Development and validation of nucleic acid tests to diagnose Aleutian mink disease virus

Highlights

• Setting up a sensitive and specific probe-based AMDV-PCR.

• Comparison and validation of three diagnostic AMDV-PCRs.

• Screening and detection of AMDV from mink and fox samples.

Abstract

Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), causes significant welfare problems to mink, and financial losses to the farmers. As there is no vaccine or treatment available, reliable diagnostics is important for disease control. Here, we set up a probe-based real-time PCR (NS1-probe-PCR) to detect all strains of AMDV. PCR was validated and compared to two other real-time PCR methods (pan-AMDV- and pan-AMDO-PCR) currently used for AMDV diagnostics in Finland. The NS1-probe-PCR had a similar detection limit of 20 copies/reaction based on plasmid dilution series, and similar or better diagnostic sensitivity, when evaluated using spleen samples from mink, and stool samples from mink and foxes. None of the three PCR tests cross-reacted with other parvoviruses. The NS1-probe-PCR also showed a significantly higher specificity than the pan-AMDO-PCR with spleen samples and the best specificity with stool samples. Furthermore, it produced the results more rapidly than the other two PCRs making it a promising tool for both diagnostic and research purposes.

Introduction

Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), is one of the most significant infectious diseases of mink. AD causes both significant welfare problems to the animals and financial losses to farmers. AMDV is secreted into urine, feces and saliva of infected mink (Gorham et al., 1964; Farid et al., 2015; Jensen et al., 2014) and is transmitted vertically through placenta or horizontally in direct contact with an infected animal or through indirect contact with contaminated environment (Gorham et al., 1976; Porter et al., 1977; Broll and Alexandersen, 1996). Clinical signs vary from subclinical to severe and fatal (Bloom et al., 1994) depending, among other things, on the age and genotype of the animal (Gorham et al., 1976; Alexandersen, 1986).

AD was first reported in Aleutian-type mink in the USA in 1956 (Hartsough and Gorham, 1956) but it has since spread to all mink producing countries and also been reported in other mink genotypes (Aasted, 1985), ferrets and several wild mustelids and carnivores (Knuuttila et al., 2015; Farid, 2013; Fournier-Chambrillon et al., 2004; Murakami et al., 2001). AMDV belongs to family Parvoviridae (Shahrabadi et al., 1977; Bloom et al., 1980) and species Carnivore amdoparvovirus 1 (Cotmore et al., 2014) and its 4.7 kb ssDNA genome (Bloom et al., 1980, 1988; Bloom et al., 1990) encodes two structural proteins (VP1 and VP2) and three non-structural proteins (NS1, NS2 and NS3) (Shahrabadi et al., 1977; Qiu et al., 2006, 2007; Huang et al., 2014).

Despite eradication efforts, AMDV still causes major epidemics (Hagberg et al., 2017). As there is no effective treatment and the attempts to develop a vaccine have been unsuccessful (Aasted et al., 1998; Castelruiz et al., 2005), reliable diagnostics is important in disease control. Diagnostics is mostly based on pathological findings and serology, using antibody detection methods such as CIEP and ELISA, as well as DNA detection by PCR (Knuuttila et al., 2009, 2014; Cho and Ingram, 1972). As some mink can clear the virus, but remain antibody-positive, the best way to determine viremia (from euthanized individuals) is to perform a PCR from spleen tissue (Jensen et al., 2014). However, genetic diversity makes it difficult to set up a PCR that is sensitive and able to amplify all virus strains, while remaining specific. As false positive or negative results might have severe economic consequences to the farmers, it is important to have properly validated PCRs.

The aim of this study was to set up a diagnostic PCR that is specific, amplifies all AMDV strains, and is easy to interpret, as well as validate and compare the novel test and PCRs currently used in diagnostics in Finland.