Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 8/1/2006
Publication Date: 10/12/2006
Citation: Fulton, R.W., Elam, N., Hessman, B., Johnson, B.J., Kapil, S., Ridpath, J.F., Burge, L.J., Braziel, B., Kautz, K., Reck, A. 2006. Bovine viral diarrhea virus persistently infected and acutely infected calves: assays for viral infectivity, polymerase chain reaction analysis, and antigen detection [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 212. Interpretive Summary:
Technical Abstract: There are numerous assays for bovine viral diarrhea virus (BVDV) detecting infectious virus, nucleic material, and antigen. Persistently infected (PI) and acutely/transiently infected calves with BVDV represent two different manifestations. Diagnostic test results impact on differentiation of PI or acutely infected cattle, particularly relating to management of the two forms. The objectives of the initial study included: (1) assay for infectious virus and polymerase chain reaction (RT-PCR) in serums and nasal swab materials of acutely infected and PI calves. The second study had the objectives, using PI calves held over several months; (1) determine if PI calves remain positive over time by the antigen capture ELISA (ACE) test on notches in PBS and by immunohistochemistry (IHC) on fixed notches; (2) quantitate virus in nasal swab materials and serums of PI calves; and (3) assay for BVDV using RT-PCR in nasal swab materials and fresh notches. Samples included in these studies were from acutely infected and PI calves. Cell culture assays for BVDV were of two methods (1) qualitative cell culture assay (QCCA) based on positive antigen in bovine monolayer MDBK cultures; and (2) quantitative assay using viral titration (VT) in 96-well plates using staining for BVDV in MDBK cells. The ACE test and IHC were used on notches and the RT-PCR on serums, ear notches, and nasal swab materials. The detection limit based on dilution factors for the VT were: 10**1.6 per ml for serum and 10**2.9 per ml for nasal swabs. Nasal swab materials were obtained by placing commercial viral culturettes into 2 ml cell culture media. Acutely infected calves with positive QCCA from nasal swab were either negative by the VT (<10**2.9) or undiluted samples were toxic under cell culture conditions used in VT. The VT for three PI calves' nasal swab materials had titers of 10**3.9 to 10**4.4 per ml. There were 14 acutely infected calves positive by the QCCA in PBS (7 with positive QCCA in serum), and 13/14 were negative by the VT (<10**1.6). Only one calf had measurable titer in the serum, 10**1.6 per ml. The PI calves' serums had titers of 10**3.6 to 10**3.8 per ml. There were however, 6 acutely infected calves negative by the QCCA in nasal swabs which were positive by RT-PCR in those same nasal swabs. There were 3 PI calves with negative PCR in ear notches. In the second study there were two groups of PI calves. (1) Trial 1. 18 PI calves sampled over a 215-day period. One calf was added on day 73. Of the 19 calves 16 were infected with a BVDV1b and 3 with a BVDV1a. During the study 3 calves died; and (2)Trial 2. 12 calves sampled over a 155-day period. In this second study, all samples collected monthly were ACE and IHC positive. There were instances where there was a weak positive staining on IHC. All 31 calves had infectious virus, quantitated by the VT in the serum, ranging from 10**1.6 to 10**5.6 per ml. The VT for the nasal swab fluids ranged from 10**2.9 to 10**7.85 per ml. There were instances where the nasal swab materials were toxic to VT cultures. Nasal swabs were PCR positive in most cases; however there were calves with VT positive titers that were negative by the RT-PCR. Some fluids with VT positive PCR negative fluids inhibited the RT PCR of VT positive samples. In summary, serums and nasal swabs of BVDV acutely infected calves had no detectable or greatly reduced viral infectivity compared to PI calves. Also, positive BVDV PI calves remained ACE and IHC positive on ear notches in all cases. Also, some ear notches and nasal swab fluids were inhibitory in the RT–PCR test.