Comparison of Immunohistochemistry, Electron Microscopy, and Direct Fluorescent Antibody Test for the Detection of Bovine Coronavirus

Abstract

Abstract. Bovine coronavirus (BCV) is 1 of the major causes of calf diarrhea and has also been implicated in respiratory infections of young calves and winter dysentery of adult cattle. Currently, transmission electron microscopy (TEM), direct fluorescent antibody (DFA), and enzyme-linked immunosorbent assay (ELISA) techniques are considered standard methods for the diagnosis of BCV infection. However, these techniques are not useful if fresh tissues and intestinal contents are not available for examination. The detection of viral antigens in formalin-fixed, paraffin-embedded tissues using immunohistochemistry (IHC) is a suitable alternative. In the present study, 166 tissue specimens were tested by IHC for the presence of BCV. These tissues were from animals whose feces were positive for rotavirus and/or coronavirus by TEM. Some of these samples were also tested by DFA. Thus, TEM, DFA, and IHC were compared for the detection of BCV. There was 56% agreement among the 3 methods (overall kappa ϭ 0.368). When IHC was compared with TEM, 78% agreement was observed (kappa ϭ 0.475). Similarly, IHC and DFA had 64% agreement (kappa ϭ 0.277). These kappa values indicate a moderate degree of agreement between IHC and TEM; agreement between IHC and DFA was fair. The results of this study indicate that IHC may be a suitable adjunct for the detection of BCV because of its simplicity, ease of use, and relatively close correlation with TEM results.Calf scours is a major cause of economic losses to the cattle industry resulting in annual losses of up to $1.7 billion around the world, 28 increasing in recent years due to the adoption of intensive beef and dairy practices. 30 Bovine coronavirus (BCV) is one of the leading causes of calf scours, 32 with coronaviruses and rotaviruses estimated to account for 20-26% and 27-36% of all cases of calf diarrhea, respectively. 3 More severe diarrhea and higher mortality is caused by BCV than by rotaviruses because it involves both the large and the small intestines. 38 BCV has also been implicated in winter dysentery of adult cattle 31 and in respiratory tract infection of 2-16-week-old calves. 20 BCV has also been implicated in pneumonia in adult dairy cattle 9 and in cattle after shipping. 35 BCV can be detected by transmission electron microscopic examination of fecal samples, direct fluorescent antibody assay (DFA) on frozen sections of spiral colon, 23 and enzyme-linked immunosorbent assay (ELISA) 10 and occasionally through virus isolation in vitro. 21 Currently, transmission electron microscopy (TEM) and DFA are the most widely used methods for BCV diagnosis. 27 Results from TEM can be obtained in a few hours, but the technique has the limi- tation of detecting nonviral particles that may look like coronavirus and detecting virus only when the number of virions is Ͼ10 6 /g of feces. 12 After the onset of diarrhea, the amount of BCV in feces may fall rapidly below the detection limits of TEM. 20 Another difficulty is that a relatively small number of samples can be examined by TEM in...

Bovine Coronavirus Associated Syndromes

“…2 and 3). 117 Spiral colon is the sample of choice for detection of BECoV at necropsy because the virus persists in that specific location for the longest time after oral infection. 50 A fresh fecal sample collected directly from the rectum can be also included, as cattle with respiratory disease commonly shed the virus in the feces concomitantly.…”

Achados clínico patológicos durante um surto de disenteria de inverno em bovinos no Rio Grande do Sul

“…A marcação positiva na imuno-histoquímica anti-coronavírus bovino associada com lesões no caso fatal comprova a atuação do vírus nesse surto (Van Kruiningen et al 1987, Zhang et al 1997, Dar et al 1998, Cho et al 2000, Natsuaki et al 2007.…”

“…tem sido feita pela detecção deste nas fezes e pela soroconversão dos animais de rebanhos afetados (Saif 1990). Para tal, as técnicas que têm sido empregadas incluem, microscopia eletrônica direta (Saif et al 1991, Athanassious et al 1994, Dar et al 1998, ensaio imunoenzimático (ELISA) (Athanassious et al 1994, Smith et al 1996, Cho et al 2000, Jeong et al 2005, RT-PCR (Brandão et al 2002, Jeong et al 2005, Brandão et al 2007a,b, Natsuaki et al 2007), imuno-microscopia eletrô-nica (Athanassious et al 1994, Cho et al 2000, Jeong et al 2005 e isolamento viral in vitro (Sai 1990, Jeong et al 2005, Natsuaki et al 2007. No entanto, ainda há controvérsias sobre a relação da detecção do coronavírus nas fezes de animais com a doença clínica, visto que há relatos da presença do vírus nas fezes de animais clinicamente normais, com picos de eliminação nos meses de inverno e próximo do parto (Collins et al 1987, Clark 1993, Radostits et al 2000.…”

Detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease

“…There was moderate hepatic lipidosis with a slightly increased number of leukocytes within hepatic sinusoids. Using a monoclonal antibodybased immunohistochemical test for BVDV 4 and BCV, 5 only BCV antigen was detected in the intestines in all 3 calves. In the colon, BCV antigen was associated with necrotic epithelium in the crypts (Fig 2).…”