Fractionation of feline bone marrow with the soybean agglutinin lectin yields populations enriched for erythroid and myeloid elements: transplantation of soybean agglutinin-negative cells into lethally irradiated recipients.

Feline bone marrow cells treated with the soybean agglutinin (SBA) lectin are separated into two populations, the agglutinated SBA(+) fraction containing predominantly cells of myeloid origin and the nonagglutinated SBA(-) fraction consisting of cells primarily of the erythroid lineage. FACScan analyses revealed a clear distinction of the cells based on their light scattering properties, i.e., large cells and cells with high granularity were found in the SBA(+) fraction, whereas cells having a low forward light scatter and side light scatter were found in the SBA(-) fraction. Colony-forming assays showed colony-forming unit-granulocyte/monocyte (CFU-GM) cells to have a strong affinity for SBA because these were found almost entirely in the SBA(+) fraction; in contrast, burst-forming unit-erythroid (BFU-E)-forming cells were concentrated in the SBA(-) fraction. When the marrow was fractionated by counterflow centrifugal elutriation (CCE), a differential binding to SBA among the CFU-GM forming cells was found. The SBA(-) fractions of cells collected at 21 and 25 ml/min contained primarily BFU-E forming cells, similar to that observed with whole marrow; the later CCE fractions, those collected at 32 ml/min and the rotor off fraction, when treated with SBA showed a small but significant number of CFU-GM cells in the SBA(-) fraction. T lymphocytes were found predominantly in the SBA(+) fractions of whole bone marrow and the CCE fractions. Successful autologous marrow transplants were performed with the early CCE SBA(-) fractions. The latter cells were used for our initial transplant attempts because ongoing studies in our laboratory had shown these cells to be free of any viral-containing cells when the marrow had been obtained from animals infected with the feline immunodeficiency virus. In summary, although SBA treatment of feline marrow yields a marked separation of CFU-GM and BFU-E progenitors, select CCE SBA(-) fractions contain stem cells capable of providing hematopoietic reconstitution of lethally irradiated animals.

Characterization of paramyxoviruses isolated from three snakes.

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.

Causes of mortality in the Florida panther (Felis concolor coryi).

Panthers necropsied at the University of Florida ranged between 2 weeks and 14 years of age; there were 38 males and 17 females in the cohort. Main categories of causes of death included trauma inflicted from either vehicular collisions (43%) or territorial fights (16%). Specific endogenous diseases involved the respiratory system in 13%, the urinary system in 4%, and the central nervous system in 2%. Ostium secundum atrial septal defects (ASD) were diagnosed in 11% of the panthers necropsied. Seventeen (54%) of the 38 male panthers had either unilateral or bilateral cryptorchidism. Cause of death remained undetermined in 11% of the total cohort.

Feline bone marrow transplantation: its use in FIV-infected cats.

The use of autologous and allogenic bone marrow transplantations (BMT) in FIV-infected and uninfected cats is a novel therapy for feline hematopoietic diseases and retroviral infections. A total of 13 specific pathogen-free (SPF) cats received either autologous or allogenic BMT and seven of these cats were also infected with FIV before autologous or allogenic BMT. All BMT recipients received total body irradiation of 900 cGy just before BMT. Two FIV-infected and four uninfected cats received autologous uninfected BM cells cryopreserved before BMT. Five infected and two uninfected cats received BM cells from allogenic uninfected donors (RBC-, MHC-, and cross-matched). MHC-matching was based on mixed leucocyte reaction (MLR) and the donor-recipient combination which was compatible by MLR analysis, was used in this study. Recipients were monitored for hematology, immunology, virology, and clinical signs. All FIV-infected and uninfected recipients of autologous BMT had complete engraftment with minimal complications. Uninfected recipients of allogenic BMT had a more severe clinical episode with slower rate of engraftment. None of these BMT groups had mortality. In contrast, only two of the five infected recipients of allogenic BMT survived for a significant period of time (23 and 50 weeks) and rest of the cats succumbed to transfusion reactions. Both infected BMT groups had persistent CD4/CD8 inversion, low CD4+ cell counts, and FIV infection of engrafted peripheral blood mononuclear cells (PBMC). Overall, successful autologous and allogenic BMTs were performed in FIV-free cats. All infected recipients of autologous BMT had compete engraftment and are currently alive, with thelongest survival time being over 1 year. Thus, BMT in combination with antiviral drug therapies may be an alternative therapy against retroviral infection.

Subclinical infectious bursal disease in an integrated broiler production operation.

A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.

Immunoperoxidase detection of ophidian paramyxovirus in snake lung using a polyclonal antibody.

In a retrospective study of proliferative interstitial pneumonia in viperid and nonviperid snakes, formalin-fixed, paraffin-embedded lungs from 52 snakes were screened for immunohistochemical reactivity to ophidian paramyxovirus. All snakes were from zoological collections that experienced mortalities attributed to paramyxovirus infection. Of the 52 snakes, 47 had pulmonary lesions compatible with ophidian paramyxovirus infection. Histologic changes in affected lungs included hyperplasia and hypertrophy of septal and faveolar epithelial cells, loss of ciliated cells, mixed leukocytic interstitial infiltrates, fibrinonecrotic exudate in the lumen of proximal and distal faveolar compartments, and occasional epithelial syncytial cell formation or intraepithelial eosinophilic intracytoplasmic inclusions. Lungs were immunohistochemically stained for paramyxovirus antigens by utilizing rabbit polyclonal antibodies against a paramyxovirus isolate from a black mamba (Dendroaspis polyepis polyepis). Virus infection in 6 snakes was confirmed by virus isolation from frozen lung tissue. Of the 6 lungs from which paramyxovirus was isolated, 5 lungs stained positively for viral antigens utilizing antisera to the black mamba isolate. Altogether, 36 lungs stained positively for paramyxovirus antigens. There was multifocal to diffuse linear staining of the lumenal surface of faveolar epithelium, and there were multiple foci of granular cytoplasmic staining. Immunohistochemical staining of formalin-fixed lungs from snakes with proliferative interstitial pneumonia was helpful as a routine diagnostic test for substantiating a diagnosis of ophidian paramyxovirus infection.

Histomoniasis in Leghorn pullets on a Florida farm.

Recurrent outbreaks of histomoniasis in flocks of 4-to-6-week-old white leghorn pullets is reported. In a typical outbreak, 5% of the pullets were stunted and listless with unkempt feathers. Mortality ranged from 2 to 3%, and the cull rate was approximately 2%. Ceca of affected chickens contained caseous cores. Histological examination of the ceca revealed fibrinonecrotic ulcerative granulomatous typhlitis associated with numerous histomonad trophozoites in the cecal wall. The outbreaks of histomoniasis were associated with heavy parasitism of pullets by Heterakis gallinarum. Litter had not been completely removed from the houses for the past 10 years, providing a constant source of Histomonas-infected larvated Heterakis ova.

Cranial teratomas in two domestic ducks (Anas platyrhynchos domesticus).

Teratomas are rare tumors in domestic fowl, and none have been reported in the cranial area of ducks. Clinical signs and gross and microscopic findings associated with a cranial teratoma in two domestic ducks are reported. One tumor arose within the cranial vault and was associated with neurologic signs. The other tumor was subcutaneous and invaded the parietal bone but did not extend into the brain. Both tumors were tridermic.

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.

Soluble scute proteins of healthy and ill desert tortoises (Gopherus agassizii).

OBJECTIVES: To characterize protein composition of shell scute of desert tortoises and to determine whether detectable differences could be used to identify healthy tortoises from tortoises with certain illnesses. ANIMALS: 20 desert tortoises. PROCEDURES: Complete postmortem examinations were performed on all tortoises. Plastron scute proteins were solubilized, scute proteins were separated by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were analyzed, using densitometry. Two-dimensional immobilized pH gradient-PAGE (2D IPG-PAGE) and immunoblot analysis, using polyclonal antisera to chicken-feather beta keratin and to alligator-scale beta keratin, were conducted on representative samples. The 14-kd proteins were analyzed for amino acid composition. RESULTS: The SDS-PAGE and densitometry revealed 7 distinct bands, each with a mean relative protein concentration of > 1 %, ranging from 8 to 47 kd, and a major protein component of approximately 14 kd that constituted up to 75% of the scute protein. The 2D IPG-PAGE revealed additional distinct 62- and 68-kd protein bands. On immunoblot analysis, the 14-, 32-, and 45-kd proteins reacted with both antisera. The 14-kd proteins had an amino acid composition similar to that of chicken beta keratins. There was a substantial difference in the percentage of the major 14-kd proteins from scute of ill tortoises with normal appearing shells, compared with 14-kd proteins of healthy tortoises. CONCLUSIONS AND CLINICAL RELEVANCE: The major protein components of shell scute of desert tortoises have amino acid composition and antigenic features of beta keratins. Scute protein composition may be altered in tortoises with certain systemic illnesses.

Staining and morphologic features of bone marrow hematopoietic cells in desert tortoises (Gopherus agassizii).

OBJECTIVES: To determine optimal site for collection of bone marrow from desert tortoises, and to characterize cytologic staining and morphologic features of bone marrow hematopoietic cells. ANIMALS: 16 desert tortoises. PROCEDURE: Bone marrow was obtained at necropsy from the pelvis, proximal portion of the humerus, femur, and thickened portions of the cranial to craniolateral and caudal to caudolateral margins of the carapace and plastron for histologic and cytologic examinations. Cytocentrifuged preparations of marrow cells were evaluated for reactivity to cytochemical stains. RESULTS: Histologic sections were adequate for evaluating acidophils, acidophil precursors, and erythrocyte precursors. It was difficult to differentiate among monocytes, lymphocytes, thrombocytes, and blast cells, and eosinophils could not be differentiated from heterophils. Basophils were in rare, small clusters of 3 to 12 cells. A few lymphoid follicles were found in the pelvis and long bones. Use of cytochemical staining accomplished differentiation between agranular heterophil precursors and granulated heterophils, and between granulated eosinophils and basophils. Monocytes, azurophils, and monoblasts had similar staining features. Staining of erythrocyte precursors with Sudan black B differentiated them from lymphocytes. Only a few small cells with periodic acid-Schiff-positive cytoplasm were identified as thrombocytes. Lymphocytes did not stain with any of the cytochemical stains. CONCLUSIONS: For histologic and cytologic evaluation of bone marrow hematopoietic cells, pelvis, proximal portion of the humerus, femur, and thickened portions of the peripheral cranial and caudal regions of the carapace and plastron are suitable sites to collect specimens. There are distinct cytochemical markers for heterophil, monocyte, and erythrocyte precursors, as well as later stage heterophils, eosinophils, basophils, monocytes, and azurophils.

Respiratory and pharyngo-esophageal iridovirus infection in a gopher tortoise (Gopherus polyphemus).

A free-living adult male gopher tortoise (Gopherus polyphemus) was found on Sanibel Island, Florida (USA), on 18 February 1992 with signs of upper respiratory disease. On necropsy after euthanasia on 27 February 1992, severe, extensive necrotizing ulcerative tracheitis, multifocal necrotizing pneumonia, and multifocal necrotizing ulcerative pharyngitis and esophagitis were observed. Large ovoid to round intracytoplasmic basophilic inclusions, which appeared to displace the nucleus to the cell periphery, occurred within degenerate and necrotic epithelial cells of the above tissues. On transmission electron microscopy of formalin-fixed trachea and lung, intracytoplasmic viral particles were observed within necrotic cells in the tracheal lumen and epithelial cells of the lung. Most infected cells also had a roughly spherical granular cytoplasmic inclusion that contained clusters of viral particles. Viral particles had an electron dense spherical to icosahedral core surrounded by a less electron dense icosahedral capsid. Mature extracellular virions were surrounded by an envelope and were 150 to 220 nm in diameter. Virions and cytoplasmic inclusions were morphologically similar to those of the Family Iridoviridae.

Nasitrema sp.-associated encephalitis in a striped dolphin (Stenella coeruleoalba) stranded in the Gulf of Mexico.

An immature female striped dolphin (Stenella coeruleoalba) found dead on a northwestern Florida beach in 1988 exhibited severe inflammation bilaterally in the dorsal and mid-thalamus in association with adult trematodes (Nasitrema sp.) and trematode eggs. Numerous specimens of Nasitrema sp. also were present in the pterygoid sinuses. Pneumonia in association with a heavy growth of Vibrio damsela was observed also. This report confirms the occurrence of Nasitrema sp.-associated encephalitis in striped dolphins and in small cetaceans from the Gulf of Mexico.

Pathology of diseases in wild desert tortoises from California.

Twenty-four ill or dead desert tortoises (Gopherus agassizii) were received between March 1992 and July 1995 for necropsies from the Mojave and Colorado deserts of California (USA). Diseases observed in these animals included cutaneous dyskeratosis (n = 7); shell necrosis (n = 2); respiratory diseases (n = 7); urolithiasis (n = 3); and trauma (n = 5). In tortoises with cutaneous dyskeratosis the horn layer of shell was disrupted by multiple crevices and fissures and, in the most severe lesions, dermal bone showed osteoclastic resorption, remodeling, and osteopenia. In tortoises with shell necrosis, multiple foci of necrotic cell debris and heterophilic inflammation within the epidermal horn layer were subtended by necrotic dermal bone colonized by bacteria and fungi. Of the seven tortoises with respiratory disease, five were diagnosed with mycoplasmosis. The diagnosis of mycoplasmosis was based on the presence of chronic proliferative rhinitis and positive serologic tests and/or isolation of Mycoplasma sp. Chronic fungal pneumonia was diagnosed in one tortoise with respiratory disease. In the three tortoises with urolithiasis, two were discovered dead, and the live tortoise had renal and articular gout. Traumatic injuries consisted of one tortoise entombed within its burrow, one tortoise burned in a brush fire, two tortoises struck by moving vehicles, and one tortoise attacked by a predator. While the primary cause of illness could be attributed to one or two major disease processes, lesions were often found in multiple organ systems, and a variety of etiologies were responsible for morbidity and mortality.

Shell disease in river cooters (Pseudemys concinna) and yellow-bellied turtles (Trachemys scripta) in a Georgia (USA) lake.

A disfiguring shell disease was detected in river cooters (Pseudemys concinna) and yellow-bellied turtles (Trachemys scripta) from Lake Blackshear, Georgia (USA). The turtles used were part of a mark-recapture study conducted from September 1991 to June 1993. Histologic changes on four turtles included acute segmental necrosis of the epidermis, followed by ulceration, necrosis of the underlying dermis and dermal bone, and exaggerated remodeling of bone. Additional findings included visceral inflammatory lesions and bacterial infection, sepsis and marked trematode ova granulomatosis. The cause of the shell lesions was not determined.

Atrial septal defects in Florida panthers.

Ostium secundum atrial septal defects (ASDs) were observed in six (3 M, 3 F) of 33 (20 M, 13 F) (18%) Florida panthers (Puma concolor coryi) necropsied by veterinary pathologists between 1985 and 1998. A seventh ASD was found in a female panther necropsied in the field and is included in the pathological description but not the prevalence of ASDs in Florida panthers. One panther (FP205) with severe ASD also had tricuspid valve dysplasia (TVD). Atrial septal defects and/or TVD are believed to have caused or contributed to the deaths of three (9%) Florida panthers in this study. Mean diameter +/- SD of ASDs was 9.0 +/- 4.7 mm (range 3 to 15 mm). Gross pathological changes attributed to ASDs/TVD in severely affected panthers (ASD > or = 10 mm) (n = 4) included mild right ventricular dilatation (n = 3) and hypertrophy (n = 2), mild to severe right atrial dilatation (n = 2), and acute pulmonary edema (n = 3). Panthers with mild ASDs (ASD < or = 5 mm) (n = 3) had no other detectable gross pathological changes associated with the ASDs. Histological examination of lungs of three panthers with severe ASDs revealed mild to moderate dilatation with fibrosis and smooth muscle atrophy of the tunica media of medium to large caliber arteries (n = 2), interstitial and/or pleural fibrosis (n = 2), perivascular fibrosis (n = 1), and acute to chronic edema (n = 3). Twenty-six necropsied panthers were examined one or more times while living; medical records were retrospectively evaluated. Antemortem radiographic, electrocardiographic, and echocardiographic examinations were performed on two panthers with severe ASDs (FP20 and FP205). Thoracic radiographic abnormalities in both included right heart enlargement, and in FP205 (severe ASD and TVD), mild pulmonary overperfusion. Electrocardiographic examination of FP205 revealed a right ventricular hypertrophy pattern, while FP205 had a normal electrocardiogram. Echocardiographic examination of FP20 revealed marked right atrial dilatation; a bubble contrast study indicated regurgitation across the tricuspid valve. Echocardiographic abnormalities in FP20 included right atrial and ventricular lilatation, atrial septal drop-out, and severe tricuspid regurgitation; non-selective angiography revealed significant left to right shunting across the ASD. All panthers with severe ASDs ausculted (n = 3) had systolic right or left-sided grade I-V/VI murmurs loudest at the heart base. All male panthers with ASDs (n = 3) (100%) and 9 of 17 (53%) male panthers without ASDs in this study were cryptorchid.

Paraneoplastic leukocytosis associated with a rectal adenomatous polyp in a dog.

A dog with a rectal adenomatous polyp had extreme neutrophilic leukocytosis, monocytosis, and eosinophilia consistent with a paraneoplastic syndrome. Resolution of the leukogram abnormalities after tumor excision supported this belief. Except for a lack of circulating myeloblasts, the dog had leukogram findings consistent with a neutrophilic leukemoid reaction.

Disseminated subcutaneous Mycobacterium fortuitum infection in a dog.

A 15-month-old 27.7-kg sexually intact male Doberman Pinscher was examined because of multiple subcutaneous abscesses on the neck, trunk, and limbs that developed 2 months after a dog bite and were refractory to antibiotic treatment. Incubation of a biopsy specimen at 37 C on a Lowenstein-Jensen agar slant for 8 days yielded growth of a Runyon's Group IV mycobacterium, and disseminated subcutaneous Mycobacterium sp infection was diagnosed. The organism was identified as M fortuitum, and was susceptible to amikacin, doxycycline, cefoxitin, minocycline, trimethoprim/sulfadiazine, and sulfisoxazole. Lesions resolved after 8 months of treatment with doxycycline (5 mg/kg of body weight, PO, q 12 h). The cause of dissemination was unknown; however, delay in debridement of the bite wound and corticosteroid use in initial wound management may have potentiated dissemination.

Mercury distribution in American alligators (Alligator mississippiensis) in Florida.

Thirty American alligators (Alligator mississippiensis), including 24 wild-caught and six control captive farm-raised alligators, were analyzed for whole body mercury contamination. Wild-caught animals were collected from Water Conservation Area 3 in the Everglades ecosystem (n = 12) and from Alachua, Brevard, and Collier counties outside the Everglades (n = 12). Using cold-vapor atomic absorption spectrophotometry, samples of brain, cervical spinal cord, liver, paired kidneys, paired testes, paired ovaries, paired oviducts, heart, lungs, spleen, bile, tail and leg muscle, and tail and leg scales were analyzed on a wet weight basis to determine mercury concentration. Mercury was consistently detected in all specimens except for bile. Farm-raised alligators, fed a commercially prepared diet, contained very low mercury concentrations in all tissues analyzed. In comparison with alligators from outside the Everglades, Everglades alligators had significantly elevated concentrations of mercury in all tissues analyzed except ovaries, oviduct, bile, tail scales, and leg scales (paired two-sample Student's tau-test, P < 0.05). Muscle concentrations exceeded state (0.50-1.50 ppm) and federal (1.00 ppm) allowances for safe human consumption in alligators collected in the Everglades. No clinical signs of neurologic, hepatic, or renal toxicosis were detected. Because of the alligator's ability to bioaccumulate mercury, this species might be useful as a bio-monitor for environmental mercury contamination.