| Salmonella belongs to the Enterobacteriaceae family and is a parthenogenic anaerobic Gram-negative bacillus.Salmonellosis is one of the important foodborne diseases,poultry is the most common storage host of Salmonella,and contamination of poultry and their products is closely related to the outbreak of human salmonellosis.Salmonella Enteritidis,the most common serotype,has a broad host spectrum and chickens of all ages are susceptible.Usually chicks infected with Salmonella Enteritidis will cause acute inflammation of the digestive tract and death in serious cases,resulting in significant economic losses;adult chickens are often recessively infected and will discharge bacteria into the environment,becoming a source of infection in the flock;in addition,breeder chickens infected with Salmonella Enteritidis will spread vertically to their offspring flocks,causing serious harm to breeder farms.Therefore,the prevention,control and decontamination of Salmonella in breeding birds are of particular importance to the poultry industry and public health safety.Salmonella detection by pathogen isolation and identification and serum antibody testing are often used in production,but studies have shown that the rate of Salmonella antibody positivity is not consistent with pathogen positivity,leading to incomplete decontamination and thus affecting the decontamination process.At present,commercial antibody detection reagents mainly include plate agglutination antigen and ELISA kit.The two types of commercial detection reagents have different principles and different detection effects.Therefore,it is particularly important to evaluate and screen the detection reagents with strong specificity,high sensitivity and good stability to improve the detection and purification efficiency of Salmonella and reduce the production cost.In this project,sera and swabs were collected from artificially infected SPF chickens at different time points for serum antibody detection and isolation and identification of Salmonella Enteritidis respectively,in order to study the antibody production pattern and excretion pattern after Salmonella Enteritidis infection.In addition,a set of positive reference sera for Salmonella Enteritidis was developed to evaluate the commercial plate agglutinogen and ELISA kits to screen for high quality antibody detection reagents for Salmonella purification.The main elements include:1.Study on the pattern of antibody growth after artificial infection with Salmonella Enteritidis in SPF chickens.In order to study the pattern of antibody growth and decline after artificial infection with Salmonella Enteritidis in SPF chickens,16 52-day-old SPF chickens were infected with standard Salmonella Enteritidis strain(CVCC3377)by infusion at two doses of 10~7CFU/each and 10~8 CFU/each,with 8 chickens in each group,and the infection was repeated1 week later at the same dose as the first.In order to study the pattern of antibody growth and decline after artificial infection with Salmonella Enteritidis in SPF chickens,1652-day-old SPF chickens were gavaged with Salmonella Enteritidis standard strain(CVCC3377).Chickens were divided into two dose groups of 10~7 CFU/each and 10~8CFU/each,with 8 chickens in each group,and the infection was repeated after 1 week.Blood was collected and serum was isolated on days 2,5,7,9,12,14,21,28 and 35 after the first artificial infection,and the antibody levels of Salmonella Enteritidis in serum were detected by commercial B+D group ELISA kits and plate agglutinogens,and antibody extinction curves were plotted.The results of antibody detection by ELISA showed that the same dynamic changes of antibody in the high-dose infection group with the low-dose infection group,and both groups started to detect antibody-positive chickens(1/8 and 3/8)on the 7th day after the first artificial infection,with lower average antibody levels(205.37 and 447.59,respectively);thereafter,the antibody levels increased continuously until 28 days after the first infection.At the same time,the antibody levels in the high-dose infection group were higher than those in the low-dose infection group,and the antibody positivity rate in the high-dose group reached 100%before that in the low-dose group,and the maintenance time of 100%antibody positivity rate in the high-dose group was longer.The results of the plate agglutination test showed that antibody-positive chickens were detected on day 7 and day 5after the first infection in both groups(3/8 and 6/8,respectively),and the agglutination potency reached peak on day 14 after the first infection,and the agglutination potency was higher in the 9 days before and after the peak.The highest antibody positivity rates began to be detected at 12 and 9 days after the first infection,with 75%and 100%,respectively.2.Study on the pattern of Salmonella Enteritidis excretion after artificial infection with Salmonella Enteritidis in SPF chickens and correlation between pathogenic and serological test results.Oral and cloacal swabs were collected separately for pathogenic detection of Salmonella at the same age of blood collection,and swabs and serum samples from each chicken in different sampling batches were divided into 1 of 144 groups.The results showed that both groups started to excrete on the 7th day after the first artificial infection,and Salmonella was not detected during the 21 days after the first infection,with pathogen positivity rates ranging from 6.25%to 25%during the excretion period.Positive rates for pathogen isolation,ELISA and plate agglutination were 7.64%,52.78%and 67.36%,respectively.There were 102 groups tested positive by?1 method,of which 0.98%were positive by pathogenic test only,89.22%were positive by antibody(ELISA or plate agglutination)only,9.80%were positive by both pathogenic and antibody tests,and 7.84%were positive by all three methods.The consistency of the results obtained by the different assays was determined by calculating the Kappa coefficient,and the consistency of the results of both antibody and pathogenic assays was weak for ELISA and plate agglutination,and moderate consistency was found for ELISA and plate agglutination.The results showed that a single serological or pathogenic test could not detect all positive chickens,and the test results should be considered to prevent missing detection.3.Preparation and application of reference serum for Salmonella Enteritidis.In order to develop reference sera and establish common ELISA kits and plate agglutinogen methods for evaluation and screening,the average antibody elongation curve was used as the standard curve,and the antibody elongation curve of individual chickens was observed.Besides,the chicken antisera with high agreement between the antibody elongation curve and the standard curve were finally selected,and the sera of the screened chickens were mixed in equal proportions at the early stage of antibody production,the rising stage,the peak stage and the falling stage,respectively,to obtain a set of reference sera consisting of five mixed sera.Two commercial Group B+D ELISA kits and one commercial Group D ELISA kit were used to test the five mixed sera,and the prepared reference sera were calibrated.A total of seven commercial platelet agglutinogens and three commonly used ELISA kits from different batches of different manufacturers were evaluated for specificity,sensitivity and stability using prepared reference sera.The results show that this method can effectively evaluate the specificity,sensitivity and stability of different assay kits and become a powerful tool for evaluating the screening of commonly used ELISA kits and plate lectins.In this experiment,we studied the pattern of excretion of Salmonella Enteritidis after artificial infection of SPF chickens and the pattern of antibody growth,analyzed the correlation between pathogenic and serological detection results,and developed a reference serum for Salmonella Enteritidis,and initially established a quality assessment method for avian Salmonella plate agglutinogen and ELISA kits to provide theoretical support and screen high-quality detection reagents for salmonellosis purification. |
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