Detection and confirmation of reptilian adenovirus infection by in situ hybridization.

Adenovirus infections are documented in at least 12 different species of reptiles. In contrast to their mammalian and avian counterparts reptilian adenoviruses are not well characterized as to their pathogenic potential and their ability to cause primary disease. In the diagnostic setting, fresh tissues are often not available for virus isolation, and the confirmation of reptilian adenovirus infections is dependent largely upon electron microscopy for the identification of intranuclear viral inclusions associated with histopathologic changes. The diagnosis of adenovirus infection in 2 different species of snake was confirmed by the application of DNA in situ hybridization. Using an aviadenovirus specific oligoprobe, adenoviral DNA was observed in the nuclei of hepatocytes, Kupffer cells, endothelial cells, and enterocytes. Electron microscopy of the liver confirmed the presence of intranuclear viral particles morphologically consistent with an adenovirus. DNA in situ hybridization on formalin-fixed tissues can serve as a suitable alternative to electron microscopy in the diagnosis of reptilian adenovirus infections. Both affected snakes had other concurrent diseases, suggesting that the adenovirus may not have been the primary pathogen.

Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants.

A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.

Effects of dietary protein on glomerular mesangial area and basement membrane thickness in aged uninephrectomized dogs.

The primary objective of this study was to determine the effects of diets containing 18% or 34% protein on glomerular mesangial area (GMA) and basement membrane thickness (GBMT) in uninephrectomized aged dogs. A secondary objective was to determine the combined effects of aging and uninephrectomy on GMA and GBMT in dogs. Ten clinically healthy, pure-bred dogs were unilaterally nephrectomized at about 8 y of age. After 2 mo, 5 dogs were fed an 18% protein diet and 5 dogs were fed a 34% protein diet for 48 mo. At month 48, the dogs were euthanized and the remaining kidney was collected. Samples of kidney from both times of collection were used to measure GMA and GBMT using electron microscopy. The effects of diet on GMA and GBMT were analyzed (student's t-test) using necropsy/nephrectomy score ratios. The effects of time-nephrectomy were determined by comparing nephrectomy values for GMA and GBMT with necropsy values (paired t-test). Dogs fed 34% dietary protein did not have a significant increase in GMA and GBM thickness when compared to dogs fed the 18% protein diet. A significant increase in GMA and GBMT occurred with time-nephrectomy (P = 0.011 and 0.018, respectively). Although dietary protein intake was not a significant factor in causing structural changes to glomeruli in uninephrectomized aged dogs, the power to detect a difference was low. However, significant effects of aging and nephrectomy were detected despite the low power of the study. These results suggest that the increases in GMA and GBMT that occur over time are not markedly influenced by dietary protein intake. However, subtle protein effects cannot be eliminated as a possibility based on this study.

Lafora bodies associated with neurologic signs in a cat.

Lafora bodies (polyglucosan deposits) were identified in the brain of a young adult cat with neurologic signs characterized by intermittent but progressively worsening head and body tremors. The cerebellar cortex was the most severely affected area of brain, and the deposits were identified within Purkinje cell bodies and processes and throughout the neuropil. The association of Lafora bodies with neurologic signs, occurrence of deposits within neuronal perikarya, and distribution primarily within the cerebellar cortex are features distinct from the more commonly recognized situation in which Lafora bodies occur as incidental lesions in cats.

The occurrence of ambient temperature-regulated adhesins, curli, and the temperature-sensitive hemagglutinin tsh among avian Escherichia coli.

Escherichia coli establishes a secondary respiratory tract infection in birds following inhalation of contaminated dust and litter particles. Escherichia coli express adhesins under conditions reflective of the ambient temperatures present in poultry houses. These microbial adhesins allow E. coli to attach to cell types that it initially encounters in the respiratory tract. Ambient temperature-regulated adhesins represent a new class of bacterial hemagglutinins that include pili and the thin, aggregative, flexible filaments known as "curli." This study examines the occurrence of the ambient temperature-regulated adhesins, curli (crl, csgA), and an avian-specific, temperature-sensitive hemagglutinin, tsh, among avian and mammalian E. coli isolates. The avian hemagglutinin gene tsh was present in approximately 46% of clinical avian E. coli isolates. This gene was not detected among commensal E. coli isolated from healthy broiler chickens. Unlike tsh, curli genes were ubiquitous among E. coli. However, curli were observed in only half of the avian E. coli examined by electron microscopy. Curli were not present among several nonavian E. coli positive for crl and csgA. Approximately 25% of avian E. coli isolates agglutinated chicken erythrocytes when bacteria were grown at room temperature. Hemagglutination was not specific to E. coli isolated from poultry. Presence of either tsh or curli genes was not indicative of an isolate's ability to agglutinate chicken red blood cells. No discernible structures were observed mediating attachment of the bacteria to chicken red blood cells. An additional avian-specific hemagglutinin appears to be present among avian E. coli.

Colonization of the chicken trachea by an avirulent avian Escherichia coli transformed with plasmid pHK11.

Recombinant plasmid pHK11 was transformed into an avirulent, wild-type avian Escherichia coli (E. coli Av) in order to study the plasmid's effect on colonization of the chicken trachea. The transformant (E. coli Av + pHK11) produced colicin V (ColV), had type F1 fimbriae, and was motile. The E. coli Av recipient possessed type F1 fimbriae but was nonmotile; it did not produce ColV. Four-day-old chicks were inoculated in the trachea with 100 microliters of an overnight culture (approximately 10(8) colony-forming units) of E. coli Av, E. coli Av + pHK11, or sterile brain-heart infusion (BHI) broth. A group of uninoculated chicks was also included. Samples of the trachea were taken on days 4 and 10 postinoculation and compared histologically and bacteriologically. Birds inoculated with E. coli Av + pHK11 had enhanced tracheal colonization and showed increased histologic changes as compared with those inoculated with E. coli Av or BHI broth or uninoculated controls. These results indicate that production of ColV and motility enhance the colonization of the trachea and may be involved in the cause of pathologic lesions.

Occurrence of Loma cf. salmonae in brook, brown and rainbow trout from Buford trout hatchery, Georgia, USA.

During a 6 mo study of moribund trout from Buford hatchery, Buford, Georgia, USA, a Loma cf. salmonae microsporidian parasite was studied in the gills of brook trout Salvelinus fontinalis, brown trout Salmo trutta, and rainbow trout Oncorhynchus mykiss. This parasite was morphologically similar to L. salmonae and L. fontinalis but differed in spore size. Scanning and transmission electron microscopy demonstrated that xenomas were embedded in gill filaments. Transmission electron micrographs prepared from fresh tissue showed mature spores with 12 to 15 turns of their polar tube. Spore diameters for the Georgia strain from formalin-fixed gill tissues measured 3.5 (SD +/- 0.1) by 1.8 (SD +/- 0.1) microns. Electron micrographs of formalin-fixed, deparaffinized tissues of rainbow trout from Pennsylvania and West Virginia show spores with a diameter of 3.5 (+/- 0.2) by 1.7 (+/- 0.1) microns and 3.4 (+/- 0.2) by 1.8 (+/- 0.1) microns, respectively. Transmission electron micrographs of spores from Pennsylvania and West Virginia show that mature spores from both states had 13 to 15 turns of their polar tubes. Measurements from transmission electron micrographs prepared from alcohol-fixed tissues from Virginia fish contained spores with a diameter of 3.0 (+/- 0.3) by 1.1 (+/- 0.3) microns and 12 to 15 turns of their polar tubes. These measurements are consistent with L. salmonae and therefore suggest that the parasite is present on the east coast of the United States. During the height of the Georgia epizootic, the percentage of fish with observed xenomas reached 62.2% (N = 87), and the highest number of xenomas counted per 10 gill filaments was 133 (N = 87). The microsporidian epizootic occurred either during the autumn months or when intake river water quality reached combined iron-manganese concentrations as high as 1.01 (mean 0.44, SD +/- 0.42) mg-1.

Malignant chromatophoroma in a canebrake rattlesnake (Crotalus horridus atricaudatus).

An adult female canebrake rattlesnake (Crotalus horridus atricaudatus) at Zoo Atlanta (Atlanta, Georgia, USA) had a subcutaneous mass on the left lateral abdomen. Microscopically, the tumor contained a pleomorphic population of cells with abundant intracytoplasmic brown to gold nonrefractile pigment (chromatophores), large stellate cells resembling neurons, and small stellate cells whose cytoplasmic processes formed a fibrillar matrix. The pigment stained black with the Fontana-Masson technique and was positive with the periodic acid-Schiff technique (prior to and after diastase treatment). Neuron-specific enolase was detected in the large stellate cells using an immunohistochemical staining technique. In addition, glial fibrillary acidic and S-100 proteins were detected in the chromatophores with immunohistochemical staining. The smaller stellate cells were strongly S-100 positive. Ultrastructurally, chromatophores contained intracytoplasmic structures composed of concentric lamellar membranes bordered by a triple-layer outer membrane. The morphology of these structures was compatible with pterinosomes. Three fluorescent pigments were isolated from the neoplasm by one-dimensional chromatography and characterized by spectrophotometry and spectrofluorometry.

Immunogold for detection of avian leukosis/sarcoma virus in formalin-fixed heart and kidney.

Immunoprobes are used in the qualitative and semi-quantitative demonstration of antigens in clinical samples. In the present study, an immunogold method has been used for routine and rapid detection of avian leukosis/sarcoma virus (AL/SV) in sections of formalin-fixed chicken heart and kidney. Application of this ultrastructural indirect immunogold labelling technique (IILT) will enable pathologists to obtain a definitive diagnosis of avian AL/SV infection when inclusions or putative viral particles are observed by light or electron microscopy. IILT also may be used in research on AL/SV infections.

Transmission electron microscopic demonstration of abnormalities on the tracheal cilia of chicks exposed to formaldehyde during hatching.

Formaldehyde gas used to fumigate hatcheries for control of microbial contamination has an adverse effect on tracheal cilia function and morphology. Evaluation of the changes revealed alterations in the ultrastructure of the axoneme with the absence of B subfibers and the production of additional A subfibers. Spikes and vesicular blebs in the cilia walls were evident in formaldehyde-exposed cilia. These changes could result in ciliastasis and cilia loss.

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.

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.

Fringed membranous particles and viruses in faeces from healthy turkey poults and from poults with putative poult enteritis complex/spiking mortality.

The purpose of the present study was to use clinical epidemiologic tools to define the relationship between production performance data, virus particles, and intestinal fringed membranous particles (FMPs) in healthy turkey poults and in poults that were experiencing an outbreak of poult enteritis complex (PEC) and spiking mortality syndrome (SMS). Small and large intestines from flocks of healthy poults and poults with PEC/SMS were collected, processed, and examined for viruses. Production performance parameters were collected and analyzed. Hocks of turkeys with PEC/SMS in the present study had low survival expectancy (livability) and poor growth compared to their healthy turkey counterparts. The only significant association between sickness and intestinal virus was the presence of coronavirus.

Clinical and histologic characterization of cutaneous reactions to stings of the imported fire ant (Solenopsis invicta) in dogs.

Four adult dogs received experimentally controlled stings in the dorsolateral abdominal skin by imported fire ants (Solenopsis invicta). The sites were examined grossly 15 minutes and at 1, 2, 3, 4, 5, 6, 24, 48, and 72 hours and histologically 15 minutes and 6, 24, 48, and 72 hours after stinging. The initial gross lesions at 15 minutes were swelling and erythema, and the microscopic changes were vascular congestion and superficial dermal edema. By 6 hours, the lesions consisted of bright erythematous pruritic papules characterized microscopically by a band of full thickness dermal necrosis and inflammation. By 24 hours and continuing to the end of the study at 72 hours, the sites appeared completely normal grossly. Biopsies taken 24, 48, and 72 hours after stings contained microscopic changes similar to those present at 6 hours after stings. These histologic changes are unlike those described for human beings stung by imported fire ants. In human beings, fire ant stings are characterized histologically by an initial superficial vesicle that evolves into a sterile pustule.

Evidence for a common epitope on the surface of Mycoplasma gallisepticum defined by monoclonal antibody.

An antigen containing a common epitope in most strains of Mycoplasma gallisepticum was purified by isoelectric focusing and used in the production of monoclonal antibodies (mAb). Of several mAb produced, only one mAb reacted with focused component and with all six strains of M. gallisepticum except strain 6/85. This mAb was designated MG3D6.A5, and it was subsequently purified with immobilized rProtein Atm. The MG3D6.A5 mAb recognized a common epitope on a molecule with relative molecular weight of 98 kilodaltons (kDa), termed p98. No binding was observed when the MG3D6.A5 mAb was reacted against antigens extracted from other mycoplasma species, indicating its species-specificity. Physicochemical studies revealed that p98 had an isoelectric point of 5.2, was stable to heat, and was resistant to periodate oxidation but sensitive to trypsin treatment, suggesting that p98 is a nonglycosylated protein. Furthermore, ultrastructural studies with colloidal gold revealed that M. gallisepticum cells were selectively stained with MG3D6.A5 mAb to p98. The latter was focally distributed on the surface of a mycoplasma cell membrane near the attachment organelle. These results suggest that p98 is a highly conserved protein in M. gallisepticum strains, is immunogenic, and is surface-accessible; its binding specificity to MG3D6.A5 mAb could be used to identify M. gallisepticum in multiple cultures.

Attachment and entry of Legionella pneumophila in Hartmannella vermiformis.

Legionella pneumophila is an intracellular parasite of Hartmannella vermiformis. Attachment to the amebae and entry of L. pneumophila were studied by two quantitative assays: One used plate counts to measure the number of bacteria attaching to amebae at 4 degrees C; the other determined the number of intracellular bacteria by use of transmission electron microscopy (TEM). The attachment assay showed that L. pneumophila are inefficient in attachment to amebae. About 0.05% of the bacteria were bound after 1 h with a 10- to 40-fold increase over the next 11 h. Attachment of both virulent and avirulent strains of L. pneumophila occurred at a similar rate. Uptake of L. pneumophila was measured by counting intracellular bacteria using TEM. Limited numbers of virulent L. pneumophila were found intracellularly before 4 h, but the numbers increased logarithmically after this time. The number of amebae containing virulent L. pneumophila increased linearly during the 12-h co-incubation. Avirulent L. pneumophila were rarely detected within amebae throughout the 12-h incubation. Results indicate that entry, not attachment, of virulent L. pneumophila is the limiting step in infection of axenically grown H. vermiformis.

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.