To date there are few reports in the world that use the molecular technique as a diagnosis for Eimeria species in cattle, and to our knowledge there are no studies in Mexico. Of the 57 positive microscopy samples used in this study, 26 (45.6%) of them were identified as Eimeria species using PCR. These results are consistent with those presented by other authors such as Lee et al. (2018) with 53.4% in the Republic of Korea, followed by Hastutiek et al. (2022) with 52% in Madura, Indonesia, Ekawasti et al. (2022) with 25.1% in Indonesia, and in the same authors reported a 30%. Most of the reported studies have been based on the methodology described by Kawahara et al. (2010), who have been pioneers in the development of genus-specific primers to identify six Eimeria species using the ITS-1 molecular marker (E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis, and E. zuernii), which in turn has been used in recent years for different investigations (Lee et al., 2018; Pyziel et al., 2020; Ekawasti et al., 2021; Ekawasti et al., 2022; Hastutiek et al., 2022).
First of all, these authors use the generic primer and from the positive samples, they use it for the specific primer. This is possibly associated with a limitation to exclusively identify these six species at the molecular level, and there may be unidentified mixed oocysts (Lee et al., 2018). In this study, it was decided to perform a PCR through a primer at the genus level to subsequently obtain genetic information by means of sanger sequencing. In this way, data were obtained for two more species, E. bukidnonensis and E. canadensis, which will allow the creation of more specific primers that, can be used globally.
Olivares-Munoz et al. (2022) reported the presence of ten species of Eimeria using taxonomic keys, which are circulating in the state of Veracruz: E. canadensis, E. bovis, E. zuernii, E. ellipsoidalis, E. cylindrica, E. bukidnonensis, E. alabamensis, E. wyomingensis, E. auburnensis and E. subspherica. This study confirms the presence of six species in cattle: E. bovis, E. zuernii, E. ellipsoidalis, E. bukidnonensis, E. canadensis and E. cylindrica. The other species were not identified by molecular technique. The authors suggest further studies to obtain genetic information on Eimeria spp. affecting cattle in Mexico. These previous studies also showed that a significant percentage of microscopy-positive samples were negative for species identification by PCR. In our study, 31/57 oocyst concentrations were negative in agarose gel. There are some factors, such as the quantity and quality of DNA, the presence of mixed Eimeria oocysts, contamination with PCR inhibitors, resistance of the oocyst wall to rupture or restricted access to template DNA, which can interfere with the molecular diagnosis of Eimeria in cattle (Oliveira et al., 2011; Lee et al., 2018; Ekawasti et al., 2022; Melo et al., 2022). Regarding the quantity and quality of the DNA, failures in the molecular identification of Eimeria spp. have been associated with insufficient numbers of oocysts of these species in feces (< 10) for successful DNA extraction using PCR (Hastutiek et al., 2022), or with the presence of inhibitors because the samples were collected from the environment and fecal matter (Lee et al., 2018). According to Oliveira et al (2011), it is possible to detect Eimeria spp. with a DNA amount of 500 fg to 1 ng. In this study, DNA samples were obtained from at least 200 oocysts, and a mean of 10–15 ng was obtained for PCR. Although normal DNA ranges were obtained, there is a possibility that the amount of DNA indicates fecal debris rather than coming directly from the oocyst; therefore, this cannot be ruled out (Lee et al., 2018). Another reason may be due to a deficiency in the mechanical disruption of the oocyst wall. This is related to the problem of wall resistance that has a double layer. The outer layer is thick and elastic and is composed of a chitin-like material, as well as tanned quinone proteins which hinder its disruption (Melo et al., 2022). Several techniques have been proposed for oocyst rupture, either mechanically or chemically, but a reproducible method for disrupting the oocyst wall and recovering high yields of DNA has not yet been developed (Oliveira et al., 2011). In order to create PCR tests, emphasis should be placed on the purity of the sample, the number of oocysts and the characteristics of the primers to validate the geographical origins of the strains and verify that they can be used in the region studied (Ekawasti et al., 2022).
In this study, the prevalence of E. canadensis previously reported in Mexico by Alcala-Canto et al. (2020) was higher than 62%, while the PCR technique showed a prevalence of less than 4%; however, the samples used in this work contained mixed oocysts from multiple species, which resulted in amplification inhibition of some species, such as E. canadensis (Pyziel et al., 2020). If there are less than 10 oocysts per species in the sample, there will be no amplification (Hastutiek et al., 2022). Despite the difference in percentages between both techniques, the high prevalence of E. canadensis observed in previous research should not be overlooked, and in vivo and in vitro experimental studies should be carried out to reassess whether it is still considered a non-hazardous species for livestock. The presence of Eimeria maxima, a common species in poultry, was also observed in calf samples. The presence of emerging diseases in animals can be a threat to both biodiversity and public health. Domestic animals are important sources of new pathogens for populations and the spread of disease is facilitated by the invasion of livestock into natural areas and their movement (Ferreira – Junior et al., 2018). Coccidia of the Eimeriidae family are usually monoxenic; however, some species such as Cystoisospora spp. can have paratenic hosts (i.e. intermediate hosts) (Salgado-Miranda &Soriano- Vargas, 2021). A paratenic host is one that serves as a "disseminator" and vehicle for access to the definitive host. The parasite does not evolve in this host, so it is not essential in order to complete the life cycle (Thrusfield, 2012). In the cattle farms of the state of Veracruz, there is interaction of various domestic animals such as bovines, horses, canines, felines, birds, etc., as well as wild animals, ruminants of other species and vice versa. This information is very valuable from an epidemiological point of view because it shows the presence of hosts that represent vehicles and transport in those animals susceptible to this parasitosis, since the interaction between poultry and cattle was demonstrated. Kawahara et al. (2010) conducted a study where they confirmed that the ITS-1 gene was efficient for the differentiation of Eimeria species due to the heterogeneity it shows among the sequences of different species, being an ideal marker for the design of specific primers with a close phylogenetic similarity between coccidian species. In this study, the phylogenetic analysis based on the ITS-1 region of Eimeria clearly differentiated E bovis, E. zuernii, E. canadensis, E. cylindrica, E. ellipsoidalis, E. bukidnonensis, and E. maxima from other Eimeria species, but did not show monophyletic characteristics among them.
Of all the species analyzed, their genetic information coincides with that reported in different countries of North America, Arkansas, Japan, and Turkey. The traceability observed in the phylogenetic tree of Eimeria species found in this study is related to the fact that in recent years there has been a greater mobilization and transport of animals, allowing a higher diversity of Eimeria species in cattle (Lassen & Ostegaard, 2012). Similarly, if sequences from different regions were obtained, this study could be used to evaluate the geographic diversity in the country and/or Latin America.
Since not many molecular studies on coccidiosis have been conducted, further investigations with more cases are required to elucidate the molecular characteristics of Eimeria species in Mexico.