Epizootic cutaneous pythiosis in beef calves

Enzootic bovine granulomatosis (EBG) is a disease that affects beef calves in the flooded Savanna regions of Venezuela. Although Pythium insidiosum was originally the suspected etiologic agent, nothing was done to demonstrate its presence in the infected animals. The objective of this study was to investigate the etiologic agent causing cutaneous lesions in a group of 63 calves diagnosed with EBG. The collected samples were analyzed by histopathology, immunohistochemistry, serology, and mycological techniques. The clinical manifestations of BEG included ulcerated and swollen granulomatous lesions on the limbs of young calves. Histopathological studies revealed an eosinophilic inflammatory reaction with neutrophils, giant cells and branching hyphae with the Splendore-Hoeppli phenomenon. Immunofluorescence analysis identified the branching hyphae as elements of the fungal-like organism P. insidiosum. Wet mounts in 10% KOH revealed hyaline slender branching hyaline coenocytic hyphae, but cultures from those samples were negative. Anti-P. insidiosum immunoglobulins were also demonstrated in the collected sera of the affected bovines using latex agglutination, immunodiffusion, ELISA, and Western Blot. The histopathological, immunohistochemical, and serological tests showed for the first time that P. insidiosum could be involved in epizootic bovine pythiosis.     

Cutaneous pythiosis in a nestling white-faced ibis

A nestling white-faced ibis (Plegadis chihi) with multifocal skin ulcerations on the wings, neck, head, and limbs was found in a wetland agricultural region of the central valley in California. Pathologic, microbiologic, and molecular findings were consistent with restricted, cutaneous infection by the oomycete Pythium insidiosum. The microscopic features of the disease, including intense, necrotizing eosinophilic and granulomatous inflammation, are similar to those previously described in mammals. Pythiosis, which is most typical in tropical and subtropical climates, has recently emerged in California as a cause of cutaneous and enteric disease in horses and dogs, respectively. Environmental stability and persistence of a "water-mold" in the arid central valley of California could be associated with agricultural and community watering practices. To the best of the authors' knowledge, this is the first published report of pythiosis in birds.     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

Esophagitis Due to Pythium insidiosum Infection in Two Dogs

Chronic esophagitis due to Pythium insidiosum infection caused weight loss, dysphagia, and hypersalivation in 2 dogs from rural Tennessee. Although dog 1 presented for evaluation in December 1984 and dog 2 in October 1992, infection likely occurred during the previous summer by drinking fresh pond water containing zoospores. Hematologic testing revealed eosinophilia and hypergammaglobulinemia in one dog. An etiologic diagnosis was not made until postmortem evaluation when hyphae within necrotic and granulomatous cellular infiltrates in the esophageal wall were identified as P insidiosum by immunoperoxidase staining. Failure to culture the organism from the esophageal wall in dog 2 was attributed to refrigeration of the tissues, since refrigeration decreases the survivability of P insidiosum. J Vet Intern Med 1996;10:139–742. Copyright © 1996 by the American College of Veterinary Internal Medicine .     

Gastrointestinal pythiosis in two cats.

Two young adult male Domestic Shorthair cats living in the southeastern United States were evaluated for signs attributable to partial intestinal obstruction. Physical examination indicated a palpable abdominal mass in each animal. Exploratory laparotomy revealed a large extraluminal mass involving the ileum and mesentery with adjacent mesenteric lymphadenopathy in cat No. 1 and an abscessed mass in the distal duodenum in cat No. 2. Mass resection and intestinal anastomosis were performed in both cats. Histologic evaluation indicated that the intestinal lesions involved primarily the outer smooth muscle layer and serosa and consisted of eosinophilic granulomatous inflammation with multifocal areas of necrosis. In Gomori methenamine silver-stained sections, broad (2.5-7.5 microm), occasionally branching, infrequently septate hyphae were observed within areas of necrosis. A diagnosis of Pythium insidiosum infection was confirmed in both cats by immunoblot serology and by immunoperoxidase staining of tissue sections using a P. insidiosum-specific polyclonal antibody. Cat No. 1 was clinically normal for 4 months after surgery but then died unexpectedly from an unknown cause. Cat No. 2 has been clinically normal for at least 9 months after surgery and appears to be cured on the basis of follow-up enzyme-linked immunosorbent assay serology.     

Canine pythiosis--isolation and identification of Pythium insidiosum

Pythium insidiosum was isolated from the subcutaneous tissue of a 1-year-old tan crossbreed dog and from the intestinal tract of an 18-month-old Samoyed male. Gomori's methenamine silver stain was superior to hematoxylin and eosin in demonstrating the organism in tissue sections. The agent was identified as P. insidiosum by zoospore formation in an aqueous yeast extract solution containing grass blades. Exoantigens produced in culture were shown to be identical to known P. insidiosum antigens by microimmunodiffusion.     

Duodenal obstruction caused by infection with Pythium insidiosum in a 12-week-old puppy

Pythium insidiosum is an aquatic fungus-like organism that causes a serious chronic granulomatous disease called pythiosis in animals and humans in tropical and subtropical regions of the world. In North America, pythiosis is most often diagnosed in the Gulf Coast states. Early recognition of the disease is crucial to successful treatment, which includes surgical resection of granulomatous lesions and administration of antifungal agents. Despite increasing availability of diagnostic tests, intestinal pythiosis is insidious and is often not detected until lesions are extensive. Intestinal pythiosis was diagnosed in a 12-week-old puppy from South Carolina examined because of vomiting, diarrhea, and anorexia. Pythiosis was not initially suspected because of the young age of the patient and because pythiosis is uncommon in this area.     

Aspergillus fumigatus infection in two wild Eurasian black vultures (Aegypius monachus Linnaeus) with carbofuran insecticide poisoning: a case report

Aspergillus spp. are opportunistic pathogens which cause pulmonary aspergillosis in animals and humans with compromised immune systems. Two Eurasian black vultures (Aegypius monachus Linnaeus) were found dead or clinically ill from carbofuran insecticide during the winter of 2004. Carbofuran was detected in the stomach contents by gas chromatograph-mass spectrometry. Gross lesions showed severe granulomatous pneumonia and serofibrinous pleuropneumonia in both birds, with most lesions restricted to the pulmonary system. Histological lesions included pyogranulomatous pneumonia and suppurative parabronchiolitis/pleuritis/air sacculitis with a number of septated fungal hyphae, suggesting severe pulmonary aspergillosis. Fungal isolates from each vulture were identified as Aspergillus fumigatus by both lactophenol cotton blue staining and genetic analysis. This is the first report of pulmonary aspergillosis caused by A. fumigatus in wild Eurasian black vultures and suggests that Aspergillus infection could be an important cause of death in these birds which migrate from Mongolia to Korea during the winter. The incidence of the disease may be related to impaired immunity caused directly or indirectly by carbofuran poisoning.     

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.     

Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.

The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.     

Development and evaluation of an enzyme-linked immunosorbent assay for the serodiagnosis of pythiosis in dogs

Pythiosis (caused by the aquatic oomycete Pythium insidiosum) is a devastating and often fatal cause of either severe transmural gastroenteritis or locally invasive subcutaneous disease in dogs living in the southeastern United States. Although early diagnosis is essential for successful treatment, tools available for this task are limited. Therefore, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P insidiosum antibodies in canine serum. A soluble mycelial extract of P insidiosum was utilized as antigen in the ELISA, which was used to evaluate serum from 43 dogs with pythiosis, 8 dogs with lagenidiosis (another canine oomycosis), 16 dogs with nonoomycotic fungal or algal infections, 22 dogs with nonfungal gastrointestinal or skin disease, and 55 healthy dogs. Results were expressed as percent positivity (PP) relative to a strong positive control serum run on each plate. Medians and ranges for each of the 5 groups were as follows: pythiosis (81.7%, 50.6-98.5%), lagenidiosis (17.3%, 11.3-29.2%), other fungal or algal infections (8.2%, 4.7-15.4%), nonfungal gastrointestinal or skin disease (6.2%, 3.9-20.7%), and healthy dogs (6.7%, 3.0-15.2%). When using a cutoff value of 40% PP, the sensitivity and specificity of the ELISA both were 100%. In addition, ELISA values measured after successful surgical therapy in 2 dogs showed a decrease of anti-P insidiosum antibody concentrations into the normal range as early as 2 months after treatment. We conclude that the ELISA is a sensitive and specific test for the diagnosis of canine pythiosis, and may be a useful tool for monitoring response to medical or surgical therapy.     

Pathological findings in dogs naturally infected with Angiostrongylus vasorum in Newfoundland and Labrador, Canada.

Fifty-six dogs from St. John's, Newfoundland, Canada, were evaluated for Angiostrongylus vasorum infection. Small numbers of nematodes were found within pulmonary arteries of 6 dogs. Larvae were identified in fecal samples in 2 of 6 dogs. All 6 dogs had multifocal granulomatous pneumonia and sometimes foci of chronic thrombosis, which varied from very mild to severe. One dog had extensive pulmonary lesions resulting in cor pulmonale. Right heart failure was characterized by right ventricular hypertrophy, hepatic congestion, ascites, and hydrothorax. Microscopically, in most cases, eggs, larvae, and sometimes intravascular adults, were present within lung tissue sections. Small foci of granulomatous inflammation with and without larvae were present in kidney and brain in 4 dogs. An additional dog, diagnosed antemortem with angiostrongylosis via fecal examination, was also examined. Pathological findings consisted of severe pyogranulomatous interstitial pneumonia with myriad eggs, larvae, and numerous intravascular pulmonary adult nematodes with extensive arterial thrombosis. Five hundred and seventy-two adult worms were removed from pulmonary arteries. Foci of granulomatous inflammation, often associated with larvae and/or eggs, were present in tracheobronchial lymph nodes, adrenal gland, brain, and kidneys. Severe seizuring noted antemortem was attributed to several large, discrete areas of acute hemorrhagic infarction within the cerebrum and cerebellum. Natural A. vasorum infection in domestic dogs in eastern Newfoundland causes lung pathology of variable severity, which in some cases, may progress to cor pulmonale and which may be associated with extrapulmonary lesions and clinical signs.     

Experimental infection of dexamethasone-treated goats with Pasteurella haemolytica A2.

Sixteen goats either subjected to transport stress or without transport stress were treated with dexamethasone for 3 days prior to infection with P. haemolytica serotype A2 intranasally. The transport-stressed and dexamethasone-treated goats in the first group had various degrees of pulmonary lesions and the organism was re-isolated from the nasal cavity, lymph nodes and lungs. None of the goats treated with dexamethasone only were infected with P. haemolytica and had no lesions of pneumonic pasteurellosis. Treatment with dexamethasone alone failed to induce experimental infection by P. haemolytica except in combination with another stress factor.     

Pulmonary hyalohyphomycosis caused by Fusarium spp in a Kemp's ridley sea turtle (Lepidochelys kempi): an immunohistochemical study

AIM: To describe the microscopic and immunohistochemical findings in a case of pulmonary hyalohyphomycosis in a Kemp's ridley sea turtle (Lepidochelys kempi). METHODS: Samples of lung, liver and kidney from a stranded, dead Kemp's ridley sea turtle were routinely processed for histopathological studies. Two monoclonal antibodies that reacted specifically with antigens of Aspergillus spp and the Mucorales (Zygomycetes) group, and a panel of polyclonal antibodies raised against Aspergillus fumigatus, Candida albicans, Geotrichum candidum, Fusarium solani, and Scedosporium apiospermum were used for immunohistochemical or immunofluorescence staining. RESULTS: Histologically, a severe multifocal granulomatous pneumonia associated with fungal infection was diagnosed. All hyphae were identified as Fusarium spp because a strong and uniform reactivity was obtained only with a heterologously-absorbed polyclonal antibody raised against somatic antigens of Fusarium solani. CONCLUSIONS: Fusarium spp should be included in the differential diagnosis of mycotic pneumonia in Kemp's ridley sea turtles. CLINICAL RELEVANCE: This is the first report of a pulmonary mycotic infection in a sea turtle diagnosed using immunohistochemistry. Wildlife rehabilitators and pathologists should be aware of the availability of immunohistochemical techniques for identifying fungi in sea turtles.     

Multinodular pulmonary fibrosis,pancytopenia and equine herpesvirus‐5 infection in a Thoroughbred gelding

A 21‐year‐old Thoroughbred gelding was examined for intermittent fever, lethargy, inappetance and weight loss. Initial diagnostic evaluation revealed pancytopenia (anaemia, thrombocytopenia and neutropenia), hyperfibrinogenaemia and multifocal pulmonary nodules. Bacterial and fungal culture and viral isolation on transtracheal aspirate samples were negative, consistent with either pulmonary neoplasia, idiopathic granulomatous pneumonia or equine multinodular pulmonary fibrosis (EMPF). Bone marrow evaluation was consistent with pancytopenia due to a combination of peripheral consumption/destruction and suppression/destruction of progenitor cells at the level of the marrow. Pancytopenia resolved and clinical signs improved transiently with immunosuppressive corticosteroid therapy, but the horse deteriorated rapidly one month after presentation and was subjected to euthanasia. Necropsy findings were consistent with EMPF, and equine herpesvirus‐5 (EHV‐5) DNA was found in both the lung and bone marrow. The specific role of EHV‐5 in the development of the pulmonary pathology in EMPF and the pancytopenia in this horse is unclear, but an aberrant host immune/inflammatory response to EHV‐5 infection may be important.     

Extrapulmonary granulomatous lesions in canine paragonimiasis

Extrapulmonary granulomatous lesions associated with a naturally acquired Paragonimus kellicotti infection were observed in a dog with severe pulmonary paragonimiasis. Lung lesions were similar to those previously described. Extrapulmonary granulomatous lesions were found in the liver, mediastinal lymph nodes, spermatic cord and the tunica vaginalis. Extravascular deposition of eggs in the spermatic cord and tunica vaginalis caused a perilymphatic granulomatous inflammation and partially occlusive lymphangitis with dilatation of the external lymphatic vessel of the right spermatic cord.     

Concurrent pulmonary adenocarcinoma and silicate pneumoconiosis in a red-legged partridge (Alectoris rufa)

An adult red-legged partridge (Alectoris rufa) presented with concurrent pulmonary carcinoma and severe silicosis. The animal was submitted to the Veterinary Teaching Hospital of the University of Córdoba (Spain) because of respiratory signs, and it died during clinical examination. At postmortem examination, numerous firm, whitish to yellowish nodules involving the lungs, mainly the right lobe, were found. The histopathologic study revealed numerous peribronchiolar large granulomatous lesions composed of macrophages, which showed abundant cytoplasm containing numerous birefringent crystals identified as silicates by transmission electron microscopy. An epithelial neoplasm showing papillary, acinar, and solid patterns occupied large areas of the pulmonary parenchyma. The histopathologic and immunohistochemical features were consistent with a pulmonary carcinoma. Small tumor nests were often located close to the granulomatous lesions. This is the first report of concurrent pneumoconiosis and pulmonary carcinoma in a nonhuman species.