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An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.

Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.