Marine Mammal Bacteriology Flashcards

1
Q

Causative agent of brucellosis in cetaceans

A

Brucela ceti

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2
Q

What bacteria is associated with unusual mortality events in sea otters? What lesions are found on necropsy?

Most cases of sepsis in marine mammals are attributable to which organisms?

A

Septicemia

  • Immune response trigger by infection may cause septic shock
  • Most cases of septic shock result from endotoxin-producing gram-negative bacteria (ie E. coli and Pasteurella)
  • Use of antibiotics with slow bactericidal activity may exacerbate the situation
  • 1/3 described unusual mortality events involving sea otters caused by infected with Streptococcus bovis/equinus 🡪 sepsis and cardiac damage
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3
Q

What is the most common bacterial agent in cetacean pneumonia?

What about pinniped pneumonia?

What clinical signs are seen?

How is pneumonia treated?

A

Respiratory disease

  • Pneumonia is a common finding in stranded marine mammals
  • Upper and lower respiratory anatomy of cetaceans designed for rapid exchange of large volumes of air 🡪 can predispose to infections
  • Retrospective study of pneumonia in bottlenose dolphins at the Navy
    • 50% of dolphins evaluated had pneumonia confirmed by histopath
    • Bacterial pneumonia in 43% of cases
      • Staphylococcus aureus most common
      • Others: Erysipelothrix rhusiopathiae, Proteus, Pseudomonas aeruginosa, Streptococcus zooepidemicus
    • Many involved disseminated disease
    • Lungs were consistently most severely affected
  • Pneumonia was 2nd most common finding in stranded California sea lions
  • Pneumonia epizootics in harbor seals along the New England coast
    • Mycoplasma phocidae, M. phocarhinis, M. phocacerebrale
    • Isolated from both healthy and diseased animals
    • *Mycoplasma may be the most commonly transmitted pathogenic bacterium from seals to humans – “seal finger”*
  • Cetaceans
    • Coughing – frequently observed in pinnipeds, infrequently observed in dolphins until a significant portion of the lung field is damaged
    • Unilateral pneumonia or large pulmonary abscesses in cetaceans 🡪 listing to side of damaged lung
    • Nasal or blowhole swabs and exhalation plates + cytological analysis of sputum + bronchoalveolar lavage aid in diagnosis in cetaceans
  • Treatment
    • Antibiotics with broad spectrum and strong activity against gram-negative organisms often used initially (before test results are back)
    • Pinnipeds – oxygen supplementation, bronchodilators, mucolytic agents have been helpful
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4
Q

Marine mammal abscesses are often caused by what bacterial organism?

Cutaneous nodules in Galapagos sea lions have been associated with what bacterial organism?

What other bacterial infections have been seen to affect marine mammal skin?

A

Dermatological disease

  • Bacterial skin disease often follows a primary viral, parasitic, or traumatic insult
  • Arcanobacterium phocae from harbor and gray seals and Arcanobacterium pluranimalium from harbor porpoises found
    • Phylogenetically similar to Trueperella pyogenes, a common cause of pyogenic infections in terrestrial mammals
    • Isolated from abscesses, wounds, ear canal, ocular/nasal/tracheal discharges
  • Epizootic in Galapagos sea lions with suppurative cutaneous nodules containing Pseudomonas aeruginosa
  • Of bacteria isolated from ulcerative dermatitis, panniculitis, diamond skin disease, and slow-healing abscesses and ulcers:
    • Aeromonas sp., M. marinum, Pseudomonas spp., and Vibrio spp – normally present in marine environment
    • E. rhusiopathiae and Streptococcus iniae – fish pathogens that may also infect dolphins
  • Most can be opportunistic pathogens
  • Etiologic agents of bowhead skin lesions: Rodococcus equi, Corynebacterium pseudotuberculosis, Moraxella spp.
  • Dermatophilus congolensis – cutaneous disease in South American sea lions
    • Prominent layered scabs
    • Mortality low, morbidity high
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5
Q

What two bacterial infections are common in the urogenital systems of marine mammals?

A

Urogenital disease

  • Leptospirosis and Brucella more common
  • Pyometra and metritis in wild California sea lions
  • High prevalence of urogenital carcinomas
    • Associated with OtHV-1, genetics, contaminant exposure, and beta-hemolytic Streptococcus in females
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6
Q

What is an important cause of enterotoxemia and myositis in managed marine mammals?

What other bacterial pathogens are isolated from marine mammal GI tracts?

A

Gastrointestinal disease

  • Clostridial enterotoxemia and myositis in managed cetaceans and pinnipeds
    • Clostridium perfringens
    • Managed Steller sea lions, northern fur seals, belugas, pilot whales
    • Enteritis in wild harbor seals
    • Widespread in terrestrial and aquatic ecosystems
    • Seasonal changes and type of nutrition are important risk factors
    • Ideal condition: devitalized tissue, anaerobic conditions, high glucose concentrations found in diving mammals
    • Sudden change to abundant food with large amounts of carbs and/or proteins
    • Vaccination performed in sea lions, fur seals, killer whales, belugas
      • None licensed for use in marine mammals
  • Salmonella
    • Often isolated
    • S. enteritidis considered significant cause of fur seal pup mortality on Pribilof Islands of Alaska
  • Gastritis and gastric ulcers well-recognized in cetaceans
    • Helicobacter spp. isolated from gastric mucosal samples/gastric fluid of bolphins, beluga, and an Australian sea lion
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7
Q

What type of bacteria is Brucella?

What are the two marine mammal brucella species?

What is the geographical distribution of these diseases?

How is brucellosis diagnosed?

A

Brucella spp. and brucellosis

  • Gram-negative, nonmotile, facultative intracellular, coccobacilliary bacteria
  • Zoonosis
  • Worldwide distribution
  • Brucella ceti – preferred host of cetaceans
  • Brucella pinnipedialis – preferred host of pinnipeds
  • No reports of isolation from marine mammals in the southern hemisphere
  • Diagnosis
    • Gold standard diagnostic: isolation of bacterium
      • No ideal tissue
      • Majority of cetacean isolates appear on Farrell’s medium
      • Recommend incubation period of 14 days on a nonselective medium as well as Farrell’s
    • Serological tests
      • Validation of these tests with comparison to a bacteriological gold standard is recommended
      • Absence of antibodies does not exclude exposure
      • Terio pinnipeds – lack of consistency in serological assays used makes valid comparisons difficult
    • PCR
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8
Q

What brucella species affects cetaceans?

What are the lesions associated with the CNS?

What about the reproductive tract?

How is this disease transmitted? Are there possible vectors?

A
  • Cetaceans
    • B. ceti – zoonotic
    • CNS
      • Isolation from the CNS in association with neurological signs reported many times, often in stranded striped dolphins
      • Pathological changes: spinal discospondylitis, meningoencephalitis, meningitis, choroiditis, altered cerebrospinal fluid, remodeling of the occipital condyles
    • Reproductive tract
      • Isolated from aborted fetuses and reproductive organs of bottlenose dolphins with placentitis
      • Reproductive organs, milk, and fetuses of stranded striped dolphins
      • Found in a genital ulcer, uterus, mammary gland, and milk of a stranded harbor porpoise
      • In association with mastitis and endometritis and from a testicular abscess
    • Transmission
      • Poorly understood
      • Horizontal transmission via aborted, infected material is possible
      • Could be via close contact or by vertical transmission
      • Has been isolated from lungworms – transmission via parasites may be possible
    • Terio Cetacea
      • Exposure is common in free-ranging populations, significant proportion of animals overcome infection
        • May act as carriers and potential sheddings
      • Possible lungworm vectors: Halocercus and Pseudalius
      • Most important lesions in reproductive, CNS, and musculoskeletal systems
        • Females – endometritis, placentitis, abortions
        • Males – epididymitis and orchitis
      • Most common lesions of cetacean brucellosis in the CNS
        • Harbor porpoises, white-beaked dolphins, white-sided dolphins, striped dolphins, bottlenose dolphins, common dolphins
        • CSF was increased in volume and cellularity
        • Acquired hydrocephalus a common sequela
        • Predilection sites: cerebellum, brainstem, spinal cord, medulla oblongata with less involvement of cerebral cortex
      • Isolated from bones and joints
        • Discospondylitis, fibrinopurulent osteoarthritis, degenerative joint changes in a white-sided dolphin, similar in bottlenose dolphins and white-beaked dolphins
        • Blubber abscesses and pneumonia
      • Diagnosis: immunohistochemistry highlights antigens in phagocytic cells
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9
Q

What Brucella affects pinnipeds?

How does brucellosis in pinnipeds differ from cetaceans?

Are there any potential vectors?

What public helath concerns are there?

A
  • Pinnipeds
    • B. pinnipedialis
    • Phocids
      • In harbor and hooded seals, it did not multiple in vitro in human, murine, or hooded seal macrophages
        • In other species, brucellosis usually persists in macrophages, resulting in chronic infections
      • As NOT been isolated from any Brucella-associated pathology in true seals
        • Instead isolated in association with a wide range of different organs and disease conditions
        • Usually, Brucella is characterized by bacterial replication in the reproductive system of primary hosts
      • Age-dependent serological pattern in true seals
        • Low probability of seropositive in pups, higher probability in yearlings, and decreasing probability with age
          • Not transferred from mother to newborn pup
          • May be clearing infection prior to reaching age of primiparity
        • Regularly isolated from juvenile true seals
      • All of these disparities suggest that true seals may not be the primary host of B. pinnipedialis (may be spillover)
      • Isolated from lungworms in seals
        • Terio pinnipeds - Lungworms associated with Brucella found in lungs of pinnipeds – whether they are primary route of transmission or incidentally infected is unknown
      • Hooded seal strain has survived in Atlantic cod
        • May play a role in transmission of this pathogen to hooded seals
    • Otariids
      • Number of isolates and PCR-positive cases are few
      • Those reported have been recurrently associated with pathology in the reproductive organs
      • Transplacental transmission has been shown to take place
      • Zoonotic strain type (ST27) has been detected in a CA sea lion
      • Terio pinnipeds – northern fur seal with suppurative and necrotizing placentitis
    • Terio pinnipeds
      • Fish or vertebrate vector possible
      • Zoonotic concern for those who consume seal or whale meat, but none of the reported human cases had direct exposure to marine mammals
      • Majority of infections in pinnipeds are subclinical
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10
Q

How do vibrio bacteria affect marine mammals?

What are the typical lesions in cetaceans? What vibrio species are commonly implicated?

What are the typical lesions with pinnipeds?

A

Vibriosis

  • Gram-negative motile bacteria in marine and brackish water
  • Temperature: 20-30C
  • Mesohaline (low to medium salinity) (<5-30%)
  • Marine species V. vulnificus and V. parahemolyticus are responsible for noncholera Vibrio disease in humans
    • Ingestion of undercooked seafood or infection of wounds
  • With global temperature increases, ranges of marine Vibrio may increase
  • Increasing resistance of several isolates
    • Early treatment with aminoglycosides, quinolones, or 3rd-generation cephalosporins indicated to prevent fatal septicemias (but be selective about which animals to treat…)
  • Cetaceans
    • V. alginolyticus isolated from blood, liver, and lungs of an Atlantic white-sided dolphins with acute focal bronchopneumonia and acute necrotizing hepatitis
    • Occurrence of skin lesions linked to Vibrio in both dolphins and whales
    • Have been isolated from blowhole, anus, and colon of healthy cetaceans, and from internal organs of stranded animals with unknown cause of death
    • Isolated from porpoises in the North and Baltic seas (populations assume to be more exposed to anthropogenic factors) but not from porpoises inhabiting waters less exposed to anthropogenic stressors
      • Suggests increased anthropogenic activity and concomitant stress may influence total bacterial load
    • Human pathogen V. cholerae found in Indo-Pacific humpbacked dolphins from heavily polluted waters
      • Sewage waste being the most likely origin of infection
  • Pinnipeds
    • Fewer reports than in cetaceans (In CA sea lions, elephant seals, harbor seals, Hawaiian monk seals, hooded seals)
    • Common species: V. cholera (non-O1), V. parahemolyticus, V. alginolyticus
    • Associated with gastritis, enteritis, septicemia, abscessation, pneumonia in elephant seals, harbor seals, CA sea lions
    • Prevalence of fecal shedding greater in wild-caught compared to stranded harbor seals
      • Location, turbidity, and salinity all influenced prevalence
  • Sirenia
    • Isolated from dugongs
    • Mixed infections with Vibrio and Aeromonas associated with pneumonia or pleuritis
  • Sea otters
    • Found in feces of sea otters, associated with water contact or consumption of food harvested from contaminated environments
    • Pathological significance unclear
      • Dead otters found to be more likely to test positive for P. parahaemolyticus than live otters
      • Otters from more urbanized coastlines and areas with high freshwater runoff were more likely to test positive for Vibrio and other enteric bacterial pathogens
      • Most commonly isolated from otters and mussels (prey species): V. parahaemolyticus, V. alginolyticus, V. cholera (Non-O1)
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11
Q

What diseases have been documented with pasteruella infections in marine mammals?

A

Pasteurellosis

  • Potential pathogens on the mucosal surfaces of birds and mammals
  • Primarily colonize the upper respiratory tract and lower reproductive tract
  • Diagnosis
    • Bacterial isolation and identification, PCR, specific hybridization probes, serological tests
  • Cetaceans
    • Cultured from cetaceans at Mystic Aquarium a few times as incidental findings
    • Enteritis due to P. multocida involving bottlenose dolphins and short-beaked common dolphins has been reported
      • Acutely ill and died due to bacteremia and intestinal hemorrhage
    • P. haemolytica associated with septicemia in a group of managed bottlenose dolphins
  • Pinnipeds
    • Isolated from both healthy and diseased phocids and otariids
      • Diagnosed in both phocids and otariids at Mystic Aquarium
    • Most frequently isolated from pinnipeds: P. multocida
    • New species: Otariodibacter oris
      • Oral cavity of healthy sea lions, fur seals, walruses
    • Bisgaardia hudsonensis from lungs of ringed seals in association with an investigation into death of 21 seals
    • Isolated from conjunctiva and wounds of stranded harbor seals
    • Vaccines
      • Attempt to use commercial P. multocida vaccines for cattle at Mystic Aquarium did NOT provide protection
      • Polysaccharide vaccine developed from a necropsy isolate appears to have provided long-term immunity after administration of primary and booster vaccinations
  • Sirenian
    • Cultured in a mixed infection with Morganella morganii and Serratia marcescens from lungs of an adult dugong with pneumonia
  • Otters
    • Isolated from feces of a Eurasian otter
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12
Q

What type of bacteria is Erysipelothrix rhusiopathieae?

How is it transmitted to marine mammals?

Describe vaccination attempts.

What are teh two forms of the disease?

A

Erysipelothrix

  • Erysipelothrix rhusiopathiae
  • Gram-positive, non-acid-fast, small bacillus
  • Ubiquitous in the marine environment
  • Worldwide distribution
  • Most commonly associated with disease in pigs, turkeys, chickens, and ducks
  • Survives for long period in the environment
  • Grows on mucous layer in fish skin
    • Should be considered an occupational hazard in humans
  • Diagnosis
    • Clinical signs, isolation of the bacterium, presence of antibodies
  • Cetaceans
    • One of several infections associated with pneumonia in Navy bottlenose dolphins
    • Study of 54 bottlenose dolphins vaccinated using an off-label porcine bactrin component
      • Generated humoral immunity against E. rhusiopathiiae in dolphins
      • Efficacy influenced by number of vaccine administration but not by gender, age, or history of natural infection
  • Terio Cetacea
    • Common clinical concern for managed cetaceans, identified in stranded free-ranging cetaceans
    • Presumptive route of oral exposure or through rake marks or ulcers
    • 2 forms
      • 1) acute fatal septicemic form
        • Sudden death with few, if any, premonitory signs
        • Ascites with mottled livers
      • 2) milder cutaneous form
        • Seen in belugas and bottlenose dolphin calves
        • Rhomboid shaped patch of discoloration with loss of appetite and inflammatory leukogram
        • Responsive to antibiotics
        • Lesions regress +/- sloughing of affected skin
        • Development of disease in calves from vaccinated dams in enzootic populations – suggest role of species-susceptibility or antibody transfer that is protective against the acute form
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13
Q

What type of bacteria are mycobacteria?

What mycobacteria cause disease in pinnipeds, cetaceans, and sirenians?

What is the primary difference between pinniped mycobacteriosis and cetacean or sirenian mycobacteriosis?

What clincial signs and lesions are typically seen?

A

Mycobacterial infections

  • Acid-fast, gram-positive rods
  • Aerobic and non-spore-forming
  • Colored red by Ziehl-Neelsen staining (diagnostic tool)
  • M. pinnipedii causes tuberculosis in pinnipeds
    • Mycobacterium tuberculosis complex (MTBC) also includes
      • M. tuberculosis- most common cause of TB in humans
      • M. bovis – etiologic agent for bovine TB
  • Non-MTBC mycobacteria cause disease in marine mammals
    • M. chelonae, M. fortuitum, M. chitae, M. marinum
  • Cetaceans
    • M. abscessus isolated from 23yo Atlantic bottlenose dolphin
      • Pyogranulomatous pneumonia and associated coughing and blood respiratory discharge
    • M. marinum isolated from a managed beluga
      • Dermatitis, panniculitis, chronic pleuritis
    • M. chelonae caused disseminated panniculitis in a bottlenose dolphin
    • Should be a differential in whales with generalized dermatitis and/or panniculitis
    • Terio Cetacea
      • Atypical mycobacteriosis
        • Panniculitis, pneumonia, pleuritis in managed odontocetes
        • Infections via percutaneous inoculation or secondary contamination of cutaneous lesions
        • Animal-animal or animal-human infection is uncommon
        • Cannot pass through mucosa or integument
        • Occurs due to reduced host immunity or debilitation
        • Unique anatomy of respiratory tract may predispose
  • Pinnipeds
    • Used to think that many causes were M. bovis, then described M. pinnipedii
    • M. marinum also present as an opportunistic pathogen, can affect humans
    • Seals may not show clinical signs, or may have anorexia, weight loss, lethargy
    • Subantarctic fur seals had thoracic lesions and a large volume of yellowish exudate in the lungs
    • South American fur seals had purulent exudate adherent to the trachea and enlarged lymph nodes
    • Granulomas may also be found in kidneys, spleen, liver, peritoneum
    • Diagnosis
      • Serological testing, radiography, CT
        • CT will help find calcified tubercles in lungs and other organs
      • Tuberculin skin testing has been tried – LOW sensitivity and specificity, lacks test validation
      • If shedding, a sputum or tracheal wash sample can be examined by acid fast or fluorescent antibody staining and microscopy, or by cultivation
      • Gold standard: cultivation
        • May take up to 8 weeks
        • PCR is faster (will also detect dead bacteria)
    • Most isolates are susceptible to isoniacide, rifampin, streptomycin, ethambutol, and pyrazinamide
      • Treatment is controversial and effect is uncertain
    • Transmitted easily between individuals
    • Zoonotic
      • Test suspicious cases and their contact animals, maybe euthanize positive cases
      • Several reports of transmission of mycobacteria to humans from marine mammals in managed care
    • Terio pinnipeds
      • Reported in a number of managed otariids worldwide
      • Most frequently affects Southern sea lions
      • Infection of free-ranging pinnipeds confined to southern hemisphere
      • Not reported in any phocid species (M. pinnipedii)
      • Most likely route of infection = aerosolized bacteria shed in respiratory secretions of infected pinnipeds
      • Clinical signs
        • Poor body condition
        • Thoracic organs most often affect, supporting inhalation as the dominant route of infection
        • 2 gross presentations: 1) granulomatous to pyogranulomatous pleuropneumonia, 2) multifocal caseating granulomas within the lungs and thoracic lymph nodes
      • Diagnosis
        • Confirmation requires culture and biochemical assays (slow) or PCR
      • Sirenia
    • Two reports in Amazonian manatee
      • Managed 1yo with pyoderma and pustules, complete healing never achieved, died 4 years later
        • Abscesses found in both lobes of lungs
        • M. chelonei
      • Systemic infection with M. marinum
        • Anorexia and skin lesions that lasted for 4 years until the animal died
        • SC edema, thickened skin, disseminated tubercles filled with caseous material in the testes, lungs, pleura
    • Terio Sirenia
      • M. marinum, M. fortuitum, M. kansaii, M. chelonei have been reported
      • Multiple coalescing caseous nodules present throughout the lungs with pleural, lymph node, testicular, cutaneous involvement
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14
Q

Leptospirosis in california sea lions is caused by what serovar?

Describe typical clinical signs and pathology.

What other species can be affected?

How is this disease diagnosed?

How is it treated?

A

Leptospirosis

  • Geographically widespread
  • Zoonotic
  • Leptospires enter body through small skin scratches via mucosal membranes, or through wet skin 🡪 disseminate into the blood
  • Acute leptospirosis
    • Immune phase
    • Organism disappears from bloodstream, antibodies appear
    • Spirochetes responsible for both human and animal leptospirosis
    • Signs range from mild febrile illness to severe multi-organ failure (especially pulmonary hemorrhage and renal failure)
  • Terio pinnipeds
    • Most commonly reported in free-ranging pinnipeds including CA sea lions, harbor, and northern elephant seals along Pacific cast
  • Diagnosis
    • Direct observation of leptospires by darkfield microscopy is untrustworthy
    • Isolation can take months
    • Often by serology
      • Antibodies are lacking in the acute phase
      • PCR may confirm diagnosis in early phase
  • Most frequently reported in 2 otariid species from eastern Pacific: CA sea lions and northern fur seals
    • Reports in phocids restricted to Pacific harbor seals and northern elephant seals
    • Single account from a cetacean
  • Pinnipeds
    • Periodic leptospirosis outbreaks causing morbidity and mortality in CA sea lions over the last 4 decades
      • Cyclical epizootics associated with Leptospira interrogans serovar Pomona
      • Pacific coast of Canada and the US
      • 90% of cases from CA
      • Most often seen July to December
      • Serologic evidence shows exposure of the youngest age classes each year
      • Likely enzootic in the population
      • Terio pinnipeds
        • Epizootics every 3-5 years and result in large numbers of CA sea lions stranding
    • Mechanism for persistent circulation of leptospires in population
      • Asymptomatic urinary shedding in free-ranging CA sea lions
      • Asymptomatic seroconversion as well as chronic asymptomatic urinary shedding in a rehabilitated sea lion
    • Clinical signs
      • Depression, dehydration, polydipsia, anorexia, fever, vomiting, icterus, abortion, oral ulcerations, reluctance to use rear limbs
      • Marked leukocytosis and elevated serum creatinine, Na, Ca, P, BUN
      • Terio pinnipeds –
        • May cause abortion
        • Consistent with renal failure: PD, elevated BUN, creatinine, P
    • Necropsy
      • Kidneys appear swollen
      • Liver may be enlarged and friable
      • Renal cortex and medulla often pale with loss of renicular and corticomedullary differentiation and sporadic infarcts
        • Subcapsular hemorrhages and hemorrhaging at the corticomedullary junction
      • Aborted fetuses and seal pups may have SC hemorrhages
      • Tubulointerstitial nephritis
        • Abundant associated spirochetes within the tubular epithelium and lumen
      • Terio pinnipeds
        • Confirmed histologically by demonstrating spirochetes with silver stains or IHC
        • Kidneys are swollen, pale tan, exhibit lack of renule and corticomedullary differentiation
        • PCR can be used for diagnosis
        • Zoonotic!
    • Treatment
      • PO or SC fluids and tetracycline 22 mg/kg PO TID or long-acting tetracycline 11mg/kg IM q24hr
      • Study with 66 sea lions treated successfully with tetracycline 22mg/kg PO TID or potassium penicillin G 44,000 U/kg PO or IM BID for 10-14 days
    • Vaccine
      • Fur seals were vaccinated twice yearly with 5-serovar commercial livestock vaccine (Lepto-5)
        • Considerable variability in antibody titer levels achieved against different serovars
        • Incidence of leptospirosis cases decreased
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15
Q

Describe nocardiosis in marine mammals.

What are teh most common clinical signs?

What is the most common form in cetaceans? Where does infection usually start?

What is the disease typically like in pinnipeds?

A

Nocardiosis

  • Opportunistic, noncontagious disease
  • Gram-positive, facultative intracellular, aerobic bacteria in order Actinomycetales
  • Ubiquitous in soil and water, identified in marine sediment
  • Tends to be more common in certain geographic areas and climates
  • Most common in cetaceans: N. asteroids, N. farcinia, N. brasiliensis, N. cyriacigeorgica, N. levis
  • Most common in pinnipeds: N. asteroids, N. farcinia, N. brasiliensis, N. otitisdiscavarium
  • Clinical signs: abscesses, cutaneous/SC lesions, mastitis, pneumonia
    • Granulomatous inflammatory reactions can be acute or chronic
    • Terio Cetacea
      • Pyogranulomatous inflammation in multiple organs, predilection for lung
      • Reported in Atlantic bottlenose dolphins, belugas, killer whales
      • Most common presentation: systemic form (2+ organs)
      • Most frequently affected organs: lung, thoracic lymph nodes
        • Infection in brain is the site most commonly associated with death
  • Diagnosis
    • Identification of the organism from infected tissue
    • Culture can be challenging
    • Histology or cytology showing acid-fast (or partially acid-fast) branched filaments, with clinical signs, may provide presumptive diagnosis
    • Serologic tests – no single test used routinely
    • Terio Cetacea
      • Bacterial culture, gross/histologic findings, molecular diagnostic tests
      • Branching, rod-shaped bacteria
      • Gram-positive, positive with modified acid fast and Grocott’s methanamine silver (GMS) special stains
      • Juvenile animals more affected than adults
  • Reported in both managed and free-ranging
  • Can become airborne 🡪 managed housing might enhance exposure to terrestrial pathogens via aerosol (esp dust)
  • Cetaceans
    • Harbor porpoise, bottlenose dolphin, spinner dolphin, striped dolphin, beluga, false killer whale, killer whale, Scammons pilot whale
    • Vulnerable to pneumonia; first site of infection often pulmonary
    • Cutaneous and SC infections described
    • Systemic form is the most common presentation in cetaceans
    • Pyogranulomatous pneumonic nocardiosis often reported
    • Vertebral osteomyelitis, granulomatous lesions in the cerebrum, pleura, thyroids, spleen, adrenal, heart, and mediastinal lymph nodes observed
    • Some cases reported shortly after violent, dust-spreading weather
    • Treatment
      • Aminoglycoside amikacin
      • Recurrence reported, despite repeated and apparently successful chemotherapeutic treatment
  • Pinnipeds
    • Few cases
      • Hooded seal, leopard seal
    • Thoracic disease as part of systemic infection is primary in hooded seals
    • Pyogranulomatous lesions in lungs, thoracic lymph nodes, adrenals, and brain of leopard seal
    • Terio pinnipeds
      • Most commonly reported in juvenile stranded hooded seals
        • Stranded along east coast of US and Canada, found outside of their normal range
        • Underlying immunosuppression, environmental stressors, and decreased prey availability secondary to extralimital ranging may increase susceptibility
      • Lesions: pyothorax and abscesses or pyogranulomas
      • Often systemic at death
      • Necropsy: pyogranulomatous inflammation in the skin, brain, other tissues
      • Diagnosis: culture or PCR; organisms demonstrated using GMS and acid-fast stains
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16
Q

Describe the epidemiology of marine brucellosis.

How is Brucella transmitted?

What are the potential vectors?
- For B. ceti?
- For B. pinnipedialis?
- What species is isolated in freshwater fish?

What pathology has been associated with B. pinnipedialis?

What pathologies have been described with B. ceti?
- What are the most susceptible species?
- What are the common clinical signs?
- What are the common findings of neurobrucellosis on necropsy?

What coinfections are common?

A

Marine Brucellosis Epidemiology

Marine host
* Potential MM Brucella definitive hosts include 130 species from rivers, lakes, oceans
– Most: Odontoceti & Mysticeti (dolphins, porpoises, whales)
– Less: pinnipeds (phocidae, otariidae, odobenidae)
– May also be infected: otters (sea/marine/European otters), polar bears, manatees, dugongs
* Species w/ pathologies
– Many reports of pathologies: cetaceans
– 1 single cases of pathologies: pinnipeds, sea otters
– May be seropositive by consumption of Brucella infected seals: polar bears

Transmission
* Like TM, B. ceti isolated from placenta, fetal tissues, amniotic/allantoic fluids, umbilical cord, mammary gland, milk, semen, uterine/vaginal fluids (after abortions)
* Vertical & horizontal transmission
* Potential vectors
– Lung nematode
– B. ceti found in cetacean lungworms (Pseudalius inflexus, Halocercus spp)
– B. pinnipedialis found in seals lungworm (Parafilaroides spp.)
* Fish
– B. melitensis isolated from fresh-water fish.

Pathology of Brucellosis in MM
- Isolation of bacteria or its DNA
- B. ceti = cetaceans + harbor seals, harp seals
- B. pinnipedialis = pinnipeds, mustelids, beluga whale, narwhal, minke whale

Pathologies related to B. pinnipedialis infections
- Recorded in 1 pinniped species & sea otter
- No associated pathology
- Just 1 sea otter w/ chronic granulomatous osteoarthritis with Brucella positive culture & intense positive immunolabeling

Pathologies related to B. ceti infections
- Recorded in 12 cetacean species
- Highly susceptible species: striped & bottlenose dolphins
- Commonly found in CNS w/ severe neuro. signs (seizures & death). Pathophysiologic mechanism unknown.
- Neurobrucellosis which include 2ary hydrocephalus => causes weakness, buoyancy, motor malfunctions => erratic swimming and tremors.
- Empty stomachs of stranded cetaceans => disease impairs their ability to successfully forage
- Also, co-infection in striped dolphin: meningoencephalitis with Toxoplasma gondii, Listeria monocytogenes, Morbillivirus
- Before necropsy, can use techniques such as virtopsy using CT scan => improves accuracy for collection of infrequent samples (cerebrospinal fluid from dilated lateral brain ventricles)

17
Q

Describe the diagnosis of marine brucellosis.

What serological assays exist?
- What do positive results indicate?
- What is the use in the field?
- What sample types may result in false positive results?

What ELISA tests exist?
- Are they validated?
- What test errors are common?

How is Brucella cultured?
- What mediums and what conditions are recommended for culture?
- What are some of the problems with commercially available media?

What is the molecular characterization of marine brucella?
- What sequence types are common in seals?
- What about porpoises?
- What about odontocetes & ziphiids?
- What is the most common and zoonotic one?

A

Diagnosis
- Serological assays = Rose Bengal Test (RBT)
– Simple, economic, stable reagent
– Produced with killed B. abortus cells stained with Rose Bengal stain at pH 3
– Agglutinating antibodies could be IgG, IgM, IgA => positive results w/ previous & current infections
– Validated for odontocetes + results in 4 minutes
– Can be used under field conditions, rehabilitation, postmortem (if dead < 16 hours)
– Used on field for biosafety purpose before necropsy
– NOT recommended in highly hemolyzed plasma or sera => may give false-positive results
- Enzyme linked immunosorbent assays (ELISAs)
– Not available commercially for marine brucellosis diagnosis
– BUT in-house competitive & indirect ELISAs developed for pinnipeds, cetaceans
– No validation: not the same cut-off as ruminants + protein G ELISA assays ≠ than ungulates
– False positive common
- Culture of Brucella
– Commercially media have too many antibiotics => limit growth of some strains or samples
– Noncommercially media with less antibiotics => good sensitivity & specificity for B. ceti isolation
– Use CITA and Farrell’s medium, under 10% CO2 atmosphere, at 37°C for 15 days
– On fresh to moderate decomposition carcasses

Molecular Characterization of Marine Brucella
- Several molecular methods employed to Brucella typing, include the next ones
- Whole-genome sequencing (WGS)
- Recommended for unravelling taxonomic complexity of Brucella isolated from MM worldwide.
- Multilocus sequence typing (MLST)
- B. pinnipedialis: ST24 & ST25 => seals
- B. ceti strains are clustered in 3 distinct paraphyletic clusters
- ST23 => porpoises
- ST26 => odontocetes & ziphiids
- ST27, zoonotic => bottlenose dolphins, California sea lions, dwarf sperm whales
- Isolated in humans with neurobrucellosis (Peru) & osteomyelitis (New Zealand)
- Multiple loci variable number of tandem repeats analysis (MLVA)

18
Q

Describe the risks of Marine Brucella as a zoonosis.
- What cases have been documented in people?
- What were the suscpected sources?
- What is the incubation period like?
- What are the most common brucellosis symptoms in people?
- What are some of the more rare or severe forms of disease?

What are some of the limitations of studying brucellosis in stranded marine mammals?

A

Marine brucellosis as a zoonosis
- No official reports of natural transmission from animals to humans => just few non-conventional cases
- 1 laboratory worker w/ mild symptoms (sinusitis, headaches, fatigue) => ST23 isolated
- 3 humans w/ naturally acquired marine brucellosis - ST27 infection (source of infection unknown)
- (1) intracerebral neurobrucellosis granulomas
- (1) spinal osteomyelitis, fever, rigors, lumbar spinal tenderness
– Source unknown: no direct contact w/ MM but regular unpasteurized milk & raw seafood
- Incubation periods unknown for ST27 brucellosis in MM & human
– But brucellosis in terrestrial animals: weeks - several months/years
- Common brucellosis symptoms in humans:
– Flulike symptoms (undulating fever, headache, malaise, chills, myalgia, back pain).
– Severe presentations: arthritis, spondylitis, sacroiliitis, osteomyelitis, bursitis, tenosynovitis, lymphadenopathy, peritonitis, pancreatitis, splenomegaly, hepatomegaly, myelitis, uveitis, optic neuritis, endophthalmitis, brain abscesses, meningitis, meningoencephalitis, vasculitis, aneurysms, endocarditis, seminal vesiculitis, prostatitis, epididymitis, orchitis, miscarriage or premature birth
– Rare: cutaneous or GI signs

Limitation to study stranded animals with marine brucellosis
- Real global impact on mortality & reproduction of MM unknown
- The isolation & correct identification of marine Brucella necessary for One Health concept
- Serology and culture w/ selective media not routinely done for various reason:
- Nutritional reason (Africa), culture reason (Asia) & difficult access to brain & CSF on field.