Isolation and identification of bovine coronavirus (BCoV) from diarrheatic calves in Dakahlia Governorate, Egypt

Abstract

In the current study, a total of 107 fecal samples were collected from diarrhetic calves from 1 day to 3 weeks of age from different localities in Dakahlia governorate, Egypt (Gamasa, Belkas, Sherbin, Nabaroh, Talkha, Almanzala, Algamalia, and Aga) during the period from year 2015 to year 2018. These calves were suffering from watery diarrhea, dehydration, weakness and recumbency. All samples were taken from calves which were born from vaccinated cows. The fecal samples from 107 cases were screened for the presence of BCoV by using Ag-ELISA and RT-PCR. From ELISA and PCR positive identified samples, we visualized the Coronavirus particles when negatively stained by transmission electron microscopy. From ELISA and PCR positive identified samples, virus isolation was done via MDBK tissue culture, six passages were carried out followed by identification of BCoV in harvested tissue culture via DFA test and indirect immunoperoxidase technique. Furthermore, nucleotide sequence analysis of amplified N gene of the identified BCoV nucleic acid was done and compared with published reference sequences on GeneBank. The results of ELISA and RT-PCR revealed that: out of 107 tested fecal samples, 4 samples showed positive results (3.7%) and 103 samples showed negative results (96.2%). The viral particles showed pleomorphism with characteristic radial projections forming a corona, by transmission electron microscopy. All tested samples gave positive results with DFAT as yellowish green coloration was detected in stained MDBK cells which increased gradually with increased passages. All tested samples gave positive results with IP as brownish coloration was detected in stained MDBK cells which increased gradually with increased the period of incubation. further studies is required for better understanding the genetic diversity among BCoV circulating in Egyptian farms based on full genome sequencing.