Abstract
Background
Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described.
Findings
Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells.
Conclusions
Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.
Findings
Bovine viral diarrhea is contagious disease of domestic and wild ruminants and one of the economically most important diseases in cattle. Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus, within the family Flaviviridae [1]. Based on viral RNA sequence, there are at least 2 viral genotypes of BVDV that can be further divided into subgenotypes [2, 3].
Furthermore, isolates of BVDV can be separated into non-cytopathogenic and cytopathogenic biotypes based on cytopathogenic effects observed in infected cell cultures [4, 5]. Non-cytopathogenic strains have the possibility to cause persistent infection. Infection of heifers or cows with non-cytopathogenic biotype of BVDV in the first four months of pregnancy can lead to the birth of persistently infected (PI) calves [6]. PI animals act as a viral reservoir and are the main factor of the virus's continuation within herds [7]. To confirm the persistent infection, two samples should be taken within three to four week intervals. The identification and elimination of the PI animals from the herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for detection of PI animals. In diagnosis of BVDV, many different reliable methods are available. Virus or virus antigen can be detected in blood, sera, and organs from dead animals. Virus isolation (or its modification - the immunoperoxidase test) is still considered as a golden standard in diagnosis of BVDV. However, in cattle younger than six months, detection of the virus in sera samples can be adversely affected by the presence of specific antibodies [8]. Because of that, the reverse transcription polymerase chain reaction (RT-PCR) can be applied as a relative golden standard on sera samples [9] or ear notch tissue samples can be tested. Ear notch tissue samples can be tested with antigen immunoassay test (Ag ELISA), RT-PCR or immunohistochemistry (IHC). However, the distribution of viral antigen within tissue can be established only with IHC. IHC on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of PI cattle from ear notch tissue samples. However, such approach has many disadvantages due to complicated tissue fixation process and long preparation time. IHC on frozen tissue in diagnosis of BVDV is usually applied on organs from dead animals. For all of these reasons we described for the first time the IHC on frozen ear notch tissue samples from live animals. Ear notch tissue samples were collected during the period 2008-2009 from 17 positive heifers. This study was approved by ethics committee of Veterinary Faculty University of Zagreb; number: 251-61-01/139-11-72. Virus was confirmed by Ag ELISA and testing was repeated after four weeks. At the second sampling, two samples of ear notch tissue per animal were taken. A sample for the Ag ELISA and RT-PCR tests was stored at -20°C. The other sample was placed immediately into liquid nitrogen for 1 min and then stored at -20°C. To prevent contamination, samples were put in sterile cryo tubes and then placed in liquid nitrogen. Samples were transported to the laboratory on dry ice in a portable refrigerator. Delivery time to the laboratory was between 3 and 6 h. In the laboratory, the samples were stored at - 20°C until testing. The Ag ELISA for detection of viral antigen on ear notch tissue was performed using commercially available kit according to manufacturer's instructions (Herdchek BVDV Ag/Serum Plus, IDEXX, Liebefeld-Bern, Switzerland). Prior to the test, ear notch tissue samples 1 × 1 cm in size were placed in individual sterile tubes with 2 ml of provided buffer and refrigerated overnight at 4°C. All 17 samples showed positive result by Ag ELISA and were tested once again with RT-PCR in the same manner as described previously [10], targeting the 441-bp fragment of the Npro genome region. All 17 samples were also positive by RT-PCR. For IHC, ear notch tissue samples that were fixed in liquid nitrogen and stored at - 20°C were sectioned with scalpel in the shape of a trapeze, approximately 2 × 1 cm in size. Tissue sections were quickly transported in the cryostat (LEICA; CM1500S, Wetzlar, Germany) and fixed onto holders with the tissue freezing medium (LEICA, Nussloch, Germany). Tissue sections were cut at -25°C at 6
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This research was supported by grant No. 048-0481186-1183 from the Ministry of Science, Education and Sports, Republic of Croatia.
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TB: Main initiator, designer and performer of the study and author of the manuscript; NL, IL, AB, ML, JM: Contributor to design, perform and revising the manuscript. All authors read and approved the final manuscript. Please see sample text in the instructions for authors.
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Bedeković, T., Lemo, N., Lojkić, I. et al. Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle. Acta Vet Scand 53, 65 (2011). https://doi.org/10.1186/1751-0147-53-65
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DOI: https://doi.org/10.1186/1751-0147-53-65