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The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.

Outbreaks of poult enteritis mortality syndrome (PEMS) continue to cause financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, flock unevenness, and immunosuppression. PEMS is a very difficult disease to control and prevent. Depopulation of PEMS-affected flocks and thorough cleaning of the contaminated housing have failed to prevent infection (disease) in subsequent flock placements. The relationship of PEMS to other enteric disease complexes of young turkeys is unknown, partly because the causative agent of PEMS remains unknown. Recently, we isolated a unique astrovirus strain from the thymus and intestines of PEMS-infected poults. This strain is molecularly and serologically distinct from the astrovirus that circulated in turkeys in the 1980s. Mammalian astroviruses are very resistant to inactivation. In these studies, we examined the stability of partially purified PEMS-associated astrovirus to inactivation with heat, laboratory disinfectants, and commercial disinfectants used in commercial turkey houses in an embryonated egg model system. Similar to mammalian astroviruses, the PEMS-associated astrovirus is resistant to inactivation by heat, acidification, detergent treatment, and treatment with phenolic, quaternary ammonium, or benzalkonium chloride-based products. Only treatment with formaldehyde, beta-propriolactone, or the peroxymonosulfate-based product Virkon S completely inactivated the astrovirus in the embryo model. These studies provide an alternate means to potentially control at least one virus associated with PEMS through the use of specific disinfectants.

Formalin-fixed, paraffin-embedded spleen and intestinal tissues were harvested at 2 days postinfection from 4-wk-old white rock chickens infected with five different strains of Newcastle disease virus (NDV). These tissues were examined for the presence of macrophage antigen expression, virus replication, and interferon gamma (IFN gamma) production. The five strains represented all three NDV pathotypes. Viral replication and IFN gamma, as determined by riboprobe in situ hybridization, were detected only in those chickens infected with velogenic viscerotropic NDV (VVNDV) strains. Macrophage antigen expression, an indicator of macrophage activation, was determined by immunohistochemistry with a macrophage-specific antibody, CVI-ChNL-68.1. Presence of macrophage antigen was most prominent in VVNDV-infected chickens. The distribution of this antigen within tissues was far more diffuse than the staining for viral mRNA. The presence of IFN gamma mRNA was detected in the spleen and intestinal lymphoid tissue of VVNDV-infected chickens. There was also increased macrophage antigen expression in the mesogen-infected birds, but it was less dramatic than in tissues from VVNDV-infected chickens. One of two lentogen-infected birds had evidence of increased macrophage antigen expression only in the spleen.

Fifty-seven Newcastle disease virus (NDV) isolates from chickens, turkeys, a rhea, a parrot, and an anhinga were pathotyped and characterized by monoclonal antibody (mAb) inhibition profile, elution rate, and hemagglutinin thermostability. Nucleotide sequence analysis of portions of the fusion protein and matrix protein genes of the parrot isolate was done for comparison with prior sequence analysis of the anhinga isolate and NDV reference strains. Seven of the 43 chicken isolates were recovered from flocks in Canada. The remaining isolates, including 11 from turkeys, were isolated in the United States. All isolates except that of the anhinga were of low virulence by mean death time in embryos, intracerebral pathogenicity index, and/or intravenous pathogenicity index procedures and were classified as lentogens. The anhinga isolate was more virulent than the other strains and was pathotyped as a mesogen. However, nucleotide sequence analysis of the anhinga isolate had revealed a homology with the virulent cormorant isolates of 1992 rather than the classical U.S. mesogens characterized by the Roakin strain. Variability was evident among the lentogenic isolates. Two isolates from turkeys had mAb profiles that differed from B1 and La Sota reference and vaccine strains, and 38% (21/56) of the isolates had more thermostable hemagglutinins than those reference strains. There was no evidence that any of the isolates from poultry were more virulent than the lentogenic pathotype.

Four-week-old mixed-sex White Rock chickens were used in four experiments to determine the effect of negative air ion enrichment on airborne transmission of the Roakin strain of Newcastle disease virus (NDV). The experiments were conducted in specially constructed airborne disease transmission cabinets in which donor (upwind) chickens cannot contact susceptible (downwind) chickens because of physical separation by a "no man's land." Temperature and humidity were computer-controlled at 26.7 C and 50% relative humidity, and ventilation rates were manually adjusted from 0.34 to 1.36 m3/min (12 to 48 ft3/min). Donor chickens were inoculated with Roakin NDV by eyedrop and intranasal routes and placed in the upwind end of each cabinet. One day later, susceptible chickens were placed in the downwind end. Seroconversion (> or = 1:10 NDV hemagglutination-inhibition titer) was considered evidence of infection from inoculation (upwind) or airborne transmission (downwind). Commercial air ion generators were used either in the ends or in the "no man's land" of the treatment cabinets and operated at power supply voltages ranging from -8kV direct current to -15 kV direct current. The use of negative air ion generators reduced airborne transmission an average of 6.6% to 27.7% compared with the control cabinets. Significant (P < or = 0.05) reductions in transmission were obtained with some treatments. The greatest reduction in transmission was obtained with the higher power supply voltages (13.8% reduction) and when the ionizers were placed in the "no man's land" (27.7% reduction) between the upwind and downwind chickens.

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.

Replicate hemagglutination-inhibition (HI) tests of selected convalescent and hyperimmune infectious bronchitis virus (IBV) antisera were run against five IBV antigens: Massachusetts 41, Connecticut 46, H52, Arkansas 99, and JMK. Six different HI test procedures were included in this comparison to evaluate the effect of procedural differences on HI titers. The results were similar among procedures to the extent that positive and negative reactions were differentiated by each procedure with each antigen. This indicated that specificity was not affected by the procedure. However, the mean titers did differ among procedures in tests of the same serum-antigen combination, and the range of the titer differences was frequently fourfold or greater. Generally, high or low titers were associated with specific procedures, regardless of which test antigen was used.

Three-to-seven-week-old broiler-type chickens were inoculated with Newcastle disease virus (NDV) by eye-drop (ED) or intratracheally (IT), and virus isolation was attempted from oropharyngeal (oral) swabs and medium harvested from tracheal explant cultures (TEC). The TEC were maintained in screw-capped tissue-culture flasks for at least 1 month, and medium harvested at regular feeding times was assayed for NDV and NDV antibody. The earliest and latest sample times were 3 and 21 days after NDV inoculation. The three experiments done were: Expt. 1, infection of nonvaccinates with NDV strain La Sota; Expt. 2, infection of NDV vaccinates and nonvaccinates with NDV strain Largo; and Expt. 3, infection of NDV vaccinates and nonvaccinates with NDV wild-type strain Kansas-Manhattan (KM) and two temperature-sensitive (ts) clones derived by J. S. Youngner from the KM strain. All experiments yielded similar results. On day 3 postinoculation (PI), most chickens were shedding virus recoverable by oral swabs and detectable in harvests from TEC prepared on that day. On day 7 PI, there was a sharp reduction in the frequency of virus-positive oral swabs, but there was no decline in the frequency of virus-positive TEC. On day 14 PI or later, all oral swabs and TEC were virus-negative, except for one chicken in Expt. 3 that was oral-swab-positive. There was no evidence of NDV persistence in the TEC of oral-swab-negative chickens on or after day 14 PI. The results of these experiments are in contrast with previous reports of the detection of latent NDV by virus isolation from harvests of TEC prepared 18 or more days PI. The ts clones of strain KM used in Expt. 3 induced a markedly poorer antibody response and were shed for a shorter time than the KM parental virus.

Newcastle disease virus (NDV) hemagglutination-inhibition (HI) titers were determined for serum samples from eight commercial broiler breeder flocks and their progeny. The chickens sampled had been vaccinated and reared by different producers in different regions of the United States. Breeder flocks had the highest number of NDV-positive HI titers (greater than or equal to 1:10). Eighty percent or more of the samples from six of eight breeder flocks were positive; the geometric mean titers (GMTs) for those six breeder flocks ranged from 19 to 92. Only 3 of 8 broiler flocks had an increased frequency of positive titers and higher GMTs after vaccination. The frequency of positive titers was greater than 80% in only 2 of 8 of the oldest broiler flocks. The number of NDV-negative titers (less than 1:10) increased with age in most broiler flocks, even though all had been vaccinated once or more with live NDV vaccines.

Three 1986 infectious bronchitis virus (IBV) isolates were serotyped by a hemagglutination-inhibition method and were found to be serologically different from the seven strains currently used in regular and specially licensed vaccines in the United States (Mass 41, H52, H120, Conn 46, Fla 18288, JMK, and Ark 99) and from 13 other reference IBV strains. The recent isolates were from layer flocks that had histories of egg-production declines and shell-quality problems. Two of the isolates, one each from Maine and Ohio, were serologically related to a 1983 IBV isolate from layer replacements in Pennsylvania. The third isolate, from Korea, was serologically different from any of the reference strains and the other new isolates.

Serum samples were collected from broiler breeders and their 1-day-old, 2-week-old, and 5-week-old progeny from different regions of the United States. Individual samples were tested by hemagglutination-inhibition (HI) against six infectious bronchitis virus (IBV) strains: Massachusetts 41 (Mass), H52, Connecticut 46 (Conn), Arkansas 99, SE17, and JMK. The use of multiple strains to test broiler flocks resulted in the detection of seroconversions to Conn and JMK vaccination that were not detected with the IBV Mass HI test. Further, HI titers were detected to IBV strains not used for flock vaccination. In some cases, those titers could be due to cross reactions to antigens common to each of the virus strains. In two breeder flocks, the highest HI titers were to heterologous strains.