3.3. Occurrence of C. parvum in Neonatal (1-4-Week-Old) Calves
During the outbreak, 27 of 40 (67.5%) fecal specimens collected from neonatal calves were positive for Cryptosporidium spp. by PCR, including C. parvum (n = 24) and C. bovis (n = 3). After the outbreak, 35 of 56 (62.5%) fecal specimens collected from neonatal calves were positive for Cryptosporidium spp., including C. parvum (n = 17), C. bovis (n = 17) and C. ryanae (n = 1) (Fig. 2). The infection rate of C. parvum during the outbreak (60.0%, 24/40) was significantly higher (χ2 = 8.38, df = 1, P = 0.004) than after the outbreak (30.4%, 17/56) (Fig. 2). Animals older than 4 weeks had no C. parvuminfection during and after the outbreak. Instead, they were mostly infected with C. bovis (n = 56) and C. ryanae (n = 39), together with some occurrence of C. occultus (n = 6) and C. andersoni (n = 5) (Table 3). Therefore, during the outbreak, C. parvum was the dominant species in neonatal calves, while after the outbreak, C. bovis and C. parvum occurred at the same frequency (Table 3).
Altogether, 39/41 specimens positive for C. parvum were successfully subtyped at the gp60 locus. The sequences obtained were all identical to the reference sequence KU248815 of the IIdA20G1 subtype from GenBank.
3.4. Correlation between Cryptosporidium Spp. and Diarrhea Occurrence as Indicated byχ2Analysis
In neonatal (1-4-week-old) calves, Cryptosporidium infection rates were 66.7% (26/39), 69.2% (9/13) and 61.4% (27/44) in animals with watery diarrhea, moderate diarrhea and no diarrhea, respectively. The differences among groups were not significant in χ2 analysis (χ2 = 0.40, df = 2, P = 0.820) (Tables 4). ByCryptosporidium species, C. parvum infection rates were 61.5% (24/39), 38.5% (5/13), and 27.3% (12/44) in animals with watery diarrhea, moderate diarrhea and no diarrhea, respectively. The difference in C. parvum infection rates between animals with watery diarrhea and those with no diarrhea was significant (χ2 = 9.88, df = 1, P = 0.002) (Tables 4). In contrast, the infection rate of C. bovis in animals with no diarrhea (31.8%, 14/44) was significantly higher than those with watery diarrhea (5.1%, 2/39) (χ2 = 9.46, df = 1, P = 0.002). Infection rates of C. ryanae were similar among the three groups (χ2= 1.19, df = 2, P = 0.550) (Tables 4).
No C. parvum infection nor watery diarrhea was observed in calves older than 4 weeks. In this age group, the infection rates ofCryptosporidium spp. were 58.8% (30/51) and 60.6% (40/66) in animals with moderate diarrhea and those without diarrhea, respectively (χ2 = 0.04, df = 1, P = 0.845) (Tables 4). By Cryptosporidium species, the infection rates ofC. bovis (χ2 = 0.078, df = 1,P = 0.780) or C. ryanae (χ2 = 0.005, df = 1, P = 0.941) were not significantly different between the two groups (Tables 4).