Abstract.A reproducible, experimental model of columnaris disease was developed to study the pathogenesis of cutaneous disease associated with Flavobacterium columnare infection in koi (Cyprinus carpio). In experimental infections, lesions were usually restricted to skin and fins; gill necrosis was not a consistent finding. Cytologic and histopathologic examinations provided a presumptive diagnosis of columnaris disease. Specific detection of F. columnare was done using the polymerase chain reaction and DNA in situ hybridization (ISH). Polymerase chain reaction allowed the detection of F. columnare in fresh biological material and in formalinfixed, paraffin-embedded tissues. The DNA ISH technique allowed the identification and localization of F. columnare in formalin-fixed, paraffin-embedded tissues. Using these molecular techniques, F. columnare was readily detected in skin specimens from infected fish; however, the bacterium was infrequently detected in specimens of liver, kidney, and spleen. These observations suggest that columnaris disease generally presents as a cutaneous disease that is unassociated with systemic infection in koi. Hematologic studies indicated that most infected koi developed microcytic, normochromic, nonregenerative anemia and leukopenia characterized by lymphopenia, mild neutrophilia, and monocytosis. Biochemical changes in diseased fish included significant hyperglycemia, hyponatremia, and hypochloridemia.

A novel siadenovirus was identified in the Sulawesi tortoise (Indotestudo forsteni). A group of 105 Sulawesi tortoises was obtained by the Turtle Survival Alliance. Many of the tortoises were in poor health. Clinical signs included anorexia, lethargy, mucosal ulcerations and palatine erosions of the oral cavity, nasal and ocular discharge, and diarrhea. Initial diagnostic tests included fecal testing for parasites, complete blood count and plasma biochemical analysis, mycoplasma serology, and polymerase chain reaction (PCR) testing for intranuclear coccidia and chelonian herpesvirus. Treatment included administration of antibiotics, antiparasitic medications, parenteral fluids, and nutritional support. Tissue samples from animals that died were submitted for histopathologic evaluation. Histopathologic examination revealed systemic inflammation and necrosis associated with intranuclear inclusions consistent with a systemic viral infection in 35 tortoises out of 50 examined. Fecal testing results and histopathologic findings revealed intestinal and hepatic amoebiasis and nematodiasis in 31 animals. Two of 5 tortoises tested by PCR were positive for Chlamydophila sp. Aeromonas hydrophila and Escherichia coli were cultured from multiple organs of 2 animals. The mycoplasma serology and PCR results for intranuclear coccidia and chelonian herpesvirus were negative. Polymerase chain reaction testing of tissues, plasma, and choanal/cloacal samples from 41 out of 42 tortoises tested were positive for an adenovirus, which was characterized by sequence analysis and molecular phylogenetic inference as a novel adenovirus of the genus Siadenovirus. The present report details the clinical and anatomic pathologic findings associated with systemic infection of Sulawesi tortoises by this novel Siadenovirus, which extends the known reptilian adenoviruses to the chelonians and extends the known genera of reptilian Adenoviridae beyond Atadenovirus to include the genus Siadenovirus.

Abstract. Histologic sections of crop tissue were evaluated for the presence of lymphoplasmacytic infiltrates within mesenteric ganglia. All birds with proventricular dilatation syndrome that had lymphoplasmacytic infiltrates in crop ganglia had similar infiltrates in the proventricular and/or ventricular ganglia. False-negative crop biopsy results occurred approximately 24% of the time. More invasive procedures, such as proventricular or ventricular biopsy, may be necessary if the crop biopsy is nondiagnostic in a bird with clinical signs of proventricular dilatation syndrome.

Important indicators of population health needed for large-scale sea turtle population recovery efforts include demographics, disease and mortality trends, condition indices, and baseline blood data. With this comprehensive health assessment of adult female green sea turtles Chelonia mydas nesting on Juno Beach, Florida, USA, we (1) established comprehensive baseline health indices; (2) identified individuals with evidence of infection by chelonid alphaherpesviruses 5 and 6 (ChHV5, ChHV6), which are implicated in fibropapillomatosis and respiratory and skin disease, respectively; and (3) compared measured health indices between turtles that did versus those that did not test positive for ChHV5 and/or ChHV6. All 60 turtles included in the study were in good body condition with no external fibropapillomatosis tumors. Hematological and biochemical reference intervals were established. Via quantitative PCR (qPCR), 5/60 turtles (8%) tested positive for ChHV5, and all turtles were negative for ChHV6. Of 41 turtles tested for antibodies to ChHV5 and ChHV6, 29% and 15% tested positive, respectively, and 10% tested positive for antibodies to both viruses. Notably, there were no statistically significant differences between health variables for nesting turtles that tested positive for ChHV5 DNA versus those that tested negative; and also no differences between turtles that tested positive for ChHV5 or ChHV6 antibodies and those that did not. This suggests that these viruses are enzootically stable in Florida’s adult green turtles. This study provides a health profile of nesting green turtles in southeastern Florida applicable to temporal and spatial investigations of this and other populations.

Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.

Abstract. 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.Adenoviruses have been reported in at least 12 different species of reptiles of the orders Crocodylia (crocodiles, alligators, caimans) and Squamata, including both the Serpentes (snakes) and Sauria (lizards) suborders. The reptiles in which adenoviruses have been identified include those belonging to zoologic collections and commercial breeders. In these species, adenoviruses have been incriminated as the cause of gastroenteritis, hepatitis, nephritis, pneumonia, and encephalitis. 1,[5][6][7][8][9]11,12,15,16 Adenovirus infection has been reported in reptiles with 5,6,12 and without 5,7-9,15,16 concurrent disease, and in the majority of communications, only individual animals have been affected. 15 Therefore, the pathogenic potential of the adenoviruses and their ability to cause primary disease in reptiles remains uncertain.Confirmation of adenovirus infection in the majority of the cases is based upon morphologic features of the virus or viral inclusions as determined by electron microscopy. In these accounts, tissues suitable for virus isolation were not available. In 3 reports, isolation and propagation of a reptilian adenovirus in cell culture was successful, with a virusinduced cytopathogenic effect. 7,11,15 However, in only 1 investigation were Koch's postulates fulfilled by the reproduction of disease, specifically identifying the isolated adenovirus as the primary etiologic agent of hepatitis and subsequent death in a common boa. 7 In contrast to reptilian adenoviruses, the pathogenesis and disease-causing potential of human adenoviruses and those of mammalian (mastadenovirus) and avian (aviadenovirus) origin are well characterized. In the diagnostic setting, DNA in situ hybridization (ISH) is a useful adjunct to microscopic and ultrastructural identification o...

Fibropapillomatosis is associated with chelonid alphaherpesvirus 5 (ChHV5) and tumor formation in sea turtles. We collected blood samples from 113 green (Chelonia mydas) and 112 loggerhead (Caretta caretta) turtles without fibropapillomatosis, including 46 free-ranging turtles (20 green turtles, 26 loggerheads), captured in Core Sound, North Carolina, and 179 turtles (93 green turtles, 86 loggerheads) in rehabilitative care in North Carolina. Blood samples were analyzed for ChHV5 DNA using quantitative polymerase chain reaction (qPCR), and for antibodies to ChHV5 peptides using an enzyme-linked immunosorbent assay (ELISA). None of the samples from foraging turtles tested positive for ChHV5 by qPCR; ELISA was not used for foraging turtles. Samples from 18/179 (10.1%) rehabilitating turtles tested positive for ChHV5 using qPCR, and 32/56 (57.1%) rehabilitating turtles tested positive for antibodies to ChHV5 using ELISA. Five turtles that tested positive by qPCR or ELISA at admission converted to being undetectable during rehabilitation, and five that initially tested negative converted to being positive. Both sea turtle species were significantly more likely to test positive for ChHV5 using ELISA than with qPCR (p < 0.001). There was no difference in the proportions of green turtles versus loggerheads that tested positive for ChHV5 using qPCR, but loggerheads were significantly more likely than green turtles to test positive for ChHV5 using ELISA. This finding suggests that loggerheads infected with ChHV5 at some point in their life may be more able than green turtles to mount an effective immune response against recrudescent infection, pointing to species-specific genetic differences in the two species’ immune response to ChHV5 infection. This is the first study to analyze antibodies to ChHV5 in loggerhead turtles and represents the most complete dataset on ChHV5 DNA detection in sea turtles encountered in the more northern latitudes of their western Atlantic habitat.

An adult male Rio Fuerte beaded lizard (Heloderma horridum exasperatum) was examined because of a history of anorexia and lethargy of one week duration. Diagnostic tests included a physical exam, complete blood cell count, plasma biochemistries, whole-body radiographs, and ultrasonography. The physical exam revealed the presence of a large mass in the midcoelomic cavity. Radiographs confirmed the presence of the midcoelomic mass and showed a smaller mass in the right cranial lung field. The ultrasonogram showed a homogeneous mass with soft tissue echogenicity. A fine-needle aspirate was collected, and the cytology results were suggestive of a melanophoroma. Exploratory surgery revealed a large mass (10×6 cm) within the right lung, with extensive adhesions to the caudolateral margin of the right liver lobe. The smaller mass (2×3 cm) was within the cranial aspect of the right lung. A right pulmonectomy and partial hepatectomy were performed to remove the tumors. The animal died 3.5-yr postsurgery, and histopathologic evaluation did not show evidence of melanophoroma in any of the tissues evaluated.