2.1. Animals and Specimens
The investigated farm is located in Hebei Province in northern China. There were ~2,000 cattle altogether, including about 350 calves on the farm. Among them, the majority of animals were of the Holstein breed, together with fewer than 200 cattle of the Simmental breed and 40 adult cattle of the Japanese Wagyu breed. There were no vaccinations against rotaviruses, coronaviruses, and Escherichia coli prior to the occurrence of the outbreak of diarrhea.
From December 2018 to April 2019, an outbreak of diarrhea was noticed in neonatal calves on the study farm (Fig. 1), with watery diarrhea occurring in all ~200 neonatal calves born during the period. Forty-one calves died of diarrhea at 10 – 30 days of age despite antibiotic therapy. They experienced severe diarrhea before death, with the excretion of watery feces. Most of them also had other clinical signs such as poor appetite, weight loss and dehydration (Table 1). To identify the cause of the diarrhea, 402 fecal specimens were collected from the farm during the outbreak in January 2019 (n = 173) and after the outbreak in June 2019 (n = 229). Among the specimens collected during the outbreak, 40 were from neonatal calves (1 - 4 weeks in age) and 133 were from older cattle (> 4 weeks in age). Similarly, 56 and 173 specimens collected after the outbreak were from neonatal calves and older cattle, respectively. Altogether, 213 specimens were from calves under 24 weeks and 189 from older cattle. The specimens from calves included formed feces from animals with no diarrhea (n = 110), loose feces from animals with moderate diarrhea (n = 64), and liquid feces from animals with watery diarrhea (n = 39). The older animals were mostly healthy.
Fecal specimens were collected directly from the rectum of each animal using disposable gloves into 50-mL centrifuge tubes. Approximately 1.0 g of each fecal specimen was transferred into a 1.5-mL tube and kept frozen at -80 ℃, while the remaining fecal material was stored in 2.5% potassium dichromate at 4 ℃.