Meningoencephalitis in a polar bear caused by equine herpesvirus 9 (EHV-9).

A 12-year-old female polar bear (Ursus maritimus) developed a sudden onset of muscle tremors, erratic circling, increased blinking, head shaking, and ptyalism, which progressed to partial and generalized seizures. Ancillary diagnostic tests were inconclusive, and the only significant laboratory finding was nonsuppurative pleocytosis of cerebrospinal fluid. Euthanasia was elected. Microscopic evaluation demonstrated multifocal, random nonsuppurative meningoencephalitis involving most prominently the rostral cerebral cortex, as well as the thalamus, midbrain, and rostral medulla. Lesions consisted of inflammation, neuronal necrosis, gliosis, and both neuronal and glial basophilic intranuclear inclusion bodies. Immunohistochemistry with a polyclonal antibody reactive to several equine herpesviruses was positive within affected areas of the brain, and polymerase chain reaction conclusively demonstrated the presence of only equine herpesvirus 9. The clinical and morphologic features of this case resemble other fatal herpesvirus encephalitides derived from interspecies transmission and underscore the need for extreme caution when managing wild or captive equids.

Histologic, immunohistochemical, and electron microscopic features of a unique pulmonary tumor in cockatiels (Nymphicus hollandicus): six cases.

A unique form of pulmonary malignancy develops in cockatiels. This report describes the gross, histologic, electron microscopic, and immunohistochemical features of this tumor in 6 cockatiels. DNA in-situ hybridization for polyomavirus in the neoplasm was also performed. The tumor was comprised predominantly of compact sheets of anaplastic round to polygonal cells. All tumors had a high mitotic index, and had occasional large clear to slightly basophilic intranuclear inclusions that caused peripheral dispersal or complete masking of chromatin. Tumors were invasive but convincing metastases were not observed. Transmission electron microscopy identified intracytoplasmic intermediate filaments, desmosomes between cells, and intranuclear cytoplasmic invaginations corresponding to the intranuclear inclusions in light microscopic sections. Neoplastic cells stained positive for vimentin, lysozyme, and in 1 bird, pan cytokeratin. All 6 pulmonary neoplasms were negative for avian polyomavirus using the FN-19/FN-40 cocktail and the long VP-1 probe. We propose that these tumors may be poorly differentiated carcinomas of pulmonary or thymic origin.

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.

Cluster of cases of malignant schwannoma in cattle.

Between 1998 and 2001, several cases of ataxia and paresis followed by recumbency and death were reported in cows from different farms in a restricted area of the Argentinian Patagonia. Five cases of this cluster were studied and a diagnosis of malignant schwannoma was established. Electron microscopy (em) of tumour samples from three of the animals revealed intracytoplasmic or interstitial structures resembling retroviral particles. Attempts to isolate a viral agent from the tumours were unsuccessful but the epidemiological data and the em findings suggest a viral aetiology.

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.

Fatal coxiellosis in Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus).

Three Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus), ranging from 6 to 8 months of age, presented with lethargy, emaciation, and progressive neurologic signs. The first one died 24 hours after the onset of clinical signs, and the other two were euthanized 10 to 14 days after the onset of progressive neurologic disease. Clinical signs in these lorikeets included head pressing, hemiparesis, seizures, obtunded mentation, weakness, and lethargy. Two of the lorikeets had hepatomegaly, and one had splenomegaly on gross examination. Histopathology revealed disseminated microgranulomas in the liver, spleen, and brain, and lymphohistocytic perivascular encephalitis and cephalic vasculitis. Electron microscopic examination of macrophages in brain lesions revealed spherical to rod-shaped prokaryotic organisms with a trilaminar cell wall. Molecular analysis revealed a novel species of Coxiella. This is believed to be the first report of a Coxiella sp. causing disease in a lorikeet.

Renal myxozoanosis in weedy sea dragons, Phyllopteryx taeniolatus (Lacepède), caused by Sinuolinea phyllopteryxa n. sp.

Renal myxozoanosis was diagnosed histologically in 11 captive, wild caught, adult weedy (common) sea dragons, Phyllopteryx taeniolatus, from three separate public aquaria in the United States. Myxozoan spores were visible in wet mounts of kidney tissue and were associated with renal tubular dilatation and tubular epithelial cell hypertrophy. Light and electron microscopy revealed spore morphology consistent with the genus Sinuolinea. Spores were spheroidal, slightly dorso-ventrally compressed, length (L) 17.1 x width (W) 16.4 x thickness (T) 15.6 microm, with two shell valves joined at a distinct, sinuous sutural ridge, and had two nearly spherical polar capsules, L 5.5 x W 5.0 microm, with five to seven turns of the polar filament. There were no extra-valvular ridges or protrusions. DNA sequencing required the design of three new primers that yielded 1740 bp of 18S ribosomal DNA sequence. The parasite was determined to be novel based on morphological and molecular data, and was given the name Sinuolinea phyllopteryxa after its vertebrate host.

Myofasciitis in the domestic ferret.

Since late 2003, an inflammatory disease of muscle and fascia has been diagnosed in several ferrets at Northwest ZooPath, and this report describes the condition in 17 ferrets. It is a disease of young ferrets, characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure, and death (or euthanasia). Gross lesions include atrophy of skeletal muscle; red and white mottling and dilatation of the esophagus; and splenomegaly. Histologically, moderate to severe suppurative to pyogranulomatous inflammation is in the skeletal muscle and the fascia at multiple sites, including esophagus, heart, limbs, body wall, head, and lumbar regions. Myeloid hyperplasia of spleen and/or bone marrow also is a prominent feature. Ultrastructural lesions include mitochondrial swelling, intracellular edema, disruption of myofibrils and Z bands. Bacterial and viral cultures, electron microscopy, immunohistochemistry, and polymerase chain reaction were negative for a variety of infectious agents. The clinical presentation and distribution of lesions suggests that polymyositis in domestic ferrets is likely a distinct entity. The etiopathogenesis if this condition is not known.

Disseminated encephalitozoonosis in captive, juvenile, cotton-top (Saguinus oedipus) and neonatal emperor (Saguinus imperator) tamarins in North America.

Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.

Renal myxozoanosis in crowned river turtles Hardella thurjii: description of the putative agent Myxidium hardella n. sp. by histopathology, electron microscopy, and DNA sequencing.

Chelonian myxozoanosis is rarely reported and has previously not been documented to cause disease. This report describes myxozoanosis associated with significant renal disease in two Crowned River turtles (Hardella thurjii). One turtle presented with emaciation and died. The cage mate presented with emaciation and was euthanized. Histologically, renal intratubular myxozoan spores were associated with renal tubular necrosis, tubular mineralization, and chronic interstitial nephritis, with membranoproliferative and mes-angioproliferative glomerulopathy. Both turtles also had disseminated metastatic mineralization. On the basis of these findings, chronic renal insufficiency from myxozoanosis and subsequent metastatic mineralization were considered the primary problems. By light and electron microscopy, the myxozoan spores had features of the genus Myxidium. Maximum parsimony analysis of small-subunit rDNA sequences placed the turtle myxozoan basal to a clade containing Myxidium truttae and a Myxidium sp. with strong bootstrap support. This myxozoan agent appears to be a significant pathogen in H. thurjii on the basis of morphologic changes in the kidneys of in the infected turtles.

Comparison of the 5.8S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity.

Sequence analysis of the 5.8S rRNA gene and the internal transcribed spacer regions (ITSRs) was used to compare trichomonadid protozoa (n = 39) of varying morphologies isolated from the bovine preputial cavity. A multiple sequence alignment was performed with bovine isolate sequences and other trichomonadid protozoa sequences available in GenBank. As a group, Tritrichomonasfoetus isolates (n = 7) had nearly complete homology. A similarity matrix showed low homology between the T. foetus isolates and other trichomonads recovered from cattle (<70%). Two clusters of trichomonads other than T. foetus were identified. Eighteen isolates comprised 1 group. These isolates shared >99% homology among themselves and with Pentatrichomonas hominis. The other non-T. foetus cluster (n = 14) did not exhibit a high degree of homology (<87%) with other bovine isolates or any of the trichomonad sequences available in GenBank. The sequence homology among isolates in that cluster was >99%, except for 1 isolate that varied from the others in both ITSRs (approximately 2% dissimilarity). Sequence analysis of the 5.8S rRNA gene and ITSRs was useful for comparing trichomonadid protozoa isolated from the bovine preputial cavity and demonstrated that 2 distinct types of trichomonads constituted the non-T. foetus isolates recovered from the bovine preputial cavity.

Equine abortion associated with the Borrelia parkeri-B. turicatae tick-borne relapsing fever spirochete group.

Direct amplification and sequencing of the 16S rRNA gene and a variable region of the flagellin gene from fetal liver-associated spirochetes belonging to the Borrelia parkeri-B. turicatae tick-borne relapsing fever spirochete group with a late-term abortion in a mare are described.

Experimental adenovirus hemorrhagic disease in white-tailed deer fawns.

Infection with a newly described endotheliotropic adenovirus was the cause of a 1993 epizootic reminiscent of hemorrhagic disease in California mule deer (Odocoileus hemionus columbianus and O. hemionus hemionus). Pulmonary edema and intestinal luminal hemorrhage, or necrotizing stomatitis associated with systemic or localized vasculitis, respectively, were common lesions seen in animals that died during the epizootic. In order to determine if white-tailed deer (Odocoileus virginianus) also are susceptible to infection and fatal disease with the deer adenovirus, eight white-tailed deer fawns (4- to 6-mo-old) were inoculated with purified deer adenovirus. Four were inoculated intravenously and four were inoculated through the mucous membranes. Seven days post-inoculation, one of the fawns inoculated intravenously died. Pulmonary edema and hemorrhagic enteropathy were associated with pulmonary and intestinal vasculitis with systemic multiorgan distribution of endotheliotropic adenovirus as demonstrated by transmission electron microscopy and immunohistochemistry. Adenovirus was reisolated from lung homogenates of the fawn that died of adenovirus hemorrhagic disease.

A disease resembling inclusion body disease of boid snakes in captive palm vipers (Bothriechis marchi).

Between April 1998 and June 1999, 8 palm vipers (Bothriechis marchi) were diagnosed with a disease similar to inclusion body disease (IBD) of boids. Six palm vipers were captive bred, and 2 were wild caught. All of the vipers were adults at the time of death. Three palm vipers were found dead with no premonitory clinical signs, and 5 had anorexia plus possibly 1 of the following clinical signs: regurgitation, paresis, and dehydration. Histologically, all snakes had intracytoplasmic, round to oval, single to multiple eosinophilic inclusion bodies in hepatocytes and renal tubular epithelial cells. Inclusion bodies were distributed among other organs with varying frequency. Common concurrent histologic lesions were urate nephrosis, septic thrombi, and hepatocellular degeneration. Ultrastructurally, inclusions had features similar to inclusions in boid snakes with IBD.

Morphological and molecular characterization of Encephalitozoon hellem in hummingbirds.

Microsporidiosis was identified as a cause of enteritis in wild, migratory hummingbirds (Calypte anna). Electron microscopic examinations of parasites showed microsporidian spores with a double spore coat and a polar filament containing four to six coils, compatible with the genus Encephalitozoon. Molecular analysis of ribosomal RNA genes further identified the parasites from droppings and small intestinal segments as Encephalitozoon hellem, genotype I. Microsporidial spores were identified in 19% of droppings from C. anna, Archilochus alexandri and Selasporus sasin using Gram or modified trichrome staining methods. Since E. hellem is an opportunistic pathogen in immunocompromised humans, the pathogenic potential in avian hosts, the zoonotic potential of this parasite, and the role of birds as reservoirs needs to be further explored.

A Herpesvirus Associated with Mass Mortality of Juvenile and Adult Koi, a Strain of Common Carp.

A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks-one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney-spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80-100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.

Lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns.

Adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California during the latter half of 1993. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. To study transmission of adenovirus infection in deer and susceptibility of black-tailed deer (Odocoileus hemionus columbianus) fawns to adenovirus infection, six 3-6-month-old black-tailed fawns were divided into two treatment groups. One group was inoculated intravenously and the other group was inoculated through the mucous membranes of the eyes, nose and mouth with purified adenovirus. Each treatment group also included two additional fawns (four total) that were not inoculated but were exposed to inoculated animals (contact animals). One fawn served as a negative control. Between 4 and 16 days postinoculation, 8/10 fawns developed systemic or localized infection with lesions identical to lesions seen in animals with natural disease that died during the epizootic. Transmission was by direct contact, and the route of inoculation did not affect the incubation period or the distribution of the virus (systemic or the localized infection). Immunohistochemical analysis using polyclonal antiserum against bovine adenovirus type 5 demonstrated staining in endothelial cells of vessels in numerous tissues in animals with systemic infection and endothelial staining only in vessels subtending necrotic foci in the upper alimentary tract in animals with the localized form of the disease. All inoculated or exposed animals had staining in the tonsillar epithelium. Transmission electron microscopic examination of lung and ileum from two fawns with pulmonary edema and hemorrhagic enteropathy demonstrated endothelial necrosis and adenovirus virions in endothelial cell nuclei. Adenovirus was reisolated in black-tailed deer pulmonary artery endothelial cells using lung homogenate of the first fawn that developed systemic adenovirus infection. Serum virus neutralization test results suggest that this deer adenovirus is a new serotype.

Experimental adenovirus hemorrhagic disease in yearling black-tailed deer.

An apparently novel adenovirus was associated with an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California (USA) during 1993-1994. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. Six black-tailed yearling deer (O. hemionus columbianus) were inoculated with purified adenovirus isolated from a black-tailed fawn that died of acute adenovirus hemorrhagic disease during the epizootic. Three of six inoculated deer also received intramuscular injections of dexamethasone sodium phosphate every 3 days during the study. Eight days post-inoculation, one deer (without dexamethasone) developed bloody diarrhea and died. Necropsy and histopathologic findings were identical to lesions in free-ranging animals that died of the natural disease. Hemorrhagic enteropathy and pulmonary edema were the significant necropsy findings and there was microscopic vascular damage and endothelial intranuclear inclusion bodies in the vessels of the intestines and lungs. Adenovirus was identified in necrotic endothelial cells in the lungs by fluorescent antibody staining, immunohistochemistry and by transmission electron microscopy. Adenovirus was reisolated from tissues of the animal that died of experimental adenovirus hemorrhagic disease. Similar gross and microscopic lesions were absent in four of six adenovirus-inoculated deer and in the negative control animal which were necropsied at variable intervals during the 14 wk study. One deer was inoculated with purified adenovirus a second time, 12 wk after the first inoculation. Fifteen days after the second inoculation, this deer developed severe ulceration of the tongue, pharynx and rumen and necrotizing osteomyelitis of the mandible which was associated with vasculitis and thrombosis of adjacent large vessels and endothelial intranuclear inclusions. Transmission electron microscopy demonstrated adenovirus within the nuclei of vascular cells and immunohistochemistry demonstrated adenovirus antigen within tonsilar epithelium and in rare vessels.

Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique.

A protocol for routine 4-hour microwave tissue processing of clinical or other samples for electron microscopy was developed. Specimens are processed by using a temperature-restrictive probe that can be set to automatically cycle the magnetron to maintain any designated temperature restriction (temperature maximum). In addition, specimen processing during fixation is performed in 1.7-ml microcentrifuge tubes followed by subsequent processing in flow-through baskets. Quality control is made possible during each step through the addition of an RS232 port to the microwave, allowing direct connection of the microwave oven to any personal computer. The software provided with the temperature probe enables the user to monitor time and temperature on a real-time basis. Tissue specimens, goat placenta, mouse liver, mouse kidney, and deer esophagus were processed by conventional and microwave techniques in this study. In all instances, the results for the microwave-processed samples were equal to or better than those achieved by routine processing techniques.