Henry D. Stone

Evaluation of the Efficacy of Oil-Emulsion Bacterins for Reducing Fecal Shedding of Salmonella enteritidis by Laying Hens

Two replicate experiments were conducted to test the efficacy of two different Salmonella enteritidis oil-emulsion bacterins (an experimentally prepared acetone-killed vaccine and a commercially available vaccine) for protecting laying hens against intestinal colonization following oral exposure to S. enteritidis. Each vaccine was administered twice (4 weeks apart), and all hens were challenged with 10(8) cells of a nalidixic-acid-resistant S. enteritidis strain 2 weeks after the second vaccination. Fecal samples from vaccinated and unvaccinated control hens were cultured at three weekly intervals post-challenge to determine the incidence of intestinal colonization and the numbers of S. enteritidis shed into the environment. Both vaccines significantly reduced the incidence of intestinal colonization (P < 0.05) and the mean number of S. enteritidis cells shed in the feces (P < 0.01) at 1 week post-challenge. However, the degree of protection afforded by vaccination was only partial, as more than half of the vaccinated hens still shed substantial numbers of S. enteritidis. If used in conjunction with other flock sanitation and infection-monitoring strategies, vaccination with bacterins could potentially reduce the overall level of environmental contamination and thereby also reduce the horizontal transmission of S. enteritidis within and between laying flocks.

Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines

The influence of vaccine strain and antigen mass on the ability of inactivated avian influenza (AI) viruses to protect chicks from a lethal, highly pathogenic (HP) AI virus challenge was studied. Groups of 4-week-old chickens were immunized with inactivated vaccines containing one of 10 haemagglutinin subtype H5 AI viruses, one heterologous H7 AI virus or normal allantoic fluid (sham), and challenged 3 weeks later by intra-nasal inoculation with a HP H5 chicken-origin AI virus. All 10 H5 vaccines provided good protection from clinical signs and death, and produced positive serological reactions on agar gel immunodiffusion and haemagglutination inhibition tests. In experiment 1, challenge virus was recovered from the oropharynx of 80% of chickens in the H5 vaccine group. In five H5 vaccine groups, challenge virus was not recovered from the cloaca of chickens. In the other five H5 vaccine groups, the number of chickens with detection of challenge virus from the cloaca was lower than in the sham group (P < 0.05). Reductions in the quantity of challenge virus shed from the cloaca and oropharynx were also evident in some H5 vaccinate groups when compared to the sham group. However, there was no positive correlation between the sequence identity of the haemagglutinin gene from the vaccine strain and challenge virus, and the ability to reduce the quantity of challenge virus shed from the cloaca or oropharynx. As the quantity of AI antigen in the vaccines increased, all parameters of protection improved and were virus strain dependent. A/turkey/Wisconsin/68 (H5N9) was the best vaccine candidate of the H5 strains tested (PD50= 0.006 μg AI antigen). These data demonstrate that chickens vaccinated with inactivated H5 whole virus AI vaccines were protected from clinical signs and death, but usage of vaccine generally did not prevent infection by the challenge virus, as indicated by recovery of virus from the oropharynx. Vaccine use reduced cloacal detection rates, and quantity of virus shed from the cloaca and oropharynx in some vaccine groups, which would potentially reduce environmental contamination and disease transmission in the field.

Preparation of Inactivated Oil-Emulsion Vaccines with Avian Viral or Mycoplasma Antigens

The influence of the composition of water-in-oil emulsions on their physical characteristics was determined by preparing experimental emulsions with various water-to-oil ratios and various emulsifiers. Emulsions containing Tween 80 in the aqueous phase and Arlacel A or Arlacel 80 in the oil phase were lower in viscosity than emulsions containing only an oil-phase emulsifier. Viscosity decreased as the concentration of oil increased. Oil-emulsion vaccines prepared with aqueous- and oil-phase emulsifiers had low viscosity, were stable for more than 12 weeks at 37 C, and induced a marked primary antibody response in chickens.

Evaluation of the Efficacy of an Oil-Emulsion Bacterin for Protecting Chickens Against Salmonella enteritidis

To assess the potential protective efficacy of a Salmonella enteritidis bacterin, an acetone-killed oil-emulsion vaccine was prepared from a phage type 13a S. enteritidis strain and administered subcutaneously to hens in two experiments. Hens were housed individually, and every other hen was vaccinated (at 23 weeks of age in one experiment and at 45 weeks in the other). A second (booster) bacterin injection was administered 6 weeks later in both experiments. Three weeks after the second vaccination, all hens were challenged with an oral dose of approximately 10(9) cells of a heterologous (phage type 14b) S. enteritidis strain. In both trials, S. enteritidis was isolated from fewer internal organs (spleens, ovaries, and oviducts) and pools of egg contents from vaccinated hens than from unvaccinated control hens. Vaccination did not, however, affect the percentage of hens that shed S. enteritidis in feces in either experiment.

In ovo vaccination of chicken embryos with experimental Newcastle disease and avian influenza oil-emulsion vaccines.

Inactivated oil-emulsion (OE) Newcastle disease (ND) and avian influenza (AI) vaccines were injected into 18-day-old white rock (WR) and white leghorn (WL) chicken embryos to evaluate their immunologic efficacy and their effects on hatchability. Embryonating eggs were inoculated at 1.5 inches depth with various vaccine volumes and antigen concentrations. Serum hemagglutination-inhibition (HI) titers were first detected in chickens at 2 wk posthatch. Protection against morbidity and mortality was demonstrated in all of 10 chickens vaccinated as embryos and challenged with viscerotropic velogenic ND virus at 53 days of age and also in all of eight in ovo- vaccinated chickens challenged with highly pathogenic AI virus at 34 days of age. All of five unvaccinated control chickens for each respective ND- and AI-vaccinated group died. In pooled groups from successive hatches, the hatchability of WR or WL embryos injected with 100 microliters of vaccine was not significantly different (P > 0.05) from unvaccinated hatchmate controls when needle gauges of 22, 20, and 18 were used. Seroconversion rates of chickens vaccinated as embryos ranged from 27% to 100% with ND vaccination and 85% to 100% for AI vaccination. For ND, geometric mean HI titers of chickens per vaccine group ranged from 11 to 733, and in pooled groups, the range was 49 to 531. Titers for AI vaccine groups ranged from 156 to 1178. This study demonstrated that acceptable hatchability, seroconversion rates, and protective immunity can be attained with in ovo inoculation of ND or AI OE vaccines if the vaccines are prepared with sufficient antigen and administered properly.

Simple and rapid methods for detecting Salmonella enteritidis in raw eggs

The Centers for Disease Control and Prevention estimates there were 300,000 cases of Salmonella enteritidis (SE) in 1997. Egg products were associated with many of the cases. To address this problem, many producers implemented flock surveillance of the SE situation at their facilities. A rapid and simple method for detecting SE from poultry samples is critical for the effective implementation of such testing strategies. A lateral flow device for the detection of SE utilized in this study was manufactured by Neogen, Lansing, MI. The test panel is a presumptive qualitative test system that detects only members of Group D1 Salmonella species. A series of studies were conducted to optimize the test procedure for raw eggs with different sample preparations. A novel antigen extraction method was developed for use with the test panel kit. The detection limit of the test panel kit was increased approximately tenfold when the extraction method was used. Detection of SE was 100% in raw egg pools inoculated with 10 SE cells per ml of egg and incubated at a 1:10 ratio in buffered peptone water (BPW) or tetrathionate brilliant green broth (TBG) for 24 h at 37 degrees C. The developed lateral flow test kit could provide a simple, rapid, and inexpensive method for egg producers and processors to test specifically for Salmonella group D1 serovars, such as SE, in egg samples.

Efficacy of Experimental Animal and Vegetable Oil-Emulsion Vaccines for Newcastle Disease and Avian Influenza

Acceptable oil-emulsion vaccines were sought to replace mineral oil-emulsion vaccines that, by regulations, require a 42-day minimum holding period for poultry between injection and slaughter for consumption. Water-in-oil emulsions were prepared using animal or vegetable oils in a ratio of 4 parts oil to 1 part Newcastle disease or avian influenza aqueous antigen. Beeswax particles suspended in the oil at the 5% or 10% level (wt:vol) served as the oil-phase surfactant. Hemagglutination-inhibition titers induced by mineral-oil vaccines were not significantly different from those induced by the most efficacious formulations prepared from animal and vegetable oils. Tissue reaction from injection of animal- and vegetable-oil vaccines was less than that induced by mineral-oil vaccines. An inactivated avian influenza vaccine formulated from peanut oil induced protection against morbidity and death when vaccinated chickens were challenged with a virulent isolate of avian influenza virus.

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.

Mucosal Humoral Immunity to Experimental Salmonella enteritidis Infection in the Chicken Crop

SUMMARY. In this report, we show that chickens, infected with Salmonella enteritidis (SE) by oral gavage, produce secretory immunoglobulin As (sIgAs) that specifically bind to numerous SE antigens. Chickens infected with SE showed strong sIgA response against flagella in both bile and crop. The optical density values of enzyme-linked immunosorbent assay (ELISA) tests in positive bile and crop were 1.17 and 0.38, respectively, and were significantly different from those of negative samples. Western immunoblotting revealed that ∼13.5-, ∼56-, ∼62-, ∼80-, and ∼143-kD polypeptides were immunodominant proteins in bile, whereas ∼56-, ∼62-, and ∼80-kD polypeptides were found to be strong antigens in crop. These results indicate that the crop may function as another site for mucosal immunity, and the SE flagella-based ELISA of crop samples can provide a useful screening test of SE exposure in chickens.

Molecular characterization of the hemagglutinin gene and oral immunization with a waterfowl-origin avian influenza virus.

Vaccination against highly pathogenic (HP) subtypes of avian influenza (AI) virus in poultry has been prohibited in the United States. Recently, policy has been changed to potentially allow use of inactivated vaccines in emergency programs to control HP H5 and H7 AI. Vaccination with inactivated virus against non-highly pathogenic AI viruses has been allowed in the U.S. turkey industry since 1979 (1) but requires expensive handling of individual birds for parenteral inoculation. Oral immunization would provide a less expensive method to protect commercial poultry from AI. Prime candidates for oral vaccines are waterfowl-origin (WFO) isolates, which have a tropism for the alimentary tract. One WFO isolate, A/mallard/Ohio/556/1987 (H5N9) (MOh87), was characterized by determining the complete nucleotide sequence of its hemagglutinin (HA) gene. The HA protein of this isolate possessed a deduced amino acid sequence nearly identical to the consensus amino acid sequence for all published H5 genes, indicating that it has potential as a broadly effective vaccine. Experimental results demonstrated measurable serum antibody responses to orally delivered live and inactivated preparations of MOh87. Oral vaccination also protected chickens from diverse, lethal H5 AI virus challenge strains and blocked cloacal shedding of challenge virus.