3R-Project 45-96

Replacement of mouse and rat antibody production test by polymerase chain reaction assays

Felix R. Wolf
Research Animal Resources Center, Cornell University, New York 100021, USA.
hombergf@mskcc.org

Keywords: viruses; molecular biology: pcr; reduction; refinement; diagnostic approaches: viruses

Duration: 1 year Project Completion: 1998

Background and Aim
Murine viruses may be transmitted from one animal to another other through contaminated biological material such as transplantable tumours, cell lines or hybridoma lines. Such inadvertent transmission may cause enzootic infections in the recipient colony resulting in clinical disease and/or compromised research results.

To avoid this, biological material is screened for pathogens before introduction into an animal colony. Historically, this has been done using the mouse antibody production (MAP) or the rat antibody production (RAP) test, by inoculating the material into naive mice or rats and subsequently testing the animals for seroconversion. The goal of this project was to establish an alternative assay using PCR technology that can detect all the relevant murine viruses directly in the biological material. Our aim is to replace the MAP/RAP test completely (see also 3R project 74-00).

Method and Results
PCR assays were developed to detect the Sendai virus, pneumonia virus of mice (PVM), minute virus of mice (MVM), Kilham rat virus (KRV), Toolan's H-1 virus, reovirus type 3, epizootic diarrhea of infant mice (EDIM), mouse adenoviruses type I and II, ectromelia virus, polyoma virus, K virus, Theiler's murine encephalomyelitis virus, lymphocytic choriomeningitis virus (LCMV) and the Hanta virus group. These tests, combined with PCR assays to detect the coronaviruses of mice and rats and the lactate dehydrogenase elevating virus (LDV) as published previously, cover the complete spectrum of agents detectable by the traditional MAP/RAP test. Routine samples submitted to our diagnostic lab for MAP testing, as well as deliberately spiked samples, were compared with the newly developed PCR screening test (1). The PCR approach was more sensitive and had a much shorter turnover time (two days) than the MAP test (one month to obtain seroconversion plus a few days for the serology results).

Conclusions and Relevance for 3R
This PCR screening assay eliminates the need of animals or animal tissue to screen for murine viruses in biological materials. It will eventually replace the MAP test. Additionally, the method contributes to the refinement of many other animal experiments by ensuring that research results are not compromised by inadvertent (subclinical) infections of experimental animals with viral pathogens. Since most diagnostic labs offering serology are equipped with the equipment necessary to perform PCR, this new assay can be easily implemented by other labs, and could become the regulatory standard in the future.

References
1. Tischhauser, M. E. (1999). “Replacement of mouse and rat antibody production (MAP/RAP) test by polymerase chain reaction.” (Inaugural Dissertation, University of Zürich).

2. Tischhauser, M.E., Popovic, D. and Homberger F.R. (1999) Replacement of mouse and rat antibody production (MAP/RAP) test by polymerase chain reaction assays. Lab. Anim Sci., submitted.

3. Sieber, I.U. (2001) “Ersatzmethode für den Maus-Antikörper-Produktionstest. Ein Sensitivitärsvergleich verschiedener PCR-Assays mit dem Tierversuch.” (Inaugural Dissertation, University of Zürich).1.

4. Bootz F., I. Sieber, D. Popovic, M. Tischhauser, F.R. Homberger. 2003. Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials, Lab. Anim. 37: 341-351.