Comparison of reverse-transcription real-time PCR and immunohistochemistry for the detection of canine distemper virus infection in raccoons in Ontario, Canada

Abstract

Canine distemper virus (CDV) is a widespread morbillivirus that causes subclinical to fatal infections in domestic and wild carnivores. Raccoons ( Procyon lotor) are CDV reservoirs and suffer from associated disease. Aspects of pathogenesis may lead to difficulty in the interpretation of commonly used testing modalities, such as reverse-transcription real-time (RT-rt)PCR and immunohistochemistry (IHC). The reliance upon such tests is greater for wildlife, which are often submitted as carcasses with no clinical history. We compared CDV RT-rtPCR results to immunohistochemistry (the gold standard) in tissues from 74 raccoons. These tests had high kappa agreement (lymph node: 0.9335; lung: 0.8671) and a negative correlation between IHC score and threshold cycle (Ct) value for lymph node and lung (Spearman rank correlation coefficient [ r_s] = -0.8555 and -0.8179, respectively; p < 0.00001). An RT-rtPCR Ct value of 30 in lung and lymph node with sensitivity and specificity of 92.3 and 92.6% and 86.8 and 96.4%, respectively, was suitable for determining CDV involvement. Conjunctival swabs provide an alternative for distemper diagnosis, as there was a strong correlation between Ct values of conjunctival swabs and tissues ( r_s = -0.8498, p < 0.00001, n = 46). This information will aid in more efficient and accurate diagnoses in individuals, small-scale outbreaks, and epidemiologic investigations in wildlife.

Keywords: Detection test; distemper; morbillivirus; raccoon; wild animal.

Figure 1.

Representations of canine distemper virus (CDV) reverse-transcription real-time (RT-rt)PCR and immunohistochemistry (IHC) data for tissue (lung and lymph node) and conjunctival swab samples from raccoons in Ontario, Canada. Samples with no replication within 45 cycles were considered negative by RT-rtPCR and assigned a threshold cycle (Ct) value of 45.01 (panels C and D). A. Comparison of sensitivity (Sn) and specificity (Sp) calculated for each raccoon tissue (Lu = lung, LN = lymph node) at each CDV RT-rtPCR Ct value using IHC immunoreactivity as the gold standard. The proposed positive threshold Ct value for CDV positivity is represented by the dotted line. B. Calculated kappa statistic at each CDV RT-rtPCR Ct value for lung and lymph node using IHC as the gold standard. The proposed threshold Ct value for CDV positivity is represented by the dotted line. C. CDV IHC immunoreactivity scores (0–3) of lung, lymph node, and pooled tissue samples compared with corresponding RT-rtPCR Ct values; there is strong correlation between Ct values and the IHC score (r_s = −0.85233; p < 0.00001). D. Scatter plot depicting RT-rtPCR Ct values of pooled tissues and conjunctival swabs; there is strong correlation between sample types (r_s = −0.8498; p < 0.00001). Trend lines and corresponding R² values on panels C and D demonstrate goodness of fit.