Charles W. Beard

Protection of Chickens Against Highly Pathogenic Avian Influenza Virus (H5N2) by Recombinant Fowlpox Viruses

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.

Avian influenza antibody detection by immunodiffusion.

Convalescent levels of influenza antibodies were demonstrated by an agar gel precipitin (AGP) test in turkey sera after field outbreaks. The AGP test was positive with chicken sera for at least 8 weeks after laboratory exposure to any of 3 influenza strains, and positive with turkey sera for at least 12 weeks after exposure. The AGP test facilitates a diagnosis of avian influenza using a suspect virus or paired sera without the influences of strain-to-strain antigenic differences, which limit the usefulness of the hemagglutination-inhibition test.

The Influence of Test Conditions on Newcastle Disease Hemagglutination-Inhibition Titers

Replicate samples of serum from chickens immune to Newcastle disease were titrated to determine the influence of certain test conditions on hemagglutination-inhibition (hi) titers. The test conditions studied were those most likely to vary in normal laboratory operations. The most marked effect on magnitude of HI titers was incubation time of twofold serum dilutions in antigen-saline; the average titer increase after incubation of the serum-antigen mixture for 1 hr at 37 C was log2 2.3 (fivefold). Twofold increases in virus concentration of the antigen-saline diluent caused an average titer reduction of log2 0.8. Shifts in HI titers were only minor with twofold changes in erythrocyte concentration (log2 0.3), with variations of test reading times from 0.5 to 2.0 hr (log2 0.1), and with variations in the period between preparation of the initial 1:10 serum dilution in antigen-saline and the subsequent serum dilutions (log2 0.3).

Effect of Route of Administration on the Efficacy of a Recombinant Fowlpox Virus Against H5N2 Avian Influenza

A recombinant fowlpox vaccine virus containing the H5 hemagglutinin gene of avian influenza virus was administered to susceptible chickens via wing-web puncture, eye drop, instillation into the nares, and drinking water. Even though there was a negligible hemagglutination-inhibition (HI) serologic response, all 10 chickens vaccinated by wing-web puncture remained without obvious signs of disease and survived challenge with a highly pathogenic strain of H5N2 avian influenza virus. All unvaccinated chickens and those vaccinated by nasal and drinking-water routes died following challenge. Eight of 10 chickens vaccinated with the recombinant by eyedrop died. All vaccinates were negative on the agar gel precipitin (AGP) test, and only one chicken had a positive HI titer before challenge. All chickens that survived challenge had high levels of HI antibody and were positive on the AGP test, indicating that they were infected by the challenge virus.

Viscerotropic Velogenic Newcastle Disease In Pigeons: Clinical Disease and Immunization

SUMMARY Racing homer pigeons were exposed to viscerotropic velogenic Newcastle disease virus (VVNDV) to further characterize the disease in pigeons. Mortality was 7 of 21 juvenile birds and 1 of 10 adult birds. Virus shedding was detected until 21 days postinoculation in some birds. Pigeons infected experimentally shed VVNDV before the onset of clinical disease and were able to transmit VVNDV to chickens and pigeons by contact exposure. Pigeons were also susceptible to infection with VVNDV by contact exposure to an infected chicken. In experiments in which 3 commercial Newcastle disease virus (NDV) vaccines were used to evaluate several vaccination programs, only 1 of the programs completely prevented morbidity and mortality following VVNDV challenge 6 weeks after the final vaccination. Pigeons in that program were vaccinated with viable vaccine and revaccinated at 6 weeks postvaccination with inactivated oil-emulsion vaccine.

Influence of Formulation on the Efficacy of Experimental Oil-Emulsion Newcastle Disease Vaccines

Twenty-one experimental oil-emulsion vaccines with different emulsifier contents, aqueous-to-oil ratios, and antigen concentrations were compared by immunization of 4-week-old chickens. Vaccines that contained oil-phase (Arlacel 80) and aqueous-phase (Tween 80) emulsifiers induced 2-to-4-fold higher hemagglutination-inhibition titers than vaccines with only the oil-phase emulsifier. The emulsion vaccines containing both emulsifiers were also more stable at 37 C and less viscous than those containing only the oil-phase emulsifier. Vaccines that had different aqueous-to-oil ratios and contained different quantities of allantoic-fluid antigen (1.2% to 50% of the vaccine volume) induced similar protection against challenge, but hemagglutination-inhibition titers were proportional to the amount of antigen added. Vaccines that had different aqueous-to-oil ratios but contained equal amounts of antigen induced similar hemagglutination-inhibition titers and similar protection against challenge.

Influence of Dietary Calcium Stress on Lethality of Avian Influenza Viruses for Laying Chickens

The effect of calcium stress was studied in an attempt to reproduce lethal infections in laying chickens with A/Chicken/Alabama/75 (H4N8) influenza virus and with two nonpathogenic H5N2 influenza viruses from the 1983-84 outbreak in the eastern United States. Hens were fed calcium-deficient or standard diets for 7 to 14 days; then the calcium-deficient feed was replaced with standard feed supplemented with ad libitum oyster shell, and both groups of hens were inoculated with virus. When hens were infected with the H4N8 virus, respective mortalities of those on the calcium-deficient and standard diets were 19% (27/141) and 5% (7/143). The H5N2 viruses did not kill hens fed either diet. In standard pathogenicity tests, Alabama H4N8 viruses reisolated from the hens that died generally were more lethal for 4-week-old chickens than the stock virus. These results argue for characterization of the Alabama H4N8 virus as pathogenic rather than nonpathogenic as originally determined.

Evaluation of inactivated Newcastle disease oil-emulsion vaccines.

Three commercial European inactivated oil-emulsion (OE) vaccines and a U.S.-licensed inactivated AI(OH)3 vaccine were evaluated by primary vaccination of susceptible broilers and by secondary vaccination of layers previously immunized with live virus. A commercial live-virus vaccine was also used as a primary and secondary vaccine control. Evaluation was based on the hemagglutinationinhibition (HI) response and protection against challenge with neurotropic or viscerotropic velogenic (VV) Newcastle disease virus (NDV). OE vaccines induced higher and generally more sustained HI antibody titers than AI(OH)3 or live vaccines. With one exception, all challenged OE vaccinates were protected against clinical disease, a drop in egg production, and a decrease in egg quality. Vaccination did not prevent infection with the challenge viruses; however, the infection rates were lowest in LaSota vaccinates and highest in Al(OH)3 vaccinates.

THE EGG-BIT TECHNIQUE FOR MEASURING NEWCASTLE DISEASE VIRUS AND ITS NEUTRALIZING ANTIBODIES

SUMMARY The egg-bit technique was modified and adapted for measuring Newcastle disease virus (NDV) and its antibodies. Infectivity of 3 strains of NDV generally increased with the incubation period. Infectivity titers did not differ greatly with bits from embryos either 10, 11, or 12 days old. Virus-neutralization tests with NDV were accomplished successfully with egg bits. Results from egg bits were compatible with results from conventional techniques. Data are presented demonstrating the role of HI antibodies in the egg-bit neutralization test.

NEWCASTLE DISEASE VIRUS: EVALUATION OF AN AVIRULENT ENTERIC ISOLATE AS A VIABLE VACCINE

SUMMARY An avirulent enteric isolate of Newcastle disease virus from Northern Ireland was evaluated as a viable vaccine. Viable virus was recovered for at least 3 days when it was diluted in water containing powdered skimmed milk and held at 37 C. Chicks were infected by administering the virus in drinking water. Eight experiments with parentally immune and unimmune chicks demonstrated antibody formation in nearly all birds following administration of the virus in drinking water. The chicks received the virus when they were as young as one day old. When their immunity was challenged with the virulent GB strain of NDV by contact exposure, a high percentage of birds previously exposed to the N. Ireland isolate survived. They were, however, infected by the challenge strain, resulting in signs of respiratory disease, virus shedding, and a boost in antibody titers. Several considerations for and against this type of vaccine are discussed.