Effectiveness of chlorine dioxide in disinfection on two soft denture liners.

STATEMENT OF PROBLEM: Soft tissue denture liners frequently require replacement that necessitates complete removal from the denture base. A high speed lathe located in a "clean laboratory" is often used to facilitate removal of these materials, but it is unclear whether routine disinfection procedures reduce bacterial contamination sufficiently to prevent contamination of the laboratory. PURPOSE: The first phase of this study evaluated the effectiveness of 3-minute chlorine dioxide spray and immersion disinfection procedures on 2 denture liners (Coe Soft and Coe Comfort) and stainless steel specimens used as controls. The second phase evaluated the effectiveness of spray disinfection at time intervals of 1, 3, and 10 minutes. MATERIAL AND METHODS: Specimens made of soft denture liners attached to acrylic resin bases (10 per group) were contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans. Colony-forming units were counted after different disinfection techniques were applied. Kruskal-Wallis 1-way analysis of variance on ranks and an all pairwise multiple comparison procedures (Dunn's method) were used to test for significant differences among test groups at the P <.05 level of significance. RESULTS: Chlorine dioxide was effective against nonporous stainless steel specimens but was inadequate for denture liners at the recommended 3-minute time of disinfection. The immersion technique was more effective than the spray technique, but the difference was not significant. Increasing the time of disinfection did not significantly reduce the numbers of microorganisms. CONCLUSION: Coe Soft and Coe Comfort denture liners should be removed before entering the laboratory. These materials contain sufficient viable bacteria after routine disinfection procedures to cause contamination of the "clean laboratory."

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.

Diagnosis of avian adenovirus infections using DNA in situ hybridization.

Three DNA oligonucleotide probes designated FN-23, FN-48, and FN-96 were evaluated for the diagnosis of aviadenovirus infections by DNA in situ hybridization. Paraffin-embedded tissues were obtained from birds with confirmed adenovirus infection, birds with putative adenovirus infections, and birds with intranuclear inclusions caused by herpesvirus and polyomavirus. In birds with confirmed adenovirus infection, probes FN-23 and FN-96 identified 78% and 72% of diseased individuals, respectively. Only probe FN-48 detected chickens with group II adenovirus infection. In birds with putative adenovirus infection, the DNA probes confirmed aviadenovirus infections in 76% of the population. Probes FN-23, FN-96, and FN-48 detected 85%, 74%, and 18% of adenovirus-infected birds, respectively. None of the DNA probes cross-hybridized with tissues from polyomavirus-infected psittaciform birds or with tissues from a chicken with infectious laryngotracheitis. In contrast, probe FN-23 did cross-hybridize to herpesvirus-infected tissues from two of eight psittaciform birds with Pacheco's parrot disease. Probes FN-48 and FN-96 did not react with these tissues.

Polyomavirus inclusion bodies in chicken caecal epithelium.

In this case report we describe the light microscopic and transmission electron microscopic appearance of polyomavirus inclusions in caecal epithelial cells from a chicken with diarrhoea and intestinal parasites. The DNA in situ hybridization technique demonstrated that the polyomavirus reported here is different from polyomaviruses observed in psittaciformes. The chicken polyomavirus described may share some homologous base sequences with psittaciforme polyomavirus; however, the viruses are different. Clinicians and pathologists should be aware that polyomavirus can infect chickens and that polyomavirus inclusion bodies can be found in chicken caecal epithelial cells.

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.

Polyomavirus encephalopathy in a Ducorps' cockatoo (Cacatua ducorpsii) with psittacine beak and feather disease.

Necropsy tissues were examined from an adult wild-caught Ducorps' cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.

Viral proventriculitis in chickens.

The objectives of the present study were to examine proventriculi of broiler chicks for lesions, and to classify and record the incidence of these lesions. Deep non-purulent necrotizing proventriculitis (accompanied by adenoepithelial hypertrophy and hyperplasia) was the most common (109/220 = 49.5%) light microscopic diagnosis in the proventriculi examined. Degenerating and necrotic alveolar secretory cells had amorphous, granular or vacuolated cytoplasm. Nuclei usually were either pyknotic, karyorrhectic or karyolytic; however, fewer attached or sloughed cells had swollen nuclei with marginated chromatin and clear centres. Basophilic inclusion bodies were never seen. In five cases examined ultrastructurally, hexagonal virus particles were found in intact nuclei (average size = 68.9 nm, n = 89), associated with unbound condensed chromatin, and within vacuolated spaces in the cytoplasm (average size = 62.3 nm, n = 109). DNA in situ hybridization failed to detect adenovirus or polyomavirus nucleic acids. The presence of intralesional virus suggests that a causal relationship might exist between the virus and the proventricular lesions.

Detection of eastern equine encephalomyelitis virus RNA in formalin-fixed, paraffin-embedded tissues using DNA in situ hybridization.

Eastern equine encephalomyelitis (F.EE) virus was detected in infected formalin-fixed horse and emu tissues and in infected chicken embryo fibroblasts. Results of in situ hybridization using a digoxigenin-labeled 40-base DNA probe complementary to a conserved region of the EEE virus RNA compared favorably with results of both virus isolation and serum neutralization tests. This technique may be useful for diagnosis of EEE virus infection in various animal species, especially when fresh tissues are not available for analysis, and also will provide a means for studying the involvement of alphaviruses in pathogenesis studies.

Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization.

The evaluation of the usefulness of DNA probes in a diagnostic setting to identify nuclear inclusions in selected viral infections (psittacine beak and feather disease viral infection, avian polyomavirus infection, adenovirus infection and Pacheco's parrot disease) is reported. A DNA in situ hybridization method was used to detect viral nucleic acid in sections of paraffin-embedded tissues coming from birds naturally and/or experimentally infected. It is concluded that DNA probes used for polyomavirus (FN-19) and adenovirus (FN-23) are able to identify nucleic acid of each virus in the cells with nuclear inclusions, and when used for psittacine beak and feather disease virus (FN-8), and Pacheco's parrot disease virus (FN-49) are able to detect viral nucleic acid in cells with or without inclusions.

Diagnosis of polyomavirus infection in seedcrackers (Pyrenestes sp.) and blue bills (Spermophaga haematina) using DNA in situ hybridization.

Avian polyomavirus (APV) infection was diagnosed in a closed research colony of seedcrackers (Pyrenestes sp.) and blue-bills (Spermophaga haematina) using DNA in situ hybridization. The DNA probe was a 1-kbp double-stranded PCR-generated probe that recognized a conserved nucleotide sequence within the VP-1 gene. Using this technique, APV infection was diagnosed, in 25 of 45 birds based upon examination of formalin-fixed paraffin embedded tissues. Birds infected with APV apparently had a higher incidence of hepatic necrosis, hepatitis, bacterial infections and parasitism than did birds without APV.

Particles resembling circovirus in the bursa of Fabricius of pigeons.

Histological examination of the bursae from 12 pigeons under 4 months old revealed basophilic globular inclusion bodies, 5 to 25 microns in diameter, in the cytoplasm and the nuclei of the various bursal follicular cells. Electron microscopy of these inclusions revealed large electron-dense areas containing non-enveloped icosahedral viral particles, 14-19 nm in diameter, either loosely arranged or in paracrystalline array. Similar basophilic globular inclusion bodies were seen in the spleen and cecal tonsils of a few pigeons and in the duodenum of one pigeon. There were various degrees of lymphoid depletion in the bursa, spleen, and bone marrow. The morphology of the inclusions in the bursa and size of the viral particles are most consistent with circovirus. Preliminary studies on the bursae of two pigeons were negative for psittacine beak and feather disease (PBFD) viral antigen and nucleic acid by immunoperoxidase staining, DNA in situ hybridization, and polymerase chain reaction techniques, suggesting that this virus differs from PBFD virus. Most of the pigeons had concurrent infections such as paramyxovirus-1, salmonellosis, herpesvirus, and hepatic and cerebral trichomoniasis associated with adenovirus.

Diagnosis of polyomavirus-induced hepatic necrosis in psittacine birds using DNA probes.

Liver sections from 32 psittacine birds with multifocal to coalescing hepatocellular necrosis were examined to determine the cause of disease. Avian polyomavirus (APV) infection (19 of 32 birds), bacterial hepatitis (5 of 32 birds), and chlamydiosis (3 of 32 birds) were major causes of hepatic disease. The presence of APV inclusions or nucleic acid was demonstrated using hematoxylin and eosin (HE) staining, DNA in situ hybridization, and DNA amplification with Southern blotting. Amphophilic intranuclear inclusions, suggestive of APV infection, were observed in HE-stained liver sections from 5 of 32 birds. Hepatocellular karyomegaly was present in liver tissues from 10 birds (5 birds with typical APV inclusions and 5 birds without discernable inclusions). DNA in situ hybridization recognized intranuclear APV nucleic acid in liver sections of 18 of 32 birds. DNA amplification with Southern or dot blots also identified APV nucleic acid in processed, paraffin-embedded livers of 18 of 32 birds. This study demonstrates that acute APV infection is a frequent cause of multifocal to coalescing hepatocellular necrosis in psittacine birds. Furthermore, APV infection is best diagnosed using DNA probes, especially when typical intranuclear inclusions are not observed microscopically.

A retrospective study of circovirus infection in pigeons: nine cases (1986-1993).

Circovirus infections were diagnosed in 12 pigeons from the United States 4 pigeons from Australia, and 1 pigeon from Canada (1986-1993). Circovirus was identified by electron microscopic examination of basophilic botryoid cytoplasmic inclusions that had a histologic appearance similar to that of psittacine beak and feather disease virus inclusions. Inclusions were seen in splenic, bursal, gut-associated, and bronchus-associated lymphoid tissue macrophages and in bursal epithelial cells. Inclusions were composed of paracrystalline arrays of tightly packed, nonenveloped icosahedral virions 14-17 nm in diameter. Histologic changes in the spleens ranged from lymphofollicular hyperplasia with mild discrete lymphocellular necrosis to lymphoid depletion and diffuse histiocytosis. Lesions in the bursa of Fabricius ranged from mild lymphocellular necrosis to severe cystic bursal atrophy. Remaining histologic findings coincided with concurrent bacterial, viral, fungal, and parasitic infections. Immunoperoxidase staining and DNA in situ hybridization demonstrated that pigeon circovirus is distinct from psittacine beak and feather disease virus; however both viruses apparently share some homologous DNA sequences. Clinical and diagnostic findings indicate that pigeon circovirus may be similar to psittacine beak and feather disease virus with respect to acquired immunodeficiency and subsequent multiple secondary infections.

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.