Avian Infectious Bronchitis Virus Infections in chickens: Virus Exccretion and Immune Response

Avian infectious bronchitis virus (AIBV), a coronavirus, causes acute respiratory disease in chickens. A rapid diagnostic method was developed for the detection of AIBV in infected chickens using a modified indirect double antibody sandwich (MIDAS) enzyme-linked immunosorbent assay (ELISA). The system was compared with isolation of virus by the conventional use of chicken embryos. The MIDAS ELISA was as sensitive as isolation of virus with the use of chicken embryos. However, the MIDAS ELISA was more rapid and easier to apply than the use of chicken embyos. In detecting antibody against indirect (MI) ELISA was also developed. AIBV, a modified The MI ELISA was used to study the immune response of experimentally infected chickens. Results indicated that antibody was not detected in chickens prior to infection, however, the antibody increased in titer as the infection progressed. With increased antibody levels in chickens, shedding of the virus in the trachea and the intestines gradually decreased and disappeared, except in two of eleven chickens whereby

A rapid diagnostic method was developed for the detection of AIBV in infected chickens using a modified indirect double antibody sandwich (MIDAS) enzyme-linked immunosorbent assay (ELISA).
The system was compared with isolation of virus by the conventional use of chicken embryos.
The MIDAS ELISA was as sensitive as isolation of virus with the use of chicken embryos. However, the MIDAS ELISA was more rapid and easier to apply than the use of chicken embyos.
In detecting antibody against indirect (MI) ELISA was also developed.    Avian infectious bronchitis virus (AIBV) causes a disease which has emerged within the last four decades from the melange of the "respiratory disease complex" of poultry to a well-defined and separate entity (1).
In April of 1930, Schalk and Hawn (2) were the first to describe and report "an apparently new respiratory disease of baby chicks" in North Dakota and throughout the  (20).
The polypeptide composition of AIBV has been reported to contain as few as three and as many as sixteen structural proteins in the virion (16,22,23,24,25).

Prevention and Control.
A. Management.
Strict isolation of the flocK is the best prevention, along with sound management practices such as adding only day-old chicks as replacement stock and rearing them in isolation. However even with sound management practices, AIB may occur. This has brought on the necessity or using immunization programs in order to control the disease.
The immunization against AIBV has been reviewed by Hofstad (84 The grid was then stained as above.
In the final procedure, a pseudoreplication method was used. -20°c.
The tubes were then centrifuged at The samples were labelled and stored at The second experiment was similar to the first, except 1.5 ml of HBSS was used and the tubes were centrifuged at 180 x g for 10 min.
The lung tissue that was taken from the chickens were washed once in PBS and then placed in a small beaker of PBS.
The tissue was minced with a pair of ~terile scissors.
The fluid was collected in a tube and centrifuged at 260 x g for 10 min to remove any red blood cells. The supernatant was collected in a sterile tube, labelled, and stored at -20°c for future use.
The blood collected was prepared for sera the same way as described in the antisera production section, except that it was centrifuged at 260 x g for 15 min, due to the

Statiiti~ analy~.
A one-tailed Students' t test was used to analyze the data (see Appendix G).
Ten fractions ranging in densities of 1.07 to 1.27 g/ml were 5.5 collected.
Peak infectivity (10 ELD ) correlated with 50 fraction five, which contained a visible band at a density of 1.17 g/ml. Virus was also detectable at densities between 1.13 and 1.23 g/ml. (Fig 1. Various dilutions of a chicken anti-AIBV were used to coat the microplates. Due to a low non-specific binding and 37 high specificity, a chicken anti-AIBV dilution of 1:1,000 containing 51 mcg/ml of protein was chosen. (Fig 2.)  Of non-specific binding but high sensitivity. (Fig 9.) .ss.andardizallQ.n reference serum The titration depicted of a known positive and negative antisera is Mm ELISA. Table 2 shows the results of the detection of viral antigen in the trachea and fecal swabs of the infected chickens. isolation.
These results were comparable with those of virus was detected after day fourteen.

No virus
No virus was isolated from the control chickens.
MIDAS ELISA. In Experiment 2, there was a peak antibody titer at day fourteen followed by a slow decline in titer.
Antibody was detected by the modified indirect enzymelinked immunosorbent assay b. Chickens were infected by intratracheal inoculation with c.
10 3 ELDsolml of AIBV Chickens were three-weeks of age d.
[P] = Pre-infection serum e. Antibody titer expressed as the reciprocal of serum dilution a. Chickens were infected by intra tracheal inoculation with 1 0 3 ELD SO /ml b.
[F] = Fecal swabs number of positive isolations/ number of total tested [NT]= Not tested Table 8.
Detection of AIBV antigen from the throat and feces of infected chickens a ,b by means of a MIDAS ELISA.
10 ELDso/ml of AIBV Chickens were three-weeks of age c.
[F] = Fecal swabs e. number of positive detections I number Of total tested a Table 9.
Comparison of antibody response in noninfected and AIBV infected chickensb, c from Experiment 1 and Experiment 2.
mean titer :I: standard error (total number of chickens) h.
[••] = p~ 0.001  15) showed AIBV had five separate infectivity peaks at 1.14, 1.16, 1.17, 1w19, and 1.21 g/ml respectively. In this study, though only one peak was shown at 1.17 g/ml (Fig 1) (97) was encountered at low serum dilutions at the begining of immunization. The early IgG antibodies were not able to resist washing procedures when crowded and also were not able to aligned on the antigen site at low serum dilutions.
They did resist washing when they were not as crowded.
During later immunization, the antibodies had a high affinity for the antigenic sites and were able to "hang-on" through washing even though crowded and poorly aligned. Express the mortality rate (No. affected/total) 6/6 2/4 0/6 Express % mortality 100 % 50 % a = most concentrated dilution that shows 100 % affected b = the next dilutions between "a" and "b" c = most dilute level that shows 0 % affected 0 % d = the number of embryos affected/total per dilution tested To compute the acutal end point dilution use the following formula: oer Qent mQrtality next abQve 50 % -50 % Per cent mortality next Per cent mortality next above 50 % below 50 % = proportionate distance.
Find the log of that dilution in which per cent mortality is next about 50 %.
Multiply the proportionate distance X log of the dilution factor 10 to obtain the real value of the Proportionate distance (round to the nearest tenth). 95 I APPENDIX B Plate I.
AIBV from infected AAF of 10-day-old embryonated chicken eggs was harvested after 30 hours PI and clarified at 7,650 x g for 10 minutes. The virus was then stained using pseudoreplication.
The stained grids were viewed with a Hitachi HS-9 EM at 75 Kv.
The corona are indicted by an arrow.
The ELISA antigen-antibody complex. AIBV from infected AAF of 10-day-old embryonated SPF eggs was harvested after 36 hours PI and clarified at 7,650 x g for 10 min.
The virus suspension was then pelleted at 76,700 x g for 1 1/2 hours.
The pellet was resuspended in PBS.
Chicken anti-AIBV was added to the virus suspension and incubated for 30 min at 37o ·c.
The virus-antibody mixture was then stained using the agar block method.
The stained grid was view with a Hitachi HS-9 EM at 75 Kv.
Corona are indicated by arrows. (Mag 286,000 X) APPENDIX C Determination of protein concentrations of various ELISA test reagent was carried out.