ENUMERATION OF ISOTYPE-SPECIFIC ANTIBODY-SECRETING CELLS DERIVED FROM GNOTOBIOTIC PIGLETS INOCULATED WITH PORCINE ROTAVIRUSES

In order to evaluate mucosal antibody responses to rotavirus, an enzyme-linked immunospot (ELISPOT) assay was adapted to enumerate antibody-secreting cells (ASC) in the mesenteric lymph nodes (MLN), lamina propria (LP) of the small intestine and spleens of gnotobiotic pigs orally inoculated with porcine rotaviruses (SB1A and Gottfried). Rotavirus-specific IgM ASC occurred by post-inoculation day (PID) 3, and numbers peaked in spleen and MLN tissues by PID 7 and in intestinal LP by PID 7-14. Numbers of rotavirus specific IgA and IgG ASC in these tissues peaked at PID 14-21. Rotavirus specific IgA ASC were predominant in the gut and IgA to IgG rotavirus specific ASC ratios were highest for all rotavirus antigen coatings in the gut LP, However, the relative-ratios of specific IgA to IgG ASC were lower (ratios of 5 to 7) against combined structural and nonstructural viral antigens (rotavirus-infected fixed cell ELISPOT plates) than ratios (13 to 46) against only viral structural antigens (rotavirus-coated ELISPOT plates), indicating that there were proportionately more specific IgG ASC to the nonstructural viral antigens in the LP, the tissue adjacent to the site of rotavirus replication in the intestine. In the node cells (spleen and MLN) rotavirus-specific IgA to IgG ASC ratios were lowest and against the various ELISPOT rotavirus coatings ranged from 0.7 to 4. Gnotobiotic piglets inoculated at different ages with porcine rotaviruses generally showed similar specific immunoglobulin (Ig) ASC responses to rotavirus infection, along with similar diarrhea and virus shedding patterns in the different age groups. However, the numbers of specific IgA ASC in the MLN of 3-4 week old pigs were higher than those of 3-5 day old pigs. Although challenge of SE 1A or Gottfried rotavirus-inoculated pigs with SB1A (G4P7) or Gottfried (G4P6) rotavirus revealed a high degree of protection from diarrhea and virus shedding, greater numbers of specific IgM ASC were observed in spleen after challenge of SB1A-inoculated pigs with Gottfried rotavirus (same G type, distinct P type). Thus, by using the ELISPOT technique, we successfully measured intestinal mucosal antibody-related responses to rotavirus in gnotobiotic pigs. Moreover, our results support the use of gnotobiotic piglets as an animal model to evaluate active antibody responses and protection against rotavirus infection and disease.-