Comparison Of Ear Notch Immunohistochemistry Against E2 (Gp53) Protein And Antigen-Capture Elisa In Sera, For The Detection Of Calves Persistently Infected With Bovine Diarrhea Virus
E M., E S., F G, F S., J G
Keywords
bovine viral diarrhea, calves, elisaag., immunohistochemistry, persistent infection
Citation
E M., E S., F G, F S., J G. Comparison Of Ear Notch Immunohistochemistry Against E2 (Gp53) Protein And Antigen-Capture Elisa In Sera, For The Detection Of Calves Persistently Infected With Bovine Diarrhea Virus. The Internet Journal of Veterinary Medicine. 2009 Volume 8 Number 1.
Abstract
The objective of this study was to compare two methods of detection of calves persistently infected (PI) with bovine viral diarrhea virus (BVDV): immunohistochemistry (IHC), using ear skin biopsies, and antigen capture enzyme-linked immunosorbent assay (ELISAAg) using sera. We also aimed to determine the site of immunopositivity. Samples were taken from 80 Holstein calves of up to 3 months of age. For IHC, the streptavidin-biotin-peroxidase complex method was used, along with a monoclonal antibody binding to E2 (gp53) protein of BVDV type 1 and 2. For ELISAAg, a commercial kit was used based on the Erns (gp44–48) protein of BVDV type 1 and 2. Twelve of the 80 skin biopsies (15%) were IHC positive. Immunopositivity was observed in the epidermis, hair follicles and dermis mononuclear cells being similar to the reported in previous studies. This study confirms that 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. All sera were negative in ELISAAg. Statistical analysis showed a significant difference between the two methods in their capacity to detect PI animals, P (<0.01), indicating that IHC was more sensitive than ELISAAg, identifying 15% of infected animals.
Introduction
Bovine viral diarrhea virus (BVDV) is classified in the
Material and methods
Sample collection
Ear skin biopsies and sera were taken simultaneously from 80 Holstein calves of up to 3 months old, from herds with a BVDV infection background in the Complejo Agropecuario Industrial de Tizayuca, Sociedad Anonima (CAITSA), located on the Mexico-Pachuca highway in the state of Hidalgo, Mexico.
Immunohistochemistry
For skin biopsies, ear notches were used to obtain a tissue fragment of 1.0 × 0.8 × 0.4 cm approximately. Biopsies were fixed in 10% formalin, at pH of 7.4 for 24 h. Tissues were processed by routine histological technique and cut to a thickness of 3 mm to be examined by IHC using the streptavidin-biotin-peroxidase complex method (Haines et al., 1992). Cut tissue samples were mounted on slides with poly-L-lysine, deparaffined for 1 h at 60ºC and rehydrated in decreasing concentrations of ethanol.
Then, endogenous peroxidase inhibition was carried out using a 3% H2O2 in methanol solution, followed by antigenic retrieval using Target Retrieval Solution (Dako Corporation), according to the manufacturer's instructions, along with heat treatment for 3 min and 30 sec in a microwave oven. Endogenous avidin and biotin were blocked using an Avidin/Biotin Blocking Kit (Zymed Laboratories Inc.), according to the manufacturer's instructions, and the samples were then ready for antibody treatment. A monoclonal primary antibody that binds to E2 (gp53) of BVDV (genotypes 1 and 2) (VMRD, Inc.), was diluted 1:150 in phosphate-buffered saline (PBS) and incubated with the tissue sample for 1 h in a humid chamber (HC) at ambient temperature (AT). After a short rinse with PBS, a biotin conjugated protein G (Rockland Immunochemicals Inc.) was then applied as a secondary antibody, diluted at 1:100 in PBS, and incubated for 1 h in a HC at AT. After washing the slides three times for 5 min in PBS, streptavidin (Zymed Laboratories Inc.) was then applied for a further 30 min incubation in a HC at AT. Finally, the reaction was developed using aminoethylcarbazole (AEC) and the Histostain SP Kit (Zymed Laboratories Inc.), to contrast with Mayer’s hematoxylin. Positive and negative tissue controls were included.
Blood samples were obtained from calves by coccygeus vein puncture and obtained sera were treated with antigen capture enzyme-linked immunosorbent assay (ELISAAg) using the IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit (IDEXX Laboratories Inc.), following the manufacturer's instructions. This assay is based on the detection of protein Ems (gp44–48) of BVDV genotypes 1 and 2.
Statistical analysis
Statistical analysis was carried out using the chi-square test of homogeneity and the SAS program.
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 ELISAAg test. Statistical analysis found that the capacity to detect PI animals by means of ELISAAg and IHC was significantly different (P <0.01), with 15% of animals diagnosed as positives by IHC.
Figure 1
Figure 2
Figure 3
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
The results of this study are similar to the results carried out by Njaa et al
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