Development of an ELISA for Detection of Myxoma Virus-Specific Rabbit Antibodies: Test Evaluation for Diagnostic Applications on Vaccinated and Wild Rabbit Sera

Abstract

Abstract. An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.Myxomatosis is a specific viral disease of the European rabbit (Oryctolagus cuniculus), causing severe or attenuated clinical manifestations in domestic and wild rabbit populations. 4,6,7,13 The etiologic agent of myxomatosis is a large double-stranded DNA virus in the Leporipoxvirus genus of the Poxviridae family. It was released in Australia and Europe in 1950 and 1952, respectively, as a biological control for wild rabbits. Myxomatosis is now endemic in European and Australian rabbit populations. Diagnosis is generally based on clinical signs alone, but the occurrence of atypical cases 11 with an absence of myxoma lesions has revealed the need for specific and sensitive diagnostic tests.The immune response in infected rabbits is characterized by the early appearance and persistence for several months of specific antibodies that may be detected by complement fixation (CF) 2,5,7 virus neutralization, 5 immunodiffusion, 17,18 or immunofluorescence 3,10 tests. These tests are adequate for most purposes but are time consuming and not really suitable for the estimation of disease prevalence in wild animals. Furthermore, they do not allow precise quantification of the antibody levels in animal serum. Previously, an enzyme-linked immunosorbent assay (ELISA) was developed using virus extracted from the skin tissue of infected rabbits as antigen. 20 The interFrom the Laboratoire associé de Microbiologie Moléculaire, Ecole Nationale Vétérinaire, Toulouse and Institut National de la Recherche Agronomique, 23, chemin des Capelles, F-31076 Toulouse cedex-3, France.Received for publication April 30, 1997. assay reproducibility of the test was unsatisfactory, probably because of the poor quality of the antigen. 20 The ELISA described here was based on purified myxoma virus antigen and is compared with other serologic methods. It proved to be sensitive and specific for the detection of myxoma antibodies both in fresh serum and in dried blood on a blotter. The method was evaluated for diagnosis with sera from vaccinated and wild rabbit. Material and meth...

Early infections by myxoma virus of young rabbits (Oryctolagus cuniculus) protected by maternal antibodies activate their immune system and enhance herd immunity in wild populations

“…The serological responses against MYXV were evaluated using enzyme-linked immunosorbent assays (ELISA), as previously described [54]. Briefly, two 6 mm-diameter discs of blotting paper impregnated with blood were rehydrated in 100 μL of phosphate-buffered saline (PBS) in a 96-well microplate and stored at 4 °C overnight.…”

Myxomatosis, a continually dangerous viral disease of rabbits: current state of knowledge

“…Obecnie metody serologiczne, jak np. ELISA stosuje się tylko w ograniczonym zakresie do identyfikacji zakażeń lub oceny skuteczności szczepień profilaktycznych (30,33,44). Biorąc pod uwagę fakt częstego występowania zakażeń MYXV u królików, a także trudności diagnostyczne związane z atypową postacią choroby do diagnostyki laboratoryjnej zakażeń zostały wprowadzone metody molekularne (50).…”

Viral Diseases

“…Rabbits that survive infection develop antibodies to the virus, which can be detected by an enzyme-linked immunosorbent assay (ELISA) that has been shown to be more sensitive and specific than agar gel immunodiffusion, complement fixation (CF), or indirect immunofluorescence (Gelfi et al, 1999). Serum neutralization has also been used to detect antibodies (Marlier et al, 1999).…”