Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza.

In 1997, highly pathogenic (HP) H5N1 avian influenza virus (AIV) caused infections in poultry in Hong Kong and crossed into humans, resulting in a limited number of infections including 18 hospitalized cases and six associated deaths. The unique ability of this, AIV to infect both poultry and people raised a concern for the potential of humans to be biological as well as mechanical vectors of this AIV to poultry. The current study was undertaken to determine if existing vaccines and their technologies could be used during an outbreak to protect poultry. Commercial and experimental inactivated whole H5 AIV and baculovirus-expressed AIV H5 hemagglurinin protein vaccines provided protection from clinical signs and death in chickens after lethal challenge by human-origin HP H5N1 Hong Kong strains 156/97 and 483/97. The commercial and experimental inactivated vaccines had mean protective doses ranging from 0.25 to 0.89, which represents the milligrams of viral protein in the vaccines that provided protection from death in half of the birds. Furthermore, the vaccines reduced the ability of the challenge AIV to replicate in chickens and decreased the recovery of challenge AIV from the enteric and respiratory tracts, but the use of a vaccine will nor totally prevent AI virus replication and shedding. Existing vaccines will protect poultry from mortality and reduce virus replication from the new HP AIV strain that can infect both poultry and humans.

Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus.

In 1997, a devastating outbreak of foot-and-mouth disease (FMD) in Taiwan was caused by a serotype O virus (referred to here as OTai) with atypical virulence. It produced high morbidity and mortality in swine but did not affect cattle. We have defined the genetic basis of the species specificity of OTai by evaluating the properties of genetically engineered chimeric viruses created from OTai and a bovine-virulent FMD virus. These studies have shown that an altered nonstructural protein, 3A, is a primary determinant of restricted growth on bovine cells in vitro and significantly contributes to bovine attenuation of OTai in vivo.

Effective poultry programming in the next century. Poultry research: basic versus applied.

The debate over the relative merit of basic vs applied research has been going on for as long as there has been research. This debate could be resolved in many instances by better communication so that those who question the need for basic studies can see how basic findings can contribute directly and indirectly to the resolution of real problems. Applied research directed at problem-solving is usually dependent upon those facts that have been revealed by basic researchers. This is a debate that should be set aside. The poultry industry must have contributions from both the basic and applied researchers to remain competitive and produce those products society depends upon.

Out on the farm with DNA vaccines.

DNA vaccination is a rapidly developing technology that offers new approaches for the prevention of disease. This technology may permit the production of new vaccines against diseases that have no current vaccine, as well as allowing the development of improved vaccines to replace existing products. We describe how DNA vaccination is being developed for use in commercial animal production, with an emphasis on viral diseases, and discuss the existing hurdles to its development and use.

Interaction of mouse adenovirus type 1 early region 1A protein with cellular proteins pRb and p107.

We demonstrated functional associations between mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein and both the mouse retinoblastoma protein (pRb) and the mouse pRb-related protein, p107. Interactions between MAV-1 E1A and mouse pRb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant MAV-1 viruses. Using a polyclonal antibody to MAV-1 E1A, exogenously added mouse pRb or mouse p107 was coimmunoprecipitated from wild-type, dIE105 (CR1 delta)-, and dIE106 (CR3 delta)-infected cell lysates. No coimmunoprecipitation was seen with cell lysates from dIE102 (CR2 delta) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutation in the initiator methionine. Introduction of mouse pRb into SAOS-2 cells resulted in a flat and enlarged cell phenotype, whereas cotransfection of mouse pRb and MAV-1 E1A resulted in a significant reduction of flat cells, presumably due to E1A binding pRb. CR1 delta and CR2 delta E1A proteins were less effective at reducing the number of flat, enlarged cells induced by pRb expression than were the CR3 delta or wild-type E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bind pRb in the in vitro coimmunoprecipitation experiments. As a measure of p107/MAV-1 E1A complex formation in MAV-1-infected cells, we used mobility shift assays to examine cell extracts for the presence of p107-containing E2F protein-DNA complexes. Mock-, dIE102-, and pmE109-infected cell extracts formed a p107-containing complex, whereas wild-type-infected cell extracts did not. Thus the formation of a p107-E2F complex in wild-type- or these mutant-infected extracts inversely correlated with the presence of E1A-p107 complexes identified in the vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 complexes forming in wild-type MAV-1-infected cells.

Analysis of early region 3 mutants of mouse adenovirus type 1.

Early region 3 (E3) of mouse adenovirus type 1 has the potential to produce three proteins which have identical amino termini but unique carboxy-terminal sequences. Three recombinant deletion viruses were constructed so that each could produce only one of the three E3 proteins. A fourth mutant that should produce no E3 proteins was also constructed. These recombinants were able to grow in mouse 3T6 cells and produced wild-type levels of viral mRNAs and proteins except for those specifically deleted by the mutations. Early mRNA production from the mutant viruses was analyzed by reverse transcriptase PCR and confirmed that each deletion mutant would be able to produce only one of the three E3 proteins. Late mRNA production was analyzed by Northern (RNA) blotting and found to be similar in wild-type and mutant viruses. Capsid morphology was unaltered in the mutant viruses as seen by electron microscopy. Immunoprecipitation of E3 proteins from infections of mouse 3T6 cells using an antiserum specific for all three E3 proteins was used to examine the effect of the introduced mutations on protein expression. Two mutants produced only one class of E3 protein as predicted from their specific mutations and mRNA expression profiles. One mutant virus failed to produce any detectable E3 proteins. The predicted E3-null mutant was found to be leaky and could produce low levels of E3 proteins. Outbred Swiss mice were infected with the E3 mutant viruses to determine if the E3 proteins have an effect on the pathogenicity of the virus in mice. All of the mutants showed decreased pathogenicity as determined by increased 50% lethal doses, indicating that the proteins of the E3 region are important determinants of the pathogenesis of mouse adenovirus in its natural host.

Characterization of an 11K protein produced by early region 3 of mouse adenovirus type 1.

Early region 3 (E3) of mouse adenovirus type 1 produces three mRNAs that can encode three proteins with unique carboxy-terminal exons. A bacterial fusion protein encoding the unique terminus of one the three predicted proteins was used to generate antiserum in rabbits. This antiserum detected a 14K protein on a Western blot of infected cell lysates. Immunoprecipitation and endoglycosidase H digestion revealed that the 14K protein was a glycoprotein with a core molecular weight of 11K, and we are designating this protein E3 gp 11K. Through in vitro translation experiments we determined that the previously predicted signal sequence of gp11K was cleaved when the protein was expressed during an infection. Biochemical and immunofluorescence microscopy data indicated that E3 gp11K was localized to the endoplasmic reticulum of infected cells. Biochemical experiments further indicated that gp11K is a peripheral membrane protein.

Research to understand and control Salmonella enteritidis in chickens and eggs.

When it became evident that the association of human Salmonella enteritidis (SE) outbreaks with the consumption of contaminated Grade A eggs posed a threat to public health and to the economic viability of the egg industry, research programs were rapidly initiated to investigate the many unanswered questions about SE in eggs and chickens. Research efforts have focused on the dynamics of deposition, survival, and growth of SE in eggs, the pathogenesis of SE in chickens, strategies for detecting SE-infected flocks, opportunities for intervening to prevent infection, the sources of SE in laying flocks, options for effectively cleaning poultry houses, and the epidemiology of SE infections of humans and chickens. This research has provided a substantially better understanding of the SE problem in poultry, but many further questions about the basis for and the prevention of eggborne transmission of SE remain to be answered.

Comparative efficacy of the B-1 and VG/GA vaccine strains against velogenic viscerotropic Newcastle disease virus in chickens.

Groups of eight 1-day-old white rock chickens were vaccinated with either B-1 or VG/GA strain of Newcastle disease virus (NDV) by eyedrop instillation. Some of the chickens were vaccinated a second time at 17 days of age. Eight groups of chickens vaccinated either once or twice were challenged with the California 1083 strain of velogenic viscerotropic Newcastle disease virus (VVNDV) at 30 days of age by either intramuscular injection or eyedrop instillation. One group of unvaccinated control chickens was challenged by eyedrop instillation. All eight unvaccinated controls, two of the 16 B-1 vaccinates, and none of the 16 VG/GA vaccinates died following challenge. There were no obvious differences in pre-challenge serum antibody levels among the vaccinates. Only the twice-vaccinated chickens that were challenged by eyedrop and the unchallenged vaccinates failed to show a marked rise in serum antibody titers. The VG/GA strain of NDV provided protection against the mortality associated with VVNDV challenge similar to that provided by the B-1 strain within the conditions of this experiment.

A feather-trap system for the removal of chicken feathers from laboratory sewage.

A simple feather-trap system is described for use on the drain lines of buildings housing poultry for research or other purposes where floors are frequently washed. The trap uses disposable plastic-mesh bags that can efficiently remove almost all feathers from the water, preventing sewer lines from being blocked by compacted feathers. Critical measurements and operational procedures are described.

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.

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.

Evaluation of a chick mortality model for predicting the consequences of Salmonella enteritidis infections in laying hens.

The effects of four domestic Salmonella enteritidis (SE) isolates were compared in experimentally infected chicks and laying hens. The pathogenicity of each strain for 1-day-old chicks was determined by recording postinoculation mortality. The effects of the SE strains on adult hens were measured in terms of changes in total egg production, the frequency of production of SE-contaminated eggs, the dissemination of SE to internal organs, and the elicitation of a specific antibody response. Significant differences in the consequences of infection with different SE strains were observed in mortality rates among chicks and in total egg production, the frequency of production of contaminated eggs, and the serum antibody response among laying hens. The usefulness of a chick mortality model for predicting the probable frequency of production of contaminated eggs by laying hens infected with particular SE strains was then further evaluated by infecting chicks and laying hens with four other field isolates of SE. Although significant differences between SE strains were observed in both chick mortality and the frequency of production of contaminated eggs by hens, a strong correlation between these two parameters was not evident.

Detection and Enumeration of Salmonella enteritidis in Fresh and Stored Eggs Laid by Experimentally Infected Hens.

Laying hens were orally inoculated with a phage type 13a strain of Salmonella enteritidis (SE). Eggs laid by the infected hens were collected daily between the 4th and 14th d postinoculation and randomly allocated into three groups. One group of eggs was sampled on the day of collection, one group was held for 7 d at 7.2°C before sampling, and one group was held for 7 d at 25°C before sampling. The frequency and level of detectable contamination of egg contents by SE were determined for each group. Only 3% of the freshly laid eggs and 4% of the eggs held for 7 d at refrigerator temperature were identified as having SE-contaminated contents, whereas SE was isolated from the contents of 16% of eggs held for 7 d at room temperature. Enumeration of SE in contaminated eggs indicated greater numbers of SE in eggs held for 7 d at 25°C than in eggs from the other two groups, although most contaminated eggs in all three groups contained relatively small numbers of SE (generally less than 10/ml and rarely exceeding 100/ml).

Detection of Salmonella serogroup D-specific antibodies in the yolks of eggs laid by hens infected with Salmonella enteritidis.

Eggs laid by hens experimentally infected with Salmonella enteritidis were assayed for the presence of Serogroup D-specific yolk antibodies. Yolk antibodies were detected with S. enteritidis and Salmonella pullorum antigens in the microantiglobulin test as early as 9 days after inoculation of hens with S. enteritidis. Yolk antibody titers reached peak levels at 3 to 5 wk postinoculation and remained at detectable levels for at least 7 wk postinoculation in eggs from both orally inoculated and horizontally contact-exposed hens. Eggs laid by hens from commercial flocks implicated in epidemiological investigations of human S. enteritidis outbreaks were also tested. Serogroup D-specific yolk antibodies were detected in 5 to 22% of eggs from hens in houses identified as infected by bacteriological culturing of internal organs of hens for S. enteritidis.

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.

Amantadine resistance among hemagglutinin subtype 5 strains of avian influenza virus.

Several avian influenza virus strains of hemagglutinin subtype 5 were assayed for sensitivity to the antiviral drug amantadine. Most strains exhibited little sensitivity to the drug as measured by plaque reduction. The A/Chicken/Scotland/59 (CS59), however, was highly sensitive, making it easily distinguishable from the other H5 strains. Drug sensitivity of the viruses was also assayed in chicken embryos. The in ovo patterns of amantadine sensitivity differed from those detected in cell culture. The CS59 isolate could not be distinguished from all the other strains on the basis of its response to amantadine in ovo. Although amantadine protected chickens inoculated with CS59 from morbidity and mortality, drug-resistant viruses were readily isolated from the infected birds. As found with other amantadine-resistant variants, the structure of the matrix gene was altered in the resistant isolates. These results demonstrate that amantadine resistance is widespread among avian influenza viruses of the H5 subtype, that drug sensitivity in cell culture does not necessarily reflect responses to amantadine in ovo and in vivo, and, as previously found, amantadine-resistant derivatives of H5 strains may be isolated from birds protected by the drug.

Early region 4 sequence and biological comparison of two isolates of mouse adenovirus type 1.

The DNA sequence of 88-100 map units of mouse adenovirus type 1 (MAV-1) was determined. One translational open reading frame showed 48% sequence similarity to a human adenovirus type 2 early region 4 protein. Based on the protein similarity, genome location, and transcriptional polarity, we concluded that this region of MAV-1 corresponds to early region 4. A 241-bp sequence consisting of 10 imperfect direct repeats with sequence similarity to minisatellite DNA was found in this region. Two virus isolates with different passage histories were examined and were found to have a sequence polymorphism within this region. The two viruses were compared for growth in cell culture and mice and small quantitative differences were observed only in vivo.

Isolation of Salmonella enteritidis from internal organs of experimentally infected hens.

Tissues from experimentally infected hens were examined for the presence of Salmonella enteritidis (SE). SE was recovered from internal organs of both orally inoculated hens and hens infected by horizontal contact transmission. SE was isolated from 58% of the ceca, 51% of the livers, 47% of the spleens, 17% of the ovaries, and 17% of the oviducts of hens sampled during the first 5 weeks after exposure. SE was recovered at a low frequency from all internal organs sampled for as long as 22 weeks after exposure.

Serological detection of experimental Salmonella enteritidis infections in laying hens.

The antibody response of laying hens to experimental Salmonella enteritidis infection was evaluated in microagglutination, tube agglutination, and rapid whole-blood plate agglutination assays. Hens of three different ages were infected by either oral inoculation or horizontal contact transmission. Blood was collected at weekly intervals, and the presence of specific antibodies was assessed by reaction with antigens prepared from strains of S. enteritidis and S. pullorum. The sensitivity of detection of infected hens did not vary significantly between the assays, as all three tests effectively identified most exposed hens as seropositive. Within each test, however, variation was observed in the detection sensitivity when different antigens were used. The microagglutination titers of serum samples were determined by serial dilution. Antibody titers peaked at 1 to 2 weeks postinoculation and declined steadily, although most birds were still identified as seropositive at 10 weeks postinoculation. The mean microtest titers obtained with S. enteritidis antigens were higher than with an S. pullorum antigen, indicating greater test sensitivity. However, use of the S. pullorum antigen resulted in fewer false positives when sera from uninfected control hens were tested. The titers of contact-exposed hens peaked later and at lower values than did those of inoculated hens, but these two groups of hens had similar antibody titers after the third week postinoculation.