Development of an antigen-capture enzyme-linked immunosorbent assay for diagnosis of Aleutian mink disease virus.

Aleutian mink disease (AMD), caused by Aleutian mink disease virus (AMDV), is a very important infectious disease of mink. Currently, elimination of antibody- or antigen-positive animals is the most successful strategy for eradicating AMD, but the claw-cutting method of blood sampling is difficult to perform and painful for the animal. In this study, we aimed to establish an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) method for the efficient detection of AMDV antigens using fecal samples. A purified mouse monoclonal antibody (mAb) was used as the capture antibody, and a rabbit polyclonal antibody (pAb) was used as the detection antibody. The assay was optimized by adjusting a series of parameters. Using a cutoff value of 0.205, the limit of detection of the AC-ELISA for strain AMDV-G antigen was 2 µg/mL, and there was no cross-reaction with other mink viruses. The intra- and inter-assay standard deviations were below 0.046, and the correlation of variance (CV) values were 1.24-7.12% when testing fecal samples. Compared with conventional PCR results, the specificity and sensitivity were 91.5% and 90.6%, respectively, and the concordance rate between the two methods was 91.1%.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus.

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/µL of AMDV plasmid or 3 pg/µL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

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

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.

Aleutian Mink Disease Virus in the breeding environment in Poland and its place in the global epidemiology of AMDV.

Aleutian mink disease (AMD) leads to an increase in mortality of animals and causes losses in mink farming. The study investigated the presence of AMDV in tissue and environmental samples from farmed mink in Poland, and selected samples were genetically characterized. Blood, spleens and swabs from the breeding environment were collected on 27 farms in seven voivodeships in Poland (n = 250). DNA was isolated, amplified by PCR and subsequently subjected to sequencing to reveal information on the molecular epidemiology of the samples. A qPCR method was used to determine the viral load in test samples. The presence of AMDV was confirmed in tissues and the farm environment on 26 of the 27 farms. The average viral load in spleens was 108 copies. The virus was also present in the blood (average - 105 copies) and the farm environment (average - 103 copies). Isolates from the West Pomeranian Voivodeship showed high similarity within the voivodeship (over 99%). Variants from the Lublin and Podlaskie Voivodeships differed 5% from any of the AMDV isolates present in the NCBI database. Isolates from the Greater Poland, Pomeranian, Podkarpackie and Lesser Poland Voivodeships formed heterogeneous clades, showing over 97% similarity to variants previously isolated in Poland, the Netherlands and Lithuania. A high degree of genetic variation was identified among the majority of the samples, which indicates that AMDV has been introduced to Poland multiple times. However, the results within one area showed high identity between isolates, suggesting that one common ancestor was the source of these outbreaks.

A comparison between intraperitoneal injection and intranasal and oral inoculation of mink with Aleutian mink disease virus.

Intranasal, with (INS) and without (IN) sedation, and oral inoculation were compared with intraperitoneal (IP) injection for establishing infection with a local isolate of Aleutian mink disease virus (AMDV) in 35 American mink. Blood samples were collected on 0, 21, 36 and 56 day post-inoculation (dpi). Antiviral-antibodies and viral DNA in plasma and tissues were measured by counter-immunoelectrophoresis (CIEP) and PCR, respectively. The presence of AMDV DNA was tested by PCR in saliva, rectal and fecal samples collected on 0, 6, 10, 15, 21, 28, 36 and 56 dpi. Animals were killed at 56 dpi, samples of six organs were tested for antibody and AMDV DNA, and samples of the lungs, liver, kidneys and heart were subjected to histology. Viral DNA was detected in the spleen, lungs and lymph nodes of all inoculated mink on 56 dpi, indicating that all inoculation routes caused infection in mink. Viral DNA and antibodies were detected in plasma of all IP and INS inoculated mink by 36 dpi, but some animals which were inoculated orally or via IN remained seronegative by 56 dpi. It was concluded that INS route was the most effective method for establishing infection in mink without breaking the integrity of the animals' anatomical barriers. Viremia was short-lived in some mink, whereas antibody production persisted in seroconverted animals during the duration of the experiment. Saliva, rectal and fecal samples did not accurately detect infection. Histologic lesions of AD were observed on the four organs of most mink.

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection.

The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (105) and the farm with the subclinical course (103). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen-host relationship.

Development of a Peptide ELISA for the Diagnosis of Aleutian Mink Disease.

Aleutian disease (AD) is a common immunosuppressive disease in mink farms world-wide. Since the 1980s, counterimmunoelectrophoresis (CIEP) has been the main detection method for infection with the Aleutian Mink Disease Virus (AMDV). In this study, six peptides derived from the AMDV structural protein VP2 were designed, synthesized, and used as ELISA antigens to detect anti-AMDV antibodies in the sera of infected minks. Serum samples were collected from 764 minks in farms from five different provinces, and analyzed by both CIEP (a gold standard) and peptide ELISA. A peptide designated P1 (415 aa-433 aa) exhibited good antigenicity. A novel ELISA was developed using ovalbumin-linked peptide P1 to detect anti-AMDV antibodies in mink sera. The sensitivity and specificity of the peptide ELISA was 98.0% and 97.5%, respectively. Moreover, the ELISA also detected 342 early-stage infected samples (negative by CIEP and positive by PCR), of which 43.6% (149/342) were true positives. These results showed that the peptide ELISA had better sensitivity compared with CIEP, and therefore could be preferable over CIEP for detecting anti-AMDV antibodies in serological screening.

Progression of experimental chronic Aleutian mink disease virus infection.

BACKGROUND: Aleutian mink disease virus (AMDV) is found world-wide and has a major impact on mink health and welfare by decreasing reproduction and fur quality. In the majority of mink, the infection is subclinical and the diagnosis must be confirmed by serology or polymerase chain reaction (PCR). Increased knowledge based on a systematically description of clinical signs, pathology and histopathology might be a tool to reduce the risk of infection from subclinically infected mink to AMDV free herds. The aim of this study was to give a histopathological description of the progression of a chronic experimental infection with a currently circulating Danish strain of AMDV, Saeby/DEN/799.1/05. These results were compared with the pathogenesis of previously published AMDV stains. RESULTS: This experimental AMDV infection resulted in only decreased appetite and soft or discolored feces, primarily within the first 8 weeks after AMDV inoculation. Gross pathology revealed few and inconsistent findings mainly associated with the liver, spleen and kidneys. The majority of the AMDV inoculated wild type mink (n = 41) developed various histopathological changes consistent with AMDV infection in one or more organs: infiltrations of mononuclear cells in liver, kidney and brain, reduced density of lymphocytes and increased numbers of plasma cells in lymph nodes and spleen. Natural infection, as occurred in the sentinel sapphire mink (four of six mink), progressed similar to the experimentally inoculated mink. CONCLUSIONS: Experimental AMDV inoculation mainly resulted in subclinical infection with unspecific clinical signs and gross pathology, and more consistent histopathology appearing at any time after AMDV inoculation during the 24 weeks of observation. Thus, the observed histopathology substantiates AMDV infection and no correlation to time of inoculation was found. This confirms that diagnosing AMDV infection requires serology and/or PCR and the Saeby/DEN/799.1/05 AMDV strain results in histopathology consistent with other AMDV strains.

Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV.

[Research into the antibody detection technology of mink plasmacytosis and its current applications].

Mink plasmacytosis, caused by Aleutian Mink Disease Virus (AMDV), poses a threat to the development of the animal fur industry. Neutralizing antibodies against AMDV may result in a persistent infection rather than providing protection for minks. To date,no specific methods to prevent or cure this disease have been developed. In order to eliminate mink plasmacytosis, antibody detection technology has been used globally as a dominant approach to screen for AMDV-positive minks. This paper introduces the classical technology, counterimmunoelectrophoresis and emerging technology in terms of AMDV antibody detection,and provides a glimpse into the future development of these technologies.

Validation of an automated ELISA system for detection of antibodies to Aleutian mink disease virus using blood samples collected in filter paper strips.

BACKGROUND: Aleutian mink disease virus (AMDV) is the cause of a chronic immune complex disease, Aleutian disease (AD), which is common in mink-producing countries. In 2005, implementation of an AMDV eradication programme in Finland created a need for an automated high-throughput assay. The aim of this study was to validate an AMDV-VP2 -recombinant antigen ELISA, which we developed earlier, in an automated assay format for the detection of anti-AMDV antibodies in mink blood and to determine the accuracy of this test compared with the reference standard (counter-current immunoelectrophoresis, CIEP). METHODS: A blood sampling method based on filter paper 12-strips (blood combs) and a device to introduce these strips to an ELISA plate for elution of the samples were developed. Blood and serum samples were collected from 761 mink from two farms with low (2%) and high (81%) seroprevalences of AMDV infection in 2008. ELISA sensitivity and specificity were estimated with a Bayesian 2-test 2-population model that allowed for conditional dependence between CIEP and ELISA. Agreement between the two tests was assessed with kappa statistic and proportion agreement. RESULTS: The sensitivity and specificity of the automated ELISA system were estimated to be 96.2% and 98.4%, respectively. Agreement between CIEP and ELISA was high, with a kappa value of 0.976 and overall proportion agreement of 98.8%. CONCLUSIONS: The automated ELISA system combined with blood comb sampling is an accurate test format for the detection of anti-AMDV antibodies in mink blood and offers several advantages, including improved blood sampling and data handling, fast sample throughput time, and reductions in costs and labour inputs.

Monitoring chronic infection with a field strain of Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) readily spread within farmed mink and causes chronic infections with significant impacts for welfare and economy. In the present study a currently circulating Danish AMDV strain was used to induce chronic experimental infection of farmed mink. PCR was used to detect viral DNA in full blood, organs, faeces and oro-nasal swabs weekly for the first 8 weeks and then biweekly for another 16 weeks after AMDV challenge inoculation of wild type mink. The mink (n=29) was infected and seroconverted 2-3 weeks after AMDV inoculation and AMDV antibodies persisted during the maximum experimental period of 24 weeks. Viraemia and faecal excretion of viral DNA was detected in the mink (n=29) at various and intermittent time intervals. Excretion of viral DNA in oro-nasal swabs was detected for 1-8 weeks in 21 mink. This highlights the risk of transmitting AMDV between infected farms. PCR was successfully used to detect viral DNA in organs 8, 16 and 24 weeks after AMDV inoculation with only minor differences between these weeks which is of diagnostic interest. This AMDV challenge model was also used to mimic natural infection of susceptible sapphire mink. Four of 6 sapphire mink were infected indirectly via the AMDV inoculated wild type mink whereas the other 2 sapphire mink remained uninfected.

Diagnosing Aleutian mink disease infection by a new fully automated ELISA or by counter current immunoelectrophoresis: a comparison of sensitivity and specificity.

Aleutian disease (AD) is a severe disease characterized by hypergammaglobulinemia causing multiple symptoms such as acute renal failure, arteritis, reduced reproductive performance and pneumonia in mink. AD is caused by the parvovirus Aleutian mink disease virus (ADV) and diagnosed primarily based on ADV serology sometimes supplemented by organ PCR analysis. In Denmark, approximately 3.5-4 million serum samples are tested every year for the presence of anti ADV antibodies as part of a national eradication program. The present study compares the diagnostic performance of the two most commonly used assays for serological screening for Aleutian disease: counter current immunoelectrophoresis (CIEP) and ELISA. In total, 3810 mink were sampled in doublets and analyzed by CIEP and a newly developed fully automated ELISA. The results show that the two assays have a comparable diagnostic performance with the ELISA having a higher sensitivity but lower specificity than the CIEP assay. The ELISA has been approved by the Danish authorities for diagnosing Aleutian disease in mink.

Evaluation of two enzyme-linked immunosorbent assays for serodiagnosis of Aleutian mink disease virus infection in mink.

BACKGROUND: Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard. RESULTS: When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n = 364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n = 359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%. CONCLUSIONS: The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant VP2 capsids for the detection of antibodies to Aleutian mink disease virus.

Aleutian disease (AD), a common infectious disease in farmed minks worldwide, is caused by Aleutian mink disease virus (AMDV). Serodiagnosis of AD in minks has been based on detection of AMDV antibodies by counterimmunoelectrophoresis (CIE) since the 1980s. The aim of this study was to develop and evaluate an enzyme-linked immunosorbent assay (ELISA) based on recombinant virus-like particles (VLPs) for identifying AMDV antibodies from mink sera. AMDV capsid protein (VP2) of a Finnish wild-type strain was expressed by the baculovirus system in Spodoptera frugiperda 9 insect cells and was shown to self-assemble to VLPs (with an ultrastructure similar to that of the actual virion). A direct immunoglobulin G ELISA was established using purified recombinant AMDV VP2 VLPs as an antigen. Sera from farmed minks were collected to evaluate the AMDV VP2 ELISA (n = 316) and CIE (n = 209) based on AMDV VP2 recombinant antigen in parallel with CIE performed using a commercially available traditional antigen. CIE performed with the recombinant antigen had a sensitivity and specificity of 100% and ELISA a sensitivity of 99% and a specificity of 97%, with reference to CIE performed with the commercial antigen. The results show that the recombinant AMDV VP2 VLPs are antigenic and that AMDV VP2 ELISA is sensitive and specific and encourage further development of the method for high-throughput diagnostics, involving hundreds of thousands of samples in Finland annually.

Infection with Aleutian disease virus-like virus in a captive striped skunk.

CASE DESCRIPTION: A 5-month-old captive female striped skunk (Mephitis mephitis) was evaluated because of lethargy, signs of depression, azotemia, and erythema of the skin around the eyes. CLINICAL FINDINGS: Antemortem diagnostic tests revealed renal disease but failed to identify an etiologic agent. A diagnosis of severe nonsuppurative interstitial nephritis was made on the basis of results of histologic examination of renal biopsy specimens. TREATMENT AND OUTCOME: The skunk was administered isotonic fluids SC daily and later every other day because of the handling-related stress. Because of the skunk's deteriorating condition, it was euthanized after 24 days of supportive care. Aleutian disease was diagnosed on the basis of positive results of a PCR assay that targeted the DNA from Aleutian disease virus (ADV); positive results for ADV were also obtained by use of plasma counterimmunoelectrophoresis and an ELISA. Genetic sequencing of the 365-base pair PCR product revealed 90% sequence identity with mink ADV. CLINICAL RELEVANCE: In the skunk of this report, infection with a skunk-specific parvovirus resulted in clinical signs and pathologic changes similar to those associated with ADV infection in mink. For skunks with signs of renal failure, differential diagnoses should include parvovirus infection. In confirmed cases of infection with this ADV-like virus, appropriate quarantine and biosecurity measures should be in place to prevent spread to other susceptible animals within a zoological collection.

Aleutian disease in two domestic striped skunks (Mephitis mephitis).

This report describes the use of polymerase chain reaction and DNA in situ hybridization to diagnose Aleutian mink disease parvovirus DNA in various tissue specimens from 2 companion striped skunks. Clinical, laboratory, and microscopic findings also support a clinical diagnosis of Aleutian disease in these mink.

Immunohistochemical detection of 3 viral infections in paraffin-embedded tissue from mink (Mustela vison): a tissue-microarray-based study.

Immunohistochemical (IHC) assays were developed and tested for the detection of 3 viral infections in archived paraffin-embedded mink tissue. Specimens had been obtained from mink with diagnoses of acute Aleutian disease (AD), mink parvoviral enteritis (MVE), or canine distemper (CD) made by means of routine diagnostic procedures. To improve the efficiency and reduce the costs of IHC analyses, tissue microarray (TMA) technology was used. Representative cores 2 mm in diameter from each tissue specimen and from positive- and negative-control specimens were collected in a TMA block. Immunohistochemical reactions to viral antigens were assessed and graded. Positive reactions were found in 91% of the 32 specimens from mink with AD, 53% to 80% of the 60 specimens from mink with MVE, and all 66 of the specimens from mink with CD. To validate the use of TMAs, the IHC methods were applied to whole-mount paraffin-embedded sections of 10 of the positive specimens for each disease, together with whole-mount sections of small intestine and lung tissue from 2 healthy mink. The IHC grading of the TMA cores and the whole-mount sections from the same animal corresponded completely. These results suggest that IHC demonstration of viral antigen allows rapid and reliable diagnosis of the 3 viral infections in mink and is a potential supplement to histologic diagnostic procedures. The TMA technique proved useful for screening large numbers of samples for expression of specific viral antigens, while reducing overall costs.

[Prokaryotic expression and detective application of the main antigenic region of VP2 protein of Aleutian mink disease parvovirus].

To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD.