In the present study, Sarcocystis spp. were identified in synanthropic (Muridae) and wild (Cricetidae) rodents from cattle dairy farms in the Humid Pampa region of Argentina, using histopathological analysis, direct microscopic observation and molecular techniques. This represents the first molecular identification of Sarcocystis spp. in naturally infected Oxymycterus rufus, Akodon azarae, Necromys lasiurus, and Mus musculus from South America.
The above mentioned wild rodent species are native from South America, they are omnivorous with a preference for grains, leaves and insects. There are several reports on helminths and arthropods detection in these rodent species mainly in Argentina (Lareschi et al. 2003, 2010; Robles 2008, 2016; Guerreiro-Martins et al. 2014, 2020, 2022; Gómez-Muñoz et al. 2020; Colombo et al. 2023). However, studies aiming to identify protozoan infections are scarce. On the other hand, Mus musculus is an omnivorous and cosmopolitan rodent and is involved in the transmission of several zoonotic diseases (Bitam et al. 2010). Infections with several helminths (Hancke 2016) and with protozoa such as Toxoplasma gondii, Neospora caninum have also been reported in this species in Argentina (Dellarupe et al. 2019, Salcedo et al. 2021). However, despite numerous parasitological studies conducted on all these rodent species in Argentina, no reports of Sarcocystis spp. infections have been documented to date.
Histopathological analysis revealed a low overall prevalence (8.7%, 31/356) of Sarcocystis spp. in the captured rodents. However, when the prevalence was compared, it was significantly higher in wild rodents (Cricetidae) (15.9%) than in synanthropic rodents (Muridae) (3.8%) and any of the Rattus spp. specimens were detected as positive (0/19). In Lithuania, Grikienienė and Mažeikytė (2000) found a higher frequency of infection in Cricetidae (2%; 2/104) than in Muridae (0%, n = 30) using histological techniques and a muscle tissue compression method; however, the prevalences were lower than in the present study. In contrast O'Donoghue et al. (1987) reported a high prevalence (40%; 16/40) of Sarcocystis spp. in Muridae rodents in Indonesia. Additionally, Muridae rodents (mainly Rattus spp.) captured in agricultural areas from the Philippines, also showed a high prevalence of Sarcocystis spp. (45%; 31/69) (Cabanacan-Salibay et al. 2020). Considering that the rats in our study were captured in highly anthropized environments, we can speculate that human interference may reduce the presence of definitive hosts, thus reducing Sarcocystis spp. infections. This hypothesis is supported by other studies showing a negative correlation between Sarcocystis infection burden in rats and human disturbance (Paperna et al. 2004). Similarly, in Singapore, a lower percentage of rats were infected in an industrial area (27.5%) compared to a forested area (76%) (Paperna and Martelli, 2001). On the other hand, in two Calomys species (C. musculinus and C. laucha) no sarcocysts were detected; however, due to the low number of animals no further comparisons are plausible. In our study, the higher Sarcocystis spp. prevalence found in adult rodents could be related to a higher chance of contact with sporocysts over time. Studies in other intermediate hosts have reported similar results. For example, Castro et al. (2004) and Rodriguez et al. (2023) reported that age was a risk factor for Sarcocystis infection in alpacas, with higher prevalence observed with increasing age. Similarly, Calero-Bernal et al. (2015) and Luzón et al. (2015) described a higher probability of infection in older cattle and pigs. In addition, the prevalence of Sarcocystis spp. infection was not influenced by the host sex, which is consistent with several studies on protozoa and helminths in rodents (Bajer et al. 2001; Behnke et al. 2001; Svobodová et al. 2004).
Most intermediate infected hosts with Sarcocystis spp. remain asymptomatic. This may be due to innate immunization that occurs in free-ranging animals as a result of recurrent low doses (Caspari et al. 2011). However, sarcocystosis can manifest as a serious or even life-threatening condition, depending on the specific Sarcocystis species, the infective dose, and the host immune response (Basso et al. 2020). In relation to these aspects, no inflammatory responses associated with sarcocysts were observed in this study, suggesting an efficient host-parasite adaptation. On the other hand, there is no standardization of rodent tissues when selecting samples for histological analysis. Cabanacan-Salibay et al. (2020) examined tissues of muroid rodents and reported a higher prevalence of Sarcocystis cysts in the diaphragm (27.5%; 19/69) than in the tongue (8.7%; 6/69). In our study, the semitendinosus and masseter were identified as the most frequently infected muscles. Furthermore, sarcocyst measurements from histological sections could vary significantly depending on the cysts age and the type of section (transverse, oblique or longitudinal) being not always accurate for proper descriptions (Ambu et al. 2011). Although a higher amount of muscles per animal was analyzed by direct microscopy than by histology, a lower prevalence was detected. It is plausible that small rodent tissues and cysts are destroyed during homogenization and not further detected under direct microscopy. Similar results were informed by Canova et al. (2023) performing the same procedures in plains vizcachas muscles.
From 31 samples positive by histology, 24 resulted positives in the screening PCR targeting the 18S rRNA gene fragment, corresponding to four rodent species (the two samples from O. flavescens evidencing sarcocysts by histology were PCR negative) (Table 2). The seven negative results may be attributed to (i) a low cyst burden in the sampled muscles rendering in DNA amounts below the detection limit of the conventional PCR; (ii) primer binding regions from some rodent Sarcocystis species might be not conserved as generally assumed (More et al. 2011); (iii) presence of small cysts from other coccidia, resembling Sarcocystis. Most of the 18S rRNA consensus sequences obtained in this study from homogenized muscle DNA showed low identities with other sequences reported in GenBank. Therefore, all the sequences were submitted to GenBank as 'Sarcocystis sp.' On the other hand, most of the chromatograms were clear and consensus sequences were achieved, indicating the presence of a single species in each sample. Only in 3/24 cases, double peaks and background signals were present, and they were considered potential mixed infections (Moré et al. 2016). Four different 18S rRNA sequences or sequence groups with high intra-group identities (99.6–100%) were detected. Eight sequences showed 99.5–99.7% identity with a S. dispersa sequence (AF120115). Sarcocystis dispersa was identified in the barn owl (Tyto alba) as DH and in the house mouse (Mus musculus) as an experimental IH (Cerná et al. 1976). In the present study, S. dispersa-like sequence was identified in 5/6 M. musculus, 1/11 O. rufus, 1/2 A. azarae and 1/5 N. lasiurus which were positive by PCR, suggesting that this parasite occurs more frequently in M. musculus than in wild rodents. According to our phylogenetic analysis, a carnivorous bird as DH may be assumed. Eleven further sequences showed 95.9–96.4% identity with S. mucosa (AF109679) and Sarcocystis sp. (MW542200) from the California kingsnake (Lampropeltis californiae). This sequence was detected in 1/6 M. musculus, 8/11 O. rufus, 1/2 A. azarae and 1/5 N. lasiurus. Despite occurring in several rodent species, it seems to be more frequent in O. rufus. All these sequences are positioned in a separate branch from all other Sarcocystis spp. along with the sequence from one O. rufus (OQ924662_R10). One sequence from a N. lasiurus (OQ924675_R303) showed only 95.3% identity with a Sarcocystis sp. from the Persian horned viper (Pseudocerastes persicus), and positioned in the phylogenetic tree along with sequences from S. nesbitti, which uses snakes as definitive hosts (Lau et al. 2013; Abe et al. 2015). Levels of identity < 97% are below the accepted thresholds for specific identification of Sarcocystis spp. (Gjerde 2013; Moré et al. 2013). This fact could represent the detection of new Sarcocystis species and/or the lack of reported sequences from some of the previously described species. Our study might suggest that the 18S rRNA sequences obtained in Argentinian rodents correspond to species that use birds and snakes as DH. Nevertheless, positioning on the phylogenetic tree of some sequences should be interpreted carefully due to the low comparative sequence identity previously mentioned. On the other hand, all nine coxI sequences obtained in this study from different rodent species were almost identical among them, despite the 18S rRNA region amplified from the same samples suggested the occurrence of at least two different species (six sequences S. dispersa-like and other two without high identity in BLAST comparison). The use of the coxI gene as a genetic marker for discriminating Sarcocystis species has been proposed in 2013; however, there is a limited number of available coxI gene sequences from different rodent Sarcocystis species for comparison (Gjerde 2013). Moreover, the high homology observed in our sequences could suggest that this marker is not useful to discriminate among Sarcocystis spp. using rodents as IH. These cox1 sequences showed a high similarity with S. strixi (99.2–99.5%) and S. lutrae (99.1%). Sarcocystis strixi has been identified in the barn owl (Strix varia) as its DH, but the natural IH remains unknown (Verma et al. 2017) while S. lutrae has been identified in otters (Lutra lutra) and several mustelids and carnivores as IH and birds such as the white-tailed eagle (Haliaeetus albicilla) as DH (Maca and González-Solís 2022). The similarity and positioning in the phylogenetic tree of coxI sequences, in relation to S. strixi and S. lutrae sequences may suggest that the Sarcocystis spp. in rodents in our study could use raptor birds as DH. In the study area, several raptors birds are described, such as the barn owl (Tyto alba) (Romano et al. 2002; Teta et al. 2010, 2012) and the white hawk (Elanus leucurus) (Leveau et al. 2002). Predation on Akodon azarae by owls (Asio flammeus) and owlets (Athene cunicularia) has been reported, but also by canids (Lycalopex gymnocercus), felids (Leopardus geoffroyi) and mustelids (Galictis cuja) (Romano et al. 2002; Kittlein 2009; Guidobono et al. 2016). Nevertheless, the lack of sequences reported from several Sarcocystis spp., make further comparisons and descriptions difficult, and we cannot confirm whether our results represent the detection of new species or the molecular detection of previously described ones.
Despite obtaining suitable products using primers validated for Sarcocystis spp. ITS1 (Gjerde 2014), our sequences were longer than those available in GenBank, and only low coverage was obtained (corresponding to the short 18S rRNA fragment, flanking the ITS1 region, which is also amplified with this PCR). The sequences reported here represent one of the fist ITS1 sequences from Sarcocystis spp. using rodents as IH.
Our multilocus approach allowed the identification of new Sarcocystis spp. sequences, specially from the 18S rRNA and ITS1. However, the specificity of the used primer combinations to differentiate Sarcocystis species from rodents is uncertain. Moreover, since the DNA was obtained from muscle homogenates, the amplification of more than one species in each sample with each primer combination could not be completely excluded (Canova et al. 2023). Future studies employing single-cyst PCR to obtain unique species amplifications for all markers, as previously performed for other rodent Sarcocystis spp., would shed more light on this issue (Prakas et al. 2019; Rudaitytė-Lukošienė et al. 2022).