Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox.

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.

Hemorrhagic enteritis of turkeys in California: serologic study of hemorrhagic enteritis virus antibody with an enzyme-linked immunosorbent assay.

The incidence of hemorrhagic enteritis (HE) infection in California turkeys was studied by testing 2220 turkey blood samples from 173 flocks for HE virus (HEV) antibody by the enzyme-linked immunosorbent assay (ELISA). Maternal antibody was detected at 1 day of age in all flocks tested, and it vanished after 3 weeks. Acquired HEV antibody appeared at 8 to 10 weeks, and 100% of the meat and breeder turkey flocks were positive after 11 weeks of age. HEV infection occurred earlier in the meat flocks than in the breeder flocks, and it also occurred earlier during summer than during the fall and winter months.

Paramyxovirus type 3 (PMV-3) in California turkeys: serologic study of PMV-3 antibody with an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting paramyxovirus type 3 (PMV-3) antibody. This test had a higher sensitivity than the hemagglutination-inhibition test, and no cross-reactivity with various paramyxoviruses or avian pathogens was detected when the sera were tested diluted 1:200. The incidence of PMV-3 infection in California was studied by testing 2037 turkey sera from 174 meat and breeder flocks for the presence of PMV-3 antibody using the ELISA. The age at which the infection occurs was around 5 to 8 weeks for meat flocks and 10 to 12 weeks for breeder flocks. Infection with the PMV-3, as determined serologically, was more frequent than manifested cases of disease, and 95.2% of the flocks aged over 11 weeks had PMV-3 antibody. No typical manifestations of PMV-3 disease (respiratory disease plus drop in egg production) were observed, probably because of the early infection which occurred before laying age.

Measurement of tetracycline levels in parakeets.

Parakeets were fed hulled millet seed containing 0.5% chlortetracycline (CTC) or minocycline. Blood concentrations of more than 1 micrograms CTC/ml and more than 5 micrograms minocycline/ml were obtained. Equivalent drug assay results were obtained from blood specimens collected by venipuncture or by use of treated filter-paper discs. The latter is a fairly simple method for assaying CTC concentrations in blood of treated psittacines.

Optimizing the enzyme-linked immunosorbent assay for evaluating immunity of chickens to Newcastle disease.

Experiments employing the various steps and reagents used in the enzyme-linked immunosorbent assay (ELISA) were conducted to produce an ELISA with the highest sensitivity and specificity possible for detecting Newcastle disease antibodies in chicken sera. Of the four types of antigen tested, crude antigen gave inconsistent results. However, an alcohol-precipitated antigen prepared from infectious allantoic-amniotic fluids was as satisfactory as more highly purified virus preparations. Other factors found to be extremely important were a 0.5M concentration of NaCl in the diluent and wash solutions used in the procedure, and a pH of 13 for sensitizing solution for maximum specific binding of the antigen to the microplate plastic wells. A comparison was made between the hemagglutination-inhibition (HI) titers of 550 known chicken sera and the corresponding ELISA values. Although the ELISA is much more sensitive than the HI test, there was a general but not a direct correlation between the two tests. The ELISA did not give more information than the HI test concerning protection against an NDV-induced drop in egg production. Preliminary observations indicated that this ELISA procedure is also applicable for turkey sera.

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis.

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.

Application of the egg-bit technique in poultry-disease research.

Experiments were carried out to determine the usefulness of the egg-bit technique (EBT) in poultry virus research with various agents available on the market. The EBT was found satisfactory for propagating 9 strains of avian influenza virus of different antigenic groups, with only minor differences resulting when the procedure used two sources of gelatin, presence or absence of CaCl2, or commercial or specific-pathogen-free embryos.

Host range comparisons of five serotypes of caliciviruses.

Two new serotypes of San Miguel sea lion virus (SMSV), designated SMSV-4 and SMSV-5, were studied in vivo and in vitro. The host cell spectrums were compared with SMSV-1, SMSV-2, and vesicular exanthema of swine virus type A-48. Based on the result of these broad host spectrums, a numerical scoring system was devised for ranking each virus on the basis of its potential for infecting terrestrial mammals, including the important domestic species.

Antibody response and resistance of turkeys to Newcastle disease vaccine strain LaSota.

Studies were undertaken to determine the influence of repeated revaccination on the immune response in immuno-competent turkeys as measured by humoral antibody and resistance to challenge. Protection was better in turkeys given the LaSota spray vaccine at 4 weeks and 30 days later than in turkeys given one vaccination by spray or intramuscular route or exposed 4 times at 10-day intervals by the aerosol route. The anamnestic response, as measured by the HI tests to revaccination with the same immunogen, was not evident by the 3rd day postrevaccination but was observed on the 7th day. The interval between primary and secondary vaccination was found to be important to a true and optimal anamnestic response. Response was greater, however, in vaccinated turkeys exposed to VVND, a more virulent virus antigenically different from the vaccine strain. Exposure to LaSota vaccine by the intramuscular route gave a poorer HI response than LaSota given by aerosol.

In vitro DNA synthesis in lymphocytes from turkeys vaccinated with LaSota, TC, and inactivated Newcastle disease vaccines.

In vitro cultures of peripheral blood lymphocytes from turkeys vaccinated and revaccinated with Newcastle disease (ND) vaccines were stimulated to transformation when exposed to the homologous and heterologous strains of ND virus. The mitogenesis was measured by the uptake of 3H-thymidine into newly synthesized DNA. There was considerable difference in DNA synthesis by lymphocytes drawn 0, 2, 5, and 10 days after vaccination and revaccination with the three vaccines. Stimulation of DNA synthesis, evident as early as the 2nd day, was highest in lymphocytes from turkeys vaccinated or revaccinated with TCND intramuscularly. Stimulation was least in lymphocytes from turkeys vaccinated and revaccinated with LaSota vaccine by aersol. Stimulation was intermediate from an inactivated vaccine given subcutaneously. DNA synthesis was greater with the homologous than with the heterologous strains of NDV. Synthesis was even greater when the same strain was used as a viral suspension in allantoic or cell-culture fluid than the commercial vaccine. The bovine paramyxovirus (PI3) resulted in a minimum DNA synthesis or completely inhibited it. A many-fold (order of magnitude) stimulatory effect was observed when PHA was used as an antigen. The stimulation of DNA synthesis did not parallel the HI antibody response.

Persistence of velogenic viscerotropic Newcastle disease virus in litter.

Litter in a room which had housed chickens and turkeys actively infected with velogenic viscerotropic Newcastle disease was no longer infectious for susceptible chickens placed there 10 to 14 days later.