1.11.26
PRODUCTION OF POLICYLONAL ANTISERA SPECIFIC TO PLANT VIRUSES BY RABBIT ORAL IMMUNIZATION

JAA LIMA, RCA LIMA and MI FLORINDO

Universidade Federal do Ceara, Lab. Virologia Vegetal, CxP 12.168, Fortaleza-CE, 60.356.000, Brazil

Background and objectives
A major limiting factor in the use of serological techniques for plant virus identification is the requirement of a purified virus preparation to be used in rabbit immunization to produce antiserum. The chemical purification of plant viruses is, usually, an expensive and complicated process that involves several stages, from virus propagation in the greenhouse to different types of chemical treatment, followed by low- and high-speed centrifugation to separate virus particles from the plant components. Sometimes the virus is lost during the purification process or is purified in a non-satisfactory concentration to be used in rabbit immunization. The objective of the present research was to produce polyclonal antisera specific to cowpea mosaic virus (CPMV; family Comoviridae, genus Comovirus), and to papaya lethal yellowing virus (PLYV; possible family Tombusviridae, genus Tombusvirus) by rabbit oral immunizations with clarified infected plant extracts.

Material and methods
An isolate of CPMV obtained from cowpea, Vigna unguiculata, was increased in cowpea Pitiuba plants maintained under greenhouse conditions, and 10 g of systemically infected leaf tissues were collected. The leaf tissue was ground in a mortar and pestle with 0.15 M saline solution at 1:1 (w/v), and the extract obtained was clarified by centrifugation at 10,000 g for 10 min. The clarified extract containing CPMV was given orally to a New Zealand rabbit with a syringe in two series of five daily doses of 1.0 ml each. Similar immunization was repeated in another rabbit, using extracts of PLYV infected papaya, Carica papaya, leaves. Fifteen days after the last immunizations, the rabbits were bled to obtain the respective polyclonal antisera. The antisera were evaluated by double immunodiffusion and ELISA techniques against the respective virus antigens and extracts from healthy cowpea and papaya plants.

Results and conclusions
The polyclonal antisera obtained were shown to be very specific to their respective viruses. The antiserum for CPMV reacted with extracts of CPMV infected plants, with a titre of 1:124 in double immunodiffusion testing without showing any reaction with extracts of healthy plants. The antiserum for PLYV was shown to be reactive to extracts of PLYV- infected papaya in double immunodiffution and ELISA tests, with titres of 1:124 and 1:2,000, respectively. No reaction was observed with the PLYV antiserum and extracts of healthy plants in double immunodiffusion, and a very weak reaction in ELISA was easily absorbed. These seem to be the first antisera specific for plant viruses obtained by rabbit oral immunization. This is a simple, fast and inexpensive method for production of plant virus-specific antisera, when compared with the traditional techniques that involve rabbit injections with purified virus preparations. These results open up some possibilities for the production of antisera for those plant viruses that are difficult to purify

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