Screening of the T- and B-cell antigenicity in neonatal calves of the His-tagged Cryptosporidium parvum antigens CP15, CP15/60, P23 and TRAP-C1.

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Cryptosporidium parvum Tyzzer, 1912 is the most commonly found enteropathogen during the first weeks of the life of calves, lambs and goat kids and is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome. Because of the limited availability of effective drugs, hygienic measures and good management are the most effective disease control measures (de Graaf D.C., Vanopdenbosch E., Ortega-Mora L.M., Abbassi H., Peeters J.E. 1999: Int. J. Parasitol. 29: 1269-1287). Immunoprophylactic control of Cryptosporidium infection is still under development and most studies focused on the passive transfer of colostral immunoglobulins from immunized dams to susceptible neonates, with sporozoite surface and microneme antigens being the target molecules of choice (de Graaf D.C., Spano F., Petry F., Sagodira S., Bonnin A. 1999: Int. J. Parasitol. 29: 1289-1306). Although the key position of CD4 T-cell dependent interferon-gamma (IFN-γ) in acquired immunity against C. parvum (Ungar B.L., Kao T.C., Burris J.A., Finkelman F.D. 1991: J. Immunol. 147: 1014-1022) and C. muris (McDonald V., Robinson H.A., Kelly J.P., Bancroft G.J. 1994: Infect. Immun. 62: 2289-2294) is well documented, the development of a T-cell vaccine to be administered to the newborns themselves has so far received only little attention. In previous papers we described the specificity of the IFN-γ response in the Cryptosporidium-infected bovine host (de Graaf D.C., Peeters J.E. 1997: Int. J. Parasitol. 27: 131-134) and the involvement of CD4 T-cells and a low molecular mass antigen preparation (de Graaf D.C., Walravens K., Godfroid J., Peeters J.E. 1998: Int. J. Parasitol. 28: 1875-1880). Further efforts to identify and purify a single parasite-derived antigen that evokes this immune response failed because of the limitations and constraints of the biochemical approach. Here we report the cloning of four known C. parvum surface or microneme antigens in a pBADTOPO-TA expression vector (Invitrogen, Carlsbad, CA, USA) and the evaluation of their Tand B-cell antigenicity in the bovine host. Cryptosporidium parvum oocysts were purified from faeces of infected calves by biphasic diethyl ether/phosphate buffered saline (PBS) extraction and differential centrifugation on Percol. Sporozoites were liberated in prewarmed excystation fluid (37°C) consisting of 0.75% (w/v in PBS) sodium taurocholate with 0.25% bovine trypsine. Cryptosporidium sporozoite DNA was prepared according to a method optimized for the preparation of genomic DNA from bacteria (Ausubel F.M., Brent R., Kingston R.E., Moore D., Seidman J.G., Smith J.A., Struhl K. 1990: Current Protocols in Molecular Biology. Greene Publishing Associates, Brooklyn). PCR primers were designed for the amplification of the complete coding region (stop codon excluded) of the known C. parvum proteins CP15, CP15/60, P23 and TRAPC1 (see Table 1). Amplification was performed in a PerkinElmer model 9600 thermocycler by 30 cycles of denaturation at 94°C for 60 s, annealing at 51°C for 60 s and extension at 72°C for 4 min, with a supplementary annealing step at 72°C for 20 min after the last cycle to ensure that all PCR products were full length and 3’ adenylated. The resulting amplicons had the expected lengths and were subsequently cloned in pBAD-TOPO-TA according to manufacturer’s instructions. Cloning was confirmed by DNA sequence analyses of the retained pBAD-CP15, pBAD-CP15/60, pBAD-P23 and pBAD-TRAP-C1 clones. Indeed, we found a perfect match