Survival of exotic Newcastle disease virus in commercial poultry environment following removal of infected chickens

During the first weeks of 2003, after exotic Newcastle disease (END) was confirmed in commercial layer flocks in Southern California, it became apparent that the virus survival information in the literature varied widely and was difficult to extrapolate to current local conditions. The END Task Force used the information available in the literature and the recommendations of research scientists to establish protocols for safely handling manure from infected and depopulated premises. In an attempt to gain more applicable knowledge in the management of contaminated poultry manure in the course of the END outbreak, this virus survival study was designed and implemented. Environmental drag swabs were tested for END virus from two of the early-infected commercial ranches that consisted of several houses following immediate removal of the infected flocks. A total of 293 samples, composed of 168 manure drag swab pools, 72 dropping board swab pools, and 38 compost swab pools from 3 houses (ranch 1), and 180 manure belt scraper swab pools from ranch 2 were analyzed for ND virus isolation and characterization for 21 consecutive days postdepopulation. Thirteen manure drag swab pools (from houses 1 and 3) and two manure dropping board swab pools (from house 3) collected from ranch 1 were positive for END virus at 0, 1, 2, 3, 4, 7, 10, 12, and 16 days postdepopulation. No END virus was isolated after the 16th day following depopulation from any of the samples. All samples from ranch 2 were negative during the entire observation period.     

Pathological changes of tracheal mucosa in chickens infected with lentogenic Newcastle disease virus

Forty-day-old specific-pathogen-free chickens were exposed by aerosol to lentogenic Newcastle disease virus (NDV) and observed for 24 days for pathological changes in the tracheal mucociliary system. Specific fluorescence of NDV antigen was observed through day 5 postexposure (PE) in the cytoplasm of the tracheal epithelium and desquamated epithelium in the lumen. On day 1 PE, scanning electron microscopy revealed hypertrophy of goblet cells and small patches of the deciliated epithelium scattered mainly around the openings of mucous glands. The deciliated area of tracheal surface increased through day 4 PE. Light microscopy showed small vacuoles containing lymphocytes and heterophils in the epithelial layer. Immature epithelium proliferated in some areas. On days 5 and 6 PE, ciliated areas of the trachea tended to increase as a result of regeneration of the epithelium, still leaving many nonciliated patches of various sizes. On and after day 8 PE, there remained plaques with nonciliated flat epithelium, but most areas were covered with well-ciliated epithelium. Non-ciliated plaques were observed until day 24 PE, but they gradually decreased in size. These plaques were covered by a single layer of flat epithelium and were formed upon lymph follicles in subepithelial tissue.     

Comparison of electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus and virulent avian influenza virus

Electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus (NDV) or virulent avian influenza virus (AIV) were characterized and compared. The ECG were monitored by radiotelemetry and were recorded twice daily before virus infection and during the course of the infection. Thirteen lead II intervals, segments, and amplitudes were measured and analyzed. The ECG of NDV-infected chickens were characterized by lengthened (P less than or equal to 0.05) ST segments and increased (P less than or equal to 0.05) P amplitudes. The ECG of AIV-infected chickens were characterized by lengthened (P less than or equal to 0.05) RS intervals, ST segments, TP intervals, and PR segments and by increased (P less than or equal to 0.05) P amplitudes. The TP intervals and PR segments of ECG of AIV-infected chickens were significantly (P less than or equal to 0.05) longer than those of NDV-infected chickens. The pronounced conduction delays indicated in the ECG of AIV-infected chickens may have diagnostic importance.     

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.     

Intranuclear inclusions in cells infected with Newcastle disease virus.

Cells infected by Newcastle Disease Virus were observed to contain both intracytoplasmic and intranuclear inclusion bodies. Ultrastructurally, they consisted of twisted strands of about 18-20 nm diameter resembling nucleocapsids. The presence of these inclusions was detected irrespective of host cell or pathogenicity of the virus. In immunofluorescence and immunogold labelling experiments, these structures were tagged by an anti-P protein monoclonal antibody. In summary, we show that intracytoplasmic and intranuclear inclusion bodies, hitherto used as a taxonomic characteristic for the genus Morbillivirus of the Paramyxoviridae, also occur in a member of the genus Rubulavirus.     

Scanning electron microscopy of tracheal epithelium of chickens infected with velogenic viscerotropic Newcastle disease virus

Ultrastructural changes in the tracheal epithelium of chickens infected intranasally with velogenic viscerotropic Newcastle disease virus were examined by scanning electron microscopy. Hypertrophy of the mucus-secreting, or goblet, cells was the first sign of change, followed by disoriented and deformed cilia, hemorrhage, and hyperplasia of goblet cells accompanied by an increase in mucus. By day 7 postinfection, there was a marked decrease in the number of ciliated cells. Submucosal glands and some collagen fibers were exposed to the surface, an indication of loss of the epithelial cells. Macrophages and cell debris were abundant, and hyperplasia of the basal cells was evident in the later stages of infection, probably in an attempt to regenerate the lost epithelium. However, all chickens died 10 days postinfection, before any further work could be done.     

Detection of a macrophage-specific antigen and the production of interferon gamma in chickens infected with Newcastle disease virus

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.     

Serological response of chickens to oral vaccination with Newcastle disease virus

Conventional Newcastle disease vaccines are not suitable for application to village chickens in tropical countries of Asia. Trials with food-based vaccines are being initiated and the following experiments were performed to evaluate oral vaccination with Newcastle disease virus. Experimental chickens were vaccinated orally with the avirulent V4 strain of Newcastle disease virus and haemagglutination-inhibition antibody responses were measured. V4 virus was introduced into the crop by tube and total faecal output was collected daily and assayed for Newcastle disease virus. Virus was recovered on Days 5 and 6 after vaccination from most chickens that had received 10(7.4) and 10(6.4) 50% egg-infectious doses (EID50) of virus. There was no recovery of virus from birds receiving a lower dose of vaccine. Groups of chickens kept in cages with wire floors were given various doses of vaccine into the crop. Higher antibody titres were achieved with higher doses of virus. This dose responsiveness was not observed when various doses of vaccine were presented on food pellets and the groups of chickens were kept on concrete floors. Similar antibody responses were then seen with nominal doses of 10(5.2) and 10(8.2) EID50 per bird, possibly as a result of excretion and re-ingestion of the vaccine virus. Spread of the vaccine virus was demonstrated when control chickens and chickens receiving 10(7.7) EID50 of V4 virus on food pellets were housed together on a concrete floor. Similar antibody titres were achieved in both vaccinated and in-contact chickens.     

Persistence of exotic Newcastle disease virus (ENDV) in laboratory infected Musca domestica and Fannia canicularis

House flies (Musca domestica) and little house flies (Fannia canicularis) were examined for their ability to take up and harbor a velogenic strain of exotic Newcastle disease virus (ENDV) (family Paramyxoviridae, genus Avulavirus). Laboratory-reared flies were allowed to feed on evaporated milk containing ENDV at a virus concentration of 10(8.3) egg infectious dose (EID)50/0.1 ml or on poultry feces containing an ENDV titer of 10(5.8) EID50/0.1 g. Flies exposed to either infectious food source for 24 hr became transiently infected with virus. Virus persisted predominantly in the mid- and hindgut, with relatively little virus isolated from the remainder of the fly body. Virus persisted similarly in both fly species that were fed evaporated milk containing ENDV, with a maximum ENDV titer of 10(5.98) EID50/fly for the house fly and 10(4.78) EID50/fly for the little house fly at 1 day postexposure; titers decreased on subsequent days to 10(2.38) EID50/fly for house fly and > or = 1 EID50/fly for little house fly at 5 days postexposure. Both fly species acquired viral titers greater than the infective dose for a susceptible chicken (10(3.0) EID50-10(4.0) EID50). In addition, flies fed evaporated milk containing a high titer of ENDV maintained viral titers above the infective dose for up to 4 days postexposure to the infectious food source. Flies fed on infective feces retained a chicken infective dose for only one day. The decrease in viral titer over time was significantly explained by logistic regression for both fly species (P < 0.05). The slope of the regression line was not different for the two fly species (P < 0.05), indicating a similar rate of virus loss.     

Pathogenesis of Newcastle disease in chickens experimentally infected with viruses of different virulence

Groups of 4-week-old White Rock chickens were inoculated intraconjunctivally with nine isolates of Newcastle disease virus representing all pathotypes. Birds were monitored clinically and euthanatized sequentially, with collection of tissues for histopathologic examination and in situ hybridization using an anti-sense digoxigenin-labeled riboprobe corresponding to the sequence of the gene coding for the matrix protein. Disease was most severe with velogenic viscerotropic pathotypes and was characterized by acute systemic illness with extensive necrosis of lymphoid areas in the spleen and intestine. Viral nucleic acid was detected in multiple tissues but most prominently in macrophages associated with lymphoid tissue. Velogenic neurotropic isolates caused central nervous system disease despite minimal amounts of viral nucleic acid detected in neural tissue. Mesogenic and lentogenic pathotypes caused no overt disease; however, viral nucleic acid was present in myocardium and air sac epithelium following infection with these isolates. Compromise of air sac and myocardium may predispose mesogen- and lentogen-infected chickens to secondary infection and/or decreased meat and egg production.     

Virulence of pigeon-origin Newcastle disease virus isolates for domestic chickens.

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.     

interactions between escherichia coli and Newcastle disease virus in chickens

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.