Identification and localization of surface sialylated glycoconjugates in Actinobacillus pleuropneumoniae by direct enzyme-colloidal gold cytochemistry.

Electron microscopic examination of ultrathin sections of 5 strains of Actinobacillus pleuropneumoniae stained for polysaccharides with ruthenium red revealed considerable variability in the amounts of preserved capsular material among the 5 serotypes studied. The amount of capsule was inversely related to the extent of outer membrane-associated sialylated glycoconjugate as evidenced by the degree of binding by colloidal gold-labelled neuraminidase at the cell surface. Serotypes 1, 3, and 5 possessed a well-developed and continuous capsular layer. In serotypes 2 and 7, the capsule consisted of a broken patchy layer that left much of the underlying outer membrane exposed. Morphometric analyses of the mean frequencies of neuraminidase-conjugated gold particles over the perimeters of the A. pleuropneumoniae cells showed that the lowest mean frequencies were observed in serotypes 1, 3, and 5, whereas the second highest and highest mean frequencies were observed in serotypes 7 and 2, respectively. Evidence suggested a serotypic difference in the amount of capsule present and this correlated inversely with the number of sialylated glycoconjugates, which appear to be localized in the outer membranes of the A. pleuropneumoniae cells.

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.

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.

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.

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.

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.

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.

Evaluation of chicken heterophil adherence.

Adherence of chicken heterophils was evaluated at 37 C using preconstructed columns containing various weights of nylon fiber (75 mg, 100 mg, or 125 mg) and whole blood anticoagulated with sodium heparin or 10% disodium ethylenediamine tetraacetic acid (EDTA). Additionally, 50-mg and 75-mg nylon fiber columns incubated at 41 C were used to evaluate heterophil adherence at an increased temperature. The mean percent adherence for heparin-anticoagulated blood applied to 75-mg, 100-mg, and 125-mg nylon fiber columns at 37 C was 76%, 92% and 97.4%, respectively. Samples applied to 50-mg and 75-mg columns at 41 C had adherence values of 27% and 85%, respectively. When paired samples of blood anticoagulated with EDTA or heparin were evaluated, the EDTA samples had significantly decreased heterophil adherence (paired t-test). Results indicate that increased or decreased adherence of chicken heterophils would best be detected using 75-mg nylon fiber columns incubated 37 C and whole blood collected in sodium heparin.

Diarrhea associated with intestinal Cryptosporidiosis in turkeys.

Turkey poults were suffering from diarrhea on a farm in which several previous grow-outs of turkeys had experienced a clinically identical problem. Upon necropsy, significant gross lesions were restricted to the gastrointestinal tract. Segments of small intestine were pale and distended with cloudy mucoid material and a few gas bubbles. The ceca contained fluid and gas. Fresh organ portions were collected and fixed in 10% neutral buffered formalin and submitted for histological processing and examination (light microscopy and scanning and transmission electron microscopy). Small (2-4 micron) basophilic bodies identified as Cryptosporidium sp. were present in enterocytes of the middle and lower small intestine. The villi were moderately atrophic, the crypts were hypertrophic, and the lamina propria was infiltrated by large numbers of lymphocytes, heterophils, and fewer macrophages and plasma cells. Numerous intraepithelial leukocytes and exocytosing inflammatory cells also were present.

Skeletal lesions associated with a naturally occurring poult enteritis.

One-day-old poults were placed on contaminated litter on which poults previously had developed an enteric disease characterized by diarrhea, increased mortality, and stunting. These exposed birds were examined for clinical signs and pathologic changes in bone and parathyroid glands compared with controls. Intestinal and fecal samples were examined for potential pathogens. Exposed poults varied in size as early as day 8 and had significantly decreased weight gains and reduced shank lengths on days 8, 12, 16, and 21. The proximal tibial growth plate was narrowed. The mineralized hypertrophy zone was decreased in length and contained multiple non-mineralized bands on days 8, 12, 16, and 21. Metaphyseal trabeculae were reduced in amount on days 16 and 21. Parathyroid glands were hyperplastic on days 16 and 21. The bone and parathyroid gland lesions indicated that mineral homeostasis was being maintained at the expense of the skeleton during the enteric disease. A specific etiology for the enteric disease was not determined. Cryptosporidium, rotavirus, paramyxovirus, and Salmonella were identified in the exposed poults, and paramyxovirus and Salmonella were identified in the controls.

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.

Leukocyte changes associated with acute inflammation in chickens.

Leukocyte changes in chickens with turpentine-induced inflammation were investigated sequentially at 0, 6, 12, and 24 hours and at 2, 3, 4, 7, and 14 days. During acute inflammation, significant leukocytosis and heterophilia developed by 6 hours and persisted through 7 days. The peak mean heterophil and leukocyte counts occurred at 12 hours and 3 days, respectively. Left shifts were present at 12 and 24 hours as detected by 100-cell leukocyte differential counts. Heterophil mean nuclear scores documented nuclear hyposegmentation (left shift) during early inflammation and nuclear hypersegmentation (right shift) during convalescence. Mean monocyte and lymphocyte counts peaked at 2 and 3 days, respectively. Basophil and eosinophil counts were erratic. Toxic changes of heterophils were most apparent during intense left shifts and consisted of cell swelling, degranulation, cytoplasmic vacuolation, and cytoplasmic basophilia. Cytoplasmic basophilia was the last aspect of toxic change to resolve. Ultrastructurally, toxic heterophils had intracellular edema, dissolution of granules, retention of ribosomes, nuclear membrane blebs, and decreased heterochromatin density. All inflammation-associated alterations in cell counts and morphology returned to baseline values and appearance by 14 days after turpentine administration.

Ultrastructure of the gastrocnemius tendon and sheath from broilers infected with reovirus.

Gastrocnemius tendon and sheath from male broilers infected with reovirus at 1 day of age were evaluated ultrastructurally at 1, 2, 3, 4, 5, 10, 15, and 20 weeks postinoculation (PI). Although virus particles were not identified, degenerative changes in sheath and tendon fibroblasts were characterized by cytoplasmic vacuolization, disruption of membranes, and loss of ribosomes from rough endoplasmic reticulum, mitochondrial degeneration, and cell disruption.

Diagnoses of infections by the so-called chick anemia agent: anemia and direct transmission electron microscopic detection of virus.

Blood from 48 chicks was examined for anemia (packed cell volume), and plasma was examined for virus particles by direct transmission electron microscopy (DTEM). There was agreement between the occurrence of anemia and the presence of CAA virus particles in plasma from anemic chicks (Kappa = 0.2425, Z = 2.096, P = 0.036). Although DTEM is a method that can be used to diagnose CAA in chicks, more sensitive, economical and less laborious diagnostic assays are needed.

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.

Infectious anemia caused by a parvovirus-like virus in Georgia broilers.

Pale chicks with necrotic dermatitis, small bursas of Fabricius (BFs), small thymuses, pale bone marrow, and watery blood were suspected of having parvovirus-like virus- (PVLV) associated disease. Histologic lesions included atrophy or hypoplasia of thymuses and BFs, and septic necrotizing clostridial dermatitis and hepatitis. Clostridium perfringens was cultured from skin and liver. A PVLV was isolated in a Marek's disease tumor cell line (MDCC-MSB1) culture and was identified by physicochemical, immunofluorescent, and morphologic features. This isolate was named GA-1 PVLV. Specific-antibody-negative chicks and embryos infected with heat- or chloroform-treated GA-1 PVLV developed anemia at the same rate. Control chicks never were anemic. This is the first isolation of PVLV from clinically ill chickens in the United States and the first report of PVLV-induced anemia in chickens in the Western Hemisphere.

Myocardial sarcocystosis in a grand eclectus parrot (Eclectus roratus) and a Moluccan cockatoo (Cacatua moluccensis).

Cardiac sarcocystosis is described in a grand eclectus parrot and a Moluccan cockatoo. Many cysts containing metrocytes were observed within cardiac muscle fibers on tissue sections stained with hematoxylin and eosin. Characteristic ultrastructural features of the cyst walls included the presence of villous projections containing microtubules. Compartmentalization of the cysts resulted from inward extensions of the cyst wall. The differential diagnosis of sarcocystosis, the life cycle of the parasite, and control measures are discussed.

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.

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.