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An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.

An immuno-enzyme assay for measuring infectious bursal disease antibodies in chickens is described. The test is performed rapidly after coating plates overnight with partially purified antigen prepared in cell culture. Coated plates can be stored for at last 4 months. The chromatographically purified rabbit anti-chicken immunoglobulin-G, conjugated to horseradish peroxidase, was used optimally at a dilution of 1:3,000. It could be stored for at least 10 months without a reduction in titer. The test is safe, highly reproducible, specific, and sensitive. Results can be read visually or by spectrophotometry. Antibodies could be detected as early as 4 days postinfection. Serum titers rose rapidly to high levels, ranging from 1:1,600 to 1:25,600 by one week postinfection. High titers persisted for up to one year. The results of this assay compare favorably with results obtained with the agar-gel precipitin and virus-neutralization tests.

The present paper describes a method for measuring enzyme-linked immunosorbent assay (ELISA) antibody titers to infectious bronchitis (IB), infectious bursal disease (IBD), and Newcastle disease (ND) viruses from a single serum dilution using the same assay procedures and analyses. A regression line equation generated for predicting NDV ELISA antibody titers at a single serum dilution was successfully used to predict ELISA antibody titers to IB and IBD antigen preparations at the same test sera dilutions. Duplicate samples of 46 field and control sera were transferred simultaneously, with the aid of a replica plating device, from a reservoir plate to three different test plates, which were coated separately with IB, IBD, and ND antigens. After similar indirect ELISA parameters were conducted for each antigen, raw absorbance values from duplicate tests and controls were transmitted directly from an ELISA reader to a microcomputer, which subsequently processed average corrected absorbance values directly into individual ELISA antibody titer for each test antigen. The computer output listed individual field and control sample titers to each antigen, as well as graphing the relative ELISA titer distribution of the entire test group to each antigen. In a comparative study, test sera were evaluated for ELISA antibody titer to NDV, IBV, and IBDV by this method in three separate assays. Consistent titer values were obtained in all assays, as 96% of the 414 replicate titers evaluated varied less than twofold.

Banked serum samples collected from an industry-wide serologic survey of eighteen Delmarva broiler flocks conducted in 1985 were evaluated for the presence of Cryptosporidium- specific antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Evaluation of sera collected at 49-days posthatch showed that 38% of the broiler flocks were serologically positive, whereas 50% of samples obtained from flocks at 63-days during an extended growout were positive. From a performance standpoint, the top nine flocks in the study were essentially negative at the 49-day sampling. In order to more firmly establish the serologic prevalence of Cryptosporidium in Delmarva broiler flocks, a second serologic survey was conducted early in 1987. In this study, an estimated 22% of the 454 broiler flocks sampled at approximately 49 days of age were positive for Cryptosporidium. The negative Cryptosporidium serology in the top 25% of these flocks was associated with better performance, but positive Cryptosporidium serology was not clearly correlated with poor performance. Remarkable differences between Cryptosporidium serologic exposure rates of the growout companies were observed, with some companies having exposure rates as high as 40%, and others rates of less than 3%.

Cross-protection tests with homologous and heterologous serotypes of infectious bronchitis virus (IBV) were used to compare ciliary activity and virus recovery from tracheas of chickens. Validation of this technique included correlating the neutralization indices of antiserum obtained from some infected birds. Chickens were inoculated intratracheally with either the JMK or Connecticut (Conn) serotype of IBV. Three weeks later, infected and uninfected groups were challenged by the same route with homologous and heterologous virus. The JMK strain provided immunity against homologous challenge and the Conn strain, as indicated by good ciliary activity and lack of challenge virus recovery. The Conn strain provided only homologous protection, as ciliostasis occurred and virus was recovered after challenge with the JMK strain. In each case, antiserum to immunizing virus neutralized only the homologous virus. Controls were uniformly susceptible and lacked neutralizing antibody. A similar experiment with the Ark 99 serotype and a recent isolate (397) of IBV revealed complete cross-protection of the tracheas. Antiserum to each virus neutralized the homologous and heterologous virus in each case in reciprocal tests. The results indicate that these two viruses are closely related. The complete agreement between ciliary activity and virus isolation indicates that ciliary activity is a reliable, objective criterion upon which tracheal immunity can be judged in cross-protection tests.

Two neutralizing monoclonal antibodies (MCAs), R63 and B69, were used in antigen-capture enzyme immunoassays to verify the presence of infectious bursal disease virus (IBDV) in infected bursal tissues. The intra-serotype-common neutralization site defined by the R63 MCA was present on all IBDV isolates and laboratory strains tested. However, the neutralization site defined by the B69 MCA was found on only classic or older IBDV strains; it was not found on recently isolated variants of IBDV or on a majority of recent field viruses examined. The data suggest that a major antigenic shift in IBDV has occurred in the field and that this shift involves, at a minimum, the deletion or alteration of one of two neutralization sites previously found on classic IBDV strains.

Two methods for collecting specimens for measuring sequential antibody activity of infectious bronchitis virus (IBV) were compared. Whole blood was collected on filter-paper strips, dried for 2 hr at 37 C, and then stored in plastic bags at 4 C or eluted overnight and tested immediately. Eluates of whole blood were paired with serum samples and tested for IBV antibody activity by enzyme-linked immunosorbent assay (ELISA) at four weekly intervals. Both sampling methods yielded ELISA antibody levels that were detectable at 7 days postinfection (PI), peaked at 21 days PI, and then began to decline by 28 days PI. The paired samples showed no significant difference (P less than 0.05) between ELISA titers at any time tested. Whole blood dried on filter paper could be stored sealed in plastic bags at 4 C for at least 2 weeks with no appreciable loss of antibody titers. Virus-neutralizing antibodies, measured in serum only, were not detectable until 14 days PI but then continued to rise through 28 days PI. It was concluded that eluates of whole blood dried on filter paper may be used as an alternative to sera in ELISA for measuring IBV antibodies.

Breeder and broiler flocks were serologically evaluated using a multiple enzyme-linked immunosorbent assay (M-ELISA). The serologic status of two commercial broiler-breeder flocks and their progeny was monitored, and 840 sera were promptly assessed for antibodies against six infectious agents using the M-ELISA. Breeder flocks were sampled at lay, and broiler chicks were hatched from fertile eggs collected on the scheduled lay date of the breeders. The broiler chicks were placed for growout as eight separate flocks (four from each breeder), and the serologic survey of broilers included sequentially sampling each flock five times between 1 day of age and market. Association of broiler vaccination schedules, mortality, and condemnation data with the temporal serologic data obtained indicated that the earlier appearance of active antibody against infectious bursal disease (IBD) in some unvaccinated flocks was associated with subsequent higher growout mortality and with the poorer overall performance that these flocks experienced. The results of this serologic survey also demonstrated that if a constant, well-timed monitoring program had not been used, major serologic differences between flocks would not have been detected. Serologic profiles of selected broiler flocks by virus-neutralization (VN) tests for infectious bronchitis virus (IBV) and reovirus or by hemagglutination-inhibition (HI) tests for Newcastle disease virus (NDV) compared favorably with the serologic profiles obtained by M-ELISA. Comparison of vaccination histories with serologic results derived from M-ELISA, VN or HI tests indicated that response to vaccination for IBV and NDV at 1 day was either blocked or significantly delayed by moderate levels of maternal antibody and/or were suppressed by an apparent field outbreak of IBD that occurred in all eight broiler flocks.

The enzyme-linked immunosorbent assay (ELISA) and the conventional hemagglutination-inhibition (HI) test were compared for their ability to measure the primary serological response of chickens inoculated by the intranasal-intraocular (IN-IO) routes with Newcastle disease virus (NDV) and the secondary response after intratracheal (IT) challenge. In addition, these responses were compared with the temporal antibody response of chickens inoculated only once by the IT route. Both tests detected NDV-specific antibody by 7 days postinoculation (PI) in the IN-IO-inoculated group, while ELISA and the HI test detected antibody at 4 and 7 days, respectively, in the IT-inoculated group. Titers measured by each test were parallel in quantifying the antibody response, and titers rose anamnestically in response to secondary IT challenge at 21 days PI. ELISA titers remained high at 42 days PI, but the HI titers began to decline at this time. There was a good agreement (R = 0.94) between the results of the two tests throughout both primary and secondary responses. Conversely, there was little agreement between the results of the two tests after 21 days PI in the absence of secondary challenge. Antibody levels were higher when inoculation was by the primary IT route, and they persisted throughout the experiment (86 days). Ciliary activity served as a measure of tracheal immunity or infection of tracheal epithelium. It was reduced as early as 2 days PI and was nearly or completely absent by 5-6 days PI.(ABSTRACT TRUNCATED AT 250 WORDS)

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.

Twelve IBV isolants from diseased chickens in Maine were studied by serum-neutralization tests in chicken tracheal culture. Six isolants were neutralized by Mass-type antisera. The serum-neutralizing characteristics of six other isolants from vaccine breaks differed from any of the standard IBV viruses and appear to comprise three new strains, Maine 209, 212,and 246. The data provide additional evidence that strains of IBV responsible for disease probably vary from region to region and that the strains present must be known for a regional vaccination program to be effective.