Clinical inspection of affected dairy cattle showed watery diarrhea, bloody diarrhea, dehydration, melena or occult blood in feces, decrease milk production, and death in some cows.
Agarose gel electrophoresis of three diarrheic fecal samples yielded that out of the 25 samples the desired amplicons that were 488 base pairs (Fig. 1). In addition, after having been purified, the three samples yielded agarose gel, and they were sent sequencing analysis, by using the BLASTn. The result showed that they possessed 97–99% nucleotide identities to spike glycoprotein of bovine coronavirus.
The three cows were positive for fecal testing and also positive for the blood testing (Table 3). All blood samples analyzed the presence of the IgG antibodies to BCoV by commercially available indirect ELISA (SVANOVA, Biotech).
The sequence alignment by ClustalW multiple alignment of the BioEdit program indicated that nucleotide and amino acid sequences of the three TWD isolated in Thailand were high similar to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and were closely related to OK-0514-3 strain (virulent respiratory strain; RBCoV).The amino acid sequence identities among TWD1, TWD2, TWD3, and OK-0514-3 strain were 96.0 to 96.6%, which of those at T3I, H65N, D87G, H127Y, and Q136R were changed (data not shown).
The phylogenetic tree of partial spike glycoprotein genes of bovine coronavirus was composed of three major clades by using the 54 sequences generated in this study from field samples, GenBank reference strains, and vaccine strains. TWD1, TWD2, and TWD3 were closely related to the isolated from three positive samples (99.8 to 100% similarity), and they also had 98.2 to 98.5% similarity with OK-0514-3 strain. In Asia, they had 95.9 to 98.5% similarity with 24 Korean strains and Kakegawa strain. Furthermore, the most distance between those of TWD and other published strains were the Mebus and Kakegawa strain (95.9 to 96.2% similarity) for the partial S1 gene by nested PCR in Thailand. TWD1, TWD2, TWD3, all of Korean calf diarrhea (KCD) and Korean winter dysentery strains (KWD), some American respiratory strains (OK-0514-3, LSU-94LSS-051-2, 232NS, 220NS), and Canadian respiratory strains (BCO43277, BCQ3994, BCO44175) were clustered on clade 3. All of the Brazilian strains (BR-UEL1, BR-UEL2, BR-UEL3),Canadian enteric strains (BCQ571, BCQ2070, BCQ9, BCQ1523, BCQ20), Canadian winter dysentery strain (BCQ7373, BCQ2590), American enteric strains (ENT), and American respiratory strains (182NS) were clustered on clade 2. The Korean vaccine strains(0501, 0502, A3, SUN5, BC94 Korean vaccine), F15, L9, Norden vaccine, Quebec, Mebus, and Kakegawa strain were clustered on clade 1, but LY-138 (American enteric strain) was clustered on a separate branch (Fig. 2).