Effects of Delayed or Prolonged Fixation on Immunohistochemical Detection of Bovine Viral Diarrhea Virus Type I in Skin of Two Persistently Infected Calves

The effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus (BVDV) antigen were evaluated in skin. Ear-notch specimens from 2 calves persistently infected with BVDV type 1 were handled in 1 of 3 ways: 1) fixed in formalin promptly and processed for immunohistochemistry (IHC) after 3–176 days; 2) held at 3–4°C in plastic bags up to 10 days, then fixed in formalin for 2–5 days before processing; or 3) exposed to room air and temperature for 1–5 days before formalin fixation. Immunohistochemical staining intensity was evaluated without the knowledge of specimen handling. Staining of specimens that had been promptly fixed in formalin was moderate to strong at all fixation periods through 36 days, weak or no staining was evident in specimens fixed for 176 days. Refrigerated specimens typically had moderate to strong immunohistochemical staining. Even after 10 days of refrigeration before fixation, all immunohistochemical reactions were positive. However, no immunohistochemical staining was detected in any specimen that was exposed to room air. Results indicate that prompt formalin fixation is optimal for BVDV IHC. Samples can be held in formalin at least 36 days, without loss of reactivity. A 1-day delay in fixation caused no loss of reactivity, provided the specimen was refrigerated and protected from desiccation.

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Attenuated lymphocyte activation leads to the development of immunotolerance in bovine fetuses persistently infected with bovine viral diarrhea virus†

Biology of Reproduction ◽

10.1093/biolre/ioaa088 ◽

2020 ◽

Vol 103 (3) ◽

pp. 560-571 ◽

Cited By ~ 3

Author(s):

Hanah M Georges ◽

Katie J Knapek ◽

Helle Bielefeldt-Ohmann ◽

Hana Van Campen ◽

Thomas R Hansen

Keyword(s):

Immune System ◽

Bovine Viral Diarrhea Virus ◽

Lymphocyte Activation ◽

Control Measures ◽

Bovine Viral Diarrhea ◽

Type I ◽

Persistently Infected ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Viral Diarrhea Virus

Abstract Bovine viral diarrhea virus continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for bovine viral diarrhea virus are persistently infected animals, which are infected in utero and shed the virus throughout their lifetime. The difficulty in controlling the virus stems from a limited understanding of transplacental transmission and fetal development of immunotolerance. In this study, pregnant bovine viral diarrhea virus naïve heifers were inoculated with bovine viral diarrhea virus on day 75 of gestation and fetal spleens were collected on gestational days 82, 97, 190, and 245. Microarray analysis on splenic RNA from days 82 and 97 revealed an increase in signaling for the innate immune system and antigen presentation to T cells in day 97 persistently infected fetuses compared to controls. Reverse transcription quantitative polymerase chain reaction on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 persistently infected fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with hematoxylin and eosin staining and immunohistochemistry. Data collected indicate that fetal immunotolerance to bovine viral diarrhea virus developed between days 97 and 190, with mass attenuation of the immune system on day 190 of gestation. Furthermore, lymphocyte transcripts were initially unchanged then downregulated, suggesting that immunotolerance to the virus stems from a blockage in lymphocyte activation and hence an inability to clear the virus. The identification of lymphocyte derived immunotolerance will aid in the development of preventative and viral control measures to implement before or during pregnancy.

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Screening of Neonatal Calves for Persistent Infection with Bovine Viral Diarrhea Virus by Immunohistochemistry on Skin Biopsy Samples

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.4.898-900.2002 ◽

2002 ◽

Vol 9 (4) ◽

pp. 898-900 ◽

Cited By ~ 5

Author(s):

Daniel L. Grooms ◽

Eric D. Keilen

Keyword(s):

Skin Biopsy ◽

Bovine Viral Diarrhea Virus ◽

Immunohistochemical Staining ◽

Bovine Viral Diarrhea ◽

Persistently Infected ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Neonatal Calves ◽

Viral Diarrhea Virus

ABSTRACT Detection and elimination of cattle that are persistently infected with bovine viral diarrhea virus (BVDV) is important for controlling the transmission of this virus. Colostrum-derived antibodies make the detection of persistently BVDV-infected neonatal calves cumbersome and expensive. The objective of this study was to evaluate the use of immunohistochemical staining of skin biopsy samples from neonatal calves as a method for the early detection of persistent BVDV infection. Three hundred thirty-two 1- to 4-week-old dairy calves were screened for BVDV as part of a routine control program. Formalin-fixed skin biopsy samples were stained for BVDV antigen by immunohistochemistry (IHC), and the results were compared to those of virus isolation (VI) from white blood cell preparations. Six calves were positive by both IHC and VI and remained positive for BVDV upon subsequent follow-up testing; thus, they were classified as persistently infected with BVDV. One calf was positive by VI but negative by IHC. On subsequent testing, the calf was negative by VI, suggesting that the initial VI result was due to an acute BVDV infection. One calf was positive by IHC but negative by VI. This calf remained negative by VI on follow-up testing. Immunohistochemical staining of skin biopsy samples is a reliable method for screening neonatal calves for persistent BVDV infection and would be a useful management tool as an aid for controlling and preventing BVDV infection.

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