Animal models of orthopoxvirus infection.

Smallpox was one of the most devastating diseases known to humanity. Although smallpox was eradicated through a historically successful vaccination campaign, there is concern in the global community that either Variola virus (VARV), the causative agent of smallpox, or another species of Orthopoxvirus could be used as agents of bioterrorism. Therefore, development of countermeasures to Orthopoxvirus infection is a crucial focus in biodefense research, and these efforts rely on the use of various animal models. Smallpox typically presented as a generalized pustular rash with 30 to 40% mortality, and although smallpox-like syndromes can be induced in cynomolgus macaques with VARV, research with this virus is highly restricted; therefore, animal models with other orthopoxviruses have been investigated. Monkeypox virus causes a generalized vesiculopustular rash in rhesus and cynomolgus macaques and induces fatal systemic disease in several rodent species. Ectromelia virus has been extensively studied in mice as a model of orthopoxviral infection in its natural host. Intranasal inoculation of mice with some strains of vaccinia virus produces fatal bronchopneumonia, as does aerosol or intranasal inoculation of mice with cowpox virus. Rabbitpox virus causes pneumonia and fatal systemic infections in rabbits and can be naturally transmitted between rabbits by an aerosol route similar to that of VARV in humans. No single animal model recapitulates all known aspects of human Orthopoxvirus infections, and each model has its advantages and disadvantages. This article provides a brief review of the Orthopoxvirus diseases of humans and the key pathologic features of animal models of Orthopoxvirus infections.

Paramyxovirus infection in caiman lizards (Draecena guianensis).

Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were imported into the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizards demonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus that was consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolate of ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity within pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards were placed in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later, syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similar syncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracytoplasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundant filamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed in culture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples were collected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determine presence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of < or =1:20 and 10 had titers of >1:20 and < or =1:80. This report is only the second of a paramyxovirus identified in a lizard and is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.

Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs.

In a series of three experiments during March-October, 1998, two species of captive-bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Microscopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batrachochytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians.

Subcutaneous atypical mycobacteriosis in captive tiger quolls (Dasyurus maculatus).

From July 1989 to October 1998, 9/37 (24%) adult captive tiger quolls (Dasyurus maculatus) were diagnosed with atypical mycobacterial infection involving the subcutis and skin. Females were more often affected than males (seven females, two males). Grossly, lesions presented as focal thickenings, plaques, and abscesses within the subcutis, often with fistulous tracts. The subcutis and skin overlying cervical and thoracic regions were the primary sites of infection. Cytology of subcutaneous impression smears from all nine affected tiger quolls revealed pyogranulomatous inflammation admixed with several acid-fast bacilli. Histologically, all tiger quolls had nodular to diffuse pyogranulomatous panniculitis and cellulitis. Small numbers of acid-fast bacilli were noted histologically in 7/9 (78%) animals. Skin cultures from seven tiger quolls were positive for one or more different Runyon group IV mycobacteria. The disease described in these tiger quolls is similar to subcutaneous atypical mycobacteriosis of humans and domestic animals.

Amphibian respiratory diseases.

This article describes the diseases that commonly affect the lungs, gills, and nasal passages of frogs, toads, and salamanders. Although these organs often are affected by systemic infections, only diseases that are limited to the respiratory system are discussed. Gills of aquatic amphibians commonly are affected by poor water quality, infestations by protozoa or trematodes, or infections by water molds. Nematodes in the genus Rhabias are significant pulmonary pathogens in frogs and toads. The nasal passages of frogs, toads, and salamanders may be parasitized by Bufolucilia fly larvae.

Isolation and experimental transmission of a reovirus pathogenic in ratsnakes (Elaphe species).

A reovirus was isolated from juvenile Moellendorff's ratsnakes (Elaphe moellendorffi) and beauty snakes (Elaphe taenuris) that died soon after importation into the USA. Viper heart (VH2) cells inoculated with tissue homogenates showed cytopathic effects consisting of large syncytia formation followed by cell detachment from the monolayer. Tissue culture supernatants failed to hemagglutinate guinea pig and chicken erythrocytes at room temperature. Electron microscopy of purified virions revealed spherical to icosahedral particles measuring 70-85 nm in diameter with a double capsid layer. Preparations of the viral genome contained ten segments of dsRNA when analyzed by polyacrylamide gel electrophoresis. A juvenile black ratsnake (Elaphe obsoleta obsoleta) was experimentally inoculated with the isolate and was found dead 26 days post inoculation. Necropsy revealed diffuse subacute interstitial pneumonia with respiratory epithelial cell hyperplasia and syncytia. Reovirus isolated from this snake was used to inoculate another juvenile black ratsnake which was euthanized 40 days post inoculation. Pneumonia and multifocal subacute proliferative tracheitis were found on necropsy. Reovirus was isolated from the lung of this snake and was demonstrated by transmission electron microscopy. This is the first documentation of a pathogenic reptile reovirus and the first report of experimental transmission of a reovirus in snakes.

Fatal mycotic dermatitis in captive brown tree snakes (Boiga irregularis).

Cutaneous fungal infections occurred in four captive brown tree snakes (Boiga irregularis). The ventral scales were most commonly affected, and lesions began as areas of erythema and edema with vesicle formation, followed by development of caseous brown plaques. Lesions usually started where ventral scales overlapped and spread rapidly. All snakes died within 14 days after clinical signs were first noted. The deaths of three of the snakes were directly attributable to the cutaneous disease; the other snake died from renal failure and visceral gout, most likely induced by gentamicin therapy. Histologically, lesions consisted of epidermal hyperplasia and hyperkeratosis, with foci of epidermal necrosis, intraepidermal vesicle formation, and subacute inflammation of the underlying dermis. These lesions were associated with bacteria and numerous septate, branched fungal hyphae within the epidermis and overlying serocelluar crusts. Hyphae that penetrated through the superficial surface of the epidermis often formed terminal arthroconidia. The same species of fungus was isolated in pure culture from the skin of three snakes, but fungal cultures were not performed on samples from the fourth snake. The fungus has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii based on its formation of solitary dermatophytelike aleurioconidia and alternate and fission arthroconidia. The source of the fungus in this outbreak was not determined; however, the warm, moist conditions under which the snakes were housed likely predisposed them to opportunistic cutaneous fungal infections.

Neoplasia in snakes at the National Zoological Park, Washington, DC (1978-1997).

Of 291 juvenile and adult snakes examined post mortem over a 20-year period (1978-1997) at the National Zoological Park (NZP) in Washington, DC, 36 (24 females and 12 males) had neoplasms. Two snakes had tumours of two or three different types, but the other 34 snakes had only one type. All affected animals were adults and their average time on exhibit at the NZP was 108-9 months. Malignant neoplasms (79.5%) outnumbered benign neoplasms (20.5%). Of the malignant tumours, 19 (61.3%) were considered to have arisen in mesenchymal tissues, 11 (35.5%) were of epithelial origin, and one (3.2%) was derived from neuroectodermal tissues. All the benign neoplasms were of epithelial origin. Neoplasms of the lymphoid and haematopoietic tissues were the most common (12 cases), followed by tumours of the liver and biliary tract (seven cases) and the gastrointestinal tract (four cases).

Reference hematologic and plasma chemistry values of brown tree snakes (Boiga irregularis).

Reference hematologic and plasma chemistry values were determined from 103 blood samples collected from 53 clinically healthy brown tree snakes (Boiga irregularis). Female snakes had significantly higher mean plasma values for total solids, total protein, calcium (Ca), phosphorus (P), uric acid, and blood monocyte percentage than did males, whereas males had significantly higher mean plasma fibrinogen values. The variances for hematocrit, monocyte percentage, azurophil percentage, plasma total solids, plasma total protein, albumin, Ca, and P also differed significantly between sexes. The higher mean values and greater variances for plasma total protein, plasma total solids, Ca, and P in the female snakes were probably associated with yolk synthesis and accumulation.

Oscars, Astronotus ocellatus, have a dietary requirement for vitamin C.

We found that vitamin C is an essential nutrient for an Amazonian ornamental fish, the oscar (Astronotus ocellatus). This was demonstrated by the absence of L-gulonolactone oxidase activity, the enzyme responsible for the biosynthesis of vitamin C, in liver or kidney of oscars and by a feeding trial in which oscars without vitamin C dietary supplementation developed clinical deficiency signs. Fish weighing 29.2 +/- 1.9 g were divided into four groups, and each group was fed a casein-based semipurified diet containing 0, 25, 75 or 200 mg ascorbic acid equivalent (AA)/kg diet for 26 wk. Vitamin C was supplemented in the diets as L-ascorbyl-2-polyphosphate, a mixture of phosphate esters of ascorbate, which is more stable to oxidation than AA. At the end of 26 wk, fish fed no AA had significantly lower weight gain than fish fed the AA-supplemented diets (P < 0.05). Oscars without dietary AA supplementation gained only 37% of their initial weight, compared with 112, 102 and 91% gained by fish fed 25, 75 and 200 mg AA/kg diet, respectively. After 25 wk without dietary supplementation of AA, fish began to develop clinical deficiency signs, including deformed opercula and jaws, hemorrhage in the eyes and fins, and lordosis. Histology indicated that fish without AA supplementation had deformed gill filament support cartilage and atrophied muscle fibers. Collagen content of the vertebral column was significantly lower in fish devoid of dietary AA (P < 0.05). Liver AA concentration varied in proportion to dietary concentration of AA. The minimum dietary AA concentration tested in this study, 25 mg AA/kg diet, was sufficient to prevent growth reduction and AA deficiency signs in oscars.

Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs (Acris crepitans).

Cricket frogs (Acris crepitans) from several different sites in Illinois were collected to assess the effects of environmental contamination on the prevalence of intersex gonads. Of 341 frogs collected in 1993, 1994, and 1995, 2.7% were intersex individuals. There was no statistically significant relationship between the chemical compounds detected and cricket frog intersexuality. However, there was an association approaching significance (p = 0.07) between the detection of atrazine and intersex individuals. A comparison of reference sites with sites that had point polychlorinated biphenyl (PCB) and polychlorinated dibenzofuran (PCDF) contamination revealed a significant relationship between sex-ratio reversal and contamination with PCBs and PCDFs. The sex ratio of juvenile frogs studied from three sites with PCB and PCDF point contamination favored males over females, which was the opposite of the sex ratio in control ponds (p = 0.0007). The statistically significant correlation between organochlorine contamination and sex-ratio reversal suggests PCBs and PCDFs can influence cricket frog sexual differentiation. The current study suggests that in cricket frogs, sex ratios and the prevalence of intersex gonads are altered by environmental contamination.

Cryptococcosis in tree shrews (Tupaia tana and Tupaia minor) and elephant shrews (Macroscelides proboscides).

Fungal infections due to Cryptococcus neoformans occurred in seven short-eared elephant shrews (Macroscelides proboscides), six large tree shrews (Tupaia tana), and five lesser tree shrews (Tupaia minor) at the National Zoological Park during a 30-mo period in 1991-1993. Clinical signs were absent or included weight loss, shivering, dyspnea, and/or neurologic disease. Definitive antemortem diagnostic techniques included tracheal lavage and serum cryptococcal antigen latex agglutination titers. Thirteen cases were diagnosed solely by postmortem examination. The source of infection for these animals was uncertain, but C. neoformans is commonly found in soil and other organic material. Two lesser tree shrews and one large tree shrew received antifungal therapy and converted to a negative serum cryptococcal antigen titer.

Melanotic ependymoma in a Goeldi's marmoset (Callimico goeldii).

A spontaneous melanotic ependymoma was observed in the brain of an adult female Goeldi's marmoset (Callimico goeldii). The mass completely occupied the left lateral ventricle, rupturing the fornix and corpus callosum, and compressing the adjacent neuropil. Special histochemical techniques, including melanin bleach, periodic acid-Schiff, Perls iron and phosphotungstic acid hematoxylin, demonstrated the neoplasms to be an ependymoma with a rare melanotic differentiation.

Eosinophilic meningoencephalitis due to Angiostrongylus cantonensis as the cause of death in captive non-human primates.

Fatal eosinophilic meningoencephalitis due to Angiostrongylus cantonensis is reported in captive non-human primates. A howler monkey (Alouatta caraya) at the Audubon Park and Zoological Gardens, New Orleans, LA, died 21 days after initial clinical symptoms. A white-handed gibbon (Hylobates lar) died at the Ardastra Gardens and Zoo, Nassau, Bahamas, 17 days after onset of symptoms. Both had access to free-ranging gastropods within the zoos. These are the first reported cases of natural infection by A. cantonensis in non-human primates in the western hemisphere.

Parelaphostrongylus tenuis in captive reindeer and sable antelope.

Parelaphostrongylus tenuis caused neurologic disease in 6 reindeer (Rangifer tarandus tarandus) and 2 sable antelope (Hippotragus niger) that were housed at the National Zoological Park Conservation and Research Center in Front Royal, Va. Progressive hindlimb ataxia and weakness were seen in all affected animals. The parasite was recovered from the subdural space of one reindeer and was seen histologically within the neuropil of another reindeer and a sable antelope. In the other reindeer and sable antelope, diagnoses of parelaphostrongylosis were based on clinicoepidemiologic findings and microscopic lesions. Because parelaphostrongylosis is a density-dependent disease and because the number of white-tailed deer probably will increase, the incidence of P tenuis infections in domestic and nondomestic animals probably will increase as well. Zoos and wildlife parks especially should be cautious when introducing exotic hoofstock to areas with Parelaphostrongylus tenuis-infected white-tailed deer.

Babesia odocoilei Emerson and Wright, 1970 in white-tailed deer, Odocoileus virginianus (Zimmermann), in Virginia.

Pooled blood samples from six white-tailed deer from the Great Dismal Swamp in Virginia were inoculated into two splenectomized deer. A moderately severe clinical reaction ensued, characterized by a hemolytic anemia, and a Babesia found in both recipient animals was presumptively identified as B. odocoilei. This is the first reported identification of this parasite in white-tailed deer in Virginia.