Assessing the Effect of Sample Handling on the Performance of a Commercial Bovine Viral Diarrhea Virus Antigen-Capture Enzyme-Linked Immunosorbent Assay

Abstract

Handling practices of specimens may affect the sensitivity or specificity of diagnostic tests. In this study, as part of the Voluntary Iowa Bovine Viral Diarrhea Virus Screening Project held in 2006, 2 sample-handling practices were evaluated to determine how they affect the sensitivity and specificity of the antigen-capture enzyme-linked immunosorbent assay (ACE) for bovine viral diarrhea virus (BVDV). The null hypotheses investigated were 1) that maintenance of samples at room temperature would not be associated with decreased sensitivity, and 2) that continued use of a single pair of ear notchers would not be associated with cross-contamination of virus from 1 notch to another and reduce specificity. These hypotheses were tested in 2 studies by collecting known positive and negative samples and giving groups of samples different treatments. The first study used ACE on 4 groups of skin samples, all from a known-positive animal. Each group was subjected to different lengths of time at room temperature, from 24 to 96 hours at 24–hour intervals. No difference in test results was found between specimens subjected to different lengths of time at room temperature. The second study tested the effects of giving 3 different treatments to an ear notcher in between sample collecting (water rinse, Nolvasan solution rinse, or no treatment) on ACE results. No effect on sensitivity or specificity of ACE was observed. No difference in test results was found between the 3 ear-notcher treatment groups. The sample handling practices evaluated appeared to have little impact on test sensitivity or specificity of ACE for BVDV.

Diagnostic laboratories frequently provide guidelines for sampling and sample handling; however, the impact on test results because of the failure to follow these guidelines is often unclear. In 2006, the Voluntary Iowa Bovine Viral Diarrhea Virus Screening Project (VIBSP) was conducted using ear notches collected by producers to test for bovine viral diarrhea virus (BVDV) using a commercially available antigen-capture enzyme-linked immunosorbent assay (ACE) kit. a As part of investigating the accuracy of the ACE, a pilot study was conducted to evaluate the impact of 2 sample handling practices on the VIBSP testing protocol. The 2 null hypotheses investigated were 1) that maintenance of samples at room temperature would not be associated with decreased sensitivity, and 2) that continued use of a single pair of ear notchers would not be associated with cross-contamination of virus from 1 notch to another and reduce specificity.

Study 1 involved assessment of the effect of storing samples at room temperature for an extended period on sensitivity of the BVDV ACE. In February 2006, a calf was confirmed persistently infected with BVDV after 2 immu-nohistochemistry (IHC) tests performed a month apart. The owner opted to euthanize and donate the animal to the laboratory where it was stored frozen. On July 28, the carcass was thawed at room temperature, and 120 skin samples were collected from the flank using a commercially available notcher. b The samples were placed in individually numbered (1–120) test tubes c and stored without added liquid at −20°C. Ten days later on August 7, the samples were removed from the freezer and allowed to thaw at room temperature.

The samples were randomly assigned to 4, 30-sample groups using a random generation sequence. d The group assignment designated the length of time the samples would be held at room temperature: 24, 48, 72, or 96 hours. Subsequently, each day that the samples sat at room temperature for the designated period, 1.0 ml of phosphate buffered saline (PBS) e was added to each tube, and the samples were vortexed and returned to the freezer at −20°C. On August 14, all 120 samples were submitted to the Veterinary Diagnostic Laboratory at Iowa State University for BVDV detection with ACE. The laboratory staff receiving the samples was unaware of the handling of the samples. No known-negative samples were submitted with this batch of the samples.

BVDV ACE was performed according to the procedure recommended by the manufacturer. 1,2 Based on the manufacturer's instructions, samples with a sample-to-positive (S/P) ratio <0.2 were considered negative for BVDV. Samples with an S/P ratio between 0.2–0.4 were retested with a modified procedure as described by the manufacturer. Samples with an S/P ratio above 0.4 were considered positive for BVDV. All samples tested positive for BVDV, regardless of time stored at room temperature, demonstrating that the BVDV ACE was apparently not affected by storing samples at room temperature for up to 96 hours. Based on analysis of variance, with the S/P ratio as the dependent variable and the time left at room temperature as the explanatory variable, the time was associated with average S/P ratios (P < 0.001). For the samples stored for 24,48, 72, and 96 hours, the mean S/P ratio and standard deviation (SD), were 1.39 (SD, 0.11), 1.46 (SD, 0.11), 1.54 (SD, 0.08), and 1.36 (SD, 0.11), respectively. Although these data suggest a statistical difference, given that the cut-off for a positive value is 0.4, the difference is not biologically meaningful (Fig. 1). This finding is dissimilar to the effect of storage at room temperature on IHC test results described in a 2005 report. 3 When an unspecified number of pinna samples from 2 BVDV-positive calves were exposed to room air temperature for 1, 3, and 5 days then placed in formalin for 2–5 days, the samples could not be processed and evaluated for BVDV using IHC. The conclusion reached was that “exposure to room air, even for 1 day, resulted in desiccated specimens that were difficult to process into histologic sections and uniformly yielded false-negative IHC results.” 3

The second study was conducted to test the effect of equipment contamination on specificity of the BVDV ACE. Positive samples included in this study were provided by the same donor animal described in study 1. Negative samples were collected from a calf submitted to the Iowa State Veterinary Diagnostic Laboratory with a problem unrelated to BVDV. Immunohistochemistry and ACE on different ear notch samples were used to confirm BVDV-negative status.

Figure 1.

A box plot and strip chart of the sample-to-positive ratio of samples stored at room temperature for 24, 48, 72, and 96 hours. A sample-to-positive ratio >0.4 is considered positive.

Study 2 assessed 3 methods of cleaning an ear notcher between sample collections: no cleaning between collections (treatment 1); tap water rinse between collections (treatment 2); and Nolvasan Solution f rinse (treatment 3). The ear notcher was wiped dry with a paper towel between collections. For each method of cleaning, 30 samples were collected as follows:

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  Samples 1–9 from BVDV-positive calf

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  Sample 10 from BVDV-negative calf

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  Samples 11–19 from BVDV-positive calf

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  Sample 20 from BVDV-negative calf

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  Samples 21–29 from BVDV-positive calf

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  Sample 30 from BVDV-negative calf

Once collected, all samples were held at room temperature for 24 hours, then 1.0 ml of PBS was added, and the samples were vortexed and submitted to the Veterinary Diagnostic Laboratory for ACE as described in study 1. The laboratory staff was unaware of the study and the status of the samples.

In each cleaning treatment group, the 3 negative samples tested negative for BVDV based on ACE (i.e., no false positives). Of the 27 positive samples collected with no rinsing, tap water rinsing, or Nolvasan Solution rinse between notches, all were correctly identified by ACE.

Because producers frequently collect samples in less than ideal conditions, it is important to assess how those conditions affect the sensitivity and specificity of the BVDV ACE. The results of this small study indicate that sample handling may have little effect on test sensitivity and specificity for BVDV ACE under conditions present in the study. Storage at room temperature for extended periods appears to have little impact on the detection of the targeted BVD viral antigen using ACE. Further, no evidence was found that the BVDV antigen targeted by ACE accumulates on ear notchers even without any rinsing between collection, leading to false-positive test results. This is an important consideration for enterprises, such as feedlots, which test a large number of animals, and do not wait 21–28 days for confirmatory testing to remove the animal from the production system. Unfortunately, this study was only performed using samples from 1 BVDV-positive calf, and it is not clear if the results are valid for all BVDV isolates and all animals infected with BVDV. Further, the samples were collected when the animal was dead, and the presence of fluids, such as blood, may be a significant vector that was not included in the present study.