How Do We Detect Infection in a Sheep?

In order to control or eradicate MV infection from a flock, we need to be able to accurately detect infected animals.  To do this we can use a variety of tests to either detect the virus, or - what is more common - to detect the animal’s immune response to the virus, i.e. test for antibodies. 

Things to consider when selecting a test to use are:

Serological Tests

Sheep and lambs sero-convert (i.e. produce antibodies to the virus) between 2 weeks and several months (up to 8 months) after infection, with a small proportion never developing detectable antibody levels.  The level of antibody varies depending on

The genetic variability of the SRLV viruses requires that serological tests use antigens that are well-conserved (i.e. don't change over time) and present in the groups that tend to infect the sheep being tested.  It has also been noted that when animals become ill, antibodies to the core antigens (capsid gag gene) will drop so it is important that the diagnostic test also detect envelope antigens – ideally those that are well conserved between strains.

Agar gel immunodiffusion test (AGID)

AGID tests require quite a bit of labour and are interpreted by a person reading the plate. For these reasons, AGID tests are used less and less commonly. This serological test detects antibodies to the p25 core antigen and gp135 envelope antigen.  Sensitivity is variously reported but is probably  no better than 80% (compared to western blotting) with excellent specificity between 98 and 100%.  This low sensitivity makes it difficult to use in a test and eradication program – as it misses 20% of infected sheep.  Additionally, ELISA tests as described below pick up antibodies earlier than AGID (55 days after infection versus 191 days for AGID).

Enzyme-linked immunosorbent assay (ELISA)

ELISA tests are interpreted in machines and large numbers of samples can be run on a single plate making them less costly and more dependable. There are many types of SRLV ELISA tests: indirect whole virus, recombinant; competitive (monoclonal antibodies) and use a variety of antigens.  Whole virus or recombinant ELISA’s with multiple antigens are more sensitive than single antigen ELISA tests. However whole virus ELISA’s may lack specificity.   Many of these ELISA’s again use p25 and gp135 as well as other antigens. Use of recombinant antigens allows researchers to evaluate the sensitivity and specificity of the tests in different animal populations.

Details of some commercially available tests are provided in Table 2.  The CFIA test was commercially available from LEPAQ in Quebec until very recently.  It has been used for almost a decade in maedi visna control programs in Ontario and Quebec – but due to lack of continued access, a replacement test must be found. A recent study at the University of Guelph evaluated the ELISA tests listed below with the result that the Elitest (HYPHEN Biomed) appears to have the best sensitivity and so is currently offered by the Animal Health Laboratory (see protocols of the Ontario Maedi Visna Flock Status program for sampling).

Table 2.  Commercial diagnostic ELISA test kits for Maedi Visna Virus.

 
NAME OF ELISA TEST
ANTIGENS
CFIA test developed by Pasick & Simard. Previously offered by VPAQ commercially
Recombinant GST-MVVgag (330-amino acid fragment containing p16 and p25) and GST-MVVenv  (159 amino-acid fragment of the transmembrane domain) genes obtained from Icelandic strain 1514.  Can be used on milk, serum and plasma. This test has been used for over a decade and appears to perform well but is no longer commercially available.
VMRD Inc.  CAE virus antibody test kit
Competitive ELISA using competitive antibodies to the gp135 env glycoprotein.  No gag protein mentioned. In research at the University of Guelph, this test did not perform well with respect to both sensitivity and specificity and so is not recommended for use in MV programs.
IDEXX CAEV/MVV Antibody Test Kits:  Chekit Total Antibody ELISA; p28 Ab Screening and Verification.
Total antibody ELISA. P28 antibody test (TM, env gene and recombinant p28 capsid gag gene).  Can be used on milk, serum and plasma
Pourquier® ELISA Maedi-Visna/CAEV Antibody Test Kit
Indirect ELISA based on an immunogenic peptid of a transmembrane protein (TM, ENV gene) and recombinant p28
ELITEST-MVV/CAEV Aniara (USA) / HYPHEN Biomed SAS / MV Diagnostics
Recombinant ELISA using the capsid p28 core protein and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. Can be used on milk, serum and plasma. In research at the University of Guelph, this test performed best with respect to sensitivity in detecting animals infected with MVV.
ID Screen® Maedi Visna Indirect, IDVET Innovative Diagnostics
Uses a "bouquet" of peptides from the virus GAG, TM and envelope proteins. Specific information on which is not provided by the company.

Radioimmunoassay Radioimmunoprecipitation Assay and Western Blot

These tests are used as “gold standards” to confirm border-line or dubious SRLV serological tests.  If the appropriate “band” of protein is present, it is assumed that the animal is a true positive – although false positives can occur with these tests.  More work needs to be done to determine the usefulness of these supplementary tests.

Polymerase Chain Reaction (PCR)

The detection of virus using a PCR has the advantage that one doesn’t have to depend on an immune response by the animal.  However, SRLV infections tend to have only low amounts of virus in circulation and almost all of it as provirus in macrophages and monocytes.  Additionally, with so much strain variation selection of the appropriate primers is an issue.   The pol and LTR regions are more conserved compared to gag, while env has the highest variability. So, while PCR may be useful as an augment to serology to detect those animals that have not sero-converted, or perhaps to detect early infection – sensitivity is generally < 95% and so is not as good as ELISA serology.  Additionally it may be 4 to 5 times as expensive as serology.