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Apoptosis is essential in many physiological processes including wound healing and development of the immune response. Apoptosis also plays an important role in the pathogenesis of many infectious diseases including those caused by viruses. Influenza viruses induce apoptosis in cells that are permissive for viral replication and cells that do not support viral replication. The cellular pathways involved in influenza virus induced apoptosis are currently ill defined. Previous studies suggest that influenza virus infection increased the expression of the Fas antigen in HeLa cells, and that Fas antigen is partially involved in apoptosis. In these studies we examined the cellular pathways involved in avian influenza virus induced apoptosis in two cell lines that support productive viral replication: Madin–Darby canine kidney cells (MDCK) and mink lung epithelial (Mv1Lu) cells.

The spike 1 (S1) surface glycoprotein of infectious bronchitis virus (IBV) is the major inducer of the generation of virus neutralizing antibodies, and the administration of purified S1 has been shown to elicit a protective immune response against virulent virus challenge. On the basis of these observations, recombinant fowl poxvirus (rFPV) containing a cDNA copy of the S1 gene of IBV Mass 41 (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 in vito by indirect immunofluorescence staining and western blot analyses. Later, in vivo expression was demonstrated by the detection of IBV-specific serum immunoglobulin G and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histologic changes of the tracheas in virulent IBV Mass 41-challenged chickens previously receiving rFPV-S1 as compared with parental fowl poxvirus (FPV)-vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent virulent FPV challenge. As to a preferred method of immunization, wing web administration appeared to be superior to the subcutaneous route because a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowl pox and infectious bronchitis.

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.

We investigated the feasibility of using Flinders Technology Associates (FTA) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exdude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.

Boyden-type chemotactic chambers were used to demonstrate that Mycoplasma gallisepticum (MG) was capable of migrating into chemotactic membranes. Scanning electron microscopy was used to confirm that MG could penetrate the membranes. To further demonstrate the invasive ability of MG, MG was deposited on the shell membranes of 9-day-old chicken embryos, and after 6 days of incubation, the presence of MG DNA in the allantoic fluids was detected by polymerase chain reactions. These results indicate that MG can penetrate cellular membrane, possibly by going through the porous cellular surface.

Nucleic acid sequence-based amplification (NASBA) allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique was developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The described NASBA technique is a specific, rapid, and sensitive method of detection of influenza A subtype H5 viruses. More importantly, it can be used to distinguish high- and low-pathogenicity strains of the H5 subtype.

A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.

Dot-immunoblotting assay (DIA) using five monoclonal antibodies (MAbs) to infectious bronchitis virus (IBV) was used to detect and classify the viruses propagated in embryonated chicken eggs. Using a group-specific MAb 3F5, 10 reference strains and 12 Korean isolates of IBV were successfully detected by DIA, and the lowest virus titer of IBV detected by DIA was approximately less than 10(3.8) mean embryo infective dose/ml. For evaluating the diagnostic efficiency, DIA was compared with the conventional infectious bronchitis (IB) diagnostic method. IBV antigens in allantoic fluid from embryonated eggs inoculated with IB-suspected field samples were specifically detected by DIA within only one or two egg passages, whereas the conventional embryonated egg inoculation method required four to seven egg passages for confirming IBV infection. These results indicated that DIA could significantly reduce time and cost for IB diagnosis. For examining the possibility of classifying IBV by DIA, four strain-specific MAbs, 3A4, 2A3, 6F7, and 2C6, were used. According to the MAb reacting patterns to the IBV antigens, the 10 IBV reference strains were classified into six groups; seven strains belonged to three different groups, and the other three strains each belonged to an individual group. In the case of 12 Korean isolates of IBV, they were classified in six groups. Among the six groups, the MAb reacting patterns of three groups matched those of the IBV reference strains, but the others did not. These data suggest that at least three variant serotypes of IBV exist in Korea.

The finding of closely associated squamous cell carcinoma (SCC)-like lesions and pox lesions in chorioallantoic membranes (CAMs) inoculated with skin and palate samples taken from broilers is described. The samples were obtained from two broilers coming from different flocks that were not vaccinated against fowl pox. Both birds presented skin lesions, which were diagnosed in one bird as fowl pox, and in the other as SCC. After inoculation of CAMs with fresh tissues from both birds, histologic examination revealed, in all CAMs, lesions that were characteristic of fowl pox together with lesions consistent with those seen in the skin of broilers affected with SCC. This finding was unexpected and may shed some light on the etiology of SCC.

Dual infection with fowl pox (FP) and infectious laryngotracheitis (ILT) was diagnosed as the cause of acute mortality in a flock of three age groups of Hy-Line leghorn layers. The affected chickens had not been previously vaccinated against either FP or ILT. The diagnosis was confirmed by virus isolation, histopathology, and the use of specific pox and ILT genomic DNA probes in a dot-blot hybridization assay. FP and ILT vaccinations were recommended to control mortality. The use of FP- and ILT-specific DNA dot-blot hybridization may be used as a routine diagnostic tool to differentiate between the two diseases, especially in atypical cases of either infection or to confirm the existence of the two diseases as a mixed infection in a flock of chickens.

A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embryonating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses.

A monoclonal antibody (MAb)-based indirect immunoperoxidase (IP) procedure was applied to detect infectious bronchitis virus (IBV) antigen in frozen tissue sections. With this procedure, IBV antigen could be detected in chorioallantoic membranes (CAMs) within 15 hr postinoculation and in the respiratory tissues of chickens within 96 hr postinoculation. Endogenous peroxidases in chicken tissues were removed by treatment with periodic acid. The IP technique was highly sensitive, convenient, and economical. It allowed simultaneous evaluation of antigen-bearing cells and the general tissue morphology. The IP-stained slides also provided a permanent record.

This study was undertaken to demonstrate penetration of Salmonella typhimurium through the eggshell of newly laid broiler hatching eggs. Eggs were challenged either by lightly spraying the bacteria over the blunt end of the egg or by contact with contaminated dry nest litter. Exposure time for both groups was 10 minutes; afterward, all eggs were disinfected and incubated 19 days under normal conditions. Chorioallantoic membranes and yolk sacs were cultured in brain-heart infusion broth on day 19 to demonstrate penetration. Isolation of the bacteria from chorioallantoic membranes and yolk sacs, respectively, were as follows: sprayed group: 100% and 83%; contact group: 59% and 29%. These results showed that although water enhanced S. typhimurium penetration, its presence on the eggshell is not essential for penetration to occur.

A rapid-plate hemagglutination (HA) test to detect infectious bronchitis virus (IBV) in allantoic fluid of embryonated eggs was introduced into routine procedures for IBV identification. This system was tested in 468 diagnostic cases received by the Poultry Diagnostic and Research Center at the University of Georgia. Allantoic fluids from inoculated embryos were harvested and treated with commercially available neuraminidase enzyme. IBV in neuraminidase-treated allantoic fluid was identified by clear and consistent HA of chicken red blood cells within 1 min of incubation. The specificity of the neuraminidase rapid-plate HA assay was examined with other avian viruses in individual and dual embryonic infections. Sensitivity of this test was compared with embryo lesions and reverse transcriptase-polymerase chain reaction (RT-PCR). The rapid-plate HA assay of neuraminidase-treated allantoic fluid correlated with the RT-PCR during the early stages of IBV detection, identification, and isolation in embryonated eggs.

The effectiveness of egg dipping in a 6% hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes was evaluated. The first step was to assess the water uptake from broiler hatching eggs dipped in tap water once or twice using a positive-pressure differential method in order to determine the best method to use in the disinfection trials. Double dipping increased water uptake by 86% more than single dipping. Dipping S. typhimurium-contaminated eggs twice in a 6% hydrogen peroxide solution reduced the average number of organisms in eggshell membranes by 95% and the number of S. typhimurium-positive eggs by 55% compared with the infected untreated group. Dipping the eggs in a 6% hydrogen peroxide solution did not adversely affect hatchability.

The feasibility of using fragments of chorioallantois attached to egg shell (C-S) as an alternative to embryonated eggs for isolating Newcastle disease virus (NDV) was assessed. In paired titrations of 6 strains of virus, embryonated eggs detected an average of 6-to-7-fold greater amounts of virus than did C-S fragments. The efficiency of isolation of viscerotropic velogenic NDV from 240 swab samples was 97% in embryonated eggs and 84% in C-S fragments. Sensitivity differences between the 2 host systems were revealed only in tests of samples expected to contain small amounts of virus. These results suggest that C-S fragments can be reliably used in some but not all instances, thereby relieving logistic constraints sometimes associated with the use of embryonated eggs.

The use of infected allantoic fluid (AF) as precipitating infectious bronchitis (IB) antigen was investigated. The results show that unconcentrated AF harvested at the right time can be a very satisfactory precipitating IB antigen. With the majority of the virus strains used, unconcentrated AF, harvested 68 hours postinoculation (PI), showed more precipitating activity than that harvested at 20 or 120 hours PI. If further antigen concentration is required, dialysis with polyethyleneglycol MW 20,000, freeze-drying, and precipitation with polyethyleneglycol 6,000 are satisfactory methods of concentration.

Forty-two clone-purified, cell-culture-propagated type I avian adenoviruses (AAV) representing 11 serotypes and two intermediate strains were evaluated for virus replication (evidenced by embryo death and lesions) resulting from the inoculation of specific-pathogen-free chicken embryos via the chorioallantoic sac or yolk sac. Commonly observed embryonic changes were death, stunting and curling, hepatitis, splenomegaly, congestion and hemorrhage of body parts, and urate formation in the kidneys. Basophilic or eosinophilic intranuclear inclusion bodies characteristic of fowl adenoviruses were observed in hepatocytes. The magnitude and relative uniformity of intra- and interserotypic embryo mortality, gross lesions, and virus titers was greater in embryos inoculated via the yolk sac. This work identifies the yolk sac as a practical and sensitive chicken embryo inoculation route for poultry diagnosticians to employ. It is suggested that the yolk sac may be a reliable alternative to cell culture for the successful isolation of all type I avian adenoviruses.

A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17). Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF. Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation. AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains. The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material. Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g. Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer. Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers. The HA antigen preparations were stored and were stable at 4 C. Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon. Addition of merthiolate as a preservative had no deleterious effect on HA activity. Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment. Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory. An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried. Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production. Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens.