Surveillance for zoonotic and selected pathogens in harbor seals Phoca vitulina from central California.

The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Genotypic characterization of Streptococcus infantarius subsp. coli isolates from sea otters with infective endocarditis and/or septicemia and from environmental mussel samples.

Pulsed-field gel electrophoresis (PFGE) was used to type 128 Streptococcus infantarius subsp. coli isolates from sea otters and mussels. Six SmaI PFGE groups were detected, with one predominant group representing 57% of the isolates collected over a wide geographic region. Several sea otter and mussel isolates were highly related, suggesting that an environmental infection source is possible.

Characterization of beta-hemolytic streptococci isolated from southern sea otters (Enhydra lutris nereis) stranded along the California coast.

The goal of this study was to characterize the beta-hemolytic streptococci cultured from southern sea otters (Enhydra lutris nereis) stranded off the coast of California (USA) and to verify identifications made using the Lancefield system. Lancefield serotyping and biochemical analysis alone was inadequate for isolate characterization. Final identification was based on sequence analysis of a portion of the 16s ribosomal RNA gene from 12 of the 35 total isolates. The majority of isolates (10 of 12; 83.3%) were Streptococcus phocae and reacted with Lancefield group G and F antisera or were less frequently untypeable. The remaining isolates belonging to Lancefield group G were identified as S. dysgalactiae subsp. equisimilis (2 of 12; 16.7%). This is the first report of S. phocae in southern sea otters and further evidence of S. phocae expressing cell surface antigens compatible with Lancefield group G typing.

The effect of rehabilitation of northern elephant seals (Mirounga angustirostris) on antimicrobial resistance of commensal Escherichia coli.

The aim of this study was to determine if antimicrobial drug use increases resistance of commensal gastrointensinal Escherichia coli of wild northern elephant seals (Mirounga angustirostris) treated in rehabilitation, and, if so, identify the risk factors involved. Minimum inhibitory concentration (MIC) levels of twelve antimicrobial drugs were determined for 289 E. coli isolates from 99 seals sampled at admission and 277 isolates obtained at release from rehabilitation using broth microdilution. Prevalence of E. coli antimicrobial resistance, MIC(50), MIC(90), and clustering of MIC values were determined for seals and the data were analyzed using Fisher's exact test, ordinal logistic regression and negative binomial regression. At release from rehabilitation 77.8% of the seals had antimicrobial resistant E. coli compared to 38.4% of the seals at admission. The MIC(90) for amoxicillin-clavulanic acid, chloramphenicol, enrofloxacin, ticarcillin-clavulanic acid, and trimethoprim-saulfamethoxazole were at levels considered to be sensitive at admission but they increased to levels of resistance at release. E. coli were grouped into four clusters by their MIC values, with increasing levels of resistance going from Cluster 1 to 4. A primary risk factor associated with the probability of a seal having E. coli in Clusters 3 and 4 was time in rehabilitation, regardless of whether the animal received treatment with antimicrobial drugs, suggesting nosocomial infection. The results of this study provide evidence that increased levels of hygiene and appropriate use of antimicrobial therapy might be important in the rehabilitation of wild animals to prevent rise in the prevalence of antimicrobial resistant bacteria.

Capnocytophaga cynodegmi in a rottweiler dog with severe bronchitis and foreign-body pneumonia.

Capnocytophaga cynodegmi is a zoonotic, gram-negative, capnophilic bacterium that is usually seen in people with infections associated with dog or cat bites. The first reported case of C. cynodegmi infection in a dog is described here.

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity.

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.

Risk factors for infection with pathogenic and antimicrobial-resistant fecal bacteria in northern elephant seals in California.

OBJECTIVES: The goal of this study was to identify potential environmental and demographic factors associated with Campylobacter jejuni (C. jejuni), Salmonella enterica (Salmonella spp.), and antimicrobial-resistant Escherichia coli (E. coli) infection in northern elephant seals stranded along the California coastline. METHODS: E. coli, Salmonella spp., and C. jejuni were isolated from rectal swabs from 196 juvenile northern elephant seals, which were found stranded and alive along the California coast and brought to The Marine Mammal Center in Sausalito, California, for rehabilitation. Gender, weight, county where the animal stranded, month stranded, coastal human population density, exposure to sewage outfall or freshwater outflow (river or stream), and cumulative precipitation in the previous 24 hours, seven days, 30 days, 90 days, and 180 days were analyzed as potential risk factors for infection. RESULTS: The odds of C. jejuni and antimicrobial-resistant E. coli were higher in feces of seals stranded at sites with higher levels of freshwater outflow compared with lower levels of freshwater outflow. The odds of Salmonella spp. in feces were 5.4 times greater in seals stranded in locations with lower levels of 30-day cumulative precipitation, along with substantially lower odds of Salmonella shedding for seals stranded in Monterey or Santa Cruz county compared with seals stranded in regions further north or south of this central California location. CONCLUSIONS: Juvenile northern elephant seals that have entered the water are being colonized by antimicrobial-resistant and pathogenic fecal bacteria that may be acquired from terrestrial sources transmitted via river and surface waters.

Fatal pulmonary hemorrhage associated with RTX toxin producing Actinobacillus equuli subspecies haemolyticus infection in an adult horse.

A case of fatal pulmonary hemorrhage in a 6-year-old American Paint mare with a 2-week history of intermittent coughing, fever, and epistaxis is described. Significant macroscopic abnormalities at postmortem examination were restricted to the respiratory system, and microscopically, severe pulmonary hemorrhage with suppurative bronchopneumonia was found. Actinobacillus equuli subsp. haemolyticus was cultured from a transtracheal wash performed antemortem as well as from the lungs at necropsy. The presence of airway-associated hemorrhage in conjunction with bacterial bronchopneumonia suggested endothelial damage caused by a locally elaborated bacterial toxin, possibly produced by the A. equuli strain isolated from the lungs. The objective of this report was to indirectly document the presence of hemolysin repeat in structural toxin (RTX) in the lungs of the reported mare. A real-time polymerase chain reaction (PCR) assay targeting the recently described aqx gene of A. equuli subsp. haemolyticus was established and validated. Transcriptional activity of the aqx gene was used as a surrogate method to document toxin production. Real-time PCR analysis of the transtracheal fluid and lung tissue of the affected mare confirmed the presence and the transcriptional activity of the aqx gene at the genomic (gDNA) and complementary DNA (cDNA) levels, respectively. The presence of pneumonia associated with hemorrhagic pulmonary fluid and the culture of large numbers of hemolytic A. equuli should prompt the clinician to consider endothelial damage caused by bacterial toxins.

ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

Aerobic bacterial flora of the vagina and prepuce of California sea lions (Zalophus californianus) and investigation of associations with urogenital carcinoma.

To investigate the association between genital bacterial infection and urogenital carcinoma in California sea lions (Zalophus californianus), vaginal and preputial swabs for bacterial isolation were taken from 148 free-ranging and 51 stranded California sea lions including 16 animals with urogenital carcinoma. Cytological examination of vaginal or preputial smears showed a majority (65.5%, 57/87) of animals examined had mild or no inflammation. Aerobic bacteria were isolated from 116 (78.4%) wild sea lions and 100% of stranded animals. A total of 403 isolates were identified representing 51 unique bacterial species. The median number of isolates per animal increased with age in the wild group, but there was no difference in the number of isolates per animal between wild and stranded adults. The most common bacteria isolated from the wild sea lions were Psychrobacter phenylpyruvicus (39 isolates), non-hemolytic Streptococcus (35 isolates), Corynebacterium spp. (30 isolates), and Escherichia coli (20 isolates). More bacterial species were isolated from stranded animals than wild animals (33 versus 26) and there was significantly less growth of P. phenylpyruvicus, Corynebacterium spp., and Moraxella-like spp. in the stranded animals. Beta-hemolytic Streptococcus was the only bacterium significantly associated with urogenital carcinomas in California sea lions, but only in females.

Lesions associated with a novel Mycoplasma sp. in California sea lions (Zalophus californianus) undergoing rehabilitation.

From July 1999 to November 2001, Mycoplasma sp. was cultured from lesions in 16 California sea lions (Zalophus californianus) undergoing rehabilitation. The Mycoplasma sp. was the likely cause of death of four animals in which it was associated with either pneumonia or polyarthritis. The most common lesion associated with this bacterium was subdermal abscessation, found in 12 animals. Other lesions included intramuscular abscesses, septic arthritis, and lymphadenopathy. Infection was associated with a leukocytosis and left shift in 12 animals. Animals with abscesses improved clinically after surgical lancing, irrigation, and systemic antibiotic therapy. The mycoplasma isolates had a consistent 16S rRNA sequence dissimilar from other Mycoplasma spp. and represent a novel species, Mycoplasma zalophi proposed sp. nov.

Molecular characterization of Clostridium difficile isolates from horses in an intensive care unit and association of disease severity with strain type.

OBJECTIVE: To determine molecular characteristics, antimicrobial susceptibility, and toxigenicity of Clostridium difficile isolates from horses in an intensive care unit and evaluate associations among severity of clinical disease with specific strains of C difficile. DESIGN: Prospective study. ANIMALS: 130 horses. PROCEDURES: Feces were collected from horses admitted for acute gastrointestinal tract disease with loose feces and submitted for microbial culture and immunoassay for toxin production. Polymerase chain reaction assays were performed on isolates for toxins A and B genes and strain identification. RESULTS: Isolates were grouped into 3 strains (A, B, and C) on the basis of molecular banding patterns. Toxins A and B gene sequences were detected in 93%, 95%, and 73% of isolates of strains A, B, and C, respectively. Results of fecal immunoassays for toxin A were positive in 40%, 63%, and 16% of horses with strains A, B, and C, respectively. Isolates in strain B were resistant to metronidazole. Horses infected with strain B were 10 times as likely to have been treated with metronidazole prior to the onset of diarrhea as horses infected with other strains. Duration from onset of diarrhea to discharge (among survivors) was longer, systemic inflammatory response syndromes were more pronounced, and mortality rate was higher in horses infected with strain B than those infected with strains A and C combined. CONCLUSIONS AND CLINICAL RELEVANCE: Horses may be infected with a number of heterogeneous isolates of C difficile. Results indicated that toxigenicity and antimicrobial susceptibility of isolates vary and that metronidazole-resistant strains may be associated with severe disease.

PREVALENCE, PATHOLOGY, AND RISK FACTORS ASSOCIATED WITH STREPTOCOCCUS PHOCAE INFECTION IN SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS), 2004-10.

Recent studies have implicated beta-hemolytic streptococci as opportunistic pathogens of marine mammals, including southern sea otters (Enhydra lutris nereis), but little is known about their prevalence or pathophysiology. Herein, we focus on risk factors for sea otter infection by a single beta-hemolytic streptococcal species, Streptococcus phocae. Streptococcus phocae was first identified as a marine mammal pathogen in 1994, and the first report in southern sea otters was in 2009. Its broad host range encompasses fish, pinnipeds, cetaceans, and mustelids, with S. phocae now recognized as an important pathogen of marine species worldwide. We assessed risk factors and lesion patterns for S. phocae infection in southern sea otters. Using archival necropsy data, S. phocae prevalence was 40.5% in fresh dead otters examined 2004-10. Skin trauma of any type was identified as a significant risk factor for S. phocae infection. The risk of infection was similar regardless of the cause and relative severity of skin trauma, including mating or fight wounds, shark bite, and anthropogenic trauma. Streptococcus phocae-infected sea otters were also more likely to present with abscesses or bacterial septicemia. Our findings highlight the importance of S. phocae as an opportunistic pathogen of sea otters and suggest that the most likely portal of entry is damaged skin. Even tiny skin breaks appear to facilitate bacterial colonization, invasion, abscess formation, and systemic spread. Our data provide important insights for management and care of marine species.

Lower respiratory tract infection due to Capnocytophaga cynodegmi in a cat with pulmonary carcinoma.

A 10-year-old male castrated domestic shorthair cat was evaluated for coughing and lethargy. Thoracic radiographs revealed a soft tissue lung mass and diffuse peribronchial infiltrates. Bronchoscopy was performed and Capnocytophaga cynodegmi was cultured from bilateral bronchoalveolar lavage samples. Clinical signs and bacterial colonization resolved following treatment with enrofloxacin. A lung lobectomy was performed to remove the lung mass, which was diagnosed as pulmonary carcinoma. C cynodegmi is most frequently isolated from localized wound and corneal infections in humans. Specialized growth characteristics of C cynodegmi may result in low sensitivity for bacterial culture. To the authors' knowledge, this case represents the first report of C cynodegmi infection in a veterinary patient and only the second case in human or veterinary medicine where the organism has been isolated from a bronchoalveolar lavage sample. Based on this report, Capnocytophaga species should be considered as potential opportunistic pathogens.

Evaluation of aerobic bacteriologic culture of epidermal collarette specimens in dogs with superficial pyoderma.

OBJECTIVE: To evaluate a method of aerobic bacteriologic culture of epidermal collarette specimens from dogs with superficial pyoderma and compare results with those for aerobic bacteriologic culture of abdominal skin specimens in healthy dogs. DESIGN: Prospective study. ANIMALS: 22 dogs with epidermal collarettes and 24 healthy dogs. PROCEDURE: Dry sterile cotton swabs were rolled across epidermal collarettes or hairless areas of abdominal skin in healthy dogs and submitted for aerobic bacteriologic culture. Hemolytic colonies of gram-positive-staining cocci were tested for catalase production, and if results were positive, a coagulase test was performed. Colonies with coagulase activity were tested for the ability to ferment mannitol. Antimicrobial susceptibility testing was performed on all Staphylococcus spp that were isolated. RESULTS: S. intermedius was isolated from collarettes in 18 of 22 dogs with superficial pyoderma but not from healthy dogs. Estimated sensitivity and specificity of the culture method were 81.8% and 100%, respectively. There were no significant differences in the ability to culture S. intermedius, the number of S. intermedius isolates without resistance to antimicrobials, and the number of S. intermedius isolates resistant to penicillin G when comparing dogs with superficial pyoderma for the first time and dogs with recurrent pyoderma, dogs that did or did not receive concurrent antimicrobials, and dogs with and without underlying allergic disease. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture of epidermal collarette specimens was a simple and reliable method for identification of S. intermedius in dogs with superficial pyoderma, regardless of history of pyoderma or current antimicrobial use.

Corynebacterium urealyticum urinary tract infection in dogs and cats: 7 cases (1996-2003).

OBJECTIVE: To identify clinical features of Corynebacterium urealyticum urinary tract infection in dogs and cats and antimicrobial susceptibility patterns of C urealyticum isolates. DESIGN: Retrospective study. ANIMALS: 5 dogs and 2 cats. PROCEDURE: Medical records of dogs and cats for which C urealyticum was isolated from urine samples were reviewed. Isolates from clinical cases, along with previously lyophilized unsubtyped isolates of Corynebacterium spp collected between 1977 and 1995, were examined and, if subtyped as C urealyticum, tested for antimicrobial susceptibility. RESULTS: Signalment of infected animals was variable. Prior micturition disorders were common, and all animals had signs of lower urinary tract disease at the time C urealyticum infection was diagnosed. Median urine pH was 8.0; WBCs and bacteria were variably seen in urine sediment. In vitro antimicrobial susceptibility testing of 14 C urealyticum isolates revealed that all were susceptible or had intermediate susceptibility to chloramphenicol, tetracycline, and vancomycin and most were susceptible to enrofloxacin. Thickening of the bladder wall and accumulation of sediment were common ultrasonographic findings. Contrast radiography or cystoscopy revealed findings consistent with encrusting cystitis in 3 dogs. Infection resolved in 2 dogs following surgical debridement of bladder plaques and antimicrobial administration. In 2 other dogs and 1 cat treated with antimicrobials, infection with C urealyticum resolved, but urinary tract infection with a different bacterial species developed. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that preexisting urinary tract disorders are common in dogs and cats with C urealyticum infection. Treatment with appropriate antimicrobials in combination with surgical debridement might eliminate C urealyticum infection.

Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs.

Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.

Molecular and antigenic characterization of Bordetella bronchiseptica isolated from a wild southern sea otter (Enhydra lutris nereis) with severe suppurative bronchopneumonia.

Bordetella bronchiseptica was isolated in pure culture from the lung, abdomen, and intestine of a wild free-ranging southern sea otter (Enhydra lutris nereis) with severe, suppurative bronchopneumonia. Immunohistochemistry, using antiserum raised to B. bronchiseptica, revealed strong positive staining of bacteria attached to bronchial ciliated epithelia as well as scattered positive staining in affected alveoli. Western blot analysis demonstrated that virulence factors, filamentous hemagglutinin, pertactin, and adenylate cyclase toxin are produced by the sea otter B. bronchiseptica isolate. Ribotype analysis using Pvu II restriction digests indicated that this isolate is most similar to strains commonly obtained in domestic dogs and cats.

Paecilomycosis in dogs and horses and a review of the literature.

We reviewed 14 cases of paecilomycosis in a tertiary care veterinary hospital and all reports of the disease in the veterinary literature. Paecilomycosis is a rare disease primarily of dogs, horses, reptiles, and humans. Clinical manifestations in veterinary patients vary but include disseminated disease and diskospondylitis, particularly in dogs: pneumonia in dogs, horses, and reptiles; keratitis in horses; and miscellaneous local infections. It is important to have an appropriate index of suspicion because the diagnosis can be difficult, particularly in localized disease where it is difficult to determine whether a positive culture represents an etiology or a contamination with an environmental saprophyte. Spinal radiographs, transtracheal washes, histopathology, and fungal culture have proven to be valuable diagnostic tools. The prognosis for paecilomycosis is poor, although some treatment success has been reported, and success rates could improve if additional information were available regarding fungal species occurring in veterinary patients and drugs to which these fungi are susceptible.

Characterization and clinical manifestations of Arcanobacterium phocae infections in marine mammals stranded along the central California coast.

Between 1994 and 2000, 141 Arcanobacterium phocae isolates were recovered from marine mammals that stranded along the central California coast (USA). Arcanobacterium phocae was cultured from tissue sites with abnormal discharge or evidence of inflammation in 66 California sea lions (Zalophus californianus), 50 Pacific harbor seals (Phoca vitulina richardii), 19 northern elephant seals (Mirounga angustirostris), five southern sea otters (Enhydra lutris nereis), and one common dolphin (Delphinus delphis). The overall prevalence of A. phocae among cultured stranded marine mammals was 8%. This is the first report of A. phocae in animals from the Pacific Ocean. Sequence analysis of a portion of the 16S ribosomal RNA gene confirmed recent isolates as A. phocae. Prior to phylogenetic testing and the routine use of the esculin hydrolysis and motility tests, A. phocae isolates may have been misidentified as Listeria ivanovii. Arcanobacterium phocae was commonly isolated from superficial abscesses, was often present in mixed infections, and was susceptible to all antimicrobial agents tested.