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 ℃.