Historically in the United States, RVA infections were considered the more prevalent. However, recent studies indicate RVA and RVC infections are more common in piglets <21 days of age. Some RVA and most, if not all, RVC strains are extremely difficult to adapt to cell culture, and serological assays are unavailable to measurement of the immune response and antibody levels in response to natural planned exposure (NPE) protocols. The VP7 (G genotypes) is the most immunogenic, highly glycosylated, independently elicit neutralizing antibodies, and induce protective immunity. Multiple different genotypes/serotypes exist, and the neutralizing antibodies are serotype specific and provide minimal or no protection against viral infections from different serotype. In serum and colostrum, the primary antibody is IgG, which is transferred from the serum to colostrum during gestation and absorbed into the piglet’s serum upon ingestion of colostrum. The primary antibody in the intestine is IgA, which is transferred to piglets during ingestion of milk and protects against enteric infections. The consistent intake of IgA via milk protects the piglets from viral infection. The overall project’s objective was to evaluate the effect of different protocols of feedback (natural planned exposure) to rotavirus A and C on gilt immunity and passive immunity in their piglets from a study that occurred with a swine producer over the summer. Specific objectives were: 1. Develop an ELISA tests to measure neutralization of G specific surface glycoprotein against rotavirus A and C in sera, milk, and colostrum from rotavirus A and C-infected gilts, 2. Investigate the neutralizing Ab response to rotavirus A and C in gilts after feedback and passive immunity in their piglets. Currently, commercial ELISA for swine rotavirus A, B, and C are lacking. Without these RV ELISAs, the effectiveness of feedback (natural planned exposure) cannot be measured.
To understand the correlation between RVA and RVC NPE and G specific protection in piglets, 70 bred gilts were randomly divided into 3 treatment groups and a control group. Based on farrowing dates, 12 gilts per treatment remained enrolled in the study through lactation. NPE was administered according to different protocols for each treatment group. NPE material containing RVA (G5 and G4 genotypes) and RVC (G6 genotype) was mixed with a feed-water slurry and administered orally according to different protocols for each treatment group. Treatment group 1 was given NPE at five, four, and three weeks prefarrow (wpf); treatment group 2 at five and three wpf; and treatment group 3 at week five prefarrow only. The control group was not given NPE. Fecal samples were collected at 5 wpf and biweekly until 2 wpf, after which weekly sampling occurred until weaning. Serum samples were collected from the gilts at 5 and 3 wpf and at 0 and 3 post farrowing. Colostrum was collected at birth and milk was collected 1-3 weeks post farrowing. At farrowing, five piglets per gilt were enrolled, and serum were collected four times from birth to weaning (Table 1).
To achieve project objectives, VP7 sequences of G5 RVA and G6 RVC (sequenced from the feedback material) were cloned into pcDNA3.1(+) mammalian expression vector with a CD5 secretory signal for efficient secretion into the culture media. An 8-his tag and streptavidin tag was added at N and C terminals respectively for tracking protein expression and affinity purification of recombinant proteins. Expi293 cell suspension cultures were transfected with pcDNA3.1+ constructs expressing the rotavirus antigens (G5 RVA and G6 RVC). Anti-his cobalt resin (TALON, Takara Bio) was used for purification of the the recombinant antigens from culture media under native conditions. Purified proteins were validated and confirmed for purity using SDS-PAGE and immunoblotting using anti-his monolonal antibodies. Two indirect ELISAs were developed to detect rotavirus A or C VP7-specific IgG and IgA antibodies in clinical samples. Checkerboard titration revealed optimum coating concentration of G5 VP7 and G6 VP7 to be 100ng and 50ng, respectively. Other parameters such as serum dilutions, secondary antibody concentration, incubation temperature and time combinations were successfully standardized. Newly developed ELISAs were able to detect specific antibodies in the serum samples at different dilutions.
Piglets born to gilts receiving 3 doses of NPE resulted in highest serum RVA IgG titers at birth until weaning, which indicates that 3 doses of NPE provide better protection against RVA infection in piglets. For RVC, three doses of NPE resulted in highest RVC IgG and IgA titers in sow colostrum and milk. Most importantly, piglet serum RVC IgG levels were significantly low compared to serum RVA IgG titers at birth, which possibly explains the high prevalence of RVC in neonatal piglets. In particular, all three treatment groups showed low RVC titers compared to RVA titers. This project describes the antibody levels in sows and their piglets after receiving natural planned exposure. Clear differences were observed in antibody levels among study groups against rotavirus A. However, rotavirus C antibody levels were not significantly different among different treatment groups. In particular, piglet serum RVC IgG and IgA levels were not very different among different treatment groups, which could be due to the high RVC ct values (29.33 – 32.55) of the original NPE material administered to the gilts. In comparison, RVA ct values (2.46 – 24.43) of the NPE materials were significantly less. However, results of this project expand our understating of the NPE protocols used in swine farms and will assist producers to make better decision in terms of timing and dose of the NPE. Moreover, the highly efficient protein expression platform used in the project can be used to expressed G glycoprotein of other important swine RVA and RVC genotypes. Finally, this project has resulted in the development of immunoassays, which swine industry can use to assess immune response to rotavirus A and C in swine.
• Three doses of NPE resulted in highest serum rotavirus A IgG titers in gilts at farrowing
• Piglets born to gilts receiving 3 doses of NPE resulted in highest serum rotavirus A IgG titers at birth and until weaning
• Three doses of NPE resulted in highest RVC IgG and IgA titers in sow colostrum and milk
• Piglet serum RVC IgG levels significantly low compared to serum RVA IgG titers at birth