Immunoactivity of Duck Serum Recovered from Riemerella Anatipestifer Infection
|Preventive Veterinary Medicine
|Anatipestifer anatipestifer duck Micro agglutination test antibody passive immunity
Riemerella anatipestifer (RA) is a short rod, gram-negative bacterium that can cause infectious serositis (IS) of ducks, geese and turkeys and other birds. A total of21serotypes of RA was reported around the world. A number of bacteria that were identified as members of Riemerella spccies of the Flavobacterius with similar morphological and cultural charateristics to RA have been identified into RA and Riemerella columbina (RC). RA infection is the comman disease in duck farms that affects ducklings aged1-8week old with80%to100%of mobidity (Mb) and30%to70%of mortality(Mt). It has been reported that serotype1,2,5,6,14of RA are the main epidemic serotypes in duck farms. Although RA outbreaks in duck farms can be controlled and prevented with the immunization of the inactived bacterin and attenuated vaccine of RA, IS is the mainly disease in duck farms recently in China. If more and more antibiotics are prohibited in duck disease treatment, IS will become much more serious, resulting in severs losses in duck production. Reasons to control this desease difficult are no significant cross-protection against challenging among different serotypes of RA,10-14days for the immune response to the RA vaccine against RA infection, easy modification in serotype of RA in a duck farm, and the RA strain with antibiotics resistance. To improve the effect of immune protection induced by RA vaccine, this theses describes a rapid diagnostic method to distinguish different serotypes of RA infection without bacterium isolation, and the immunoactivity of the antibody in serum from recovered ducks infected.1. AF strainsStrain AF, used in this study, was a virulence bacteria isolated from the clinical sick duckling in Chongqing, southwest of China, and typed into serotype2. Two fragments sized1480bp of 16srDNA and596bp of Cohemolysin (camp) gene were amplified by PCR and sequenced from strain AF. The results showed that strain AF belongs to RA.2. Improved Micro-agglutination test (iMAT) used to diagnose the serotype of RAAgglutination test, a classical immunological method, was used to investigate the serotype of RA. The technique was carried out in a vector of96wells of PVC microplate and named to micro agglutination test (MAT), which could be used to detect the titre of antibody in serum against RA. However, the agglutination phonomena was difficulty to judge, resulting in low sensitivity. The improved MAT to detect the antibody against RA was studied. The RA cultured on rabbit blood choolate LB (RBCLB) plate was resuspeded with physical solution (PS) and inactivated by the final concentration of0.5%formalin. The killed bacterium stained by basic fuchine was iMAT antigen. The volume of iMAT reaction is50μL, serum and antigen each25μL. The aggulutination was observed when the vector reclined vertical and the red line was found distinctly in the negative result. On the contrary, the red line was not found in the agglutination. With iMAT detection,76.5%of the healthy duck without infeted RA showed zero titre, with the rest of only1log2. In the inactivated bacterin immunized group, the titre ranged from2log2to5long2from14to22days post immunization, and70%of ducks displayed2to3log2. The titre increased by1to2log2after challenging in the second day post inoculation. However, all ducks became sick and20%to70%of them died during7days observation in the control group. The serum of ducks recovered from RA infection in control group showed higher titre ranged from3log2to8log2in7days post challenging, with73.9%ducks titre maintained more than5log2. The results showed that5log2of iMAT antibody titer was a boundary value to diagnosing the ducks infected RA. Data found here showed that iMAT detection could be a method with high sensitivity and specificity. It suggests that iMAT could be used to diagnose the serotype of the RA outbreak.3. Non-agglutination antibodies were the immunoprotction antibody in the serum of ducks recoveredDucklings of22days were divided into4groups, and were injected healthy duck serum (HDS), recovered duck serum (RDS), inactivated vaccine immunized duck serum (IDS) and PS, respectively with a dosage of3.0mL each. The ducks were challenged24hours later. The data showed that ducklings became ill with100%of Mb in the PS and HDS groups, and56%(5/9) and20%(1/5) of Mt. In group RDS, however, both of Mb and Mt were zero. In group IDS, no ducklings died but20%of ducklings illed. Data from the repeated experiment displayed that ducklings became ill with100%of Mb in groups PS and HDS, and37.5%(5/14) and60%(3/5) of Mt. In group RDS, however, both Mb and Mt were zero. In group IDS, no ducklings died but100%of ducklings illed. The data demonstrated that the duck serum recovered from the RA infection showed excellent passive immunoprotection effect.Mixed RDS from several ducks with a titre of5logs by iMAT detection was divided into four groups. Three of them were treated in56℃water bath for30min (wbRDS), absorbed for4to5times to remove agglutinating antibody (zero of the iMAT titre) and in56℃water bath for30min(ar+wbRDS), diluted4times (31og2) by PS(dRDS) respectively. The passive immunoprotection in the ducklings aged23days was carried out with these sera and RDS with no treatment. The results showed that ducklings become ill with100%of Mb in groups PS and HDS and33.3%(3/9) and zero of Mt. In groups wbRDS, ar+wbRDS, and RDS, however, the Mb and Mt were zero. In group dRDS, no ducklings died but22.2%(2/9) of ducklings illed. The data demonstrated that the antibody without agglutination in sera of ducks recovered from the RA infection was the passive immunoprotection antibody which have good stable in56℃water bath. The result showed that the titre of iMAT was corrected indirectly with passive immmunoprotection.4. The growth-agglutination and deagglutination-growth PhenomenonRDS with6log2MAT titer of100μul was added to1.9mL LB broth to cultivate strain AF of RA. In2hours post inoculation (HPI), the small aggregation was founded in the cultures and the solution was transparent. The size of the agglutination reached to maximum4-5HPI and the solution was also transparent. In5-6HPI, however, the agglutination was deagglutinated and the cultures became greyish white. The agglutination completely disappeared in6-7HPI and the solution turned turbidity. The phenomenon did not appear when RDS with a titer lower than4log2. All groups with duck serum of HDS and RDS included, the growth of RA was accelerated, the maximum concentration of the RA appeared in12HPI with A525value of2.2, but24HPI in the group LB without duck serum. The phenomenon the RA was brought up in LB and agglutinated with specific antibody and deagglutinated and grew qiukly was named growth agglutination and deagglutination growth (GADAG). The mechanism of GADAG of RA will need to be study in future.The morphology of the RA in the GADAG was observed by microcopy. In the agglutination, AF bacterium was agglutinated and became shorter. In the deagglutination, the bacteria recovered with scattered spherical form. The bacteria returned to short rod-shaped when the cultures became turbidity. The virulent detection showed that the AGDGA cultures of RA with A525of0.85±0.05brought up45%of Mb and13.4%of Mt in6HPI and100%and71.5%in20HPI, respectively. There were no significant difference in bacteria from the AGDGA culture and RBCLB culture (P>0.05). The data demonstrated that GADAG had no effect on RA virulence.