The current study provided evidence-based information about the prevalence of and potential risk factors for Cryptosporidium spp. infection in working horses in Egypt. This information should be considered by the working horse owners and management practices of these horses should be modified accordingly, for example, observing personal hygiene such as hand sanitation when handling or cleaning for these animals. The results of the current study will be communicated to the veterinary surgeons in Egypt through social media groups to help them to provide informed advice to their clients.
Cryptosporidium spp. prevalence reported in the present study (28.7%) was higher than reported prevalence in farm animal species in Egypt such as cattle (10.2%), buffalo (0.5–12.3%) and sheep (1.3%) (Ibrahim, et al. 2016, Mahfouz et al. 2014). Two studies on dogs from Egypt reported comparable prevalence to the current study (30–34%) (Awadallah and Salem 2015, Gharieb, et al. 2018). These four studies that originated from Egypt had also used the modified Zielh-Neelsen staining technique and light microscopy to identify Cryptosporidium spp. oocysts in faeces.
Several studies have investigated the prevalence of Cryptosporidium spp. in foals and adult horses and reported variable prevalence estimates. The prevalence estimates reported varied according to the geographic location of the population studied and the detection methods used. Studies from China that used polymerase chain reaction (PCR) to investigate Cryptosporidium spp. prevalence in adult horses reported an average prevalence of 1.8–3.1% (Li, et al. 2019, Qi et al. 2015, Xu, et al. 2022) which is much lower than the prevalence reported here. A recent systematic review and meta-analysis reported a pooled prevalence of 13.8% from studies that used microscopy compared with only 4.7% from studies that used PCR (Hatam-Nahavandi, et al. 2019). Another recent study from Chile that investigated Cryptosporidium spp. infection prevalence in 100 adult horses using microscopy reported greater prevalence of 67% (Tuemmers et al. 2022). Earlier studies of the prevalence of Cryptosporidium spp. infection in adult horses that used microscopy for identification of oocysts reported prevalence of 36.9% in Taiwan (Guo, et al. 2014), 9.4% in Poland (Majewska et al. 1999), 18.4% in Brazil (Inacio et al. 2012) and 19.5% in Iran (Haghi et al. 2020).
Studies consistently reported greater prevalence of Cryptosporidium spp. in foals compared with adult horses (Cole, et al. 1998, Inacio, et al. 2012, Tuemmers, et al. 2022, Veronesi, et al. 2010). The horse age was negatively associated with Cryptosporidium spp. prevalence in the present study, but this association was not statistically significant. The present study population included only 14 foals that were < 6 months of age, half of which were found to be infected. The sex of the horse was not also associated with Cryptosporidium spp. infection in the current study which is consistent with published research (Tuemmers, et al. 2022).
In the current study, we attempted to extract DNA and amplify 18S rRNA genes using PCR from a random subset of positive samples but none of the PCR reactions yielded positive results. This could be attributed to low oocyst excretion by adult clinically normal horses (Kostopoulou et al. 2015) which may have resulted in a low DNA yield especially if DNA was directly extracted from faeces without concentrating oocysts. Furthermore, presence of PCR inhibitors in faecal samples could impair DNA amplification (Elwin et al. 2012). This was also consistent with prevalence studies of Cryptosporidium spp. infection which reported lower prevalence when PCR is used as sole detection method (Hatam-Nahavandi, et al. 2019, Li, et al. 2019, Qi, et al. 2015, Xu, et al. 2022). Overall, epidemiological studies of Cryptosporidium spp. prevalence should be interpreted considering the test characteristics (e.g., sensitivity and specificity) of the diagnostic method/methods used (Majewska, et al. 2004). The detection threshold of the modified Ziehl–Neelsen staining technique has been estimated to be 10 × 105 oocysts per gram of faeces (Cole et al. 1999, Weber et al. 1991). Furthermore, presence of other acid-fast microorganisms in faeces, which are comparable in size to Cryptosporidium spp. oocysts, such as yeast, fungi, and other protozoa such as Cyclospora could result in a low test specificity unless stained smears are examined by an expert technician (Nielsen and Ward 1999, Tahvildar-Biderouni and Salehi 2014). Therefore, use of modified Ziehl–Neelsen staining technique to diagnose Cryptosporidium ssp. infection may underestimate (high % of false negative) or overestimate (high % of false positive) the prevalence depending on the shedding intensity of oocysts and the level of experience of the diagnostician.
Other limitations of the current study were that samples were collected at a single time point and therefore we did not consider intermittent shedding of Cryptosporidium spp. oocysts (Xu, et al. 2022) which might have underestimated the prevalence reported. Furthermore, neither locations nor horses were randomly selected to be included in the current study which might limit the generalisability of our results. Notwithstanding these limitations, the current study provided evidence-based information about the prevalence of Cryptosporidium spp. infection in a population of working horses in Egypt. Further studies to genotype these parasites in this population of horses may be required to investigate the zoonotic potential of the infection.