Humoral and Delayed-type Hypersensitive Responses against Listeria monocytogenes Phosphatidylinositol- speci¢c Phospholipase C in Experimentally Infected Bu¡aloes S.P. Chaudhari, S.V.S. Malik and S.B. Barbuddhe* DivisionofVeterinaryPublicHealth,IndianVeterinaryResearchInstitute,Izatnagar243 122India *Correspondenceandpresentaddress:ICARResearchComplexforGoa,Ela,OldGoa 403402,India E-mail:barbuddhesb@yahoo.com Chaudhari, S.P., Malik, S.V.S. and Barbuddhe S.B., 2004. Humoral and delayed-type hypersensitive responses against Listeria monocytogenes phosphatidylinositol-speci¢c phospholipase C in experimentally infected bu¡aloes. VeterinaryResearchCommunications, 28(7), 569^579 ABSTRACT The kinetics of antibody production against phosphatidylinositol-speci¢c phospholipase C (PI-PLC) and the isolation pattern of Listeriamonocytogenes from bacteriological samples were studied following oral infection of bu¡alo calves with 3610 9 cells each of pathogenic L.monocytogenes. Antibodies to PI- PLC appeared by 4^8 days post infection (PI), with a peak between days 7 and 16 PI, when tested by indirect plate-ELISA. Subsequently, antibody titres in all the animals declined and became undetect- able on days 26^35 PI onwards until the study concluded on day 211 PI. Dot-ELISA could detect the antibodies to PI-PLC 1^2 days earlier and at higher titres as compared to plate-ELISA. L. monocytogenes could be recovered from faeces, nasal swabs and haemocultures from days 2 to 33, days 2 to 21 and days 11 to 17 PI, respectively. Antibodies to PI-PLC were detected during the course of active infection but their titres declined sharply once animals became culturally negative. Sonicated antigen elicited the highest delayed-type hypersensitivity response, followed by PI-PLC and listeriolysin O. Keywords: bu¡aloes, DTH, ELISA, Listeria monocytogenes, phosphatidylinositol-speci¢c phospholi- pase C, toxins Abbreviations: ALLO, anti-listeriolysin O; BHI, brain^heart infusion; CFA, culture ¢ltrate antigen; DEAE, diethylaminoethyl; DTH, delayed-type hypersensitivity; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; HKA, heat-killed antigen; HRPO, horseradish peroxidase; LLO, listeriolysin O; LB, Luria^Bertani; PBS, phosphate-bu¡ered saline; pI, isoelectric point; PI, post infection; PI-PLC, phosphatidylinositol-speci¢c phospholipase C; SA, sonicated antigen; SDS-PAGE, sodium dodecyl sulphate^polyacrylamide gel electrophoresis INTRODUCTION Listeric infections in animals are caused by Listeria monocytogenes and Listeria ivanovii. L.monocytogenes is a Gram-positive, facultative, intracellular bacterium that is ubiquitous in nature. L. monocytogenes is a cause of septicaemia, mastitis, VeterinaryResearchCommunications,28(2004)569^579 # 2004 Kluwer Academic Publishers. Printed inthe Netherlands 569