Comparative evaluation of diagnostic techniques for bovine viral diarrhoea virus in aborted and stillborn fetuses

Abstract

The fetal fluid samples were also tested for BVDV-specific antibodies using a blocking ELISA kit (BVD/BD p80 Blocking One-Step; LSI). IFAT was performed using a commercial poly clonal BVDV antiserum (VMRD). The spleen, ileum and lung samples from each fetus were pooled, and a 10 per cent w/v suspension was prepared and processed for virus isolation according to standard procedures (Graham and others 1998). Each pool received two passages on fetal bovine lung cells followed by IFAT for BVDV. Viral RNA was extracted from a 200 µl volume of each fetal fluid using the QIAamp Virus BioRobot MDX kit on a BioRobot Universal system (Qiagen). Real-time RT-PCR was subsequently carried out using the Quantitect SYBR Green RT-PCR kit (Qiagen) with the primer pair F2/PESTR (Letellier and Kerkhofs 2003). Briefly, 25 µl of the reaction mixture, containing 2·5 µl of extracted RNA and 300 pmol of each primer, was subjected to the following thermal conditions: 50°C for 30 minutes, 95°C for 15 minutes, then 40 cycles of 95°C for 15 seconds and 55°C for 30 seconds, followed by 72°C for 30 seconds. Thereafter, melt analysis was performed, with samples that gave both a typical amplifi cation curve and a melt temperature between 83°C and 86°C being considered positive. All kits were used according to the manufacturers' instructions. For a minority of fetuses, not all tests were performed, due primarily to insufficient sample being available.Based on the calculation reported by Rexroad and others (1974), the fetuses examined ranged from 122 to 302 days of gestation (median 210 days). One or more tests were positive from a total of 25 (17·9 per cent) fetuses (Table 1). Virus isolation gave positive results in three of 134 (2·2 per cent) fetuses, and IFAT gave a positive result in one of 134 (0·7 per cent) fetuses. Specific fluorescence was detected in all three tissues (spleen, ileum and lung) of the IFAT-positive fetus, which was also culture positive. In contrast, 20 of 140 (14·3 per cent) fetal fluids and 16 of 129 (12·4 per cent) ear skin samples were positive by antigen ELISA. The overall agreement between antigen ELISA results for fetal fluids and ear skin was 95·3 per cent. The real-time RT-PCR was positive in 18 of 138 (13 per cent) fetal fluids, with positive cycle threshold values ranging from 17·9 to 26·9 (mean 23·5). The agreement between RT-PCR results and antigen ELISA results on fetal fluids was 97·1 per cent. Three fetal fluid samples positive by ELISA were negative by RT-PCR. This could have been due to inhibitors in the samples, although this cannot be stated conclusively. Alternatively, it could reflect a greater resistance of viral proteins relative to RNA to degradation in the fetal environment. All three of the culture-positive fetuses were also positive by antigen ELISA on fetal fluid and by realtime RT-PCR, with the ear skin sample from two of these also positive by ELISA. Antibodies were detected in two of 140 (1·4 per cent) samples. No other tests for BVDV on these two fetuses were positive. A possible alternativ...

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period

“…The poor state of preserved specimens due to autolysis usually has negative impact on diagnostic test performance [16]. To assess a relationship between viral detection and abortion, it is necessary to associate such finding with compatible fetal pathology, antibody response, and herd history, while ruling out other potential reproductive pathogens [17,32]. Thus, a multi-pronged approach should be applied in order to confirm BVDV as responsible for reproductive losses.…”

“…Thus, AgELISA appears to be of limited value for archived or autolytic samples such as fetal tissues. In the present study, spleen and occasionally lymph node, brain, or lung tissues were used for AgELISA as indicated by the manufacturer, but fetal fluids [17] or skin [16,17] seems to be more suitable for this purpose. The AC 1 coefficient and the percentage of positive agreement between the RT-PCR methods were moderate in this retrospective study.…”

“…Although VI is the "gold standard" for virus detection, there is uncertainty about the selection of diagnostic methods to detect BVDV in samples from aborted fetuses and neonatal deaths. Additionally, evaluation or validation of different assays for BVDV identification in those types of samples is limited [8,16,17]. Moreover, there is no published information on the comparison of diagnostic methods and performance of phylogenetic, serologic, and pathologic studies of BVDV in specimens from cases of bovine abortion.…”

Role of Infection and Immunity in Bovine Perinatal Mortality: Part 1. Causes and Current Diagnostic Approaches

“…It is more commonly detected in suckler than dairy foetuses [38]. Virus detection rate is dependent upon diagnostic technique; antigen-detection ELISA or RT-PCR being better than IFAT, virus isolation or serology [42]. Given the low (generally <1%) prevalence of BVDv-persistently infected (PI) newborn calves internationally, unless a large number of PM calves are tested for virus it is likely that a false negative conclusion will arrived at regarding the presence of BVDv in such calves.…”

“…In contrast to the relatively limited proportion of PM attributed to infection, in bovine abortion infection is the most important diagnosed cause of death. This disparity can be obscured as many studies report findings for both abortion and PM combined (Figure 7), (e.g., [11,12,42,89]), and depending on the terminology used to describe the foetuses it can appear that infection is a major cause of PM. For example, a Dutch study detected pathogenic antigens in 28% of aborted and stillborn fetuses [89].…”