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.     

Beak and feather disease virus haemagglutinating activity using erythrocytes from African Grey parrots and Brown-headed parrots

Psittacine beak and feather disease (PBFD) is a common viral disease of wild and captive psittacine birds characterized by symmetric feather loss and beak deformities. The causative agent, beak and feather disease virus (BFDV), is a small, circular single-stranded DNA virus that belongs to the genus Circovirus. BFDV can be detected by PCR or the use of haemagglutination (HA) and haemagglutination inhibition (HI) assays that detect antigen and antibodies respectively. Erythrocytes from a limited number of psittacine species of Australian origin can be used in these tests. In South Africa, the high cost of these birds makes them difficult to obtain for experimental purposes. Investigation into the use of erythrocytes from African Grey parrots and Brown-headed parrots yielded positive results showing the haemagglutinating activity of their erythrocytes with purified BFDV obtained from confirmed clinical cases of the disease. The HA activity was further confirmed by the demonstration of HI using BFDV antiserum from three different African Grey parrots previously exposed to the virus and not showing clinical signs of the disease.     

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.     

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.     

Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition

SUMMARY Simple and sensitive haemagglutination and haemagglutination Inhibition assays were developed for psittacine beak and feather disease (PBFD) virus and serum antibody, respectively. The assays were used in the examination of samples from 73 birds clinically affected with PBFD. High antigen titres (log₂ 9 to log₂ 12) were detected In feathers, faeces and cloacal contents of PBFD-affected birds. Antigen was not detected In either faecal or feather samples from 20 normal galahs (Eolophus roselcapillus) and 9 normal sulphur crested cockatoos (Cacatua galerita). After kaolin treatment and haemadsorption of serum, haemagglutination inhibition (HI) antibody titres could not be detected in serum from 42 PBFD-affected birds, whereas serum HI titres from 64 normal psittacine birds ranged from less than log₂ 1 to log₂ 8. Serum and yolk HI antibody responses of 6 PBFD virus-inoculated layer hens were measured. Pre-inoculation chicken sera contained high concentrations of non-specific haemagglutination inhibitors (not detected in chloroform-extracted yolk), which were removed by kaolin treatment and haemadsorption.     

Vaccination and challenge studies with psittacine beak and feather disease virus

SUMMARY: Psittacine beak and feather disease virus (PBFDV) was administered to adult galahs ( Eolophusroselcapillus ) by mouth or by intramuscular injection. Concentration of PBFDV antibodies in serum and excretion of PBFDV were monitored by haemagglutlnation inhibition (HI) and haemagglutination (HA) respectively. After oral administration, 17 of 18 galahs remained clinically normal and a small rise in antibody titre was detected in 3 of 18 birds. After intramuscular administration, antibody was detected in all birds. PBFDV was not detected in the feather dander of birds in either group. One bird developed diarrhoea and high faecal HA titres within 4 days of oral administration and then died. Adult and nestling cockatoos were vaccinated with an experimental inactivated double-oil emulsion vaccine. PBFDV antibody responses are comparable to those induced by a primary-oil emulsion vaccination regimen using Freund's adjuvants. Both vaccines protected nestlings. Three sibling wild-caught sulphur-crested cockatoos were vaccinated but died of PBFD before experimental challenge despite antibody responses in all birds. Unvaccinated control chicks developed acute PBFD within 4 weeks of challenge, probably from PBFDV-induced hepatitis since high concentrations of PBFDV were detected in their livers.     

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.     

Severe leukopenia and liver necrosis in young African grey parrots (Psittacus erithacus erithacus) infected with psittacine circovirus

This paper describes the signs, clinical pathology, and postmortem findings in 14 young African grey parrots (Psittacus erithacus erithacus) that were naturally infected with psittacine beak and feather disease (PBFD) virus (psittacine circovirus). All but two of the parrots had severe leukopenia at clinical presentation. Two other parrots also had severe anemia. All birds died within 3 wk after presentation. Postmortem examination documented liver necrosis in 11 of 14 birds and secondary bacterial or fungal infections in 9 of 14 birds. Tests for Chlamydia psittaci, polyomavirus, and Salmonella sp. were negative. PBFD viral infection could be demonstrated in all birds by polymerase chain reaction. Supporting evidence of PBFD viral infection was gathered by histologic examination of the bursa of Fabricius, electron microscopy, and DNA in situ hybridization. Electron microscopic examination of both the bursa of Fabricius and liver revealed virus particles resembling circovirus. DNA in situ hybridization of six liver tissue samples confirmed the presence of PBFD virus and excluded the presence of avian polyomavirus. Our findings suggest that a specific presentation of peracute PBFD viral infection, characterized by severe leukopenia, anemia, or pancytopenia and liver necrosis in the absence of feather and beak abnormalities, may occur in young African grey parrots.     

Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes.     

Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes

Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test. K88ac always gave negative HA results with porcine red cells. However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera. K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.     

In vitro adhesion of K88ab-, K88ac- and K88ad-positive Escherichia coli to intestinal villi, to buccal cells and to erythrocytes of weaned piglets

The phenotype of 21 weaned piglets, concerning adhesion of Escherichia coli possessing K88ab, K88ac or K88ad fimbriae to pig cells, was determined in an in vitro assay. Comparison was made with adhesion of these three K88 variant strains to buccal mucosal epithelial cells and to erythrocytes (haemagglutination) in the same piglets. Whereas adhesion of the three K88 variant strains to intestinal villi was piglet specific, buccal cell adhesion (BCA) and haemagglutination (HA) were not. The K88ab strain was weakly adhesive or non-adhesive in the BCA and negative in the HA test. K88ac strains consistently gave negative and K88ad consistently gave positive results in both assays. After washing the bacteria with phosphate-buffered saline, the K88ab strain revealed a positive HA test. Neither the BCA, nor HA test can be used to determine the pig intestinal adhesive phenotype.     

Psittacine beak and feather disease in three captive sulphur-crested cockatoos (Cacatua galerita) in Thailand

Three sulphur-crested cockatoos (Cacatua galerita) were diagnosed as psittacine beak and feather disease (PBFD). Histopathology of the feather pulp and follicles showed intracytoplasmic botryoid clusters or granular inclusion bodies in epithelial cells and macrophages. Electron microscopy revealed multiple cytoplasmic clusters of electron dense viral particles corresponding to the inclusions. PBFD virus (circovirus) DNA-specific product was detected from formalin-fixed paraffin-embedded feathers by nested polymerase chain reaction (PCR) method.     

Psittacine beak and feather disease: a first survey of the distribution of beak and feather disease virus inside the population of captive psittacine birds in Germany

Psittacine beak and feather disease (PBFD) is the most common viral disease of wild and captive psittacine birds. Here, we designed the first survey to investigate the existence of subclinical infections and the distribution of the causative agent named beak and feather disease virus (BFDV) inside the population of captive psittacine birds in Germany. DNA was isolated from feathers of 146 symptom-free birds from 19 different genera (all psittaformes) taken from 32 independent breeders from all over Germany. The presence of BFDV was analysed by performing polymerase chain reaction assays. Fifty-eight (39.2%) samples were found to be positive for BFDV. As expected, there was no significant predominance of one sex to be infected with BFDV.     

Effect of heparin on a haemagglutinin of porcine reproductive and respiratory syndrome virus

Heparin inhibited haemagglutination by porcine reproductive and respiratory syndrome virus (PRRSV) and by Aujesky's disease virus, but failed to inhibit haemagglutination by parainfluenza virus type 3. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRRSV haemagglutinin ranged from 0·1 to 1 U ml⁻¹. Mouse erythrocytes failed to combine with the haemagglutination inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRRSV. The formation of a haemagglutinin-heparin complex could be observed by sedimenting heparin with the haemagglutinin. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.     

Detection of parvoviruses in dogs using the hemagglutination test

The haemagglutination activity of the causal agent of canine parvovirosis is described. Out of the tested erythrocytes of pig, monkey, cat, horse, cattle, sheep, rabbit and guinea-pig, a positive reaction was recorded only in the erythrocytes of pig and monkey at the temperature of 4 degrees C. As a result of the examination of 20 faeces samples of hospitalized dogs by the method of haemagglutination reaction, a positive reaction typical of canine parvovirosis was obtained in 17 cases. The specificity of the reaction was confirmed by antiserum against the virus of panleucopenia of cat in the haemagglutination-inhibition test.     

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.     

Detection of pigeon circovirus by polymerase chain reaction

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.     

Seroprevalence of psittacine beak and feather disease in wild psittacine birds in New South Wales

SUMMARY A haemagglutination inhibition assay was used to detect antibody to psittacine beak and feather disease virus in sera from wild sulphur crested cockatoos (Cacatua galerita), galahs (Eolophus roseicapillus), short-billed corellas (Cacatua sanguinea), eastern long-billed corellas (Cacatua tenuirostris) and other psittacine birds in New South Wales. The seroprevalence of psittacine beak and feather disease ranged from 41% to 94% in different flocks, indicating infection with the virus is widespread in wild populations.     

Characterization of encephalomyocarditis virus isolated from aborted swine fetuses.

Characteristics of 2 encephalomyocarditis virus (EMCV) isolates (MN-25 and MN-30) recovered from aborted swine fetuses were examined along with 2 other swine isolates (NVSL-MDV and NVSL-PR) and a reference ATCC strain (VR-129). All 5 EMCV isolates were found to be serologically related by cross testing, using serum neutralization and fluorescent antibody assays. Hemagglutination (HA) properties of the 5 isolates were compared, using 5 diluents. The MN-25 and NVSL-MDV strains had HA activity with guinea pig RBC in all 5 diluents, whereas MN-30, NVSL-PR, and VR-129 had HA activity only in KCl-borate buffer. The HA ability with RBC of various animal species was examined, using KCl-borate diluent. All virus isolates had high HA titer (1:512 to 1:2,048) with guinea pig, rat, and horse RBC and lower HA titer (1:16 to 1:64) with sheep RBC. The MN-25 and NVSL-MDV isolates agglutinated dogs RBC, whereas MN-30, NVSL-PR, and VR-129 strains did not. Viral replication was evident in 8 of 10 cell lines tested, although infectivity titers of each virus varied by cell line used. Plaque-forming ability was similar for all 5 isolates, but plaque size was different by virus and cell culture used. Virus isolates were found to be stable after being heated at 56 C and subjected to a wide range of pH. A viral polypeptide pattern difference for all 5 isolates was not found by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)