-like and Sarcocystis spp. in the regions where the horses were sampled, might justify the high seroprevalence observed here. Owing to the similarity among antigens targeted in immunoassays, reports on S. neurona-seropositive horses in Brazil may also derive from the exposure of horses to other Sarcocystis species. The role of other Sarcocystis species in causing neurological diseases in horses in Brazil remains unclear.


Introduction
Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona (Dubey et al., 1991).Opossums (Didelphis spp.) are the definitive hosts and sources of S. neurona infections in horses.Didelphis virginiana is the definitive host of S. neurona in North America (Fenger et al., 1995), and Didelphis albiventris is the definitive host of S. neurona in Brazil (Dubey et al., 2001).Opossums are definitive hosts also for Sarcocystis speeri, Sarcocystis lindsayi, and Sarcocystis falcatula.Coinfection with different Sarcocystis species have already been described in opossums (Dubey et al., 2016).
Serological techniques such as immunofluorescence antibody tests (IFAT), Western blot (WB), and enzyme-linked immunosorbent assays (ELISAs) based on SAGs have been used to identify the exposure of horses to S. neurona in Brazil [summarized by Gondim et al. (2021)].Sarcocystis neurona has also been evidenced in central nervous system of horses in Brazil by means of immunohistochemistry (Masri et al., 1992;Paixão et al., 2007;Henker et al., 2020) and molecular methods (Henker et al., 2020).However, S. neurona has never been isolated from horses in the country.
Due to the high similarity among SAGs in Sarcocystis shed by opossums, serological cross-reactivity between S. neurona and S. falcatula-like was hypothesized, being later demonstrated in experimentally infected Mongolian gerbils (Meriones unguiculatus) (Jesus et al., 2019).The aim of the present study was to evaluate horse sera reactivity against S. falcatula-like (strain Dal23-CG isolated from a D. albiventris from Campo Grande) and S. neurona (North American strain SN138) among horses sampled in the cities of Campo Grande, Mato Grosso do Sul state (Midwestern), and São Paulo, São Paulo state (Southeastern), Brazil.

Samples
To detect anti-S.neurona and anti-S.falcatula-like antibodies, IFAT was performed using serum samples obtained by convenience from 342 horses.Samples from 262 horses were collected in 12 properties located in the peri-urban region of Campo Grande for screening of Rickettsia, Borrelia, and Anaplasmataceae agents, and were kindly supplied by Dr. Heitor Miraglia Herrera from the Universidade Católica Dom Bosco (UCDB).Samples from 30 horses were collected for routine exams and were kindly supplied by the veterinary staff of the military police cavalry from Campo Grande.Samples from 50 horses were collected for routine exams and were kindly supplied by the veterinary staff of an equestrian club in São Paulo.The samples included males and females of diverse ages and breeds (Supplementary Table S1).

Indirect Fluorescent Antibody Test (IFAT)
Extracellular merozoites were harvested from the flasks, purified using a sterile 5 μm pore size filter, washed in phosphate-buffered saline (PBS) by centrifugation (1500 × g for 5 min), fixed in 10% formalin for 10 min, washed twice in PBS, and used to cover 12-well slides in 10 μL aliquots per well.The slides were allowed to air dry and stored at -20 °C.Prior to use, the slides were removed from storage and allowed to thaw at room temperature (approximately 22 °C) for 30 min.IFAT was performed as previously described (André et al., 2010).

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Sarcocystis spp.antibodies in horses in Brazil Briefly, serum samples were diluted at 1:25 in PBS and pipetted into each well.The slides were incubated in a humid chamber at 37 °C for 45 min, washed for 15 min with PBS, and dried at 37 °C.The delimited areas were covered with 10 μL of commercial fluorescein-labeled anti-horse immunoglobulin G (IgG) (Sigma-Aldrich ® , St. Louis, USA) at a 1:64 dilution in PBS containing 10% Evans blue.The slides were then incubated for 45 min in a dark and humid chamber, washed, and dried.For microscopic evaluation, slides were overlaid with buffered glycerin (pH 8.7), covered with glass coverslips, and examined at 400 x magnification using an epifluorescence microscope (Olympus, Japan).Only samples presenting fluorescence in the entire periphery of merozoites were considered positive (Figure 1).Positive controls consisted of sera from horses that reacted at 1:100 solely for each parasite (S. neurona or S. falcatula-like).Negative controls consisted of horse sera that tested negative for both parasites at 1:25 dilution.Positive samples at 1:25 were further diluted in two-fold increments to obtain the final titer, with the endpoint titer being the last serum dilution showing distinct whole parasite fluorescence.
Discrepancies of the results presented in comparison with previous studies may be associated with the lower cutoff titer used here, and with the presence of opossums infected with Sarcocystis falcatula-like and Sarcocystis spp. in the same regions where the horses were sampled (De Santi et al., 2023).A starting serum dilution of 1:25 was selected based on previous study from Duarte et al. (2003), who observed that a gold standard panel of horses infected with S. neurona showed seropositivity starting at 1:20 at IFAT.The authors recommended a 1:80 cutoff in IFAT for horses suspected to have EPM.Although the clinical history of the horses in this study was not available, none of the animals presented neurological signs at the time of blood collection, therefore a less conservative cutoff was used.Exposure to different Sarcocystis species may also generate different immunological responses (Borges-Silva et al., 2020).This could explain why horses in the present study reacted differently to S. neurona and S. falcatula-like antigens, with up to 8-fold increase in titers for S. falcatula-like and up to 16-fold increase in titers for S. neurona.It is not clear if the exposure to a Sarcocystis spp.could protect the horse from EPM caused by S. neurona.
Serological assays are the main tools for ante mortem diagnosis of EPM.However, they only indicate whether horses have been exposed to S. neurona, not providing certainty of current infection (Dubey et al., 2015).Furthermore, surface antigens, targeted in serological tests, are highly similar among Sarcocystis spp.shed by opossums (Gondim et al., 2021).Therefore, immunological assays that use whole merozoites as antigens, such as IFAT, and SAG-based ELISAs, may result in the false assumption that horses are seropositive for S. neurona due to seroconversion after infection with other Sarcocystis species.Confirmation of IFAT using immunoblot assays revealed a low number of "true positive" samples in horse sera tested against S. neurona (SN37R) in the state of Alagoas (Valença et al., 2019).In an IFAT survey performed with serum samples from 189 mares, 57 from the 64 samples that reacted against S neurona, reacted also against Sarcocystis cruzy, i.e., only seven samples reacted solely against S. neurona (Antonello et al., 2015).The presence of specific antibodies in serum and cerebrospinal fluid, associated with typical clinical signs, and the complete differential diagnosis, is currently used to define EPM diagnosis (Dubey et al., 2015).
The hypothesis of cross reactivity between different Sarcocystis species shed by opossums in Brazil could justify the high seropositivity observed for S. neurona in horses, despite the low number of reports on EPM in the country (Gondim et al., 2021).Cross reactivity between S. neurona and S. falcatula-like has already been observed in Mongolian gerbils (Meriones unguiculatus) experimentally infected and tested by Western blot (Jesus et al., 2019).Interestingly, cross reactivity was not observed when the same animals were tested by IFAT.However, cross reactivity between S. neurona and S. falcatula-like has not been proven in horses to the date.It is worth to note that not all horses infected with S. neurona develop clinical disease.The exact mechanisms that favor an asymptomatic infection to progress to a severe neurological disease remains unclear.
Based on the results presented here along with that from previous studies, is clear that the high seroprevalence of S. neurona reported in horses in Brazil should be interpreted with caution.Further studies are necessary to elucidate the role of Sarcocystis species other than S. neurona in infecting and causing clinical diseases in horses in the country.

Table 2 .
Titration of Sarcocystis falcatula-like and Sarcocystis neurona-seropositive horses from Campo Grande Mato Grosso do Sul state (Midwestern), and São Paulo, São Paulo state (Southeastern) tested by IFAT.Sarcocystis spp.antibodies in horses in Brazil