Michael M. Garner

Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

ABSTRACT A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses.

Evidence of Brucella infection in Parafilaroides lungworms in a Pacific harbor seal (Phoca vitulina richardsi).

Multiple isolates of Brucella sp. that differ from the recognized species within this genus have recently been isolated from viscera of 4 harbor seals (Phoca vitulina), 2 harbor porpoises (Phocoena phocoena), 1 common dolphin (Delphinus delphis), an Atlantic white-sided dolphin (Lagenorhynchus acutus), 2 striped dolphins (Stenella coeruleoalba), a hooded seal (Cystophora cristata), and a gray seal (Halichoerus grypus), 9,18 all from the Scottish coast. A similar Brucella sp. was isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) from the coast of Califomia. During routine capture operations, 18 of 102 Pacific harbor seals (Phoca vitulina richardsi) and 4 of 50 California sea lions (Zalophus californianus) from Puget Sound, Washington, had been reported with positive titers to Brucella sp. In this report, we describe the isolation, tissue location, and immunohistochemical and ultrastructural features of Brucella sp. infection in a pacific harbor seal. These findings suggest that transmission of brucellosis by infected lungworms is a possibility. On March 28, 1996, a 7-9-month-old 17.5-kg male Pacific harbor seal (105 cm length, snout to tail tip) was collected dead from Restoration Point on Bainbridge Island in the Puget Sound, Washington. The seal had been dead for 2-5 days, and there was slight decomposition. The carcass was transported to the Washington Department of Fish and Wildlife Marine Mammal Investigations Laboratory in Tacoma and necropsied. Blood drawn from the heart was separated by centrifugation, and serum was sent to the Washington Department of Agriculture Laboratory to screen for antibodies to Brucella sp. Values were compared with the standards outlined in the Brucellosis Eradication Uniform Methods and Rules (1992, revised 1994, USDA, APHIS). The sample was tested using B. abortus antigens provided by the National Veterinary Services Laboratory (NVSL), Ames, Iowa. Official protocols for

Clinico-pathologic Features of Fatal Disease Attributed to New Variants of Endotheliotropic Herpesviruses in Two Asian Elephants (Elephas maximus)

The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.

FATAL HEMOPROTOZOAL INFECTIONS IN MULTIPLE AVIAN SPECIES IN A ZOOLOGICAL PARK

Abstract Over a 3-yr span, two juvenile lesser flamingos (Phoeniconaias minor), two green jays (Cyanocorax yncas glaucescens), and two Montezuma oropendolas (Psarocolius montezuma) died peracutely with no premonitory signs at a zoological park in the southern United States. At necropsy, the birds were in excellent body condition. Except for one green jay, the coelomic cavities were filled with a dark serosanguineous fluid. Splenomegaly and hepatomegaly were present. The livers were tan to purple with numerous, randomly distributed red-to-black foci, ranging in size from 1 to 4 mm. The predominant histopathologic finding, except in one green jay, was large protozoal cysts in the hepatic parenchyma. Histologically, the protozoal cysts were restricted to the liver, and none were identified in the skeletal muscle, spleen, or other tissues. Frozen tissue samples harvested at necropsy had a nested polymerase chain reaction assay performed to amplify the mitochondrial cytochrome B gene of the protozoa. The amplified gene sequences were compared with reference cytochrome B gene sequences for avian Plasmodium spp., Haemoproteus spp., and Leucocytozoon spp. The protozoal parasite within the hepatic parenchyma from the Montezuma oropendolas and the lesser flamingos was identified as Haemoproteus spp. Both green jays had Plasmodium spp. isolated from the submitted tissue samples. The peracute nature of the infections precluded any successful medical intervention, making prevention by exclusion the principal means to control hemoprotozoal transmission. There are no reports in the literature documenting identified fatal hemoprotozoal infections in oropendolas, green jays, or lesser flamingos.

Clinicopathologic Features of a Systemic Coronavirus-Associated Disease Resembling Feline Infectious Peritonitis in the Domestic Ferret (Mustela putorius)

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus

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.

Proliferative osteoarthritis and osteoarthrosis in 15 snakes.

Abstract Fifteen snakes representing seven species with segmental, proliferative osteoarthritis and osteoarthrosis of the spine were presented for examination. All the snakes were captive, privately owned, and fed domestic rodents. Physical examination, radiography, blood culture, bone culture, necropsy, and histopathology were performed on each snake. All the snakes had similar physical examination, radiologic, and necropsy findings. There were three histologic types of lesions: active bacterial osteoarthritis, predominantly noninflammatory osteoarthrosis with multifocal inflammation suggestive of chronic bacterial osteoarthritis, and noninflammatory lesions consistent with osteoarthrosis without evidence of inflammation or bacteria. These findings suggest that all these snakes represent a single disease process, bacterial infection of the vertebrae. The different histologic lesions observed in these snakes may be a continuum of lesions, from acute to chronic. Gram-negative bacteria were isolated from the blood or bone lesions of 8 of the 15 snakes. In six of these eight snakes, Salmonella species were isolated. Gram-positive bacteria (Streptococcus sp.) were isolated from two other snakes. Blood and bone culture results were well correlated, so blood culture may be effective for detecting active bacterial osteoarthritis.

Fatal Cytauxzoonosis in a Captive-reared White Tiger (Panthera tigris):

Fatal (Panthera tigris) cytauxzoonosis was diagnosed in a 7-year-old female white tiger. The tiger presented with a 2-day history of anorexia and lethargy. She was mildly dehydrated, with a temperature of 105.2 F and a hematocrit of 26%. Over the next day, icterus developed, and her physical condition progressed to recumbency, coma, and death. Hematologic findings obtained shortly before death included icteric plasma, severe thrombocytopenia, mild anemia, hematuria, and parasites consistent with Cytauxzoon felis in circulating erythrocytes. Gross necropsy findings included generalized icterus, generalized petechiae and ecchymoses, splenomegaly, and peribronchial edema. Histologic changes included large numbers of intravascular macrophages containing developmental stages of Cytauxzoon felis that partially or completely occluded blood vessels in the lung, spleen, liver, and bone marrow. Except for an experimental infection of a bobcat, fatal cytauxzoonosis has not previously been diagnosed in felids other than domestic cats. These findings raise questions regarding the pathogenicity of this organism in felids and may impact husbandry and interstate transfer of captive large cats.

Intranuclear Coccidiosis in Tortoises: Nine Cases

Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.

Comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus.

Abstract Ferret systemic coronavirus (FRSCV) infection is associated with an emerging, highly fatal disease of ferrets. Enhanced macrophage tropism and the resulting induction of pyogranulomatous lesions are shared with feline infectious peritonitis virus (FIPV) infection in cats, but are not features of ferret enteric coronavirus (FRECV) infection. Comparative sequence analysis of the distal one-third of the genomes of one FRSCV and one FRECV strain showed that these two ferret coronaviruses share >96% nucleotide sequence identities in the membrane (M), nucleocapsid (N) and non-structural protein genes (partial polymerase, open reading frames [ORFs] 3 and 7b). The envelope (E) protein gene showed a moderate nucleotide sequence similarity of 91.6%. In contrast, nucleotide and amino acid sequence similarities observed with the spike (S) protein were only 79.5 and 79.6%, respectively. Twenty-one amino acid differences within a 195–199-amino acid C-terminal portion of the S protein were conserved between 3 strains each of FRSCV and FRECV. Both systemic and enteric strains were found to carry a single ORF 3 gene with truncated proteins observed in two out of three FRSCV strains examined. The two enteric strains analyzed each contained an intact ORF 3 gene. Phylogenetically, FRSCV is more closely related to FRECV than to other group 1 coronaviruses.