Cells and virus
The wild-type CHYJ130330 strain (GenBank Accession No. KJ020932) was isolated, sequenced and preserved in our laboratory [20]. The strain was propagated in Vero cells grown in Dulbecco's modified Eagle’s medium (DMEM; Life Technologies) supplemented with 5% fetal bovine serum (FBS; Life Technologies), penicillin (100 units/ml) and streptomycin (100 mg/ml) at 37°C in a humidified atmosphere with 5% CO2. Briefly, for viral infection, the confluent monolayer of Vero cells propagated in a growth medium in 25 cm2 flasks was washed three times with phosphate-buffered saline (PBS, pH7.4) and inoculated with 1 ml virus. After adsorption at 37°C for 2 hrs, infected cells were maintained in growth medium supplemented with 5% fetal bovine serum at 37°C in 5% CO2. The 8th generation of PEDV CHYJ strain (containing 10´ TCID50) was used for highly pathogenic PEDV challenge experiment in piglets.
Vaccine
The inactivated PEDV CHYJ vaccine contained porcine epidemic diarrhea virus at 107.5 TCID50/ml. The porcine epidemic diarrhea virus venom was added with the final content of 0.2% formaldehyde solution and inactivated at 37℃ for 48 hours, and then 1% sterilized sodium sulfite (5%) was added to terminate the inactivation. Porcine epidemic diarrhea virus venom is mixed with 50% sterilized alumina hydroxide gel brine diluent in a ratio of 7:3 to produce the inactivated vaccine. The commercial bi-combined (PEDV and TGEV) inactivated vaccine contained porcine epidemic diarrhea virus CV777 strain.
Animals, Vaccination and experimental design
15 pregnant sows tested negative for PEDV antibodies as well as screened for antigen from un-immunized PEDV healthy pigs were randomly allocated three groups. The allocation was done 40 days prior to farrowing with five sows in each group. First group sows (n=5) were intranasally vaccinated with inactivated PEDV CHYJ vaccine 40 days prior farrowing followed by same titer booster dose after 20 days,and the dose was two milliliters per head. Second group was vaccinated intramuscularly with commercial bi-combined (PEDV and TGEV) inactivated vaccine, and the dose was two milliliters per head. Along with this vaccine control group, a third negative control group was also kept as shown in Table 1. After farrowing, milk was collected from sows in all groups on day 0, 1, 5, 10, 15 and 21. From each experimental group, five suckling piglets were randomly selected for dissection as shown in Table 2. Intestine contents including duodenum, jejunum, ileum and colon of piglets were collected aseptically before and after offering milk on day 1, 5, 10, 15 and 21. Immunoglobulins (IgA and IgG) were tested in milk (sows) and intestine contents (piglets) using PED IgA Ab ELISA antibody test kit and Porcine Epidemic Diarrhea Virus Antibody Test Kit respectively.
Table 1 Experimental groups, Vaccination and milk of sows collection at different days post farrowing
Group designation
|
No. of sows
|
Type of Vaccine
|
Route
|
Milk collection
|
CHYJ vaccine
|
5
|
Inactivated PEDV CHYJ vaccine
|
Intranasally
|
Collect milk at day 0, 1, 5, 10, 15 and 21 after farrowing
|
Commercial vaccine
|
5
|
Inactivated commercial bi-combined vaccine
|
Intramuscularly
|
Negative control
|
5
|
Saline
|
Intranasally
|
Table 2 Experimental groups, Vaccination and intestine of piglets collection at different days post farrowing
Group designation
|
No. of piglets
|
Type of Vaccine
|
Route
|
Intestine collection
|
CHYJ vaccine
|
25
|
Inactivated PEDV CHYJ vaccine
|
Intranasally
|
Collect intestine of five suckling piglets including duodenum, jejunum, ileum and colon before offering milk and at day 1, 5, 10, 15 and 21 after offering milk
|
Commercial vaccine
|
25
|
Inactivated commercial bi-combined vaccine
|
Intramuscularly
|
Negative control
|
25
|
Saline
|
Intranasally
|
Sample collection
Samples including milk (from sows after farrowing) and intestine contents (from suckling piglets) were aseptically collected before milk intake and also after offering milk on day 0, 1, 5, 10, 15 and 21 and stored at -20°C. Equal sections of piglet’s intestine were scraped, collected aseptically and centrifuged at 10,000 rpm. Supernatant obtained was stored at -20°C.
ELISA
PEDV IgA antibody detection was carried out for milk obtained from sows and intestine contents from piglets using PED IgA Ab ELISA antibody test kit. The kit contained microporous plates that have been coated with PED IgA antigen.Samples were loaded to each well including positive (Ab-positive) and negative (Ab-negative) controls in duplicate for each sample type. Plates were incubated at 37℃ for 60 minutes and washed 5 times (5´). Diluted enzyme conjugate was dispensed in each well; plates were incubated for 30 minutes. In the next step, the plates were washed 5 times (5´), substrate was dispensed in each well and plates were placed for 15 minutes at room temperature (18~25℃). The stopping solution was added to each well and the absorbance was measured at 630nm using bichromatic spectrophotometer within 30 minutes upon completion of assay.
PEDV IgG antibodies in milk of sows and intestines of piglets were also tested using Porcine Epidemic Diarrhea Virus Antibody Test Kit. The kit contained microporous plates that have been coated with PEDV antigens. Each sample was loaded along with antibody positive (positive control) and negative samples (negative plate control), run in duplicate on each ELISA plate. Calculate the results. The plates were kept at 23±2℃ for 60 minutes and washed 4 times (4´) carefully. Diluted conjugate A was dispensed to each well, the plates were kept for 60 minutes at 32±2℃. After washing (4´), diluted conjugate B was added to each well and plates were kept for 60 minutes at 23±2℃. After incubation, plates were washed four times and substrate was dispensed to each well. The plates were kept in dark for 10 minutes at 23±2℃ before dispensing stopping solution to each well. The absorbance was read within 15 minutes at 630nm using bichromatic spectrophotometer.
Challenge
The virulent PEDV CHYJ strain was grown to a final titer of 102 TCID50 /mL. Five piglets were randomly selected from each group and challenged orally with 1 mL of 102 TCID50 virulent PEDV CHYJ strain at day 3 post farrowing. Piglets in the negative control group were orally sham-inoculated with 1 mL saline. All piglets were examined daily for clinical signs of illness and anal swabs were also collected each day (day 1 to 5) for detection of PED virus using RT-qPCR.
Statistical analysis
All data were expressed as the mean ±standard deviation (SD). IgA and IgG antibody titers were subjected to statistical analysis for estimating the protective levels. GraphPad Prism 5 was employed for correlation analysis and a value of p<0.05 was considered statistically significant.