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Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae).

The relationship between a retroviral infection and the development of nonviral intracytoplasmic inclusion bodies was studied in a Boa constrictor model. Twelve juvenile age- and size-matched inclusion body disease (IBD)-negative boas were randomly divided into three groups. Each group was inoculated intraperitoneally with 1 ml of an IBD virus (IBDV)-infected liver homogenate or 1 ml of normal boa liver homogenate (sham-inoculated control) or was left untreated. All boas were monitored for development of IBD by daily examination and serial liver biopsy over 1 year. The 4 IBDV-inoculated boas became IBDV and inclusion positive by 10 weeks postinoculation. The average size and density of inclusion bodies increased with the duration of infection. Ultrastructurally, inclusion bodies <2 microm in diameter consisted of intracytoplasmic aggregates of granular electron-dense material that were not membrane limited. Larger inclusions (3-6 microm in diameter) were characterized as membrane-bound aggregates of amorphous to granular electron-dense material admixed with membranelike fragments. The sham-inoculated and untreated control snakes did not become inclusion or IBDV positive. Direct comparison of the protein electrophoretograms of IBDV-infected and normal boa tissues demonstrated a prominent 68-kd protein band unique to infected inclusion-positive tissues. Monoclonal antibodies directed against the 68-kd protein band specifically labeled inclusion bodies. The results of this study demonstrate that IBD inclusions represent an intracytoplasmic accumulation of an antigenically distinct IBDV-associated protein.

Animals↗

Retroviral particles in neoplasms of Burmese pythons (Python molurus bivittatus).

Neoplastic diseases associated with retroviruses were diagnosed in four Burmese pythons (Python molurus bivattatus) from a single collection. Snake No. 1 was a 7-year-old female with recurrent undifferentiated mesenchymal round cell tumor (lymphosarcoma) of the oral cavity. At necropsy, similar neoplastic masses were evident in the uterus and ovary, and there was diffuse involvement of the spleen. Snake No. 2 was a 4.5-year-old female that was euthanatized because of complications following resection of a segmental colonic adenocarcinoma. Snake No. 3 was a 5-year-old female that was euthanatized because of a large transitional cell carcinoma of the right kidney. Snake No. 4 was a 19-year-old female that was euthanatized following recurrence of an intermandibular fibrosarcoma. Ultrastructural examination revealed few to numerous extracellular and intracellular (intravacuolar) type C-like retroviral particles in all tumors. Tumors were about 90-95 nm in diameter, with an electron-dense core and bilaminar external membrane. The relationship of the intraneoplastic viral particles to the etiology of the tumors is uncertain.

Animals↗

Type A-like retroviral particles in a metastatic intestinal adenocarcinoma in an emerald tree boa (Corallus caninus).

A metastatic intestinal papillary adenocarcinoma was diagnosed histologically in an emerald tree boa (Corallus caninus). Metastasis was detected in the liver, both kidneys, lung, and coelomic wall. Ultrastructural examination of the metastatic intestinal epithelial cells in the liver revealed the presence of a moderate number of viral particles that most closely resembled type A retroviral particles and were mainly associated with granular endoplasmic reticulum membranes. This case is the first description of type A-like retroviral particles in a neoplasm of a snake. The role of the virions in the etiology of the intestinal adenocarcinoma is uncertain. In addition, this is the first confirmed report of a metastatic intestinal adenocarcinoma in a snake.

Adenocarcinoma↗

An epizootic of chronic regurgitation associated with chlamydophilosis in recently imported emerald tree boas (Corallus caninus).

One hundred and five wild-caught emerald tree boas (Corallus caninus) were added to a collection of 15 others. in Central Florida, during a 4-mo period. Eighty-one boas (67%) developed repetitive regurgitation during the 23-mo period after the initial introduction, and 61 (75%) of these died. Regurgitation occurred 3-4 days after feeding. Prevalence of regurgitation in this population of snakes was 25%/mo (range 0-42%), and incidence was 3.52/mo (range 0-13/mo). The cumulative mortality for those boas developing repetitive regurgitation (61 of 120) during the 23-mo epizootic was 51%. Hematologic findings included anemia and leukocytosis, with lymphocytosis, monocytosis, and azurophilia. Histologic evaluation of the gastrointestinal tract showed multifocal to diffuse lymphoplasmacytic inflammation with granuloma formation and positive immunohistochemical staining for chlamydial antigen. Electron microscopic evaluation of granulomas showed organisms consistent with Chlamydophila sp.

Animals↗

Cutaneous and pulmonary mycosis in green anacondas (Euncectes murinus).

Two dead, captive green anacondas (Eunectes murinus), including one male and one female, submitted for necropsy were in poor body condition, having multiple, scattered, dark red foci on the scales and mottled lungs. Both snakes had severe mycotic dermatitis. In addition, the male snake had mycotic stomatitis, and the female snake had mycotic pneumonia. Trichophyton sp., Verticillium sp., and Alternaria sp. were isolated from the dermal lesions. The pulmonary lesions were morphologically consistent with Aspergillus sp. Bacterial organisms isolated from skin and internal organs included Chryseobacterium meningosepticum, Stenotrophomonas maltophilia, Aeromonas hydrophila, and Providencia rettgeri. Mycotic diseases can be devastating to reptiles, and suboptimal husbandry and captivity were likely the predisposing factors that led to opportunistic invasion in these snakes.

Alternaria↗

Infectious disease serologic survey in free-ranging Venezuelan anacondas (Eunectes murinus).

Reptiles can harbor pathogenic microorganisms asymptomatically and serve as potential reservoirs of infection for humans, domestic animals, and other reptiles. Infectious diseases are also problematic for free-ranging reptile populations and are an important consideration in reptile reintroduction and translocation projects. There have been limited serologic studies of free-ranging reptiles for evidence of exposure to potential pathogens. In the present study, serum or plasma samples from five male and five female free-ranging Venezuelan anacondas (Eunectes murinus) were screened for antibodies to eastern, western, and Venezuelan equine encephalitis viruses, vesicular stomatitis virus, ophidian paramyxovirus, 19 Leptospira interrogans serovars, and Cryptosporidium serpentes. Antibodies to these agents were not detected, or antibody titers were low and possibly nonspecific. These results for the limited number of anacondas surveyed suggest that they do not serve as significant reservoirs for these infectious agents at this location.

Animals↗

Meningoencephalitis in a Boelen's python (Morelia boeleni) associated with paramyxovirus infection.

An adult male Boelen's python, Morelia boeleni, presented with acute neurologic disease and was euthanatized. Histologic examination revealed nonsuppurative meningoencephalitis. Occasional eosinophilic intracytoplasmic inclusions were noted in glial cells. On the basis of clinical signs and histopathology, inclusion body disease of boid snakes was suspected, but inclusions were not seen in other organs commonly affected with the disease. Moreover, electron microscopy revealed that the inclusions contained stacks of filaments 13-14 nm wide. With the use of a generic paramyxovirus cDNA probe, sections of brain and esophageal ganglion demonstrated hybridization. The findings indicate that paramyxovirus was the likely cause of the encephalomyelitis in this python, and this virus should be included in the differential diagnosis of pythons exhibiting central nervous system disease.

Animals↗

High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia.

Molecular methods were used to identify blood parasites frequently observed in blood smears of water pythons (Liasis fuscus) captured in our study area in the Northern Territory of Australia. A nested polymerase chain reaction (PCR) using primers amplifying the 18s ribosomal RNA (rRNA) nuclear gene resulted in a short PCR product (180 bp) matching this region in the genus Hepatozoon. However, because of the short sequence obtained. 2 new primers were designed based on 18s rRNA sequences of 3 Hepatozoon taxa available in GenBank. Using these primers, approximately 600 bp of the parasite's 18s rRNA gene was amplified successfully and sequenced from 2 water python samples. The new primers were used to investigate the prevalence of blood parasites in 100 pythons. In 25 of these samples we did not observe any blood parasites when examining stained slides. All the samples revealed a 600-bp PCR product, demonstrating that pythons in which we did not visually observe any parasites were infected by Hepatozoon spp. We also analyzed the nucleotide sequences of blood parasites in 4 other reptile taxa commonly encountered in our study area. The sequences obtained from water pythons and from 1 of these taxa were identical, suggesting that the parasite is capable of infecting hosts at different taxonomic levels.

Animals↗

Partial characterization of retroviruses from boid snakes with inclusion body disease.

OBJECTIVE: To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). ANIMALS: 2 boa constrictors with IBD and 1 boa exposed to an affected snake. PROCEDURE: Snakes were euthanatized, and tissue specimens and blood samples were submitted for virus isolation. Tissue specimens were cultured with or without commercially available viper heart cells and examined by use of transmission electron microscopy (TEM) for evidence of viral replication. Reverse transcriptase activ ty was determined in sucrose gradient-purified virus. Western blotting was performed, using polyclonal antibodies against 1 of the isolated viruses. Specificity of the rabbit anti-virus antibody was evaluated, using an immunogold-labeling TEM technique. RESULTS: 3 viruses (RV-1, RV-2, and RV-3) were isolated. The isolates were morphologically comparable to members of the Retroviridae family. Reverse transcriptase activity was high in sucrose gradient fractions that were rich in virus. Polyclonal antibody against RV-1 reacted with proteins of similar relative mobility in RV-1 and RV-2. By use of immunogold labeling, this antibody also recognized virions of both RV-1 and RV-2. CONCLUSIONS AND CLINICAL RELEVANCE: A retrovirus was isolated from boid snakes with IBD or exposed to IBD. Western blot analysis of viral proteins indicated that viruses isolated from the different snakes were similar. Whether this virus represents the causative agent of IBD is yet to be determined. The isolation of retroviruses from boid snakes with IBD is an important step n the process of identifying the causative agent of this disease.

Animals↗

Pharmacokinetics and tissue concentrations of azithromycin in ball pythons (Python regius).

OBJECTIVE: To determine pharmacokinetics and tissue concentrations of azithromycin in ball pythons (Python regius) after IV or oral administration of a single dose. ANIMALS: 2 male and 5 female ball pythons. PROCEDURES: Using a crossover design, each snake was given a single dose of azithromycin (10 mg/kg) IV. After a 4-week washout period, each snake was given a single dose of azithromycin (10 mg/kg) orally. Blood samples were collected prior to dose administration and 1, 3, 6, 12, 24, 48, 72, and 96 hours after azithromycin administration. Azithromycin was quantitated by use of liquid chromatography-mass spectrometry. RESULTS: After IV administration, azithromycin had an apparent volume of distribution of 5.69 L/kg and a plasma clearance of 0.19 L/h/kg. Harmonic means for the terminal half-life were 17 hours following IV administration and 51 hours following oral administration. Mean residence times were 37 and 94 hours following IV and oral administration, respectively. Following oral administration, azithromycin had a peak plasma concentration (Cmax) of 1.04 microg/mL, a time to Cmax of 8.4 hours, and a prolonged mean absorption time of 57 hours. Mean oral bioavailability was 77%. Tissue concentrations ranged from 4 to 140 times the corresponding plasma concentration at 24 and 72 hours after azithromycin administration. CONCLUSIONS AND CLINICAL RELEVANCE: Azithromycin is well absorbed and tolerated by ball pythons. On the basis of plasma pharmacokinetics and tissue concentration data, we suggest an azithromycin dosage in ball pythons of 10 mg/kg, orally, every 2 to 7 days, depending upon the site of infection and susceptibil ity of the infective organism.

Animals↗

Use of an ELISA for detection of antibody responses in Argentine boa constrictors (Boa constrictor occidentalis).

OBJECTIVE: To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis. ANIMALS: Two 3-year-old Argentine boa constrictors. Procedure-Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 microg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity. RESULTS: A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas. CONCLUSIONS AND CLINICAL RELEVANCE: Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens.

Animals↗

Pulmonary mycobacteriosis caused by Mycobacterium haemophilum and M. marinum in a royal python.

An adult female royal python was referred with an 18-month history of chronic respiratory tract disease. Anemia and moderate leukocytosis with heterophilia and monocytosis were detected and interpreted as evidence of a chronic inflammatory condition. Evaluation of lateral and dorsoventral radiographic views revealed multiple soft-tissue opacities within the cranial lung fields. Endoscopic evaluation revealed that the normal reticulated pattern on the surface of the lung had been largely replaced by diffuse, granulomatous tissue. Histologic examination of biopsy specimens revealed classic pyogranulomas. Ziehl-Neelsen stains revealed numerous acid-fast bacilli consistent with Mycobacterium spp. Molecular methods including polymerase chain reaction restriction assays and DNA sequencing confirmed the identification of M. haemophilum and M. marinum. The snake was euthanatized. Mycobacteriosis is an uncommon and sporadic pyogranulomatous disease of reptiles. In most cases of reptile mycobacteriosis, treatment is not advised because of the chronic nature and often advanced stage of the disease, long-term and expensive nature of potential treatment regimens, and the risk of spread to other animals, including humans.

Animals↗

Recovery of ranavirus dsDNA from formalin-fixed archival material.

The extraction and amplification of nucleic acid from formalin-fixed and paraffin-embedded tissues has become an important exercise in the collection of retrospective epidemiological data. A protocol is described that enables the extraction and amplification of dsDNA from fixed tissues within paraffin blocks and from specimens stored in 10% (aq) formalin. The procedure can be used for the examination of ranavirus DNA within archival tissues thereby providing valuable data for identifying the origin and tracing the spread of ranaviruses.

Animals↗

Sarcosporidiasis in rodents from Thailand.

One to six Sarcocystis spp. were identified in the skeletal muscles of 41 (33%) of 124 wild rodents (Rattus spp. and Bandicota indica) mainly captured in the central plains of Thailand throughout the year in 1995. Included were S. singaporensis, S. villivillosi, and S. murinotechis-like cysts all of which showed a striated cyst wall at the light microscopical level, and Sarcocystis cymruensis, S. sulawesiensis, and S. zamani which possessed smooth cyst walls. The ultrastructure of the cyst wall and other morphological characteristics used to distinguish species are described. By inoculation of muscle cysts from wild-caught rodents into coccidia-free pythons (Python reticulatus, P. molurus bivittatus), we confirmed that P. reticulatus is a suitable definitive host for S. singaporensis and S. zamani in Thailand. Furthermore, we showed by fecal examination of reticulated pythons collected in the wild and subsequent experimental infection of laboratory rats that these hosts also are naturally infected with both species. Sarcocystis cymruensis is reported for the first time from Southeast Asia. This parasite was prevalent in brown rats (Rattus norvegicus) and bandicoot rats (B. indica) which were captured near human habitations; it is likely to be transmitted to rats via cats. The definitive hosts of S. sulawesiensis and S. murinotechis are unknown. Hence, at least three Sarcocystis spp. (S. singaporensis, S. zamani, S. villivillosi) are likely to cycle between snakes and rodents in agricultural areas in Thailand. Among these, S. singaporensis appears to be the most prevalent species.

Animals↗

First identification of a ranavirus from green pythons (Chondropython viridis).

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.

Amino Acid Sequence↗

Infectious dermatitis in a ball python (Python regius) colony.

Seven wild-caught ball pythons (Python regius), including six gravid females and one male, were obtained from Africa and were housed in a government animal facility in Research Triangle Park, North Carolina. Upon arrival, the snakes were found to be infested with ticks (Aponomma latus), which were manually removed. Four weeks following arrival, vesicular skin lesions began to appear on the snakes. Despite treatment of all affected female snakes with amikacin (5 mg/kg i.m., every 3 days) and cefotaxime (25 mg/kg i.m., every 3 days), the condition progressed and five of the female snakes died 7 wk after arrival. The remaining male and one female improved after an increase in environmental temperature, with ecdysis followed by healing. Physiologic stress, ectoparasites, and shipping may have predisposed the snakes to sepsis.

Amikacin↗

Partial tracheal obstruction due to chondromas in ball pythons (Python regius).

Over a 9-mo period, three adult ball pythons (Python regius) (one male, two females) were evaluated for severe dyspnea. Partial obstructions of the tracheal lumen were identified radiographically and/or visualized with a 3.0-mm rigid laparoscope inserted into the tracheal lumen in all three snakes. Administration of systemic antibiotics and nebulization resulted in partial improvement of the dyspnea. In two snakes, the tracheal lesions were removed with a rigid laparoscope and a flexible biopsy instrument inserted into the tracheal lumen. The other snake died and was necropsied. Histologically, the lesions from two snakes were determined to be benign chondromas. The chondromas were composed of a variably disorganized chondroid matrix populated by quiescent, normal-appearing chondrocytes within lacunae, although the chondrocytes were increased in density compared with normal hyaline cartilage and contained rare mitotic figures. The tracheal masses in one snake grew by expansion, not invasion, and were focally continuous with a mineralized cartilage tracheal ring, suggesting a benign nature. This is the second report of tracheal chondroma in ball pythons. Tracheal chondromas are exceedingly rare in humans and domesticated animals, suggesting a possible predisposition of ball pythons for this neoplasm.

Animals↗