Identification of Candidate Protein Biomarkers Associated with Domoic Acid Toxicosis in Cerebrospinal Fluid of California Sea Lions (Zalophus californianus).

Since 1998, California sea lion (Zalophus californianus) stranding events associated with domoic acid toxicosis (DAT) have consistently increased. Outside of direct measurement of domoic acid in bodily fluids at the time of stranding, there are no practical nonlethal clinical tests for the diagnosis of DAT that can be utilized in a rehabilitation facility. Proteomics analysis was conducted to discover candidate protein markers of DAT using cerebrospinal fluid from stranded California sea lions with acute DAT (n = 8), chronic DAT (n = 19), or without DAT (n = 13). A total of 2005 protein families were identified experiment-wide. A total of 83 proteins were significantly different in abundance across the three groups (adj. p < 0.05). MDH1, PLD3, ADAM22, YWHAG, VGF, and CLSTN1 could discriminate California sea lions with or without DAT (AuROC > 0.75). IGKV2D-28, PTRPF, KNG1, F2, and SNCB were able to discriminate acute DAT from chronic DAT (AuROC > 0.75). Proteins involved in alpha synuclein deposition were over-represented as classifiers of DAT, and many of these proteins have been implicated in a variety of neurodegenerative diseases. These proteins should be considered potential markers for DAT in California sea lions and should be prioritized for future validation studies as biomarkers.

Respiratory Tract Explant Infection Dynamics of Influenza A Virus in California Sea Lions, Northern Elephant Seals, and Rhesus Macaques.

To understand susceptibility of wild California sea lions and Northern elephant seals to influenza A virus (IAV), we developed an ex vivo respiratory explant model and used it to compare infection kinetics for multiple IAV subtypes. We first established the approach using explants from colonized rhesus macaques, a model for human IAV. Trachea, bronchi, and lungs from 11 California sea lions, 2 Northern elephant seals, and 10 rhesus macaques were inoculated within 24 h postmortem with 6 strains representing 4 IAV subtypes. Explants from the 3 species showed similar IAV infection kinetics, with peak viral titers 48 to 72 h post-inoculation that increased by 2 to 4 log10 PFU/explant relative to the inoculum. Immunohistochemistry localized IAV infection to apical epithelial cells. These results demonstrate that respiratory tissue explants from wild marine mammals support IAV infection. In the absence of the ability to perform experimental infections of marine mammals, this ex vivo culture of respiratory tissues mirrors the in vivo environment and serves as a tool to study IAV susceptibility, host range, and tissue tropism. IMPORTANCE Although influenza A virus can infect marine mammals, a dearth of marine mammal cell lines and ethical and logistical challenges prohibiting experimental infections of living marine mammals mean that little is known about IAV infection kinetics in these species. We circumvented these limitations by adapting a respiratory tract explant model first to establish the approach with rhesus macaques and then for use with explants from wild marine mammals euthanized for nonrespiratory medical conditions. We observed that multiple strains representing 4 IAV subtypes infected trachea, bronchi, and lungs of macaques and marine mammals with variable peak titers and kinetics. This ex vivo model can define infection dynamics for IAV in marine mammals. Further, use of explants from animals euthanized for other reasons reduces use of animals in research.

Diffuse large B cell lymphoma and a novel gammaherpesvirus in northern elephant seals Mirounga angustirostris.

Two emaciated male northern elephant seal (NES) Mirounga angustirostris pups were admitted to The Marine Mammal Center (Sausalito, California, USA) and treated for malnutrition. Complete blood counts showed a progressive moderate to marked leukocytosis characterized by a predominance of large monomorphic mononuclear cells of probable lymphoid origin, frequently with flower-shaped nuclei. Both seals were euthanized due to suspected lymphoid neoplasia. At necropsy, most lymph nodes in both pups were markedly enlarged, some with distinct white nodules, the spleens were diffusely enlarged, and the intestinal mucosae were thickened. Histopathologic features consistent with disseminated large cell lymphoma were identified to varying degrees of severity in lymph nodes, bone marrow, liver, tonsils, spleen, liver, intestines, kidneys, lower urinary tract, and several other organs. Immunohistochemical staining of neoplastic cells was most consistent with B lymphocyte origin, with most cells staining positively for Pax 5 and CD20 with admixed small CD3-positive T lymphocytes and CD204-positive macrophages. PCR and sequencing identified a novel gammaherpesvirus, herein called miroungine gammaherpesvirus 3, from affected tissues. This virus is in a clade outside of named genera that utilize hosts in the suborder Caniformia. The present study is the first description of diffuse large B cell lymphoma with leukemic manifestation and concomitant detection of a novel gammaherpesvirus in free-living NESs. Further research regarding the prevalence of this new gammaherpesvirus and its associated pathogenesis in this species is indicated.

Demodectic mange in threatened southern sea otters (Enhydra lutris nereis).

BACKGROUND: Southern sea otters (Enhydra lutris nereis) rely on intact pelage for thermoregulation, and thus clinically significant demodicosis and associated alopecia can cause morbidity and death. HYPOTHESIS/OBJECTIVES: This study aimed to describe lesions associated with follicular Demodex sp. infestation, estimate the prevalence and intensity of infestation, describe mite distribution across key anatomical regions, and assess mite presence or absence in relation to lesions and host risk factors. ANIMALS: Twenty necropsied, wild southern sea otters that stranded along the central California coast from 2005 to 2018. METHODS AND MATERIALS: Grossly normal and abnormal integument from the head, perineum, genitals, mamillary papillae and limbs was assessed microscopically for mites and mite-associated pathological findings. RESULTS: Intrafollicular mites were observed in the integument of 55% of otters and 20% had clinical demodicosis. Demodicosis was considered to be contributory to death or euthanasia in two cases. Although Demodex sp. mites often were observed microscopically in grossly normal skin, the presence of multiple densely-packed intrafollicular mites generally was associated with pigmentary incontinence, ectatic follicles, lymphoplasmacytic perifolliculitis, and neutrophilic and lymphoplasmacytic, dermal inflammation. Other findings included epidermal hyperplasia, orthokeratotic hyperkeratosis of epidermis and follicular epithelium, concurrent pyoderma and cell necrosis. Perioral integument, especially of the chin, had the highest prevalence of mites and the highest mite density, suggesting facial contact as a means of mite transmission. CONCLUSIONS AND CLINICAL IMPORTANCE: Our research confirmed demodectic mange as a contributor to morbidity and mortality in sea otters, with important implications for clinical care, rehabilitation and conservation.

CLINICAL AND MICROBIOLOGICAL CHARACTERIZATION OF LYMPH NODE ABSCESSATION IN PUP AND YEARLING CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) UNDERGOING REHABILITATION IN A MARINE MAMMAL CENTER.

Severe focal to multifocal abscessation of the axillary and inguinal lymph nodes is an important cause of morbidity and mortality in stranded pup and yearling California sea lions (Zalophus californianus; CSLs) at The Marine Mammal Center (TMMC). A retrospective case review was conducted of all pup and yearling CSLs with axillary and/or inguinal lymph node abscesses admitted to this California rehabilitation center between January 2015 and December 2019 (n = 162). Clinical data and cultured isolates were evaluated to (1) characterize the clinical syndrome, (2) report isolated pathogens, and (3) investigate factors associated with clinical outcome (survival to release versus death). Of the 162 CSLs admitted with at least one axillary or inguinal lymph node abscess, almost all were in poor body condition, and overall mortality rate was 63% (102 of 165). Mortality rate was lower for animals presenting with a single abscess and abscess(es) in the inguinal location only; odds of death were significantly lower for animals that had their abscesses surgically drained (P = 0.029) and those that received antimicrobials (P = 0.037). Hematology and serum chemistry parameters at the time of abscess diagnosis reflected inflammation and malnutrition. Radiographic findings associated with abscesses from 45 cases included soft tissue swelling (n = 40), intralesional gas (n = 19), and osteomyelitis (n = 3). Ninety bacterial isolates were recovered from aerobic (n = 48) and anaerobic cultures (n = 17), 48.9% of which were gram negative. The most common gram-negative organisms were Escherichia coli (n = 15), Proteus spp. (n = 8), and Bacteroides ureolyticus (n = 7), and the most common gram-positive organisms were Streptococcus phocae (n = 10) and Staphylococcus spp. (n = 9). Management of lymph node abscesses via surgical drainage and multimodal systemic antimicrobial therapy with a broad-spectrum of activity may be considered to improve survival of these cases.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

Brucellosis in Endangered Hector's Dolphins (Cephalorhynchus hectori).

Brucella spp infections of marine mammals are often asymptomatic but have been associated with reproductive losses and deaths. Zoonotic infections originating from marine isolates have also been described. Hector's dolphins (Cephalorhynchus hectori) are an endangered species with a declining population, and the role of infectious disease in population dynamics is not fully understood. In this study, 27 Hector's dolphins found dead around the New Zealand coastline between November 2006 and October 2010 were evaluated for lesions previously associated with cetacean brucellosis. Tissues were examined using histological, immunohistochemical, and molecular (polymerase chain reaction [PCR]) techniques. Seven of 27 dolphins (26%) had at least 1 tissue that was positive on PCR for Brucella spp. Lesions consistent with brucellosis were present in 10 of 27 (37%) dolphins, but in 8 of these dolphins Brucella infection could not be demonstrated in lesional tissues. Two dolphins (7%) were diagnosed with active brucellosis: 1 female with placentitis and metritis, and 1 stillborn male fetus. Brucella identified in these 2 dolphins had genetic similarity (99%) to Brucella pinnipedialis. The omp2a gene amplicon from the uterus of the female had 100% homology with ST27 genotype isolates from a human in New Zealand and a bottlenose dolphin of Pacific origin. The remaining 5 PCR-positive dolphins were assessed as having asymptomatic or latent infection. While most Brucella infections identified in this study appeared to be subclinical, the finding of 2 dolphins with reproductive disease due to Brucella infection suggests that this disease has the potential to affect reproductive success in this species.

Cetacean morbillivirus in coastal Indo-Pacific bottlenose dolphins, Western Australia.

Cetacean morbillivirus (CeMV) has caused several epizootics in multiple species of cetaceans globally and is an emerging disease among cetaceans in Australia. We detected CeMV in 2 stranded coastal Indo-Pacific bottlenose dolphins (Tursiops aduncus) in Western Australia. Preliminary phylogenetic data suggest that this virus variant is divergent from known strains.

Air leak syndrome in animals: definition and pathogenesis.

Air leak syndrome (ALS) is described in human medicine as a constellation of clinical disorders including pneumomediastinum, pneumopericardium, pulmonary interstitial emphysema, pneumothorax, pneumoperitoneum, pneumoretroperitoneum and subcutaneous emphysema. The pathogenesis of ALS depends on the anatomy of the mediastinum and its associations with thoracic, abdominal and cervical connective tissues, as well as a physical phenomenon referred to as the Macklin effect. Various animal species develop diverse combinations of these lesions, although ALS has not been recognized in animals. However, this term aids pathologists in addressing this disease compilation. The aim of this retrospective study is to illustrate examples of ALS in animals by arbitrarily selecting 13 cases in dogs, cats, pinnipeds, sea otters and harbour porpoises. ALS can be classified into three groups based on aetiology: iatrogenic, secondary or spontaneous. Iatrogenic ALS was diagnosed in two cats with tracheal laceration following endotracheal intubation. Secondary ALS was identified in two dogs, one with acute respiratory distress syndrome and the other due to grass awn migration. Secondary ALS in pinnipeds was diagnosed following severe pulmonary parasitism, uraemic pneumonia and oesophageal perforation. The other marine mammals developed ALS following trauma. Spontaneous ALS was also diagnosed in one cat and one dog without any apparent predisposing causes.

DEVELOPMENT AND VALIDATION OF A NOVEL DUPLEX PROBE-HYBRIDIZATION QUANTITATIVE PCR FOR LYMPHOMA-ASSOCIATED MIROUNGINE GAMMAHERPESVIRUS 3 IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.

The symptomatology and diagnosis of domoic acid toxicosis in stranded California sea lions (Zalophus californianus): a review and evaluation of 20 years of cases to guide prognosis.

Introduction: Domoic acid (DA) is a glutaminergic excitatory neurotoxin that causes the morbidity and mortality of California sea lions (Zalophus californianus; CSL) and other marine mammals due to a suite of effects mostly on the nervous and cardiac systems. Between 1998 and 2019, 11,737 live-stranded CSL were admitted to The Marine Mammal Center (TMMC; Sausalito, CA, USA), over 2,000 of which were intoxicated by DA. A plethora of clinical research has been performed over the past 20 years to characterize the range of toxic effects of DA exposure on CSLs, generating the largest dataset on the effects of natural exposure to this toxin in wildlife. Materials and methods: In this study, we review published methods for diagnosing DA intoxication, clinical presentation, and treatment of DA-intoxicated CSL and present a practical, reproducible scoring system called the neuroscore (NS) to help assess whether a DA-affected CSL is fit for release to the wild following rehabilitation. Logistic regression models were used to assess the relationships between outcome (released vs. euthanized or died) and multiple variables to predict the outcome for a subset of 92 stranded CSLs. Results: The largest proportion of DA-intoxicated CSLs was adult females (58.6%). The proportions of acute and chronic cases were 63.5 and 36.5% respectively, with 44% of affected CSL released and 56% either dying naturally or euthanized. The average time in rehabilitation was 15.9 days (range 0-169) for all outcomes. The best-performing model (85% accuracy; area under the curve = 0.90) assessing the relationship between outcome and predictor variables consisted of four variables: final NS, change in NS over time, whether the animal began eating in rehabilitation, and the state of nutrition on admission. Discussion: Our results provide longitudinal information on the symptomatology of CSL intoxicated by domoic acid and suggest that a behavioral scoring system is a useful tool to assess the fitness for the release of DA-intoxicated CSL.

Human influenza A virus H1N1 in marine mammals in California, 2019.

From 2011-2018, we conducted surveillance in marine mammals along the California coast for influenza A virus (IAV), frequently detecting anti-influenza antibodies and intermittently detecting IAV. In spring 2019, this pattern changed. Despite no change in surveillance intensity, we detected IAV RNA in 10 samples in March and April, mostly in nasal and rectal swabs from northern elephant seals (Mirounga angustirostris). Although virus isolation was unsuccessful, IAV sequenced from one northern elephant seal nasal swab showed close genetic identity with pandemic H1N1 IAV subclade 6B.1A.1 that was concurrently circulating in humans in the 2018/19 influenza season. This represents the first report of human A(H1N1)pdm09 IAV in northern elephant seals since 2010, suggesting IAV continues to spill over from humans to pinnipeds.

Echinococcus multilocularis in urban coyotes, Alberta, Canada.

Echinococcus multilocularis is a zoonotic parasite in wild canids. We determined its frequency in urban coyotes (Canis latrans) in Alberta, Canada. We detected E. multilocularis in 23 of 91 coyotes in this region. This parasite is a public health concern throughout the Northern Hemisphere, partly because of increased urbanization of wild canids.

Tattoo Skin Disease in Cetacea: A Review, with New Cases for the Northeast Pacific.

Tattoo skin disease (TSD) is a poxviral dermatopathy diagnosed in cetaceans. We review the literature on TSD aetiology, clinical characteristics, pathology and epidemiology and evaluate immune responses against the virus. In addition, necropsy reports for fifty-five harbour porpoises (Phocoena phocoena), twenty-two Delphinidae and four Kogiidae stranded in northern California in 2018-2021 were checked for diagnostic tattoo lesions. TSD occurs in the Mediterranean, North and Barents Seas, as well as in the Atlantic, eastern Pacific and Indian Oceans in at least 21 cetacean species, with varying prevalence. Two cetacean poxvirus (CePV) clades are recognised: CePV-1 in odontocetes and CePV-2 in mysticetes. CePV-1 isolates were recovered from six Delphinidae and one Phocoenidae in the Americas, Europe and Hong Kong. Strains from Delphinidae are closely related. Among Phocoenidae, poxviruses were sampled only in harbour porpoises around the British Isles. CePV-2 isolates were obtained from southern right whales (Eubalaena australis) and a bowhead whale (Balaena mysticetus). In healthy animals, an immune response develops over time, with young calves protected by maternal immunity. Salinity and sea surface temperature do not seem to influence TSD prevalence in free-ranging cetaceans. High concentrations of immunotoxic halogenated organochlorines may cause a more severe clinical disease. Substitution and loss of genes involved in anti-viral immunity may favour CePV entry, replication and persistence in the epidermis. Off California, Delphinidae were less often (26.3%) affected by TSD than harbour porpoises (43.6%). Male porpoises were significantly more prone (58.1%) to show clinical disease than females (25%). Among males, TSD affected a high proportion of juveniles and subadults. TSD was not detected in the Kogiidae.

Clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis.

OBJECTIVE: To describe clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis. ANIMALS: 38 free-ranging juvenile to adult California sea lions examined at a rehabilitation center in California between September 2015 and December 2017. PROCEDURES: Medical records at The Marine Mammal Center were reviewed to identify sea lions in which sarcocystosis had been diagnosed. RESULTS: Clinical signs were highly variable and associated with polyphasic rhabdomyositis attributed to Sarcocystis neurona infection. Generalized severe muscle wasting, respiratory compromise, and regurgitation secondary to megaesophagus were the most profound clinical findings. Respiratory compromise and megaesophagus were associated with a poor prognosis. Eight of the 38 sea lions were treated and released to the wild, and 2 subsequently restranded and were euthanized. Two additional animals received no targeted treatment and were released. The remaining 28 animals were either euthanized or died during treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that unlike other marine mammals, which typically develop encephalitis, California sea lions with sarcocystosis often have polyphasic rhabdomyositis with highly variable clinical signs and that extensive diagnostic testing may be required to confirm the diagnosis. Treatment with an antiprotozoal drug in combination with corticosteroids may resolve clinical disease, but the prognosis is guarded.

Exploration of serum cardiac troponin I as a biomarker of cardiomyopathy in southern sea otters (Enhydra lutris nereis).

OBJECTIVE: To compare serum cardiac troponin I (cTnI) concentrations between sea otters with and without cardiomyopathy and describe 2 cases of cardiomyopathy with different etiologies. ANIMALS: 25 free-ranging southern sea otters (Enhydra lutris nereis) with (n = 14; cases) and without (11; controls) cardiomyopathy and 17 healthy managed southern sea otters from aquariums or rehabilitation centers (controls). PROCEDURES: Serum cTnI concentration was measured in live sea otters. Histopathologic and gross necropsy findings were used to classify cardiomyopathy status in free-ranging otters; physical examination and echocardiography were used to assess health status of managed otters. Two otters received extensive medical evaluations under managed care, including diagnostic imaging, serial cTnI concentration measurement, and necropsy. RESULTS: A significant difference in cTnI concentrations was observed between cases and both control groups, with median values of 0.279 ng/mL for cases and < 0.006 ng/mL for free-ranging and managed controls. A cutoff value of ≥ 0.037 ng/mL yielded respective sensitivity and specificity estimates for detection of cardiomyopathy of 64.3% and 90.9% for free-ranging cases versus free-ranging controls and 64.3% and 94.1% for free-ranging cases versus managed controls. CONCLUSIONS AND CLINICAL RELEVANCE: Cardiomyopathy is a common cause of sea otter death that has been associated with domoic acid exposure and protozoal infection. Antemortem diagnostic tests are needed to identify cardiac damage. Results suggested that serum cTnI concentration has promise as a biomarker for detection of cardiomyopathy in sea otters. Serial cTnI concentration measurements and diagnostic imaging are recommended to improve heart disease diagnosis in managed care settings.

A Comparison of Parapoxviruses in North American Pinnipeds.

Parapoxviruses cause nodular lesions on the skin and mucosal membranes of pinnipeds and infections by these viruses have been documented worldwide. Seal parapoxvirus is currently classified as a tentative species of the Parapoxvirus genus. Tissue or swab samples were analyzed from 11 pinnipeds of different host species undergoing rehabilitation on the east and west coasts of the United States of America (USA) that were positive for parapoxvirus. The aim of the study was to compare parapoxvirus sequences of fragments of the B2L, DNA polymerase, GIF and viral interleukin-10 ortholog (vIL-10) genes and to examine the evolutionary relationship between viruses detected in different pinniped species and at different locations with other members of the Parapoxvirus genus, such as Orf virus (ORFV), Bovine papular stomatitis virus (BPSV) and Pseudocowpox virus (PCPV). The sequence analysis showed that the parapoxvirus sequences from the pinnipeds differed significantly from those found in terrestrial hosts and that they formed a separate cluster within the genus. Our results suggest that transmission of the same parapoxvirus strain is possible between different species, including between members of different families (phocids and otariids). Animals belonging to the same species but living in distant geographic locations presented genetically distant parapoxviruses. The findings of this study demonstrate that sealpox lesions in pinnipeds of different species are caused by viruses that belong to the Parapoxvirus genus but have significant genetic differences compared to the established virus species in terrestrial hosts, thus strongly supporting the classification of pinniped parapoxvirus as a new species of the genus.

Fresh water skin disease in dolphins: a case definition based on pathology and environmental factors in Australia.

A distinct ulcerative dermatitis known as "freshwater skin disease" is an emerging clinical and pathological presentation in coastal cetaceans worldwide. In Australia, two remarkably similar mortality events enabled the creation of a case definition based on pathology and environmental factors. The first affected a community of endemic Tursiops australis in the Gippsland Lakes, Victoria, while the second occurred among T. aduncus resident in the Swan-Canning River system, Western Australia. The common features of both events were (1) an abrupt and marked decrease in salinity (from > 30ppt to < 5ppt) due to rainfall in the catchments, with hypo-salinity persisting weeks to months, and (2) dermatitis characterized grossly by patchy skin pallor that progressed to variable circular or targetoid, often raised, and centrally ulcerated lesions covering up to 70% of the body surface. The affected skin was often colonized by a variety of fungal, bacterial and algal species that imparted variable yellow, green or orange discoloration. Histologic lesions consisted of epidermal hydropic change leading to vesiculation and erosion; alternately, or in addition, the formation of intra-epithelial pustules resulting in ulceration and hypodermal necrosis. Thus, the environmental factors and characteristic pathologic lesions, are necessary components of the case definition for freshwater skin disease.

Environmental persistence and disinfectant susceptibility of Klebsiella pneumoniae recovered from pinnipeds stranded on the California Coast.

Hypermucoviscous K. pneumoniae (HMV) are emergent zoonotic pathogens associated with increased invasiveness and pathogenicity in terrestrial and marine mammals. In this study, HMV and non-HMV isolates recovered from stranded pinnipeds were used to investigate: 1) their persistence in sea and fresh water microcosms at 10 and 20°C, 2) their capacity to form biofilms, and 3) the biocide efficacy of four disinfectants on their planktonic and biofilm phenotypes. Results indicated that although HMV isolates were significantly more mucoviscous, non-HMV isolates displayed significantly greater capacity to form biofilms (p < 0.05). Additionally, non-HMV isolates persisted in greater numbers in both sea- and freshwater, particularly at 20°C. These two phenomena could be associated with the greater growth observed for non-HMV isolates in in-vitro growth-curve assays (p < 0.05). Similar susceptibility to disinfectants was detected in HMV and non-HMV isolates when exposed for 24 h; however, the minimal biofilm disinfectant eradication concentration for HMV isolates was significantly higher than that for non-HMV when exposed to disinfectants for 0.5 h. This information should be taken into consideration when developing biosecurity protocols in facilities holding marine mammals in captivity.

A novel quantitative real-time PCR diagnostic assay for fecal and nasal swab detection of an otariid lungworm, Parafilaroides decorus.

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy's RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild.