4. Discussion
Cryptosporidiosis was identified as the cause for outbreak of severe diarrhea in neonatal calves in this study. The initial EIA analysis of 18 fecal specimens from ill calves at the peak of the outbreak showed that C. parvum and rotavirus were possible etiological agents rather than E. coli and coronavirus. The role of rotavirus was excluded in comparative detection of the pathogen in specimens from neonatal calves during and after the outbreak. There was no significant difference in infection rates of rotavirus during and after the outbreak, and rotavirus infection was not significantly associated with the occurrence of moderate or watery diarrhea in these animals. In contrast, the infection rate of C. parvum in neonatal calves was higher during the outbreak than after it, and the infection was significantly associated with the occurrence of watery diarrhea in these animals. In addition, all C. parvum infections were observed in calves of 1-4 weeks in concurrence with watery diarrhea, reinforcing the role of C. parvum in the occurrence of the outbreak.
The present study constitutes one of several lines of evidence for the pathogenicity of the IId subtype family of C. parvum . The pathogenicity of IIa subtypes of C. parvum has been well established, as cryptosporidiosis outbreaks have been reported in neonatal calves in many industrialized countries where they are prevalent (Holzhausen, Lendner, Gohring, Steinhofel, & Daugschies, 2019; Izzo et al., 2011; Niine, Dorbek-Kolin, Lassen, & Orro, 2018). The pathogenicity of IId subtypes of C. parvum , however, has only been indicated in molecular epidemiological investigations of Cryptosporidium spp. in China, which have shown a dominance of the IId subtype family and association of it with the occurrence of diarrhea in pre-weaned calves (Cai et al., 2017; Wu et al., 2020). Recently, the IId subtype family was implicated as the cause for two outbreaks of cryptosporidiosis in calves in China, including one by the IIdA15G1 subtype in Ningxia, leading to the death of 356 calves (Cui et al., 2014), and one by the subtype IIdA19G1 in Jiangsu, leading to the death of 360 calves (Li et al., 2019). In the present study, the IIdA20G1 subtype was identified as the cause of cryptosporidiosis outbreak in calves in China.
IIdA20G1 appears to be an emerging C. parvum subtype. In China, it has been recently reported in cattle in Heilongjiang, Hebei and Xinjiang and deer in Hubei (Tao et al., 2018; Wu et al., 2020; Xie et al., 2019; Zhang et al., 2020). Elsewhere, it has been reported in livestock and humans in Egypt, Kuwait, Qatar, Jordan, Turkey, Iran, Italy and Romania (Amer et al., 2010; Amer, Zidan, Adamu, et al., 2013; Amer, Zidan, Feng, et al., 2013; Boughattas et al., 2017; Diaz et al., 2018; Helmy, Krucken, Nockler, von Samson-Himmelstjerna, & Zessin, 2013; Hijjawi, Mukbel, Yang, & Ryan, 2016; Ibrahim, Abdel-Ghany, Abdel-Latef, Abdel-Aziz, & Aboelhadid, 2016; Imre et al., 2013; Kiani et al., 2017; Mahfouz, Mira, & Amer, 2014; Majeed et al., 2018; Naguib et al., 2018; Taylan-Ozkan et al., 2016). Most of these countries are located in the Middle East. Nevertheless, this is the first report of cryptosporidiosis outbreak caused by C. parvumIIdA20G1 subtype.
As with the C. parvum IIa subtype family, young age is associated with diarrhea caused by the IIdA20G1 subtype. In present study, C. parvum all appeared in neonatal calves under four weeks of age. This is likely due to the immature immune system in these animals, which also makes them prone to the occurrence of clinical signs after C. parvum infection (Santin et al., 2008). In the present study, neonatal calves had high occurrence of C. bovis after the outbreak. This, however, was not associated with the occurrence of diarrhea. Previously, in a few reports, C. bovisand C. ryanae had been associated with the occurrence of moderate diarrhea (Lee et al., 2016; Li et al., 2019; Silverlas & Blanco-Penedo, 2013; Silverlas, Bosaeus-Reineck, Naslund, & Bjorkman, 2013).
The role of rotavirus in causing the diarrhea outbreak appears to be minor. Although rotavirus was commonly found in neonatal calves in the present study, there was no significant difference in its infection rates during and after the outbreak. In statistical analyses, its occurrence was not correlated with the occurrence of diarrhea. Nevertheless, rotavirus is a well-known cause of diarrhea in 1-2-week-old calves (Cho, Han, Wang, Cooper, & Yoon, 2013). It has been frequently identified in co-infections with C. parvum , contributing to the occurrence of diarrhea in infected calves (Brar et al., 2017; Izzo et al., 2011). Thus, the concurrence of rotavirus in neonatal calves could have worsen the deleterious impact of C. parvum infection in the investigated outbreak of diarrhea.