Introduction

Bovine viral diarrhea virus (BVDV) is classified in the Pestivirus genus within the Flaviviridae family. It is an important pathogen of cattle that can cause reproductive failure, weak born or persistently infected (PI) calves and mucosal disease. BVDV also contributes to the bovine respiratory disease complex (Fray et al., 2000; Liebler-Tenorio et al., 2000; Cornish et al., 2005). BVDV has two genotypes, BVDV 1a 1b and BVDV 2, with each genotype presenting two biotypes, cytopathic (cp) and non-cytopathic (ncp), based on whether or not they cause cellular alterations. Persistent infection results from cows being exposed to the ncp variant of the virus before day 125 of gestation, allowing the fetus to develop immunotolerance to the virus and letting the virus persist after birth (Fulton et al., 2000; Brock, 2004; Bolin and Grooms, 2004; Zimmer et al., 2004). PI animals continuously shed great quantities of the virus even when they are clinically healthy and are therefore considered an important source of viral dissemination among the herd. One of the main strategies that has been used to eliminate BVDV in herds is to identify and remove PI calves (Fray et al., 2000; Cornish et al., 2005). For this, several diagnostic tests have been used which involve viral isolation (VI), inverse transcription, polymerase chain reaction (PCR) and real time (RT)-PCR, antigen capture enzyme-linked immunosorbent assay (ELISA_Ag), viral genome identification in peripheral blood leucocytes (PBL) and immunohistochemistry (IHC) (Liebler-Tenorio et al., 2000; Njaa et al., 2000; Malhum et al., 2002; Brodersen, 2004; Luzzago et al., 2006; Hilbe et al., 2007). In order to opportunely detect PI calves, a sensitive, economical, easy to perform diagnostic test is required, to facilitate prevention and control strategies of this infection in herds. The IHC test is based on viral antigen detection in infected animal skins. This test has been effective for PI animal detection since only a small portion of the skin, generally obtained from the ear, is required, numerous samples can be analyzed simultaneously and the results have been satisfactory. The aim of this study was to detect PI calves of up to 3 months old, by IHC in skin biopsies and ELISA_Ag in serum samples, compare the results of both tests and determine immunopositive localization in skin biopsies.

Material and methods

Sample collection

Immunohistochemistry

Statistical analysis

Results

Twelve of the 80 ear skin biopsies (15%) gave positive IHC results. Immunopositivity was observed as a red color which was distinctive in the cytoplasm of the epidermis and the keratinocytes (Figure 1), hair follicles and mononuclear cells of the dermis (Figures 2 and 3). The 80 sera were negative in the ELISA_Ag test. Statistical analysis found that the capacity to detect PI animals by means of ELISA_Ag and IHC was significantly different (P <0.01), with 15% of animals diagnosed as positives by IHC.

Figure 1

Figure 1. Ear skin biopsy, treated with the Streptavidin-Biotin-Peroxidase Complex with anti-BVDV antiserum against E2 (gp53) protein from genotypes 1 and 2, from a persistently infected (PI) calf with bovine viral diarrhea virus (BVDV). Viral antigens in keratinocytes are evident. Bar = 107 µm.

Figure 2

Figure 2. Ear skin biopsy, treated with the Streptavidin-Biotin-Peroxidase Complex with anti-BVDV antiserum against E2 (gp53) protein from genotypes 1 and 2, from a persistently infected (PI) calf with bovine viral diarrhea virus (BVDV). Viral antigens on the epidermis are evident. Bar = 107 µm.

Figure 3

Figure 3. Ear skin biopsy, treated with the Streptavidin-Biotin-Peroxidase Complex with anti-BVDV antiserum against E2 (gp53) protein from genotypes 1 and 2, from a persistently infected (PI) calf with bovine viral diarrhea virus (BVDV). This image is a close-up of the image in Figure 1. Bar = 30 µm.

Discussion

The results of this study indicate that IHC on ear tissue samples from calves is a sensitive diagnostic method for the detection of PI animals. It has been reported that not all monoclonal antibodies detect BVDV antigens in formalin-fixed, paraffin-embedded tissues (Haines et al., 1992; Brodersen, 2004). However, several studies have used monoclonal antibody (15C5) which reacts with BVDV EO (gp48) (E^rns/gp48) protein and gives good results in biopsies of skin and other tissues (Haines et al., 1992; Bazler et al., 1995; Cornish et al., 2005; Luzzago et al., 2006; Montgomery, 2007). In this work, the anti-BVDV monoclonal antibody types 1 and 2, E2 (gp53), were able to detect viral antigens in the skin biopsies, previously fixed in 10% formalin. Although it is known that formol fixation affects protein antigens, this effect could possibly be reverted during treatment with citrate buffer and heat, helping antigen retrieval.

The results of this study are similar to the results carried out by Njaa et al. (2000), who found that out of 42 animals BVDV-positive by VI, 41 (97.6%) were also positive by IHC in skin biopsies from different parts of the body. In another study carried out by Grooms and Keilen (2002), using blood samples and skin biopsies from 332 calves between 1 and 4 months old, six (1.8%) animals were BVDV-positive by VI and IHC. In these studies it was also concluded that IHC was a sensitive method, comparable with VI, for the detection of PI animals by BVDV. However, the results of our study differ from those of Cornish et al. (2005), who found that ELISA_Ag and IHC detected 100% of the PI animals. The fact that both diagnostic methods had the same sensitivity in their study could be due to the fact that ear skin biopsies were used in ELISA_Ag rather than serum. On the other hand, the results of our study also differ from those of Luzzago et al. (2006) who found that of 31 PI animals, 29 of these were positive by ELISA_Ag and IHC, and 2 animals were positive by ELISA_Ag but negative by IHC. The difference between the results of both studies could be due the fact that they were used different monoclonal antibodies for IHC. According to another study carried out by Kuhne et al. (2005) in which ELISA_Ag was carried out on sera and ear biopsy samples of 11 calves PI by BVDV, no viral antigens were detected in sera after colostrum ingestion, as was found in our study, but viral antigens were detected in ear biopsy samples. It was concluded that ELISA_Ag can be a reliable and economic method for the diagnosis of BVDV in skin samples. A possible explanation for the lack of BVDV antigen detection in sera by ELISA_Ag could be that anti-BVDV antibodies are circulating in calves as a result of a humoral or acquired passive immunity response. In this way, the antigen sites of circulating viral particles could be camouflaged by antibodies present in serum, resulting in inhibition of virus-binding during ELISA_Ag (Palfi et al., 1993; Grooms and Keilen, 2002; Brodersen, 2004; Zimmer et al., 2004; Sandvik, 2005; Hilbe et al., 2007). For this reason, it is not recommended that detection methods targeting viral antigens in serum are used in PI animals less than 3 months old, due to the presence of colostrum antibodies.

Conclusions

It is concluded that the immunopositive reaction to anti-BVDV antibodies in ear skin biopsies of PI calves was similar to the reported in previous studies (Njaa et al., 2000; Grooms and Keilen, 2002; Brodersen, 2004; Cornish et al., 2005; Luzzago et al., 2006), confirming the presence of the virus at this site. This study also confirms that IHC is an efficacious method for the early detection of PI animals, being more sensitive than ELISA_Ag, in sera. IHC can be used as a routine technique to detect BVDV and can be applied to retrospective studies using stored tissues. The identification and timely elimination of PI calves decreases economic losses, dissemination of the virus in the herd and therefore avoids the development of complete generations of PI animals.