Efficacy of spray-drying to reduce infectivity of pseudorabies and porcine reproductive and respiratory syndrome (PRRS) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma.

Three experiments were conducted to evaluate viral inactivation by the spray-drying process used in the production of spray-dried animal plasma (SDAP). In Exp. 1, bovine plasma was inoculated with pseudorabies virus (PRV) grown in PK 15 cells. Three 4-L batches were spray-dried in the same manner and conditions of industrial SDAP production but with laboratory spray-drying equipment. Presence of infectivity was determined before and after spray-drying by microtiter assay in PK 15 cell cultures. Before spray-drying, all three samples contained 10(5.3) tissue culture infectious dose50 (TCID50)/mL of PRV. After four consecutive passages, no viable virus was detected in samples of spray-dried bovine plasma. In Exp. 2, bovine plasma was inoculated with porcine respiratory and reproductive syndrome (PRRS) virus propagated previously in MARC cell culture to provide approximately 10(6.3) TCID50/mL. Three 4-L batches were spray-dried in the same manner as Exp. 1. Before spray-drying, samples contained TCID50 of 10(4.0), 10(3.5), and 10(3.5)/mL, respectively. After four consecutive passages in MARC cell cultures, no viable virus was detected in spray-dried bovine plasma. In Exp. 3, 36 weaned piglets (28 d of age) were fed a common diet for 14 d and were determined to be negative for PRV, PRRS, and porcine parvovirus titer. Afterwards, pigs were allotted to six pens with six pigs per pen and fed diets containing either 0 or 8% SDAP (as-fed basis) for 63 d. The SDAP used in the feed contained antibody (titer 1:400) against porcine parvovirus. Blood samples were collected from pigs on d 0 and 63 to determine whether feeding SDAP caused seroconversion and development of antibodies against parvovirus, PRRS, or PRV. Inclusion of SDAP in the diet improved growth of pigs without seroconversion. Spray-drying conditions used in this study were effective in eliminating viable pseudorabies and PRRS viruses from bovine plasma. In this study, feeding SDAP that contained functional antibodies did not promote seroconversion in naïve animals.

Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses.

Circoviruses are a diverse group of animal and plant pathogens with undefined relationships to one another but for their non-geminate, non-enveloped capsids and circular, single-stranded DNA genomes. The sequences of the beak and feather disease virus and porcine circovirus genomic DNAs are presented and analyzed in the context of the other members of the family. Sequence comparisons, inferred phylogenies, and geographic occurrence suggest that the ambisense circoviruses, particularly the beak and feather disease virus, represent an evolutionary link between the geminiviruses and the plant circoviruses. We propose that the family members be reclassified into three groups: The family Circoviridae consists of the animal pathogens (beak and feather disease virus and porcine circovirus) that possess ambisense genomes with striking similarities to the geminiviruses. The BBTV-like viruses include the plant pathogens (coconut foliar decay virus, banana bunchy top virus, subterranean clover stunt virus) with a geminivirus-like stem-loop element in their DNAs, and single to multiple component genomes. The chicken anemia virus is an unassigned virus possessing unique characteristics bearing little similarity to the other ssDNA viruses.

Use of an inactivated virus vaccine to control polyomavirus outbreaks in nine flocks of psittacine birds.

Nine flocks of psittacine birds were examined because of sudden death of neonates. In each flock, cause of death was determined to be polyomavirus infection, by means of DNA testing and in situ hybridization. Contaminated areas of aviaries were cleaned and disinfected, and vaccination programs, using a recently approved inactivated polyomavirus vaccine, were instituted. Use of the vaccine was found to be safe and efficacious.

Safety, immunogenicity, and efficacy of an inactivated avian polyomavirus vaccine.

OBJECTIVE: To determine safety, immunogenicity, and efficacy of an inactivated avian polyomavirus vaccine in nonbudgerigar psittacine birds that varied in age, species, and immunologic status. ANIMALS: Safety of the vaccine was evaluated in 1,823 psittacines representing more than 80 species. Immunogenicity was evaluated in 285 birds (260 of various Psittaciformes species, 25 chickens). Efficacy was evaluated in 104 birds (78 of various Psittaciformes species, 26 chickens). PROCEDURES: Safety was evaluated by vaccinating birds that were determined to be seronegative or seropositive (titer > 1:10) prior to vaccination. Birds were then evaluated for clinically detectable systemic or local reactions for 2 months to 2 years. Immunogenicity was evaluated by testing for virus-neutralizing antibodies, vaccinating each bird twice, and then testing for a significant change in antibody titer. Efficacy was evaluated by vaccinating birds, followed in 2 to 4 weeks by intramuscular or intravenous challenge exposure. After challenge exposure, protection was evaluated by attempting to recover virus from tissues or by observing birds for clinical signs of disease and testing for a significant change in titer. CONCLUSIONS: Avian polyomavirus vaccine is safe, immunogenic, and efficacious for use in multiple species of mature and immature psittacines. CLINICAL RELEVANCE: Until now, prevention of polyomavirus infection in psittacine birds could only be accomplished through strict isolation to reduce potential exposure to the virus. The USDA-registered inactivated avian polyomavirus vaccine can safely be used to protect vaccinates from infection and control spread of this virus in flocks.

An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes.

The safety and immunogenicity of adjuvanted and nonadjuvanted inactivated avian polyomavirus vaccines, administered either intramuscularly or subcutaneously (s.c.), were evaluated in a group of mixed species Psittaciformes. In 233 vaccinates representing species of macaws, cockatoos, conures, and parrots, gross reactions were limited to small scab formation at the s.c. injection site in three African grey parrots. Both vaccines stimulated a virus neutralizing (VN) antibody response, particularly in birds that were seronegative prior to vaccination. Ninety-three percent of the birds that were seronegative at the beginning of the study seroconverted (greater than fourfold increase in VN antibody titer) by 2 weeks after the second vaccination. Seventy-six percent of all the vaccinates had at least a fourfold increase in VN antibody titer at this time. There was no significant difference in seroconversion between the birds vaccinated with adjuvanted or nonadjuvanted vaccines. This study indicates that an inactivated avian polyomavirus vaccine can be used to safely immunize various species of psittacine birds in a field setting.

Antibody response to and maternal immunity from an experimental psittacine beak and feather disease vaccine.

Adult umbrella cockatoos, Moluccan cockatoos, African grey parrots, and a yellow-headed Amazon parrot were inoculated IM or SC with beta-propiolactone-treated psittacine beak and feather disease (PBFD) virus. Thirty- to 45-day-old African grey parrot, umbrella cockatoo, and sulphur-crested cockatoo chicks also were vaccinated with the same inoculum. The hemagglutination inhibition (HI) and agar-gel diffusion tests were used to assay for post-vaccination development of anti-PBFD virus antibodies. All adult vaccinates seroconverted and had increases in HI and precipitating antibodies. The vaccinated chicks had increased concentrations of HI antibodies, but precipitating antibodies could not be detected. To demonstrate that chicks from vaccinated hens are protected from PBFD virus challenge, 3 African grey parrot chicks and 2 umbrella cockatoo chicks from vaccinated hens and 1 African grey parrot chick and 1 umbrella cockatoo chick from nonvaccinated hens were exposed to purified PBFD virus. Chicks from the vaccinated hens remained clinically normal during the 50-day test period. Chicks from the nonvaccinated hens developed clinical and histologic lesions of PBFD. Infected tissues from these birds were confirmed to contain viral antigen, using immunohistochemical staining techniques. The PBFD virus was recovered from the affected birds. These findings indicate that adult and 30- to 45-day-old psittacine birds will seroconvert following vaccination with beta-propiolactone-treated PBFD virus. Also, hens inoculated with beta-propiolactone-treated PBFD virus produce chicks that are, at least temporarily, resistant to virus challenge.

Cryptosporidiosis in four cockatoos with psittacine beak and feather disease.

Cryptosporidiosis was diagnosed in 4 cockatoos with psittacine beak and feather disease. Three of the birds had cryptosporidiosis confined to the epithelium covering the bursa of Fabricius. One bird had generalized parasitism of the small intestine, large intestine, and bursal epithelium. All of the birds had intermittent to protracted diarrhea before death. Presumably, acquired immunodeficiency from psittacine beak and feather disease promoted establishment of cryptosporidiosis and other secondary diseases including septicemia, peritonitis, chlamydiosis, and mycotic ventriculitis.

Production and characterization of monoclonal antibodies to psittacine beak and feather disease virus.

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

Routes and prevalence of shedding of psittacine beak and feather disease virus.

Psittacine beak and feather disease (PBFD) virus was recovered from the feces and crop washings from various species of psittacine birds diagnosed with PBFD. High concentrations of the virus also could be demonstrated in feather dust collection from a room where 22 birds with active cases of PBFD were being housed. The virions recovered from the feces, crop, and feather dust were confirmed to be PBFD virus by ultrastructural, physical, or antigenic characteristics. Virus recovered from the feather dust and feces hemagglutinated cockatoo erythrocytes. The specificity of the agglutination was confirmed by hemagglutination inhibition, using rabbit antibodies against PBFD virus. During the test period, 26% (8 of 31) of the birds screened were found to be excreting PBFD virus in their feces, and 21% (3 of 14) of crop washings were positive for PBFD virus. Some birds in the sample group had active cases of diarrhea, whereas others had normal-appearing feces. Diarrhea was found to be the only significant indicator of whether a bird was likely to be excreting virus from the digestive tract. These findings suggest that exposure of susceptible birds to PBFD virus may occur from contact with contaminated feather dust, feces, or crop secretions. Viral particles that were morphologically similar to parvovirus (20- to 24 nm-icosahedral nonenveloped virions) also were recovered from feces of some of the birds.

Hemagglutination by psittacine beak and feather disease virus and use of hemagglutination inhibition for detection of antibodies against the virus.

Conditions for psittacine beak and feather disease (PBFD) virus hemagglutination and hemagglutination-inhibition (HI) test reactions are defined. The PBFD virus was found to hemagglutinate cockatoo and some guinea pig erythrocytes. The HI test was used to assay serum antibody titer in birds with active PBFD virus infections and in others that had been exposed to diseased birds. On the basis of HI antibody titers in psittacine birds that had been exposed to PBFD virus, but remained clinically normal, we suggest that some birds exposed to the virus are able to mount an effective immune response. Birds with active PBFD virus infections had lower antibody values than did birds that had been exposed to the virus, but remained clinically normal. On the basis of these findings, the ability to develop a suitable HI antibody response may be crucial in determining the disease status of susceptible birds exposed to the PBFD virus. If HI antibodies are found to have neutralizing activity, then the fact that a high HI titer was induced in birds inoculated with purified PBFD virus might suggest that an immunization program would be effective in preventing PBFD virus infections.

A novel DNA virus associated with feather inclusions in psittacine beak and feather disease.

The nature of feather inclusions was characterized in 32 psittacine birds (30 cockatoos, one peach-faced lovebird (Agapornis roseicollis), and one red-lored Amazon parrot (Amazona autumnalis autumnalis] with naturally-acquired psittacine beak and feather disease. Intranuclear inclusions within feather epithelial cells and intracytoplasmic inclusions within macrophages in the feather epithelium and pulp cavity contained psittacine beak and feather disease viral antigen when stained by the avidin-biotin complex immunoperoxidase technique. Ultrastructurally, inclusions were observed primarily within macrophages and to a lesser extent within epithelial cell nuclei. Macrophage inclusions appeared as paracrystalline arrays of viral particles. Intranuclear inclusions were less well defined, although scattered viral particles were present. Intracytoplasmic and intranuclear particles in ultrastructural preparations were identified by colloidal gold labeling as psittacine beak and feather disease virus. Feather epithelium was more frequently and severely involved in the disease process than was adjacent follicular epithelium. Plucked feathers with an intact epidermal collar and feather epithelium were preferred to follicular biopsies for histopathologic examination.

Comparison of three animal viruses with circular single-stranded DNA genomes.

No common antigenic determinants and no DNA sequence homologies were detected when three animal viruses, chicken anaemia agent (CAA), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFDV), all of which possess circular single-stranded DNA genomes, were compared. Negative contrast electron microscopy showed that PCV and PBFDV particles were 30% smaller than CAA particles and lacked the surface structure of CAA.

Effects of standard and variant strains of infectious bursal disease virus on infections of chickens.

T-cell-mediated and humoral immune responses were measured in chickens infected with standard and variant strains of infectious bursal disease virus. One-day-old and 3-week-old chickens were infected with these viruses and then given sheep RBC, killed Brucella abortus strain 19, and Newcastle disease virus. Appropriate serologic tests were used to monitor the primary and secondary responses to the antigens. Lymphoblast transformation assays were performed weekly. The response to the infectious bursal disease virus was determined by virus neutralization tests, microscopic examination of bursas, and bursal to body weight ratios. One-day-old chickens had T-cell-mediated and humoral immune suppression with both strains of virus, compared with controls. The lymphoblast transformation responses indicated that the variant strain was significantly (P less than 0.05) more suppressive than the standard strain. Three-week-old chickens had humoral immune suppression with the standard strain, but not with the variant strain. The lymphoblast transformation response was transiently suppressed at this age by the variant strain only. During the first week of infection, 1-day-old and 3-week-old chickens had lower neutralizing antibody titers to the variant strain than to the standard strain.

Extracutaneous viral inclusions in psittacine beak and feather disease.

Thirty-five birds that died with naturally acquired psittacine beak and feather disease (PBFD) were necropsied to identify extracutaneous viral inclusions. Inclusions were found in various tissue sections from 34 of 35 birds. By immunoperoxidase staining, intranuclear and intracytoplasmic inclusion bodies were shown to contain PBFD viral antigen. Inclusion-bearing lesions were widely disseminated but often closely associated with the alimentary tract. Lesions within the palate, esophagus, crop, intestine, bursa of Fabricius, and liver probably serve as sources for viral shedding into the feces.

Ultrastructural, protein composition, and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds.

Psittacine beak and feather disease (PBFD) virus, was purified from diseased tissues of a lesser sulphur-crested cockatoo (Cacatua sulphurea), a black palm cockatoo (Probosiger aterrimus), a red-lored Amazon parrot (Amazona autumnalis), and a peach-faced lovebird (Agapornis roseicollis). The histopathology of diseased feathers and follicular epithelium from the different species was compared; basophilic intranuclear inclusion bodies were identified in the follicular epithelium and intracytoplasmic globular inclusions were observed within macrophages located in the feather pulp from the four species. Psittacine beak and feather disease virus antigen was specifically detected by colloidal gold immunoelectron microscopy. The different preparations of purified virions displayed an icosahedral symmetry, were non-enveloped, and had a mean diameter that varied from 12 to 15 nm when negatively stained. Two major viral-associated proteins with approximate molecular weights of 26 and 23 kilodaltons (kd) were consistently demonstrated from the four viral preparations. Purified virions from the four genera were antigenically related. These findings suggest that the PBFD virus purified from numerous genera of diseased birds is similar based on ultrastructural characteristics, protein composition and antigenic reactivity.

Pathogenicity and immunosuppressive properties of infectious bursal disease "intermediate" strains.

This study was conducted to test the pathogenicity and immunosuppressive effects of seven commercially available infectious bursal disease (IBD) vaccines. These vaccine strains are intermediate in their pathogenicity in susceptible specific-pathogen-free (SPF) chickens. One-day-old and 3-week-old SPF chickens were vaccinated with these vaccines. Two weeks after IBD vaccination, they were vaccinated with Newcastle disease virus (NDV). The pathogenic and immunosuppressive effects of the IBD vaccines were evaluated by the antibody response to NDV vaccination, the bursa: body weight index, and histopathological lesions of the bursa. It was found that these strains were highly variable in their virulence and immunosuppressive properties. Three of the strains tested were found to be highly virulent and immunosuppressive; two others were moderate; and two could be classified as mild.

Immunosuppressive potential and pathogenicity of a recent isolate of infectious bursal disease virus in commercial broiler chickens.

At 15 days of age and in the presence of measurable levels of maternal antibody against infectious bursal disease virus serotype I (1:170 virus-neutralization geometric mean titer), a recent isolate (U-28) and a prototype virulent isolate (Edgar) of the same virus caused subclinical infections in commercial broiler chickens. Isolate U-28 caused a significant reduction in the size of the bursa of Fabricius, whereas the Edgar isolate produced splenomegaly. Both isolates reduced the serological response to Newcastle disease virus. The experimental immunosuppressive potential and pathogenicity of isolate U-28 in broiler chickens confirms the role of this virus in recent infectious bursal disease outbreaks.

Pathogenicity of recent isolates of infectious bursal disease virus in specific-pathogen-free chickens: protection conferred by an intermediate vaccine strain.

The pathogenicity of recent isolates of infectious bursal disease virus and the protection conferred against them by a commercial vaccine strain of intermediate virulence were examined in specific-pathogen-free chickens. Based on clinical signs, mortality, and macroscopic lesions in susceptible chickens, the isolates designated as A-Delmarva and U-28 were distinct from a previously known serotype I virulent isolate (Edgar). Histopathological analysis of the bursa of Fabricius did not establish differences between the field isolates. Although the vaccine strain produced some degree of bursal damage in antibody-free chickens, it was significantly less severe than the damage caused by the field isolates. The active immune response induced by vaccination was cross-protective against the pathological effects produced by the different isolates used in this study.

Antigenic comparisons of selected avian reoviruses by use of the plaque-reduction neutralization assay.

The antigenic interrelatedness of 3 clone-purified turkey reoviruses (NG-Turkey, 82-88, and NC-TEV) to each other and to 4 clone-purified chicken reoviruses (S1133, Co8, Fahey-Crawley, and avian type 2) was determined in reciprocal cross-neutralization tests, using polyclonal antisera and the plaque-reduction technique. The morphologic features of plaques formed under agar were studied for all 7 reoviruses, and size comparisons for turkey vs chicken isolates were made. All 3 turkey reoviruses (with the exception of NG-Turkey vs Fahey-Crawley chicken reovirus) formed plaques significantly (P less than 0.05) smaller than plaques produced by their chicken counterparts. The 3 turkey reoviruses were closely related to each other and to chicken reovirus CO8. The antigenic differences between turkey reoviruses 82-88 and NC-TEV and chicken reovirus S1133 were slight (minor subtype); however, the latter and NG-Turkey were serotypically distinct. The NG-Turkey and 82-88 turkey reoviruses were more related (minor subtype) to the Fahey-Crawley and avian type 2 chicken reoviruses, than was NC-TEV turkey reovirus (major subtype).

Characterization of infectious bronchitis virus using monoclonal antibodies.

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.