Of the 119 cattle fecal specimens, 10 (8%) were confirmed positive for Cryptosporidium infection by PCR (Additional file 1: Figure S1 and Figure S2). This finding is consistent with previous investigations, reporting low Cryptosporidium prevalence (2.5–17%) in cattle herds of mixed ages [15, 23, 29]. In addition, 7 (70%) of the 10 PCR-positive samples analyzed in the present study were collected from cattle presenting symptoms of diarrhea at the time of sample collection. Among this group, five were from calves ≤ 2 years of age, including two pre-weaned calves ≤ 6 months (Table 1). These findings are not surprising given the widespread reporting of cryptosporidiosis in diarrhetic calves (predominantly pre-weaned), with decreasing rates as calves mature into adulthood [30–33].
When data were analyzed according to animal breed, it was determined that 7 (70%) of the 10 PCR-positive samples derived from dairy cattle. Previous epidemiological investigations have also reported significantly higher rates of cryptosporidiosis in dairy cattle compared to their beef counterparts [34–35]. This phenomenon is in large due to farm management practices and sanitary conditions. In the present study, beef cattle typically roamed their pastures freely or were spaced out, while the dairy cattle were confined to small spaces to facilitate grain feeding and milking. The confinement of dairy herds to small spaces has been rationalized to predispose the animals to rapid Cryptosporidium infection and subsequent re-infection; contrary to beef cattle, which are typically kept in open ranges, spreading the infection relatively slower [29].
Also shown in Table 1, sequence analysis of the 18S rRNA gene locus, identified C. parvum from one beef calf (no. Be8) corresponding with 90% sequence identity to a strain previously reported in a calf from the Netherlands (GenBank accession number: MW947436). Similarly, C. parvum was also identified in two dairy calves (nos. 34Da and 53Da), both corresponding with 99% identity to strains previously reported in calves from China (GenBank accession no: MF074701) and Egypt (GenBank accession no: AB513881). The neighbor-joining analysis confirmed that C. parvum isolates, 34Da and 53Da, share a 96% nucleotide sequence identity (Fig. 2), suggesting they are most likely the same strain. Overall, these findings agree with literature indicating that C. parvum is the most frequently characterized species in young calves [30, 31]. Moreover, the likelihood of C. parvum spreading between cattle, other livestock animals and wildlife is highly plausible in St. Elizabeth parish due to outdoor cohabitation and neighboring rearing systems, consisting of goats, sheep, hogs, cattle and horses—all of which are known carriers of C. parvum [36].
In addition, sequence analysis of the 18S rRNA gene locus identified C. hominis from one dairy calf (no. 105Da), with an 82% sequence identity to strains previously reported in humans and non-human primates (GenBank accession nos: MN836824 and MK982514). Presence of C. hominis in dairy calf no.105Da was further confirmed by gp60 subtyping, which identified the C. hominis subtype, IbA9G2. Gp60 subtyping was only successful in 1 of the 10 samples analyzed (Additional file 1: Figure S3). Typically, C. hominis has an anthroponotic transmission cycle and is a rare occurrence in animals [38, 39]. However, in recent years, there have been increasing reports of C. hominis (in low prevalence) identified in cattle. Razakandrainibe et al. [37] for example, also characterized the same IbA9G2 subtype, found in the present study, in an impaired calf in France and suggests that the presence of C. hominis and other species, less conventionally reported in cattle, may be underestimated due to limited data on Cryptosporidium spp. in asymptomatic cattle. In addition, through highly discriminatory subtyping techniques, other investigations in Malawi [40] Korea [41], the United Kingdom [42], New Zealand [43] and Australia [44] have identified C. hominis in cattle. Moreover, a recent review on C. hominis infection in non-human hosts [45], revealed C. hominis reported in cattle are primarily genetic variants belonging to the gp60 Ib family designation. The study suggests potential transmission of C. hominis from humans to wildlife, particularly in remote and desolated areas through contaminated water. Conversely, other studies have indicated the reverse process occurring, where human-borne C. hominis transmits to wildlife then back to humans [46, 47]. Whether these transmission dynamics of C. hominis occur in the rural parish of St. Elizabeth, Jamaica requires further investigation.
There were some limitations noted in the present study. Several samples had insufficient quality of DNA for sequencing. This was likely due to the presence of inhibitors [48] and low DNA yield, especially from adult infected cattle, which typically shed 105-106-fold less oocysts per day than infected calves [13, 49]. The use of a bovine serum albumin during processing may have reduced the number of PCR inhibitors. Of the successfully sequenced isolates, most were 18S rRNA sequences, which are hypervariable, making intraspecific allelic differences indistinguishable [50]. Thus, a crude fingerprinting method, such as restriction fragment length polymorphism (RFLP) is necessary to confirm C. parvum identification in the present study. Furthermore, the mixed peaks seen in the chromatograms (Additional file 1: Figure S4) were potentially due to heterologous copies of the 18S rRNA gene within a single sample. Thus, RFLP could have also been used to distinguish co-existing species or co-infections.