Fifty microliters of samples in serial dilutions (from 1:2 to 1:512) was prepared in a 96-cell plate. RV adjusted to 200 TCID50 in 50 μL of virus diluent (10% concentrated Hanks balanced salt solution, pH buy CX-4945 7.4) was added to the cell plate containing serially diluted serum. The mixture of antibody and virus was mixed and incubated at 37°C for 1 h. Then 100 μL of MA104 cells (used for virus infection) was added to the antibody-virus mixture and incubated in a 5% CO2 incubator at 37°C for 5 days. The overlay medium was then discarded, after which the wells were washed three times with sterile PBS, pH 7.4, and stained with 1% crystal violet solution. Differences in the number

of plaques formed between treatments were examined for the level of significance by ANOVA. Statistical analysis Statistical significance was determined using ANOVA, with a P value < 0.05 considered as significant. Acknowledgements This work was supported by grants from the National Science and Technology Foundation of China (No. 2006BAD06A07) and the Program for Innovative Research Team of NEAU (No. CXZ008). The authors wish to thank Jos Seegers for providing plasmid pPG611.1 and bacterial strain L. casei ATCC 393. References 1. Paul PS, Lyoo YS: Immunogens of rotaviruses. Vet Microbiol 1993, 37:299–317.PubMedCrossRef 2. Estes MK: Rotaviruses and their replication. Fields Virology 2001, 4:1747–1785. 3. Rosen I, Parwani AV, Lopez S, Flores J, Saif L: Serotypic

differentiation of rotaviruses in field samples from diarrheic pigs by using nucleic acid probes specific for porcine VP4 and human and porcine VP7 genes. J Clin Microbiol Progesterone 1994, 32:311–317.PubMed 4. Winiarczyk S, Paul PS, Mummidi ARS-1620 in vitro S, Panek R, Gradzki Z: Survey of porcine rotavirus G and P genotype in EX 527 Poland and the United States using RT-PCR. J Vet Med 2002, 49:373–378.CrossRef 5. Gatti MS, Ferraz MM, Racz ML, de

Castro AF: Rotavirus excretion in naturally infected pigs with and without diarrhea. Vet Microbiol 1993, 37:187–190.PubMedCrossRef 6. Fitzgerald GR, Barker T, Welter MW, Welter CJ: Diarrhea in young pigs: comparing the incidence of the five most common infectious agents. Vet Med Food Anim Pract 1988, 1:80–86. 7. Will LA, Paul PS, Proescholdt TA: Evaluation of rotavirus infection in diarrhea in Iowa commercials pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases. J Vet Diagn Invest 1994, 6:416–422.PubMed 8. Shaw DP, Morehouse LG, Solorzano RF: Experimental rotavirus infection in three-week old pigs. Am J Vet Res 1989, 50:1961–1965.PubMed 9. Moon HW: Comparative histopathology of intestinal infections. Adv Exp Med Biol 1997, 412:1–19.PubMed 10. Svensmark B, Askaa J, Wolstrup C, Nielsen K: Epidemiological studies of piglet diarrhea in intensively managed Danish sow herds. IV. Pathogenicity of porcine rotavirus. Acta Vet Scand 1989, 30:71–76.PubMed 11. Gerdts V, Mutwiri GK, Tikoo SK, Babiuk LA: Mucosal delivery of vaccines in domestic animals. Vet Res 2006, 37:487–510.