Phaeohyphomycosis due to Exophiala in Aquarium-Housed Lumpfish (Cyclopterus lumpus): Clinical Diagnosis and Description.

Phaeohyphomycosis caused by Exophiala species represents an important disease of concern for farmed and aquarium-housed fish. The objective of this study was to summarize the clinical findings and diagnosis of Exophiala infections in aquarium-housed Cyclopterus lumpus. Clinical records and postmortem pathology reports were reviewed for 15 individuals from 5 public aquaria in the United States and Canada from 2007 to 2015. Fish most commonly presented with cutaneous ulcers and progressive clinical decline despite topical or systemic antifungal therapy. Antemortem fungal culture of cutaneous lesions resulted in colonial growth for 7/12 samples from 8 individuals. Amplification of the internal transcribed spacer region (ITS) of nuclear rDNA identified Exophiala angulospora or Exophiala aquamarina in four samples from three individuals. Postmortem histopathologic findings were consistent with phaeohyphomycosis, with lesions most commonly found in the integument (11/15), gill (9/15), or kidney (9/15) and evidence of fungal angioinvasion and dissemination. DNA extraction and subsequent ITS sequencing from formalin-fixed paraffin-embedded tissues of seven individuals identified E. angulospora, E. aquamarina, or Cyphellophora sp. in four individuals. Lesion description, distribution, and Exophiala spp. identifications were similar to those reported in farmed C. lumpus. Antemortem clinical and diagnostic findings of phaeohyphomycosis attributable to several species of Exophiala provide insight on the progression of Exophiala infections in lumpfish that may contribute to management of the species in public aquaria and under culture conditions.

Clostridioides (Clostridium) difficile-associated disease, epiploic foramen entrapment, and gastric rupture in a Thoroughbred racehorse: case report and literature review.

Epiploic foramen entrapment (EFE) is a common cause of small intestinal colic in horses and may lead to intestinal strangulation. Strangulating intestinal obstruction impairs the gastrointestinal outflow and can lead to secondary gastric rupture and endotoxemia. Clostridioides difficile can cause enterotyphlocolitis with colic in horses of all ages, and the process is commonly referred to as C. difficile-associated disease (CDAD). Here we report the results of the postmortem examination of a 7-y-old Thoroughbred racehorse with concurrent CDAD, EFE, and gastric rupture that was euthanized following a history of colic over several days. A segment of distal jejunum and proximal ileum had passed through the epiploic foramen, and the intestinal wall was thickened and dark-red. The remaining small intestinal loops were distended and filled with blood-tinged contents. Peritonitis had resulted from escape of gastric contents into the abdominal cavity through a tear in the major curvature of the stomach. Histologically, the incarcerated segment had acute transmural hemorrhage with congestion and mucosal necrosis; neutrophilic infiltrates with fibrin thrombi were in the mucosa of the non-incarcerated small intestinal segments. C. difficile toxins were detected in the small intestinal contents, and C. difficile was isolated from the small intestine, colon, and cecum.

Early circulation of rabbit haemorrhagic disease virus type 2 in domestic and wild lagomorphs in southern California, USA (2020-2021).

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real-time PCR on the liver, and histology and immunohistochemistry for pan-lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black-tailed jackrabbits (Lepus californicus) tested positive. All RHDV2-positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%-99.95%) to each other, and also very similar (99.0%-99.4%) to sequences obtained in other southwestern states during the 2020-2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%-96.9%) and Quebec in 2016 (92.4%-92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%-98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA.

Outbreak of rabbit hemorrhagic disease virus 2 in the southwestern United States: first detections in southern California.

An outbreak of rabbit hemorrhagic disease virus 2 (RHDV2)-associated disease occurred in the southwestern United States following its first detection in New Mexico in March 2020. The disease spread throughout several states and was diagnosed for the first time in California on May 11, 2020, in a black-tailed jackrabbit (Lepus californicus). The following day, the California Department of Food and Agriculture (CDFA) issued an order banning the entrance into California of several lagomorph species and their products from any state in which the disease had been detected in the last 12 mo. RHDV2 is a threat to wild lagomorph species in California, including the endangered riparian brush rabbit (Sylvilagus bachmani riparius). Therefore, the California Department of Fish and Wildlife (CDFW) started tracking any mortality event in wild lagomorph populations. As of August 9, 2020, RHDV2 had been detected in wild and domestic lagomorphs of several counties in southern California that were submitted to the California Animal Health and Food Safety laboratory system by the CDFA or the CDFW. These positive cases included 2 additional black-tailed jackrabbits and 3 desert cottontail rabbits (Sylvilagus audubonii). In addition, the infection spilled over to domestic populations, whereby it was confirmed on July 10, 2020, in a domestic rabbit (Oryctolagus cuniculus).

Nutritional Wasting Disorders in Sheep.

The different ovine production and breeding systems share the cornerstone of keeping a good body condition to ensure adequate productivity. Several infectious and parasitic disorders have detrimental effects on weight gains and may lead to emaciation. Flock health management procedures are aimed to prevent such conditions. Nutritional management is equally important to guarantee adequate body condition. Persistent bouts of low ruminal pH due to excess concentrate in the diet may lead to subacute ruminal acidosis. Pre-stomach motility disorders may also lead to ill-thrift and emaciation. An adequate mineral supplementation is key to prevent the effects of copper, selenium, and other micronutrients deprivation, which may include, among others, loss of condition. This review elaborates on the clinico-pathologic, diagnostic, and therapeutic aspects of some of these conditions, and highlights the necessity of considering them as contributors to states of wasting in sheep flocks.

Toxic Wasting Disorders in Sheep.

Infectious and parasitic agents have been frequently associated with debilitating and wasting conditions in sheep. The prevalence of these agents has probably undermined the role of toxic causes as contributors to such disorders. In addition, many of these intoxications frequently produce acute clinical disease with specific and characteristic lesions, thus a causal relationship with the toxic substance may be relatively easy to establish. However, persistent exposure to some of these organic or inorganic toxic substances may lead to emaciation, ill-thrift, and poor external aspect. The anti-nutritional factors and alkaloids of several plants, including pyrrolizidine alkaloids, among others, have also been associated with emaciation and/or poor general performance in sheep flocks. In this review, some of these disorders are discussed with an emphasis on clinical signs and lesions, relevant diagnostic aspects, and available therapeutic approaches. In most cases, demonstrating a history of exposure should be one of the most relevant aspects of the diagnostic approach, and removing the animals from the toxic source is the cornerstone of the majority of the treatment strategies.

Ibex-Associated Malignant Catarrhal Fever in Duikers (Cephalophus Spp).

Eight duikers, representing 3 different species cohoused in a single zoological collection, died in a 10-month period. Black, red-flanked, and yellow-backed duikers were affected, appearing clinically with a combination of anorexia, diarrhea, ataxia, tremors, and/or stupor, followed by death within 72 hours of onset of clinical signs. Consistent gross findings were pulmonary ecchymoses (8/8), generalized lymphadenomegaly (6/8), ascites (5/8), and pleural effusion (4/8). Dense lymphocyte infiltrates and arteritis affected numerous tissues in most animals. Ibex-associated malignant catarrhal fever (MCF) viral DNA was detected in all cases by polymerase chain reaction and in situ hybridization. Identical ibex-MCF virus sequence was detected in spleen of a clinically healthy ibex (Capra ibex) housed in a separate enclosure 35 meters away from the duikers.

First Isolation of a Novel Aquatic Flavivirus from Chinook Salmon (Oncorhynchus tshawytscha) and Its In Vivo Replication in a Piscine Animal Model.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers' postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species.IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Paeniclostridium (Clostridium) sordellii-associated enterocolitis in 7 horses.

Enteric disease in horses may be caused by a variety of microorganisms, including several clostridial species. Paeniclostridium sordellii (previously Clostridium sordellii) has been frequently associated with gas gangrene in humans and several animal species, including horses. However, its role in enteric diseases of animals has not been fully determined. We describe herein 7 cases of enteric disease in horses associated with P. sordellii infection. Grossly, the small and/or large intestines were necrotic, hemorrhagic, and edematous. Microscopically, there was severe mucosal necrosis and hemorrhage of the small and/or large intestine of all horses. P. sordellii was isolated and/or demonstrated by immunohistochemistry and/or PCR in the intestine of all horses. All other known causes of enteric disease in horses were ruled out in these 7 cases. P. sordellii should be considered among the differential diagnoses in cases of enteric disease in horses.

Streptococcus equi subspecies zooepidemicus septicemia in alpacas: three cases and review of the literature.

Streptococcus equi subspecies zooepidemicus septicemia of alpacas and llamas, also called alpaca fever, is characterized clinically by fever, depression, recumbency, and death, and pathologically by polyserositis. Although a few natural and experimental cases of the disease have been reported, very little information about the pathology of spontaneous cases has been published. We present a detailed gross and microscopic description of 3 spontaneous cases of alpaca fever and review the literature on this condition. Typical of spontaneous and experimental infections with S. equi ssp. zooepidemicus, the 3 animals had disseminated fibrinosuppurative polyserositis with vascular thrombosis and intralesional gram-positive cocci. In addition, 2 of the animals had severe fibrinosuppurative pneumonia, endocarditis, and myocardial necrosis; the third animal had transmural pleocellular enteritis with prominent lymphangitis. The enteric lymphangitis observed in the latter suggests that dissemination of S. equi ssp. zooepidemicus occurred through lymphatic circulation and that, at least in this animal, the portal of entry of infection was the alimentary system.

Infectious necrotic hepatitis caused by Clostridium novyi type B in a horse: case report and review of the literature.

A 14-y-old bay Quarter Horse gelding was presented with progressive neurologic signs, elevated rectal temperature, and icterus for 3 d prior to death. Postmortem examination revealed icterus, large amounts of serosanguineous fluid in the abdominal cavity, widespread petechiae and ecchymoses in several organs, and a large, pale, and well-demarcated focus of necrosis in the liver. Histologically, there was coagulative necrosis surrounded by a rim of inflammatory cells and large numbers of gram-positive rods, which were identified as Clostridium novyi by immunohistochemistry. Liver samples tested by PCR were positive for C. novyi type B flagellin and alpha toxin genes, but negative for Clostridium haemolyticum and other clostridia. Based on postmortem findings and ancillary tests, a definitive diagnosis of infectious necrotic hepatitis (INH) was made. Mostly a disease of ruminants, also known as black disease, INH has rarely been reported in horses, and a definitive etiologic diagnosis has not been achieved previously; the etiology of all cases reported to date was identified as C. novyi but the type was not determined. Animals are predisposed to clostridial hepatitis when hepatic anaerobiosis is established. Such conditions allow germination and proliferation of bacterial spores, resulting in production and release of toxins. INH, caused by C. novyi type B, and bacillary hemoglobinuria, caused by C. haemolyticum, are mechanistically and pathologically almost indistinguishable. Because these 2 microorganisms are closely related, differentiation requires molecular tools.

Diagnostic approach to catastrophic musculoskeletal injuries in racehorses.

Catastrophic musculoskeletal injuries are the most common cause of euthanasia or spontaneous death in racehorses, and the most common cause of jockey falls with potential for serious human injury. Horses are predisposed to the vast majority of these injuries by preexisting lesions that can be prevented by early diagnosis and adequate bone injury management. A thorough examination of the musculoskeletal system in racehorses often determines the cause of these injuries and generates data to develop injury prevention strategies. We describe the diagnostic approach to musculoskeletal injury, review the methodology for the examination of racehorse limbs, and provide anatomy and pathology tools to perform an organized and thorough postmortem examination of the musculoskeletal system in equine athletes.

Cluster of cases of massive hemorrhage associated with anticoagulant detection in race horses.

Five horses originating from 4 different California race tracks were submitted to the California Animal Health and Food Safety Laboratory for necropsy and diagnostic workup. The 5 horses had a history of sudden collapse and death during exercise. In all of them, massive hemoperitoneum and hemorrhages in other cavities or organs were observed. The liver from these 5 animals and from 27 horses that had been euthanized due to catastrophic leg injuries (controls) were subjected to a rodenticide anticoagulant screen. Traces of brodifacoum, diphacinone, or bromadiolone were detected in the 5 horses with massive bleeding (5/5), and no traces of rodenticides were detected in control horses (0/27). Other frequent causes of massive hemorrhages in horses were ruled out in 4 of the cases; one of the horses had a pelvic fracture. Although only traces of anticoagulants were found in the livers of these horses and the role of these substances in the massive bleeding remains uncertain, it is speculated that exercise-related increases in blood pressure may have reduced the threshold for toxicity of these anticoagulants.

New disease records for hatchery-reared sturgeon. II. Phaeohyphomycosis due to Veronaea botryosa.

A series of fungal cases in hatchery-reared juvenile and young adult Siberian sturgeon Acipenser baerii and white sturgeon A. transmontanus occurred at production facilities in Florida and California, USA, respectively. Affected fish exhibited abnormal orientation and/or buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerative skin and eye lesions. Necropsies revealed haemorrhage throughout the coelom, serosanguinous coelomic effusion and organomegaly with nodular or cystic lesions in multiple organs. Fungal hyphae were observed in 27 fish (24 A. baerii and 3 A. transmontanus) via microscopic examination of tissue wet mounts and on slides prepared from colonies grown on culture media. Histopathological examination of these infected tissues revealed extensive infiltration by melanised fungal hyphae that were recovered in culture. Phenotypic characteristics and sequencing of the fungal isolates with the use of the internal transcribed spacer region and 28S rRNA gene confirmed the aetiological agent as Veronaea botryosa. To our knowledge, this is the first documentation of V. botryosa infection in fish, although melanised fungi of the closely related genus Exophiala are well-known pathogens of freshwater and marine fishes.

Pathologic findings in Western gray squirrels (Sciurus griseus) from a notoedric mange epidemic in the San Bernardino Mountains, California.

Notoedric mange, caused by the contagious, burrowing mite Notoedres centrifera, has been associated with several large-scale population declines of western gray squirrels (Sciurus griseus) and has been a significant obstacle to population recovery in Washington State where the species is listed as threatened. In 2009, residents and wildlife rehabilitators in the isolated San Bernardino Mountains of southern California reported a dramatic die-off of western gray squirrels, in what had been a previously dense and robust population. Individuals were observed suffering from abnormal neurologic behaviors (ataxia and obtundation) and severe skin disease. Full necropsy of five squirrels from the epidemic showed that all had moderate to severe infestation with mange mites and severe dermatitis characterized by hyperkeratosis, acanthosis, intralesional mites, intracorneal pustules and superficial bacteria. Mites from affected squirrels were evaluated by light and electron microscopy and identified as N. centrifera based on morphologic criteria. Additionally, the internal transcribed spacer-2 region of the mite was cloned, sequenced and accessioned in GenBank. The cause for the abnormal neurologic behavior was not confirmed on post-mortem examination. However, we hypothesize that mange can cause incoordination and obtundation as a result of malnutrition and dehydration, and intense pruritis may induce abnormal or erratic behavior that could be mistaken for neurologic signs. While we have characterized the severe impact this disease can have on individual animals, more work is needed to understand the impact on squirrel populations, particularly in view of the anecdotal reports of dramatic population declines that may take decades to recover.

Myxobolus albi infection in cartilage of captive lumpfish (Cyclopterus lumpus).

Myxobolus albi was diagnosed in the cartilage of captive lumpfish (Cyclopterus lumpus) from 2 public aquaria. Eleven fish were affected, with the most common clinical signs being exophthalmos and grossly visible 1- to 2-mm white to tan scleral nodules. Myxozoan cysts were identified in the cartilage of the skull, branchial arch, sclera, vertebrae, tongue, all fin insertions, and the pectoral girdle. Cysts resulted in expansile, deforming, space-occupying lesions, resulting in exophthalmos but often lacking significant tissue damage or inflammation. Once cysts ruptured, free spores elicited a mild to marked inflammatory response. Spores measured 7.5 to 9.0 µm × 3.0 to 6.0 µm and contained 2 pyriform polar capsules oriented at one pole as well as occasional 1-µm-diameter basophilic nuclei. Identification was based on spore morphology together with polymerase chain reaction and sequence comparison of 18S ribosomal DNA. Isolates had 99% similarity to M. albi.

Systemic mycosis in a California sea lion (Zalophus californianus) with detection of cystofilobasidiales DNA.

A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.

Pathologic and parasitologic findings of cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) stranded on Cape Cod, Massachusetts, 2001-2006.

Necropsy reports for 28 stranded, cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) that died between 2001 and 2006 were reviewed retrospectively. Gross and microscopic lesions were compiled to describe the pathologic and parasitologic findings in turtles that were found freshly dead on the beach or that died within 48 hr of stranding. Anatomic lesions of varying severity were identified in each of the examined turtles and were identified in tissues of the alimentary, respiratory, integumentary, nervous and sensory, and urogenital systems in order of decreasing frequency. Necrotizing enterocolitis and bacterial or fungal pneumonia were the most frequently encountered lesions that were considered clinically significant. Parasites and parasitic lesions were identified primarily in tissues of the alimentary system and included intestinal cestodiasis and parasitic granulomas containing larval cestodes or nematodes. Postlarval cestodes were also found in the coelom of two turtles. In many cases, the extent and severity of lesions were judged to be insufficient to have solely caused mortality, suggesting that additional factors such as metabolic, respiratory, and electrolyte derangements; hypothermia; and drowning may be important proximate causes of death in cold-stunned turtles. Results of this study provide insight into pathologic conditions that may be of clinical relevance to rehabilitation efforts for cold-stunned sea turtles.

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.