Immunohistochemical characterization of nonhuman primate ovarian sex cord-stromal tumors.

This study evaluates the immunoreactivity of 12 sex cord-stromal tumors of nonhuman primates (11 granulosa cell tumors and 1 luteoma). The markers selected are used in the characterization of gonadal tumors in dogs and other species, including cytokeratins AE1/AE3, GATA-4, inhibin-α, neuron-specific enolase, protein gene product 9.5, and vimentin. A normal nonhuman primate ovary was used as a control and to optimize immunolabeling. Staining was graded as follows: 0 (nonstaining), 1+ (< 10% positive cells), 2+ (10%-50% positive cells), and 3+ (> 50% positive cells). Calretinin, GATA-4, neuron-specific enolase, and vimentin were the most consistently expressed markers (12 of 12). Cytokeratins AE1/AE3 were also consistently expressed (11 of 12). Inhibin-α and protein gene product 9.5 were expressed in 8 and 10 sex cord-stromal tumors, respectively. Results indicate that immunoreactivity of nonhuman primate sex cord-stromal tumors is similar to that observed in other species and that calretinin, GATA-4, and neuron-specific enolase are the most consistently expressed markers in nonhuman primate sex cord-stromal tumors.

Outbreak of mortality in psittacine birds in a mixed-species aviary associated with Erysipelothrix rhusiopathiae infection.

Erysipelothrix rhusiopathiae septicemia, associated with an increased mortality of captive psittacines in a mixed-species aviary, was diagnosed by histopathology, Gram staining, bacterial culture and sequencing, immunohistochemistry, and real-time polymerase chain reaction (PCR). Over a period of 23 days with no premonitory signs, 2 rainbow lorikeets and an eclectus parrot died. Of these birds, one lorikeet and the eclectus were submitted for necropsy. The main pathologic findings were thrombosis (2/2), bacterial embolism/thromboembolism (2/2), necrotizing hepatitis (2/2), necrohemorrhagic myocarditis (1/2), fibrinohemorrhagic and heterophilic visceral coelomitis (1/2), submandibular necrosuppurative dermatitis with necrotizing vasculitis and bacterial and fungal thromboembolism (1/2), and locally extensive rhabdomyonecrosis with bacterial embolism (1/2). Intralesional bacteria were positive by Gram staining and immunohistochemistry in both cases. E. rhusiopathiae was isolated by routine bacterial culture from the liver of the lorikeet, which was also positive by real-time PCR. This report is one of the rare descriptions of erysipelas in psittacines, and to the authors' knowledge, it appears to be the first in the described species using immunohistochemistry and real-time PCR on avian paraffin-embedded tissues for the diagnosis.

Congestive heart failure in 6 African grey parrots (Psittacus e erithacus).

Six African grey parrots (Psittacus e erithacus) were diagnosed with cardiomyopathy and congestive heart failure based on gross and microscopic findings. Ages ranged from 15 days to 8 years, and 5 of 6 parrots were either neonates or juveniles at the time of diagnosis. Two neonates and 2 juveniles came from the same breeding aviary; the 2 juveniles were born to the same breeding pair. The 2 other parrots were kept as pets. Clinical signs included distention of the coelomic cavity (4 of 6), rales (3 of 6), weakness (4 of 6), bradyarrhythmia (1 of 6), growth retardation (1 of 6), crop stasis (1 of 6), and regurgitation (1 of 6). Three parrots were euthanized and 3 died. Gross findings included cardiomegaly due to biventricular, right-, or left-sided cardiomyopathy (6 of 6); coelomic effusion (6 of 6); whitish or yellow foci in the liver (6 of 6); atrophy of the liver (particularly, the left lobe; 5 of 6); reddened or grey lungs (5 of 6); subcutaneous edema (2 of 6); hydropericardium (1 of 6); and bilateral thyroid gland enlargement (1 of 6). Relevant microscopic findings included passive hepatic congestion (6 of 6) and pulmonary congestion (2 of 6), lymphocytic thyroiditis (2 of 6), and diffuse thyroid follicular hyperplasia (2 of 6). Microscopically, the heart was unremarkable (2 of 6) or had mild lymphocytic myocarditis (2 of 6), mild multifocal cytoplasmic vacuolation of cardiomyocytes (2 of 6), mild lymphocytic myocardial (Purkinje cell) ganglioneuritis (1 of 6), and mild multifocal interstitial fibrosis and nuclear hypertrophy of cardiomyocytes (1 of 6). One parrot had concurrent proventricular dilatation disease (systemic ganglioneuritis). The cause of cardiomyopathy in these parrots was not determined.

First report on natural infection with Leishmania infantum in a domestic ferret (Mustela putorius furo) in Spain.

A pet domestic ferret (Mustela putorius furo) with a papular lesion involving the right pinna was diagnosed with chronic pyogranulomatous dermatitis by histopathologic examination. Intralesional, intracytoplasmic oval microorganisms compatible with Leishmania spp. or Histoplasma spp. were observed in macrophages and multinucleate giant cells. Leishmania infantum (L. infantum) infection was diagnosed by PCR, culture in Novy-MacNeal-Nicolle medium, and immunohistochemistry. Abnormal clinicopathological results included increased alanine transferase, alkaline phosphatase, serum gamma glutamyl transferase and polyclonal gammpathy. Anti-Leishmania antibodies were detected by enzyme-linked immunosorbent assay, immunofluorescence antibody test and western blot using L. infantum antigen. Immunoreactivity against the 16 kDa specific L. infantum antigen fraction was observed by western blot. PCR performed in blood samples obtained from this patient after positive parasite isolation detected L. infantum DNA. To the authors' knowledge, this is the first diagnosis and isolation of L. infantum in a domestic ferret naturally infected in an endemic region (Spain) where canine and feline leishmaniosis is frequently detected. According to these findings, ferrets should be included as potential reservoir hosts of L. infantum. Future investigations should analyze the epidemiological role of ferrets in L. infantum infection including the prevalence of infection.

Pheochromocytoma in six New World primates.

Six New World primates, including 2 golden lion tamarins (Leontopithecus rosalia), 2 cotton-top tamarins (Saguinus o. oedipus), 1 black howler monkey (Alouatta caraya), and 1 black-handed spider monkey (Ateles g. geoffroyi), were diagnosed with unilateral (4/6) or bilateral (1/6) adrenal or extra-adrenal (1/6) pheochromocytoma by light microscopy and immunohistochemical staining for chromogranin A. Overt invasive behavior or metastases were not observed in any primate, and thus these neoplasms were considered benign. All primates either died spontaneously (4/6) or were euthanatized (2/6) as a result of concurrent malignant neoplasia, infection, renal disease, or a combination of several disease processes. Although we did not determine whether these pheochromocytomas were functional, all 6 primates had myocardial fibrosis, and some had arteriosclerosis.

Second case of histoplasmosis in a captive mara (Dolichotis patagonum): pathological findings.

A second case of histoplasmosis in a captive mara (Dolichotis patagonum) from a colony at the wildlife park Africam Safari, Puebla, Mexico, is described, and the mara died with disseminated clinical form of the disease, affecting mostly the large intestine and adrenal. The pathological findings of this case 2 revealed severe granulomatous typhlocolitis and moderate granulomatous gastrohepatic lymphadenitis with numerous yeast-like cells, 2-4 mum in diameter, with a clear halo surrounding them inside the cytoplasm of macrophages, suggesting the parasitic form of Histoplasma capsulatum. Adrenocortical cells had abundant similar microorganisms in their cytoplasm without any associated lesion. Gomori's methenamine silver and periodic acid Schiff stained positively these microorganisms. Immunohistochemistry, using a rabbit anti-H. capsulatum serum, and transmission electron microscopy supported the diagnosis of H. capsulatum infection.

Identification of the source of histoplasmosis infection in two captive maras (Dolichotis patagonum) from the same colony by using molecular and immunologic assays.

Histoplasma capsulatum was isolated from the spleen of a first infected mara (Dolichotis patagonum) and from a second mara's liver and adrenal gland, both in the same colony at the Africam Safari, Puebla, Mexico. Studies of H. capsulatum isolates, using nested-PCR of a 100-kDa protein coding gene (Hcp100) fragment and a two-primer RAPD-PCR method, suggest that these isolates were spreading in the environment of the maras' enclosure. By using a Dot-ELISA method, sera from mice inoculated with three homogenates of soil samples from the maras' enclosed space developed positive brown spot reactions to a purified H. capsulatum antigen, which identified the probable source of the maras' infection.

Pathological findings in a captive colony of maras (Dolichotis patagonum).

This paper describes the causes of death of 54 maras (Dolichotis patagonum) in a captive colony in Mexico over a period of seven years. There were 35 adults, 11 juveniles, five neonates, two fetuses and one stillbirth--27 males, 21 females and six whose sex was not determined. Trauma was the cause of 25 deaths, and there were eight cases of fatal bacterial infection. Besnoitiosis was the only parasitic disease found frequently (13 cases), and was associated with fatal interstitial pneumonia in three juveniles. Right-sided hypertrophic cardiomyopathy attributed to high altitude was observed in 26 maras, and in three cases death was attributed to acute cardiac dysfunction. Two maras died of disseminated histoplasmosis and two of hyperthermia. Additional causes of death included one case each of uterine torsion, intestinal intussusception, aspiration pneumonia and hydranencephaly. Gastric erosions with luminal haemorrhage were found in 27 of the maras and splenic lymphoid depletion in 20, changes that were attributed to stress.

Encephalitozoon hellem infection in aviary passerine and psittacine birds in Spain.

A European goldfinch (Carduelis carduelis), a canary (Serinus canaria), and a lovebird (Agapornis roseicollis) captive-bred at three different private aviaries in Spain were submitted for necropsy with a history of weakness and ruffled feathers, weight loss associated with glossitis, and respiratory disease, respectively. Microscopically, enterocytes in the jejunum and ileum contained colonies of gram- and Stamp-positive, oval to elliptical microorganisms within parasitophorous vacuoles in the apical cytoplasm. Nested PCR using MSP primers that target microsporidian RNA genes produced amplicons of expected size for Encephalitozoon species, and analysis of forward and reverse DNA sequences confirmed the presence of Encephalitozoon hellem in all cases. The main cause of death of all three birds consisted of concurrent infections. However, intestinal encephalitozoonosis may have contributed to exacerbated catabolism. Encephalitozoonosis (or microsporidiosis) has been rarely described in passerine birds.

Outbreak of salmonellosis in a zoologic collection of lorikeets and lories (Trichoglossus, Lorius, and Eos spp.).

During August 2001, a syndrome characterized by acute lethargy and dyspnea was observed in a population of 45 lorikeets and lories in an open-air zoologic exhibit. The first death occurred on August 10, and within the next 12 days, nine more birds died (22% mortality rate). Hepatomegaly, reddening and congestion of the lungs, and injection of the serosal surface of the intestines were the common gross findings. Histologic changes, including fibrinonecrotic hepatitis and splenitis, bacterial emboli (liver, spleen, lung, kidney, proventriculus), pulmonary congestion and hemorrhage, and enteritis, were indicative of an acute, overwhelming bacterial septicemia. Salmonella typhimurium, with the same antibiogram, was isolated from four birds. Several birds had attacked and killed a snake on July 24, and Salmonella serogroup B (untypeable) was isolated from intestine and kidney samples of a garter snake caught in the open-air exhibit on August 28. Salmonella was also isolated from environmental samples of the exhibit but not from food preparation areas. After antimicrobial therapy, Salmonella spp. was not isolated from the surviving birds. The source of Salmonella in this outbreak remains unknown, but infection either directly or indirectly from snakes in the exhibit is possible. Contact between captive psittacine populations and reptiles should be avoided to prevent the risk of salmonellosis.

Hepatic sarcocystosis in a striped dolphin (Stenella coeruleoalba) from the Spanish Mediterranean coast.

Fatal hepatic sarcocystosis was diagnosed in a striped dolphin (Stenella coeruleoalba) from the northeastern Spanish Mediterranean coast based on pathologic findings and the microscopic and ultrastructural characteristics of the intralesional parasite. Main gross lesions were icterus, subcutaneous hemorrhages, and hepatic congestion. The most prominent microscopic lesions consisted of severe acute multifocal to coalescing necrotizing hepatitis with cholestasis and intralesional protozoa. There was severe chronic pancreatitis with generalized distension of pancreatic ducts by hyaline plugs and adult trematodes. Only asexual stages of the protozoa were found. The parasite in the liver divided by endopolygeny. Schizonts varied in shape and size. Mature schizonts had merozoites randomly arranged or budding peripherally around a central residual body. Schizonts were up to 22 microm long, and merozoites were up to 6 microm long. Ultrastructurally, merozoites lacked rhoptries. This parasite failed to react by immunohistochemistry with anti-Toxoplasma gondii, anti-Neospora caninum and anti-Sarcocystis neurona antibodies. The microscopic and ultrastructural morphologies of the parasite were consistent with Sarcocystis canis, so far described only from animals in the Unites States. The life cycle and source of S. canis are unknown. The present report of S. canis-like infection in a sea mammal from Spain indicates that the definitive host for this parasite also exists outside of the United States.

Disseminated toxoplasmosis in a Mediterranean pregnant Risso's dolphin (Grampus griseus) with transplacental fetal infection.

Fatal disseminated toxoplasmosis was diagnosed in a Risso's dolphin (Grampus griseus) dam and its fetus on the basis of pathologic findings, immunohistochemistry, and structure of the parasite. The dolphin was stranded alive on the Spanish Mediterranean coast and died a few hours later. At necropsy the dam was in good condition. From the standpoint of pathology, however, it had generalized lymphadenomegaly and splenomegaly, enlargement of and multifocal hemorrhage in the adrenal glands, diffuse mucosal hemorrhage of the glandular and pyloric stomach, ulcerative glossitis and stomatitis, focal erosions and reddening of the laryngeal appendix, and severe paraotic sinusitis with intralesional nematodes Crassicauda grampicola. The dolphin was pregnant, most probably in the first gestational trimester. The most prominent microscopic lesions were multifocal granulomatous encephalomyelitis, diffuse subacute interstitial pneumonia, mild multifocal necrotizing hepatitis and nonsuppurative cholangiohepatitis, gastritis and adrenalitis, mild lymphoid depletion, medullary sinus and follicular histyocitosis, and systemic hemosiderosis. The fetus had foci of coagulative and lytic necrosis in the kidneys, the lung, and the heart. Most lesions were associated with tachyzoites and tissue cysts of Toxoplasma gondii. The diagnosis was confirmed immunohistochemically. This is the first report on toxoplasmosis in a Risso's dolphin (G. griseus) and on transplacental transmission to an early-stage fetus in any cetaceans.

Antioxidant status in a squirrel monkey (Saimiri sciureus) with chronic pancreatitis and degenerative myopathy.

A squirrel monkey (Saimiri sciureus) presented with wasting, vomiting and diarrhoea. Haematology revealed elevation of creatinine phosphokinase, lactic dehydrogenase, alanine aminotransferase, amylase and lipase, together with azotaemia and hypoalbuminaemia. Prominent findings were chronic pancreatitis with acinar and ductal plugs, granulomatous and necrotizing peripancreatic steatitis, degenerative myopathy, testicular atrophy, candidiasis and bacterial necrotizing glossitis. Antioxidant analyses revealed low concentrations of serum vitamin E (and apparently A), hepatic selenium and hair zinc. Pancreatitis may have caused malabsorption and maldigestion, associated with deficiency of multiple antioxidants.

Fatal acute toxoplasmosis in three golden lion tamarins (Leontopithecus rosalia).

Fatal acute toxoplasmosis was diagnosed in three golden lion tamarins (Leontopithecus rosalia), a breeding pair and their male offspring, by histology and immunohistochemistry. The distribution and severity of lesions differed among the animals, but the small intestine and the pancreaticoduodenal lymph nodes were especially affected. Protozoal organisms consistent with Toxoplasma gondii tachyzoites, often clustered, were seen in all lesions and were specifically immunostained with a T. gondii polyclonal antibody. The infection was probably acquired orally. Several breeding groups of golden lion tamarins have succumbed to toxoplasmosis both in North American and European zoos, so this disease should be considered an important problem in this endangered species. Toxoplasma gondii can cause latent infections in New World primates and therefore could potentially induce abortions or congenital infections in wild golden lion tamarins born from latently infected reintroduced females.

Actinomycotic splenitis and intestinal volvulus in an alpaca (Lama pacos).

Morphologic, microbiologic, and polymerase chain reaction amplification techniques were used to evaluate an alpaca (Lama pacos) with splenitis and intestinal volvulus. The intestinal volvulus produced a severe necrosuppurative typhlocolitis associated with vascular thrombosis and was most likely the cause of death of this animal. In addition, this animal had multiple coalescing abscesses affecting most of the splenic tissue. The isolation of Actinomyces spp. from the spleen and the morphology of the colonies when stained with Gram and Steiner stains support a diagnosis of splenic actinomycosis.

Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 µm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification.