Pinnipeds Flashcards

Question

What are the clinical signs associated with urogenital carcinoma in California sea lions? What are the potential causes? What virus has been associated? What lesions are typically seen? What are the inclusion bodies?

Answer 1

**Urogenital carcinoma (CSLs)** * High prevalence in stranded animals along west coast * Cases reported in managed CSLs, but most born in wild * Carcinogenesis multifactorial - possible role of infectious agents, genetics (alterations in p53, HPSE2gene), organochlorine contaminants (interaction with steroid hormone receptors) * **Otarine herpesvirus-1 (OtHV-1) is associated** * CS: perineal edema, hind limb paresis, abdominal effusion, penile prolapse with necrosis * Urogenital epithelium - raised plaques, thickening/roughening or dulling of mucosa, tan firm masses * Tumors - cervix, vagina, penis, prepuce * Widespread metastasis common - inguinal ln, sublumbar ln, liver, kidney, lung * Hydronephrosis common - ureter compression by sublumbar ln masses * Can also be found incidentally - important to save repro tracts for histo * Histo: genital epithelium often thickened with dysplastic cells and disordered maturation (full thickness), increased number of mitotic figures, polygonal neoplastic cells with large nuclei, lymphoplasmacytic inflammation * Central necrosis common within masses * Intranuclear inclusions infrequently noted

Answer 2

Incidence of Lens Diseases in Pinnipeds (Fowler 9) * Lens disease under human care in 46.8% * 15% had lens luxation and cataracts; 34% had cataracts alone * CSL was 44.5%, harbor seals 90%, walrus 50% * Diagnosed in stranded pinnipeds in many retrospectives; all age groups and sexes affected * Lens disease overall prevalence 0.6%, n = 337 stranded animals * Cataracts: n=31 (25 CSL and 6 NES) * All ages groups of California sea lions, only Northern elephant seal pups * Lens luxations: n=7 (5 CSL and 2 NES) * Impossible to determine prevalence of lens disease in free-ranging population Fowler 7 * Corneal Disease * Edema, opacities, chronic keratitis common * Keratitis observed in 45% captive pinnipeds in N America, Bahamas (survey) * Flare-ups occurred 2-4x/yr in the most affected pinnipeds * Especially w/ increased exposure to sunlight (Summer), and on sunny winter days w/ snow on ground * Indoor animals had less severe disease and fewer flare-ups (in some cases) * Lens Disease * Increased frequency of cataracts and lens luxations w/ age * 21% in animals 6-10 yrs old * 58% in 11-15 yr olds * 66% in 16-20 yr olds * 87% in 21-25 * 100% (n=9) in \>25 yr old animals

Answer 3

Risk factors/ protective factors * Aging * Inadequate ciliary body support -\> middle-age to older animals predisposed to lens luxation * Otariids and phocids more likely to have anterior luxation * Walrus more likely to have posterior * Lack of sufficient access to shade * Pinnipeds without access to sufficient shade 10X more likely to develop lens diseases * History of fighting (or gunshot) * Traumatic incident contributing to cataract formation * History of nonspecific ocular disease * Keratopathy most common concurrent eye problem in pinnipeds * Not seen in wild pinnipeds * Keratopathy does not fully resolve -\> results in continuous subclinical uveitis -\> cataract formation * Associated with UV light exposure * Suspect genetic predisposition to cataracts -\> seen in younger animals

Answer 4

* 3 Stages of Corneal Disease: * 1: faint gray/white corneal opacity, perilimbal corneal edema (gray line just inside limbus), small, superficial corneal ulcers * Larger gray/white corneal opacity, irregular surface, superficial ulceration, perilimbal edema, may have mild neovascularization and pigmentation, mild to mod blepharospasm, epiphora * 3: Diffuse corneal edema, infiltrates, superficial to stromal ulcerations, severe blepharospasm, epiphora

Answer 5

_Water Quality:_ * Excessive oxidants used to keep the water clean may cause damage both directly (spikes) or indirectly by binding w/ organic material in water and creating toxic byproducts _Excessive Chemicals, Oxidants, or Noxious Byproducts in the Water:_ * Chlorine, Ozone, Bromine (less common than first two) * Most common oxidizing agents used to reduce pathogen levels in marine systems. * Byproducts of disinfection are rarely measured * Disinfection byproducts include halogenated methanes (chloroform, bromoform, bromodichloromethane and dibromochloromethane) * Repeated chlorine spikes cause corneal damage * Chlorine should stay below 1 ppm * Spikes of 0.5 ppm may cause damage even if total chlorine levels remain below 1.0 * Chlorine tablet dispensers are more likely to result in spikes (vs gas or liquid dispensers) * Municipal water is often 2-4 ppm chlorine * Ozone systems must have an efficient method to de-gas the water * Residual ozone in the water can cause ocular pain, blepharospasm, epiphora * Should be no (0) residual ozone levels * Test kits are inexpensive, but are qualitative _Salinity:_ * 1995 survey suggested fresh water as cause of corneal edema. Most facilities changed to salt water pools. * One pool filled with municipal (unsalted) water, no additives and painted dark = pinnipeds are disease free. * Therefore salinity not thought to play as big a role anymore.

Answer 6

Anesthesia for pinnipeds for ophthalmologic procedures * Typically general anesthesia required * Pre-anesthetic health assessment recommended similar to other species, especially with geariatric patients * Pinniped anesthesia depends on a combination of behavioral, mechanical, and chemical restraint * Balanced anesthetic protocol ideal and often includes iso, midazolam, butorphanol, and potentially an alpha 2 * Alpha 2s provide good sedation and are easily reversible but may be contraindicated in geriatric patients, also associated with increased risk of periocular hemorrhage * Use alpha 2s with great caution in phocids (or rather don’t use them) * Voluntary mask induction more feasible in otorids -\> less likely to breath hold compared to phocids * For non-tractible animals darting or hand injection may be necessary * Vascular access * Phocids and odobenids: epidural sinus, pelvic flipper medial saphenous vein (US guidance helpful) * Otorids: pectoral flipper (cephalic and brachial), US guided jugular catheter * BP * Non-invasive methods not validated but can be used to monitor trends * Can do direct in via US guided arterial catheter in median artery of pectoral flipper or saphenous artery of pelvic flipper in otorids * Temperature control * Hypothermia should be addressed, also hyperthermia (alcohol on flippers) * Neuromuscular blockage often needed for ophthalmologic procedures to properly centralize and immobilize the eye for surgery * Atracurium commonly used, though rocuronium becoming more common due to rapid onset and fewer adverse events * Laryngeal neuromuscular function returns slower then pelvic limb -\> ensure to keep intubated/ have supplies ready for re-intubation if needed * If needed can be reversed with edrophonium though this needs to be done in a controlled manner with close monitoring for bradycardia (also makes them feel crummy) -\> given rocuronium’s shorter duration of action (~20min in dogs) may not need to be reversed * Pinnipeds have a much higher tolerance for elevated CO2 levels * Otariids will not spontaneously ventilate until PaCO2 is 60-80mmHg, phocids are unlikely to spontaneously ventilate even if PaCOs \>90mmHg * Mechanical ventilation almost always required (phocids absolutely required) * Permissive hypercapnia can allow for improved cardiac output but must be closely monitored * Trigeminocardiac reflex (oculocardiac reflex) can result in cardiac arrest if traction is placed on extraocular muscles; exaggerated in the presence of hypoventilation, hypoxemia, and acidosis

Answer 7

Surgical candidates * Best outcomes occur in eyes with visually impairing cataracts that are not anteriorly luxated and that have not caused excessive chronic uveitis * Lensectomy is still indicated in non-visual patients (ie retinal detachment) due to pain relief and cosmetics (otherwise enucleate) Outcomes and sequelae * Ideal outcome is resolution of pain and regained sight * Retinal detachments are uncommon in pinnipeds following cataract surgery; young animals who stranded with cataracts have high risk due to their liquid vitreous

Answer 8

1. **Phocine herpesvirus-1 (PhHV-1)** - Alphaherpesevirus 1. Harbor and gray seals (Europe, North America - both coasts), can be associated with high morbidity and mortality 2. Clinical disease and fatal infection most common in neonates or young pups 3. Horizontal transmission (direct contact, aerosols) more common, but vertical transmission reported 4. **Pacific harbor seals: multifocal adrenocortical and hepatic necrosis are the most common lesions** 5. Necrosis, smudgy eosinophilic intranuclear inclusions 6. Other lesions: Adrenal necrosis (mineralization can occur), thymic atrophy in pups, lesions associated with secondary infection/sepsis (omphalophlebitis, pneumonia, meningoencephalitis 7. European seals - interstitial pneumonia, massive liver necrosis reported 8. Diagnose: virus isolation, PCR, IHC, serology (ELISA) 2. **Otarine herpesvirus-1 (OtHV-1) -** Gammaherpesvirus (*Rhabinovirus*), most common in CSLs 1. Associated with urogenital carcinoma and considered likely factor in tumor development 2. Prevalence in genital tract higher in adults 3. Sexual transmission suspected, females can also transmit to pups during birth 4. Single case of genital carcinoma in South American fur seal 5. Detected in a Steller sea lion without evidence of neoplasia

Answer 9

1. **Pox** viruses (genus *Parapoxvirus*) 1. Numerous species affected, distinct viruses in Atlantic and Pacific 2. Transmission- direct contact (rubbing, biting) 3. Outbreaks in rehab facilities - young animals, stress, concurrent disease 4. Lesions: raised, firm, +/- ulcerated nodules on head, neck and thorax; plaque-like lesions may arise on tongue and oral commissure 5. Histo: epidermal and follicular hyperplasia, ballooning degeneration in stratum spinosum, chunky eosinophilic cytoplasmic inclusions within epidermal and follicular keratinocytes, mixed inflammation, epidermal necrosis and ulceration 6. **Typically spontaneously regresses** 7. **Zoonotic** - nodular cutaneous lesions

Answer 10

1. Morbillivirus 1. Transmission: respiratory, nasal, ocular secretions 2. **Canine distemper virus (CDV) - Baikal seals** 3. **Phocine distemper virus (PDV)** 1. CS: Oculonasal discharge, conjunctivitis, keratits, coughing, dyspnea, diarrhea, abortion, head tremors, convulsions, increased buoyancy 2. Lesions; Bronchointerstitial pneumonia, pulmonary atelectasis, congestion, edema, emphysema (pulmonary, mediastinal, sq), nonsuppurative encephalitis, lymphocyte depletion 3. Intracytoplasmic and intranuclear eosinophilic inclusion bodies 4. Associated with secondary parasitic or bacterial infection and concurrent viral infections 5. 23000 harbor seal deaths in Europe in 1988; 30,000 seals in 2002 6. US (Atlantic) - stranding peaks have been linked to infections in harbor, harp, hooded and gray seals 7. Morbillivirus antibodies regularly found in Arctic seal species

Answer 11

1. Influenza A 1. Harbor seal mortality events in Northeast US - H7N7 (1980) and H4N5 (1983); additional subtypes in US and Europe 2. Avian influenza A (H3N8) - 2011 harbor seal die off in NE 3. CS: dyspnea, nasal discharge, lethargy, emphysema 4. Lesions: partially collapsed lungs, pulmonary congestion, necrotizing bronchitis, bronchiolitis, hemorrhagic alveolitis, bronchial gland adenitis, occasionally interstitial pneumonia, concurrent secondary infection lesions often present

Answer 12

1. Caliciviruses 1. San Miguel Sea Lion virus (SMSV-1-17) - Vesicular exanthema of swine 2. Walrus Calicivirus 3. Vesicular dermatitis, progressing to ulceration of primarily non haired surfaces of flippers, necrosis of underlying digits can occur 4. ulcerative stomatitis and ulcerated/nodular dermatitis of lips, nasal planum and chin reported in CSLs 5. Spongiosis of stratum spinosum followed by subcorneal vesicle formation 6. Zoonotic - vesicular dermatitis and flu-like illness 7. high variety of marine and terrestrial species affected - opal eye perch may be natural host 8. RT PCR can differentiate from reportable vesicular diseases

Answer 13

1. **Leptospirosis** - free-ranging and managed pinnipeds 1. CSLs - *Leptospira interrogans serovar* Pomona; most common between July and December, periodically (every 3-5 years) large numbers strand with clinical disease, asymptomatic carrier state suspected 2. CS: Polydipsia, abortion 3. Elevated BUN, Creatinine and phosphorus 4. Lesions: swollen pale tan kidneys, lack of renule and corticomedullary differentiation 1. Leptospira-related liver lesions are not noted 5. Histo: lymphoplasmacytic tubulointerstitial nephritis, typically with a plasma cell heavy inflammatory infiltrate 6. Zoonotic - PPE measures 7. Diagnose - histo, PCR

Answer 14

1. *Klebsiella pneumonia* (gram-negative, Enterobacteriaceae) 1. Ubiquitous, common healthy animals GI or respiratory tract 2. 2002 mass mortality of **New Zealand sea lion pups** in sub Antarctic caused by a hypermucoviscous phenotype - bacteremia, fibrinosuppurative to histiocytic inflammation of meninges, joints, ln, respiratory tract, peritoneum and subcutaneous tissues 3. **CSL** isolate - pleuritis, pyothorax, pneumonia, abscesses, septicemia, meningoencephalitis; **occurs in all ages** 4. CSLs on San Miguel Island - bacteria were transferred into abdominal cavity on cuticle of hookworms causing septic peritonitis

Answer 15

1. ***Mycoplasma*** 1. *M. phocicerebrale, M. phocirhinis, M. phocidea, M. zalophi.* 2. **Part of normal pinniped oral flora and cause of Seal Finger in humans** 3. Infection - skin wounds, abscesses, polyarthritis, lymphadenitis, necrotizing pneumonia in CSLs 4. Skin wounds - gray and harbor seals 5. *M. phocicerebrale* - in utero infection associated with myocarditis and pneumonia; abortion in Australian fur seals

Answer 16

1. *Brucella pinnipedialis* 1. Fish or invertebrate vector possible - Brucella infected lung worms found in lungs (could also be incidental) 2. Zoonotic transmission is concern for humans that consume seal or whale meat (reported cases did not have direct exposure to marine mammals/necropsy setting) 3. Majority of infections subclinical; suppurative and necrotizing placentitis in northern fur seal reported

Answer 17

1. Nocardiosis 1. Most commonly reported in juvenile stranded hooded seals along east coast of US and Canada 2. Underlying immunosuppression, environmental factors, and decreased prey availability may increase susceptibility 3. Lesions - pyothorax, abscesses, pyogranulomas in lungs and thoracic ln 4. PCR, acid fast stains

Answer 18

1. Pseudomonas (*P. aeruginosa*) 1. systemic infections, young rehab animals 2. Pneumonia most common; often systemic, septicemia 3. Fibrinosuppurative inflammation with necrosis, interlobular edema, fibrin accumulation in lungs 4. Histo: vasculitis and “swarms” of gram negative bacilli 5. associated with hemorrhagic pneumonia in cluster of free-ranging harbor seal mortalities

Answer 19

* Mycobacteriuum pinnipedii belongs to the Mycobacterium tuberculosis complex (MTBC) and therefore is of high zoonotic concern * Formerly known as “seal bacillus” * Slow growing mycobacterium, therefore can be difficult to culture * Primary culture obtained at 37C on egg-based medium and takes 3-6 weeks. Susceptibility could take an additional 2-4 weeks * Isolates usually susceptible to rifampicin, streptomycin, ethambutol, and para-aminosalicylic acid * All MTBC species share a similar 16S RNA gene sequence except M. pinnipedii, which has one single different nucleotide within this sequence. * Genotyping, especially spoligotyping, is still the most efficient way to differentiate M. pinnipedii from other MTBC species. The spoligotyping method is based on polymerase chain reaction (PCR) amplification of a highly polymorphic direct repeat locus in the MTBC genome. * Hosts: original found in otariids but has since been determined to be able to infect other marine and terrestrial mammals, either naturally or experimentally * “Natural” wildlife hosts all originate from the southern hemisphere * Epidemiology * The infection is likely to remain dormant for several years in pinnipeds * Among marine animals, the South American sea lion (Otaria byronia) seems to have the greatest risk of spreading infection, mainly because of sequential importation of infected wild animals in the 1980s and early 1990s from Chile and Uruguay to European zoos. * Transmission mainly via direct contact, water, and aerosols – use of pressurized water linked to infections in animals in different enclosures

Answer 20

* Postmortem diagnosis * Gross lesions consist of granulomas within lymph nodes and/or organs such as lung, spleen, liver, uterus, and bladder. If pregnant, can also be seen in the placenta * All organs with granulomas should be submitted for cytology, PCR, and culture * Antemortem diagnosis * Clinical signs can be non-existent or signs suggesting organ deficiencies depending on the body system affected. Most frequent signs are weight loss, muscle loss, enlarged lymph nodes, and respiratory signs * Granulomas can sometimes be detected radiographically, but CT remains a better modality when possible. AUS can also be useful, especially for guided LN aspirates * Pre mortem samples of affected tissues or swabs can be submitted for PCR * Immunology * Host immune reaction to M. pinnipedii infection may be divided between humoral and cellular immunity, as for all other MTBC infections. * Cellular immunity * May be assess through intradermal testing – tuberculin injection * Good results in cattle, low negative predictive value in pinnipeds and tapirs * Recommended site is neck or flippers for pinnipeds – ability to vasoconstrict or dilate may inhibit or over-stimulate the number of T cells in the area, leading to under or over reaction to antigenic stimulation * The use of interferon gamma release assays (IGRAs) could be considered in validated species like cattle, but not available in other species at this time * However, the concept of detecting mRNA sequence coding for interferon, instead of interferon itself,36 could theoretically be applied to pinnipeds, because these sequences are very similar among carnivores. * Humoral immunity * Antibody detection may be useful, especially in an active/replicating phase * No current specific tests for M pinnipedii antibodies * Note that the application of an intradermal test my impact any subsequent humoral immunity testing

Answer 21

* Treatment * Risk analysis most be performed, but treatment can be considered * Reported treatment in pinnipeds relied on biotherapy or tritherapy with use of oral rifampicin (7.5 mg/kg), isoniazid (5 mg/kg), and addition of ethambutol (15 mg/kg). T * here are no reported pharmacokinetic studies of any antituberculous drugs in pinnipeds, and hence these dosages do not necessarily result in therapeutic plasma levels. * Significant side effects including anorexia, abdominal pain, lethargy, and hepatotoxicity * Side effects likely to impair compliance and limit efficacy * Prevention * Full history and possible diagnostics of any incoming animals * Life support system/water quality important in preventing persistence and transmission in an enclosure – mechanical and biological filtration are not sufficient at removing MTBC organisms – sterilization phase including UV, chlorine, and/or ozone is recommended, with ozone being the most effective * Close contact between humans and pinnipeds should be avoided as much as possible * Pressure hose use should be restricted to a minimum and direction of aerosol should be monitored when used * Regular cleaning of all equipment that comes into contact with pinnipeds and their water * Keepers and trainers should not be in contact with other mammalian species during the day, but if they must then strict hygienic measures including foot baths, different clothes and tools, should be applied * Routine health monitoring of keepers and trainers should be promoted

Answer 22

1. *Coccidioides immitis* 1. Endemic in California; associated with disturbance of dry soil; transmission - inhalation 2. reported in CSLs, harbor seals (rare), northern elephant seals (rare) 3. Typically causes mild disease, but can progress to fatal infection 4. Emaciated, pyogranulomatous pneumonia, pleuritis, peritonitis, lymphadenitis 5. Spherules and endospores on histo 6. Zoonotic

Answer 23

1. Fungal dermatitis - yeasts, hyaline molds, dermatophytes 1. *Candida albicans*, *Fusarium* spp., *Microsporum* sp.,*Trichophyton* sp. 2. Low salinity and high temperatures, disruption of the skin 3. Alopecia, acanthosis, hyperkeratosis - mucocutaneous junctions, nail beds, axillae 4. **Alaskan Steller sea lions:** fungal patches (*Trichophyton* sp) common - sometimes in target pattern with healed or raw skin in center 5. Skin scrape, PCR, fungal cultures

Answer 24

1. *Parafilaroides* - small lungworms 1. Otariids and phocids in both hemispheres; subclinical infection common 2. Adults in pulmonary parenchyma shed eggs into small airways -\> larva coughed up, swallowed and shed in feces 3. Young or debilitated animals - heavy infections, secondary bacterial pneumonia 4. Histo: nematodes, occasional granulomas, epithelial hyperplasia, lymphocytic tracheitis

Answer 25

1. ***Otostrongylus circumlitus*** **- large lungworms** 1. Circumpolar region - ringed, harbor, gray, elephant, ribbon, hooded seals 2. **Primary hosts are ringed and harbor seals** 3. Larvae are ingested and migrate to right ventricle, through wall of pulmonary artery, into lungs; adults live in bronchi and bronchioles (head embedded in parenchyma) 4. Significant infection typically only young animals; heavy infection may impact diving performance and health 5. Mucus can plug airways; mucosal cell hyperplasia, lymphoplasmacytic and eosinophilic peribronchitis, mild arteritis 6. Northern elephant seals - reaction to larval migration in pulmonary arteries can cause severe disease and death 1. Harsh lung sounds, depression, neutrophilia, DIC 2. Hemorrhage in lung parenchyma

Answer 26

1. Hookworm (*Uncinaria*) 1. Otariids and phocid species - northern and southern fur seal pup mortality 2. Recent study: 94% of pups infected; cause of death in 35% 3. Pups ingest L3 larvae (transmammary), larvae mature in intestine (attach to SI mucosa and feed on blood), eggs shed in feces; larvae penetrate host skin or are ingested and can migrate to ventral blubber and lie dormant, then migrate into milk 4. Clinical signs in pups associated with blood loss, reduced growth, severe fibrinohemorrhagic enteritis, anemia, death 2. GI roundworms - *Anisakis, Contracaecum*; large ulcers 3. Heartworm (*Dirofilaria immitis, Acanthocheilonema spirocauda*) - similar to infection in dogs; managed pinnipeds routinely screened and on preventatives in endemic areas

Answer 27

1. ***Toxoplasma gondii*** 1. Free-ranging and managed pinnipeds - including arctic and subarctic seals and walrus; most common in CSLs, monk seals, and harbor seals 2. Lesions (CSLs): necrotizing and lymphohistiocytic meningoencephalitis, myocarditis, myositis, associated intralesional protozoa, aborted pups; neuro: ataxia, seizures, abnormal mentation (also reported concurrent with DA toxicosis) 3. Harbor seals - meningoencephalitis is most common lesion; coinfection with *S. neurona* is reported 4. Hawaiian monk seals - disseminated infection, very large numbers of organisms, adipose tissue severely affected 5. Confirm with PCR or IHC (cross reaction with other protozoa possible)

Answer 28

1. ***Sarcocystis neurona*** 1. Pacific coast of North America; antibody titers in harbor seals and CSLs 2. Harbor seals more commonly reported to develop severe fatal disease - subadults and adults primarily affected 3. Lesions: nonsuppurative meningoencephalitis (severe in cerebellum), rosette-form schizonts with occasionally visible residual bodies differentiate from *T. gondii,* incidental sarcocysts in skeletal or cardiac myocytes 4. CSLs can develop severe polyphasic myositis - esophagus and diaphragm; atrophied muscle; pale streaks 2. *S. pinnipedi* (novel spp) - reported to cause necrotizing hepatitis sporadically in CSLs, Stellers, monk seals and gray seals; schizonts and merozoites seen in hepatocytes and Kupffer cells

Answer 29

1. Integument 1. Poxvirus – recently weaned or rehab animals 1. Pathognomonic lesions: round, raised, firm skin nodules 0.5-1 cm diameter that gradually increase in size over the first week, and may ulcerate or suppurate. 2. Lesions on head and neck most commonly, but may occur anywhere 3. Self-limiting, regress after 4-6 weeks. 4. Animals remain active. Appetite loss with lesions around mouth and eyes. 5. No direct treatment needed. Antibiotics for secondary infections or NSAIDS if needed. 6. ZOONOTIC 2. Calicivirus, San Miguel sea lion virus 1. Vesicles on flippers. Sometimes orally. Progress to ulcerations. Possible GI signs. 2. Supportive care and antibiotic coverage. 3. PCR, virus isolation but often presumptive diagnosis from clinical signs. 3. Bacterial diseases and abscess common 1. Multiple causes: trauma, bites, septicemia 2. Mycobacteria infections occur 4. Fungal Disease 1. *Candida* and *Fusarium* infections at mucocutaneous junctions and nail beds 1. Most common in captive animals in freshwater. 2. *Trychophyton* and *Microsporum* 5. Lice – usually species specific. Alopecia, broke hair shafts, pruritus. 1. CSL = *Antarctophthirius microchi* 2. Harbor seal = *Echinophthirius horridis* 6. Traumatic skin wounds – entanglements, hooks, gunshot 7. Molting issues – Seals in abnormal photoperiods

Answer 30

1. Musculoskeletal 1. Sarcocystis myositis 1. Antibody titers, muscle biopsy for histopathology and PCR 2. Ponazuril treatment 2. Trauma – osteomyelitis, fractures, monofilament entanglement 1. Splinting and bandaging is difficult. Wound care by second intention healing common. 2. Spinal cord terminates between 8^th and 12 thoracic vertebrae.

Answer 31

1. Digestive system 1. Foreign body ingestion not uncommon 1. Displaced Arctic seals may ingest rocks 2. Gastritis and ulcers 1. Gastric nematodes, *Helicobacter* 3. Enteritis 1. Morbillivirus, herpesvirus 2. *Clostridium*, *Salmonella* 3. Parasites: *Diphyllobothrium* (cestode), Acanthocephalans, Hookworms, 1. Coccidia mild in CSL may cause severe disease in harbor seals 4. Hepatitis 1. Adenovirus in otariids. May be fatal. Hepatic necrosis and intranuclear inclusions 2. Various bacteria, fungi (*Coccidioides immitis*) 3. Sarcocystis pinnipedi 5. Hemochromatosis has been described 6. Neoplasia – SCC (lingual, esophageal) 7. Iatrogenic disease – inappropriate formulas or feeding, spoiled food, improper feeding tubes 8. Diagnostics – think of the typical: PE, rads, U/S, scope, lab work

Answer 32

1. Respiratory System 1. Canine distemper virus – epizootics in Bailak and Caspian seals 2. Phocine distemper virus – epizootics in harbor seals. Smaller mortality events in gray, harp, and hooded seals. 1. Resp signs, neuro signs, depression 2. Antibody titers, but young animals may succumb without developing titer 3. Virus isolation difficult 4. Treatment: supportive, antibiotics 5. Canine distemper virus vaccine has been used 3. Influenza 1. Epizootics in harbor seals with similar signs as CDV and PDV 2. *Mycoplasma* has been found concurrently – tetracyclines may be helpful. 4. Bacterial pneumonia – gram neg most common 5. Parasitic 1. Heavy infestations of *Parafilaroides* 1. Treatment with anthelminthics, antibiotics, steroids. Strong inflammatory response to dead/dying worms. 2. *Otostrongylus* in phocids 6. *Mycobacterium pinnipedii* 1. Pulmonary granulomas. Successful treatment not documented. 2. Similar lesions from fungal disease *Coccidioides* in California

Answer 33

1. Cardiovascular 1. Anemia 1. Hookworm (*Uncinaria*) infections. Chronic renal from lepto. 2. *Otostrongylus* in elephant seals – causes DIC 1. Diagnosis in live seals no possible because disease during prepatent period. 2. Clin path – WBC \>40K, low platelets, high ALT and GGT. Serum amyloid A. 3. Word in right ventricle and pulmonary arteries 4. Therapy rarely successful. Aminocaproic acid may be helpful. 3. Domoic Acid – degenerative cardiomyopathy 1. EKG limited to diagnose cardiac pathology – both ventricles depolarize simultaneously 4. Bacterial endocarditis – *Staph aureus* and *E. coli* 5. Canine heartworm (Dirofularia immitis) or *Acanthocheilonema spirocauda* may cause dilatation of the pulmonary artery and right ventricle 1. Micofilaria must be distinguished from nonpathogenic *Acanthocheilonema odenhali* which cause the majority of microfilaria in CSL 2. Canine heartworm tests will cross react 3. Preventive treatment with ivermectin monthly 4. Removal of lice. Seal louse *Echinophthirius horridus* has been shown to transmit *A. spirocauda*. 6. Patent foramen ovale and ductus arteriosus 1. Longer after birth than terrestrial mammals. Up to 6-7 weeks without clinical disease.

Answer 34

1. Urogenital System 1. Leptospirosis – most common in CSL in California 1. Diagnosis on clinical signs and serum chemistry consistent with renal insufficiency 1. Confirmation via PCR/culture of urine or kidney 2. To date, the only serovar isolated from CSL and N. fur seals is *L. interrogans* serovar *pomona*. 3. Supportive care and antibiotics (tetracyclines). But most animals still die. 2. Urogenital tumors – common free ranging CSL 1. Paresis, scrotal or perineal edema, ascites, prolapse. Hydroureter, hydronephrosis. 2. No treatment 3. Abortions 1. Lepto, herpes, calicivirus, Coxiella burnetiid, toxins, domoic acid 2. *Brucella* has NOT been found as a causative agent of abortion in pinnipeds. 3. Domoic acid animal often improve after abortion – fetus, amniotic fluid are reservoir for DA 1. Induction of abortion may be helpful.

Answer 35

1. Nervous System 1. Viral encephalitis 1. Morbillivirus, herpes, influenza, West Nile, EEE 2. Vaccination against WNV recommended. Some species have been vaccinated with canine distemper virus vaccine. 2. Protozoal – Sarcocystis, Toxoplasma 3. Fungal – Coccidioides, Cryptococcus 4. Biotoxins 1. Domoic acid – Hippocampus necrosis, atrophy. Seizures. 2. Lorazepam choice to control acute seizures. Phenobarb. 3. Euthanasia if seizures cannot be controlled. 5. Thiamine deficiency – frozen fish. Polioecephalomalacia. History of no supplementation 6. Hyponatremia

Answer 36

_Physical exam (Walrus CRC 42)_ * Training to allow voluntary exam, weigh on scale, voluntary ultrasound, blubber thickness measurement (Best done over the shoulders) _Specimen Collection and Diagnostic Techniques (Walrus CRC 42)_ * Blood collection: metatarsal and epidural veins. * Radiography limited to extremities * Gastroscopy can be trained * Ultrasound – limited abdominal use. Can image ovaries and kidneys. Pregnancy diagnosis.

Answer 37

Siebert, U., Rademaker, M., Ulrich, S. A., Wohlsein, P., Ronnenberg, K., & Prenger-Berninghoff, E. (2017). Bacterial microbiota in harbor seals (Phoca vitulina) from the North Sea of Schleswig-Holstein, Germany, around the time of morbillivirus and influenza epidemics. Journal of wildlife diseases, 53(2), 201-214. ABSTRACT: We present microbiologic findings in harbor seal (phoca; Phoca vitulina) carcasses collected from the North Sea of Schleswig-Holstein, Germany, 1996–2014, and interpret results in relation to potential variations caused by phocine distemper virus and influenza A virus mass mortalities. We conducted microbiologic investigations on 2,124 tissue samples from lung, liver, kidney, spleen, intestine, and mesenteric lymph nodes from 549 dead harbor seals of the German North Sea. **A large variety of bacteria, including potentially pathogenic species such as Bordetella bronchiseptica, Brucella spp., Clostridium perfringens, Escherichia coli, Erysipelothrix rhusiopathiae, b-hemolytic streptococci, and Staphylococcus aureus, were isolated. These bacteria were associated with bronchopneumonia, gastroenteritis, hepatitis, polyarthritis, nephritis, myositis, myocarditis, and septicemia.** *Bordetella bronchiseptica and Streptococcus equi subsp. zooepidemicus were significantly associated with the seal die-offs from phocine distemper in 2002 and influenza in 2014.* Many bacteria were detected in tissues of dead harbor seals, of which E. coli, b-hemolytic streptococci, and Brucella spp. might be responsible for pathologic changes. **Zoonotic bacteria such as Brucella spp. and E. rhusiopathiae are frequently isolated from harbor seals. Brucella spp. was less and Vibrio spp. more frequently found in summer. Erysipelothrix rhusiopathiae showed an almost regular 4-yr oscillating trend.** We found C. perfringens less frequently and E. coli more frequently in harbor seals from St. Peter-Ording. Because zoonotic bacteria are regularly found, handling of dead and live harbor seal specimens should be conducted carefully to prevent transmission to humans. Further investigations are needed to understand microbiota changes in relation to increasing seal populations, reintroduction of rehabilitated seals to the wild, and increasing pressure from anthropogenic activities. · C. perfringens most frequently in adults and adults had the highest mortality from intestinal dislocations · E. coli/E. coli var. haemolytica was most frequently found in young individuals · Brucella (zoonotic) most common in respiratory tract · Chemical pollutants impair the health of individuals in German waters and should be considered a permanent stressor

Answer 38

McHuron, E. A., Castellini, J. M., Rios, C. A., Berner, J., Gulland, F. M., Greig, D. J., & O'Hara, T. M. (2019). HAIR, WHOLE BLOOD, AND BLOOD-SOAKED CELLULOSE PAPER-BASED RISK ASSESSMENT OF MERCURY CONCENTRATIONS IN STRANDED CALIFORNIA PINNIPEDS. *Journal of wildlife diseases*. Abstract: Mercury (Hg) poses a health risk to wildlife populations and has been documented at relatively high concentrations in many marine mammals, including wild-caught pinnipeds along the central California, US coast. We measured total Hg concentrations ([THg]) in hair and blood of live-stranded harbor seals (HS; *Phoca vitulina*), California sea lions (CSL; *Zalophus californianus*), and northern elephant seals (NES; *Mirounga angustirostris*) in California to quantify species, temporal, and spatial variability in [THg] and assess the relationships between [THg] measured by different methods (blood vs. filter paper) and in different matrices (blood vs. hair). We compared [THg] with toxicologic thresholds of concern to aid in identification of at-risk individuals or groups and better understand how the use of different methods and matrices affects assumed toxicologic risk. There was a wide range of [THg] in blood (\<0.01–1.13 μg/g) and hair (0.45–81.98 μg/g), and NES had higher [THg] compared with HS and CSL. All three species had individuals with [THg] that exceeded the lower threshold for one or both matrices, but only HS pups had [THg] exceeding upper thresholds. Spatial differences in [THg] were detected, with higher concentrations in HS pups from areas surrounding San Francisco Bay, but differences were dependent on sampling year and matrix. The relationship between [THg] in blood and filter paper (*r**²*=0.98) was strong, and differences had little influence on comparisons with toxicologic thresholds. Blood and hair [THg] were generally in agreement (*r**²*=0.72), but large mismatches for a few seals underscore the importance of combined sampling in adverse effects studies where accurate assessment of Hg exposure is crucial. The wide range of [THg] in stranded HS pups that exceeded published thresholds of concern makes them a promising candidate for adverse effects studies, particularly because different matrices represent Hg exposure across key developmental stages. * **Adverse effects from Hg: neurologic, immunologic, reproductive impairment.** * In marine ecosystem, some MM have very high tissue total Hg [THg] because of their ecology and life history. * **Hair and blood are two matrices commonly used to determine relative Hg exposure and assess potential for adverse effects in pinnipeds.** * **Majority of Hg in hair and blood – monomethylmercury (MeHg+).** * **One of the more toxic forms.** * Pinniped pelage molt acts as an excretory route for Hg. * Samples collected at any time within a molt year are representative of the same general time period, helps minimize confounding effect of time on Hg concentrations. * MeHg+ crosses the placenta and blood-brain barrier. * There can be a temporal disconnect between Hg in blood and hair. * Often in agreement with strength of the relationship between blood and hair total mercury affected by age, class, weaning status, growth, temporal distances in sampling of the two tissues. * **Strong positive relationship between mercury in blood and FP.** * Differences between spp in blood and FP not significant. * **Total Hg in hair moderately good predictor of blood total Hg.** * **NES higher blood mercury vs HS and CSL, although HS had the highest maximum total mercury across all spp.** * HS had higher mean total mercury in hair vs CSL. * Trends in spatial and temporal differences in hair total mercury of stranded HS pups were inconsistent across stranding locations and years. * Wide range of total mercury in blood and hair of stranded pinnipeds during the study years, some values exceeding thresholds of concern. * Blood [THg] and FP methods comparable. * This method can be used across a variety of spp without further validation. * Strength of relationship between blood [THg] and hair of HS pups relatively strong. * **Important to sample blood and hair in adverse effects studies where accurate assessment of Hg exposure is crucial for study outcomes.** * **The use of both tissues unnecessary in studies focused on exposure at the population level.** * Presence of lanugo had no influence on relationship between total mercury in in blood and hair. * Stranded HS pups had higher hair total mercury than wild-caught seals from same region and year. * Stranded animals may reflect the range but not the average mercury exposure in wild populations. * **The wide range of total mercury in HS pups that exceeded published thresholds of concern makes them a promising candidate for adverse effects studies.**

Answer 39

Baker, J. D., Harting, A. L., Barbieri, M. M., Robinson, S. J., Gulland, F. M., & Littnan, C. L. (2017). Modeling a morbillivirus outbreak in hawaiian monk seals (neomonachus schauinslandi) to aid in the design of mitigation programs. *Journal of wildlife diseases*, *53*(4), 736-748. **Abstract:** We developed a stochastic susceptible–exposed–infectious–removed (SEIR) model to simulate a range of plausible morbillivirus outbreak scenarios in a randomly mixing population of 170 endangered Hawaiian monk seals (Neomonachus schauinslandi). We then modeled realistic vaccination and quarantine measures to determine the potential efficacy of such mitigation efforts. Morbillivirus outbreaks represent substantial risk to monk seals—91% of simulated baseline outbreaks grew (R0.1), and in one-third of the scenarios all, or nearly all, individuals were infected. Simulated vaccination efforts in response to an outbreak were not effective in substantially reducing infections, largely because of the prolonged interval between vaccination and immunity. Prophylactic vaccination, in contrast, could be an effective tool for preventing outbreaks. Herd immunity is practically achievable because of the small sizes of monk seal populations and the animals’ accessibility on shore. Adding realistic spatial structure to the model, as informed by movement of seals tracked in the main Hawaiian Islands with the use of telemetry, greatly reduced the simulated impact of outbreaks (10 seals were infected in 62% of spatially structured simulations). Although response vaccination remained relatively ineffective, spatial segregation allowed herd immunity to be achieved through prophylactic vaccination with less effort. In a randomly mixing population of 170 seals, 86% would need to be vaccinated to achieve herd immunity in 95% of simulated outbreaks, compared to only approximately 60% in three spatially segregated subgroups with the same combined abundance. Simulations indicate that quarantining a modest number (up to 20) of ill seals has the potential to extinguish even fast-growing outbreaks rapidly. The efficacy of quarantine, however, is highly dependent upon rapid detection and respo nse. **We conclude that prophylactic vaccination combined with a quarantine program supported by vigilant surveillance and rapid, reliable diagnosis could greatly mitigate the threat of a morbillivirus outbreak in Hawaiian monk seals.** * ~1,300 Hawaiian monk seals remain throughout the Hawaiian Archipelago * Low genetic diversity and limited exposure to disease * No exposure to morbilliviruses to date * Phocine distemper virus (PDV) and canine distemper virus (CDV) -\> mass die-offs of phocids * Disease outbreak control strategies – Vaccination, quarantine, culling * SEIR (susceptible–exposed–infectious–removed) model * Vaccination response module * Prophylactic vaccination and herd immunity module * Quarantine module * Spatial structure * Heterogenous contact * Models show nearly the entire population in 1/3 of the cases became infected * Statistically signiﬁcant differences in contact rates estimated among Hawaiian monk seal size and sex classes had no appreciable effect on simulated outbreak results * Vaccination efforts in response to an outbreak cannot be relied upon to reduce the number of individuals infected effectively * Prolonged period between vaccination and acquired immunity is problematic * Prophylactic vaccination to achieve herd immunity prior to an outbreak appears to be the only viable vaccination strategy * Requires immunizing a large proportion of the population * Outbreaks in the main Hawaiian Islands monk seal population would be less severe than in a single, randomly mixing population of equal abundance * Low rate of movement among islands relative to the plausible ranges of latency and infectious period effectively limit contact and disease spread * Substantially reduces number of seals requiring vaccination to achieve herd immunity * Simulations suggest that quarantine could be an effective complementary or back up (should prophylactic vaccination fail) strategy * Efﬁcacy of quarantine is highly sensitive to outbreak detection and response times * Conclusion - prophylactic vaccination and quarantine supported by vigilant surveillance are the best tools for mitigation of morbillivirus risks to endangered Hawaiian monk seals

Answer 40

Colitz, C. M., Saville, W. J., Walsh, M. T., & Latson, E. (2019). Factors associated with keratopathy in captive pinnipeds. *Journal of the American Veterinary Medical Association*, *255*(2), 224-230. **OBJECTIVE** To identify factors associated with keratopathy in captive pinnipeds and to provide guidance for preventive measures. **ANIMALS** 319 captive pinnipeds (229 otariids [sea lions and fur seals], 74 phocids [true seals], and 16 odobenids [walrus]) from 25 facilities. **PROCEDURES** Descriptive data collected from questionnaires completed by facilities and from medical records and physical examinations of pinnipeds were compiled and evaluated. Variables were assessed with χ² tests of homogeneity to determine potential association with keratopathy, and variables with values of *P* ≤ 0.25 were inserted into the multivariable logistic regression model. **RESULTS** **Results indicated that variables associated with significantly increased odds of keratopathy in captive pinnipeds included lighter or reflective pool color (OR, 2.11; 95% confidence interval [CI], 1.20 to 3.97), pool water salinity \< 29 g/L (OR, 3.48; 95% CI, 1.89 to 6.56), and history of eye disease (OR, 3.30; 95% CI, 1.85 to 5.98), trauma (OR, 3.80; 95% CI, 1.72 to 8.89), and having been tested for leptospirosis (OR, 3.83; 95% CI, 1.54 to 10.26). However, odds of keratopathy decreased with UV index ≤ 6 (OR, 0.39; 95% CI, 0.2 to 0.72) and age \< 20 years (OR, 0.32; 95% CI, 0.15 to 0.66).** **CONCLUSIONS AND CLINICAL RELEVANCE** **Findings indicated that odds of keratopathy in pinnipeds could be reduced by maintenance of pool water salinity ≥ 29 g/L and reduction of UV radiation exposure** (eg, with adequate shade structures and use of darker, natural colors). Because UV radiation exposure is cumulative, even small attempts to reduce lifetime exposure to it could help control keratopathy in pinnipeds. * Cornea becomes jeopardized when excessive UV radiation exposure occurs, generation of reactive oxygen spp cause cellular and extracellular damage by reacting with DNA, lipids, proteins. * Corneal disease affects pinnipeds of all spp in wild and in managed care. * Pinniped keratopathy has 3 progressive stages – 1 subtle change, 2 involves indolent ulcer that can become infected, 3 infected corneal ulcer that may abscess. * Sea lions tend to have perilimbal corneal edema. With focal ulceration or corneal opacity dorsotemporal to the flattened plateau. * Fur seas generally have temporal perilimbal edema. No corneal focal opacity in stage 1. * Walrus have axial corneal opacity that is not usually ulcerated. * Seals have variable clinical signs with corneal edema consistent in stage 1. * Pinnipeds with chronic unrelenting or recurrent keratopathy tend to develop cataracts earlier in life. * Prev studies - Spatial characteristics implicated in assoc with corneal dz in pinnipeds – number of animals (water quality), enclosure surface color and texture, directional orientation of sunlight. * This study – risk factors include greater UV exposure, older age, previous eye disease, previous trauma. UV index major factor. * Lack of shade increases the likelihood of cataracts in pinnipeds by almost 10 times. * Recommendations: Implement methods to lower total UV radiation exposure (dark or natural colors for shade structures, walls, floors), maintain salinity \> 29 g/L, limit lifetime exposure to UV to help reduce odds of keratopathy with aging, prevent fighting and trauma.

Answer 41

Phillips, B. E., Páez-Rosas, D., Flowers, J. R., Cullen, J. M., Law, J. M., Colitz, C., ... & Lewbart, G. A. (2018). Evaluation of the ophthalmic disease and histopathologic effects due to the ocular trematode Philophthalmus zalophi on juvenile Galapagos sea lions (Zalophus wollebaeki). *Journal of Zoo and Wildlife Medicine*, *49*(3), 581-590. Abstract: The Galapagos sea lion ( Zalophus wollebaeki) is an otariid species endemic to the Galapagos archipelago and is currently listed as endangered. **The ocular trematode *Philophthalmus zalophi* was recently reported to affect the survival of juvenile Galapagos sea lions on Santa Cruz Island, resulting in marked ophthalmic changes.** This study evaluated the ophthalmic disease and histopathologic effects of P. zalophi on juvenile Galapagos sea lions in the largest rookery located on San Cristóbal Island. Twenty juvenile Galapagos sea lions (10 male and 10 female) were evaluated among five sites in the rookery El Malecón. **Ophthalmic examination, including fluorescein staining and evaluation of the adnexa, cornea, and sclera, were performed on each eye. The presence, number, and location of ocular parasites were determined, and parasites were collected for identification. Conjunctival biopsy was performed on 11 animals: 2 that lacked parasites and gross lesions and 9 with both parasites and gross lesions.** All parasites collected were confirmed as P. zalophi and identified in 80% (16/20) of the study animals and 70% (28/40) of the examined eyes. Philophthalmus zalophi was most frequently found attached to the nictitating membrane but also located on the palpebral conjunctiva or cornea. The most common clinical signs were varying degrees of conjunctival hyperemia (28/40 eyes), most frequently of the nictitating membrane and mucoid ocular discharge (12/40 eyes). The number of parasites was significantly associated with the degree of conjunctival hyperemia ( P \< 0.001). Histopathology of conjunctival biopsies revealed organized lymphoid follicles and lymphoplasmacytic infiltrates. The histopathologic changes and gross lesions were likely due to the parasite's attachment to the conjunctiva. This study provides additional details of P. zalophi infection in juvenile Galapagos sea lions. Further research is warranted to detail the life cycle of this parasite, transmission to sea lions, and potential treatment protocols. * Previous disease surveys have identified *Philophthalmus zalophi* as a fluke parasite often causing pathology in the eyes of Galapagos sea lions 3-8mos of age. * CS: mild to severe blepharitis, conjunctivitis, and keratitis, purulent discharge. Infection peaks coincide with increased sea temps. * Ophthalmic exam - ocular discharge present in 10/20 animals, but no corneal ulceration, and only 1/20 with blepharospasm, Most common sign - hyperemic conjunctiva - 14/20 animals. Ocular parasites present in 16/20 pups - usually on nictitating membrane * # of parasites per eye significantly associated with degree of hyperemia (but NOT associated with ocular discharge severity). Hyperemia most commonly affecting the nictitating membrane. * Lymphocytic inflammation was most common histopath lesion * Previous report of this parasite included more severe ophthalmic lesions (blepharitis, keratitis, purulent discharge, corneal ulceration globe rupture, etc.) Most severe lesion here was conjunctivitis. Possibly due to previous study occurring in El nino year (higher sea temps) **Takeaway:** Galapagos sea lions with ophthalmic dz may have Philophthalmus zalophi ocular parasite. Degree of hyperemia correlated with number of parasites per eye, lymphocytic inflammation on histo. Other CS include keratitis, purulent discharge, blepharitis.

Answer 42

Denkinger, J., Guevara, N., Ayala, S., Murillo, J. C., Hirschfeld, M., Montero-Serra, I., ... & Cabrera, F. (2017). Pup mortality and evidence for pathogen exposure in Galapagos sea lions (Zalophus wollebaeki) on San Cristobal Island, Galapagos, Ecuador. *Journal of wildlife diseases*, *53*(3), 491-498. **Abstract:** The **Galapagos sea lion (Zalophus wollebaeki)**, an endangered species, experiences high pup mortality (up to 100%) in years when El Ni ̃no events reduce food supply in the Galapagos Islands. Mortality of pups in non-El Ni ̃no years is estimated to be 5% in undisturbed colonies. From2009 to 2012 we observed high pup mortality (up to 67%) in colonies close to the Galapagos capital, Puerto Baquerizo Moreno, where contact with humans, domestic animals, and rats is frequent. Gross postmortem findings from 54 pups included hemorrhagic lesions in liver and congestion in lungs; histopathology suggested a possible association with infectious diseases. **Evidence of. Leptospira infection was found in five out of seven samples collected in 2010**. **Canine distemper viral(CDV) RNA was detected in tissues from six sea lions (in 2011–12), four of which were confirmed by nucleotide sequencing. The absence of CDV antibodies in 109 juvenile animals tested in 2014 at urban and remote colonies could indicate that the CDV infection observed in 2011 was likely confined to a few animals**. Our results indicated that Galapagos sea lions have been exposed at least to two pathogens, Leptospira and CDV; however, the impact of these infections on the sea lions is unclear. * Annual pup mortality in the Galapagos typically 5% but can go up to 100% during El nino events (food shortage) * Several islands have large populations of dogs and rats * CDV, lepto, parvovirus, herpesvirus, calicivirus, Brucella, toxoplasma * They found lepto DNA and CDV

Answer 43

Taurisano, N. D., Butler, B. P., Stone, D., Hariharan, H., Fields, P. J., Ferguson, H. W., ... & Raverty, S. (2018). Streptococcus phocae in marine mammals of northeastern Pacific and Arctic Canada: a retrospective analysis of 85 postmortem investigations. *Journal of wildlife diseases*, *54*(1), 101-111. **Abstract:** *Streptococcus phocae* is a pathogen of marine mammals, although its pathogenicity remains poorly understood. Recovery of this bacterium from asymptomatic carriers suggests that it is an opportunistic pathogen. We investigated the role of *S. phocae* in naturally occurring disease and its significance as a pathogen based on postmortem investigations. Between 2007 and 2012, 1,696 whole carcasses, tissue samples, or both were submitted from the northeastern Pacific and Arctic Canada for diagnostic testing. *Streptococcus phocae* was cultured from phocids (*n*=66), otariids (*n*=12), harbor porpoises (*Phocoena phocoena*; *n*=5), and sea otters (*Enhydra lutris*; *n*=2). **Pathologic manifestations of *S. phocae*–associated disease included localized, as well as systemic, inflammatory lesions with common findings of suppurative bronchopneumonia (*n*=17) and bacteremia (*n*=27). Lung lesions were frequently culture-positive for *S. phocae*, suggesting commensal colonization of the oropharynx with subsequent opportunistic infection of the respiratory tract during tissue injury, coinfection, immunosuppression, or other debilitating condition**s. The presence of a positive spleen culture, and interpretations at necropsy and histopathology, were used to determine the presence of *S. phocae* bacteremia. Less frequent lesions that were culture positive for *S. phocae* included abscesses (*n*=9), meningitis (*n*=7), and cellulitis (*n*=1). The majority of cases with *S. phocae* lesions featured pre-existing conditions that presumably contributed to some degree of debilitation or immunosuppression, including emaciation (*n*=29), liver mercury accumulation (*n*=29), trauma (*n*=22), severe pulmonary or cardiovascular nematodiasis (*n*=9), concurrent bacterial or viral infections (*n*=8), or sarcocystosis (*n*=6). **These findings suggest that *S. phocae* could be characterized as an opportunistic pathogen, associated with debilitating conditions in stranded and rehabilitating marine mammals.** Wildlife investigators can use these results to draw more definitive conclusions regarding positive *S. phocae* cultures during postmortem studies in marine mammals. * *Streptococcus phocae* has been recovered from clinically healthy and diseased animals * **Considered commensal with a tropism for the oropharynx and upper respiratory tract** * Opportunistic respiratory infection seems common * Methods: investigated the presence of *S. phocae*–associated lesions at necropsy of marine mammals of the northeastern Pacific and Arctic Canada * **Lung tissue was culture-positive for most frequently, 25% of the total positive tissue cultures.** * **Bronchopneumonia, meningoencephalitis, and cellulitis were consistent lesions** * Lesions in the reproductive tract were not consistent findings in this case series * Animals with high liver mercury (Selenium:Hg \< 1): Pure culture in 41% * *S. phocae* disease common in animals with sarcocysits, sea lions with lepto * Young animals were more likely to exhibit decreased vitamin A. Older animals were significantly more affected by cardiopulmonary nematodiasis and liver Se:Hg imbalance than younger animals * **Spleen and lymph nodes were more reliable for a pure culture. Lung often had mixed culture.** * ***S. phocae* was culture from non-diseased female repro tract – suggest commensal in these tissues.** * *S. phocae* did not appear to be associated with low vitamin A concentrations. * *E. coli* commonly cultured with *S. phocae* in cases of heavy parasitic infection – relationship of two bacteria not well understood. * **Debilitated young animals were nursing age, suggesting poor nursing/malnutrition as a factor** **Take Home:** *S. phocae* most frequently isolated from lung tissue. Consistent lesions = bronchopneumonia, meningitis, cellulitis, omphalitis, and abscesses. Pure culture from the spleen/lymph nodes supported true infection if there was a mixed culture elsewhere. **Infection was secondary to debilitation, in young animals due to poor nursing/malnutrition. In older animals infection assoc. with severe parasitism and Se:Hg imbalance.**

Answer 44

Seguel, M., Gutiérrez, J., Hernández, C., Montalva, F., & Verdugo, C. (2018). Respiratory mites (**Orthohalarachne diminuata**) and β-hemolytic streptococci-associated bronchopneumonia outbreak in South American fur seal pups (Arctocephalus australis). *Journal of wildlife diseases*, *54*(2), 380-385. **Abstract:** Although mites of the *Orthohalarachne* genus are common parasites of otariids, their role as agents of disease and in causing population-level mortality is unknown. In the austral summer of 2016, there was an increase in mortality among South American fur seal (*Arctocephalus australis*) pups at Guafo Island, Northern Chilean Patagonia. Pups found dead or terminally ill had **moderate to marked, multifocal, mucopurulent bronchopneumonia associated with large numbers of respiratory mites (*Orthohalarachne diminuata*) and rare Gram-positive cocci. In lung areas less affected by bronchopneumonia, acute interstitial pneumonia with marked congestion and scant hemorrhage was evident**. Bacteria from pups dying of bronchopneumonia were isolated and identified as *Streptococcus marimammalium* and *Streptococcus canis*. **Respiratory mites obstructed airflow, disrupted airway epithelial lining, and likely facilitated the proliferation of pathogenic β-hemolytic streptococci, leading to severe bronchopneumonia and death of fur seal pups**. An abrupt increase in sea surface temperature in Guafo Island corresponded to the timing of the bronchopneumonia outbreak. The potential role of environmental factors in the fur seal pup mortality warrants further study. * **Halarachnidae**: family of mites that infect mammals and most pinniped species * South American fur seals are widespread but experienced decline in Chile * Unusual mortality in 2016 * Pups that died during period of unusual mortalities were necropsied * **Findings: *Orthohalarachne diminuata* mites, dark-red lungs, mucopurulent material in airway** * **Airway swabs yielded moderate growth of Gram-positive, b-hemolytic streptococci.** * ***Streptococcus marimammalium* and *Streptococcus canis*** * SAFS mortality event: pups died because of bronchopneumonia and sepsis associated with *S. canis* or *S. marimammalium* and a heavy burden of *O. diminuata*. * Heavy mite infection likely predisposed to bronchonopneumonia and increased severity * All pups necropsied were infected with mites, compared to 30-60% in other seasons. * **Transmission by direct contact. Nymphs in nasal cavity, adults in lung.** * **Meningeal/brain edema was also seen – possible due to Step toxins.** * Outbreak was likely brought on by environmental or other stress since mites and bacteria are likely present in colonies even during periods without mortality outbreaks.

Answer 45

Kroese, M. V., Beckers, L., Bisselink, Y. J., Brasseur, S., van Tulden, P. W., Koene, M. G., ... & van der Giessen, J. W. (2018). Brucella pinnipedialis in grey seals (Halichoerus grypus) and harbor seals (Phoca vitulina) in the Netherlands. *Journal of wildlife diseases*, *54*(3), 439-449. **Abstract:** Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of *Brucella pinnipedialis* in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of *B. pinnipedialis* and accompanying pathology found in infected animals. The overall prevalence of *Brucella* spp. antibodies in sera from healthy wild grey seals (*Halichoerus grypus*; *n*=11) and harbor seals (*Phoca vitulina*; *n*=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of *Brucella* spp. **In total, 77% (30/39) seals were found to be positive for *Brucella* by IS*711* real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable *Brucella* was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals.** Tissue samples from lung and pulmonary lymph nodes were the main source of viable *Brucella* bacteria. All isolates were typed as *B. pinnipedialis* by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. **No correlation was observed between *Brucella* infection and pathology.** This report displays the isolation and identification of *B. pinnipedialis* in marine mammals in the Dutch part of the Atlantic Ocean. * Brucellosis is a major contagious zoonotic disease, mainly caused by *B. melitensis*, *B. abortus*, *B. suis*, and occasionally *B. canis* and *B. ceti* * Transmission through inhalation or ingestion * In pinnipeds, no apparent pathology has ever been associated with the isolation *B. pinnipedialis* * *B. pinnipedialis* has also been isolated from nematodes in the lungs of pinnipeds * **Seroprevalence is geographic. High in western Atlantic, low in eastern.** * For harbor seals, 77% (30/39) tested positive for *Brucella* by PCR in tissues or lung nematodes. * *Brucella* was cultured from 40% (12/30) of the PCR-positive seals. * **Lung, LN, and nematodes were often positive.** Repro tract and kidney was negative. * Unknown if nematodes play role in transmission. * No associated pathology found with PCR or culture positive tissues. * The seroprevalence was estimated to be 25% * **Surprising age-dependent prevalence: PCR was 84% (26/31) in juveniles and 57% in adults (4/7)** * ***B. pinnipedialis* was cultured only from juveniles and not from adults.** * **Supports that *Brucella* infection in pinnipeds occurs early in life and loss of antibody is probably due to clearance of bacteria**

Answer 46

Grilo, M. L., Ziege, S., Gruber, M., Wohlsein, P., Baumgärtner, W., & Siebert, U. (2018). Idiopathic iron overload in a harbor seal (Phoca vitulina). *Journal of Zoo and Wildlife Medicine*, *49*(3), 770-773. Abstract: Iron overload has been described in various wild species. The majority of cases involve captive animals, often associated with increased dietary iron uptake**. Here a case of idiopathic iron overload in a female adult harbor seal under human care is presented**. The animal displayed a progressive anorexia, apathy, and increased serum iron levels. Radiographs showed radiopaque foreign bodies in the stomach. The seal died during an elective laparotomy. Twenty-five coins and two metal rings were removed from the stomach. Histopathologic examination revealed iron storage without cellular damage in liver, spleen, kidney, and pulmonary and mesenteric lymph nodes. Atomic absorption spectrophotometry analysis for iron revealed values thirty times above the reference ranges in spleen and liver; however, the coins only contain minor levels (parts per million) of iron. The etiology of the iron overload in this animal remains unclear. A multifactorial process cannot be excluded. * Hemochromatosis has been described in many species and is usually associated with dietary increase in iron uptake * **17 yo F captive bred harbor seal presented for anorexia and refusal to train** * **Heavy metal analysis revealed severe increase in iron (~5x upper end of RR). LE’s were normal** * **AXR showed multiple radiopaque circular fb’s in the stomach** * Gastrotomy performed to remove the object, patient died during procedure * **Histopathologic examination showed severe iron storage in Kupffer cells and hepatocytes in the liver and in the macrophages of the spleen** * **Iron also present in LN’s, and epithelium of renal tubules** * Atomic absorption spectrophotometry analysis for iron revealed values thirty times above the reference ranges in spleen and liver * 25 coins removed from stomach; however, the coins only contain minor levels of iron. * **Lack of evidence of iron deposition in macrophages from small intestinal villi in the affected seal, which is regarded as indicative for excessive dietary iron absorption, and normal iron concentrations in the serum of the other harbor seals in the collection do not support a dietary source of the iron overload.** * Possible genetic mutation, though this was not investigated * Cause remains unknown though multifactorial cause is suspected

Answer 47

Sheldon, J. D., Johnson, S. P., Hernandez, J. A., Cray, C., & Stacy, N. I. (2017). Acute-phase responses in healthy, malnourished, and Otostrongylus-infected juvenile northern elephant seals (Mirounga angustirostris). *Journal of Zoo and Wildlife Medicine*, *48*(3), 767-775. ABSTRACT: Acute-phase proteins (APPs) are utilized to detect early inflammation in many domestic and nondomestic species, but variability exists between species and inflammatory diseases as to which APPs are most useful. **Stranded juvenile northern elephant seals (NESs; *Mirounga angustirostris*) undergoing rehabilitation at the Marine Mammal Center experience high mortality rates due to severe arteritis caused by the lungworm, *Otostongylus circumlitis* (OC)**, and there are currently no effective antemortem diagnostic tools for this disease. To characterize patterns of the acute-phase response in the NES, **two APPs – serum amyloid A (SAA) and C-reactive protein (CRP) – were measured, and serum protein electrophoresis was performed to measure albumin and globulin fractions** in 81 serum samples from 58 NESs in four different health states: healthy, malnourished, preclinical for OC infection, or clinical for OC infection. **Compared to healthy NESs (median, 11.2 mg/L), SAA concentrations were significantly increased in malnourished (33.9 mg/L), preclinical (247 mg/L), and clinical OC-infected NESs (328 mg/L) (P \< 0.05). CRP concentrations were increased only in clinical OC-infected NESs** (medial 53.9 mg/L) and were below detectable limits in the other three groups (\<0.01 mg/L). **These results show that SAA and CRP are positive APPs in NESs with OC infection, and that SAA may serve as the major APP for this species.** Albumin:globulin ratios were significantly increased in malnourished NESs (median, 1.26) and decreased in clinical OC-infected NESs (0.53). As a result, **albumin is a negative APP in the NES**, similar to other mammalian species. APP monitoring can be helpful in detecting and monitoring inflammation in rehabilitating juvenile NESs. Background * APPs – acute-phase response of innate immunity, expressed during inflammation before CBC. * **SAA and CRP positive APPs**, produced in liver, increase in circulation \< 1 day in response to IL1, IL6 in humans and dogs. **CRP increases with inflammation in harbor seals.** * SAA involved in recruiting leukocytes to sites of inflammation. * CRP aids in complement binding to bacterial and opsonization, cytokines through the complement system. * **Albumin decreases in response to inflammation (negative APP).** * **APP concentrations should increase and the AG ratio should decrease in the presence of inflammatory disease.** * Variability in which APP is considered major based on species. * ***Otostrongylus circumlitis*** **(OC) – nematode lungworm, significant morbidity and mortality in NES.** Responsible for 12% NES strandings and 37% mortalities at TMMC. * **Fecal not effective for diagnosis. Fecal ELISA test using bovine lungworm antigen was validated for detection in both harbor and gray seals but not for NES.** * Death from arteritis, DIC before nematode sheds ova into GIT. * 89% mortality following clinical signs. Unlike in pacific harbor seal, NES die quickly after infection, appear to be a non-definitive host for the parasite. * Primary objective of this study was **to identify the major detectable APP in NESs by comparing SAA and CRP concentrations and measuring serum protein electrophoresis patterns between healthy NESs and those in three diseased states: malnourished, preclinical for OC infection, and clinical for OC infection.** * Secondary objective to **ID changes in APP concentrations and albumin and globulin fractions in animals that underwent rehabilitation** from being malnourished upon admission to healthy upon release, and in animals that ultimately died because of OC infection before and after development of clinical signs. M+M: 81 blood banked samples from 58 weanling NES admitted during 2012-2014. Classified into one of four groups: healthy, malnourished, preclinical OC infected, clinical OC infected. * **Malnourished NES emaciated without other obvious signs of disease.** * **Preclinical OC infected developed clinical signs of OC and later confirmed at nx.** * **Clinical OC infected exhibited clinical signs at time of sampling, diagnosed on necropsy.** * Preliminary studies conducted using samples from four clinically normal and five clinically abnormal NES to validate the SAA and CRP assays. Results/Discussion: * **SAA increased in malnourished, preclinical and clinical individuals vs healthy.** * SAA higher in samples from malnourished NESs at time of admission vs samples from healthy NESs at time of release. SAA not significantly different between NESs with preclinical and clinical infections. SAA decreased from time point of admission to release. * SAA is a nonspecific biomarker for early inflammatory disease, not a specific diagnostic tool. * **CRP concentrations significantly higher in samples from clinical OC infected vs preclinical.** * Not specific for this disease. May only be a moderate APP in this species. May be only partially cross-reactive with this species. * **AG ratios significantly lower in samples from clinical OC infected NES vs samples from preclinical.** * Albumin negative APP, decreased production in acute inflammation * Decreased AG ratio could be due to decreased albumin vs increased globulin * Reduced AG ratio may help differentiate subclinical inflammation vs malnourished * AG ratio lower at time of release vs admission, likely due to resolution of dehydration and hemoconcentration. **Takeaway:** This study shows SAA is useful as an early inflammatory marker in the NES, using OC infection as an example of a severe inflammatory dz of this spp.**APP assays and SPE are nonspecific tools, use in conjunction with other dx techniques.**

Answer 48

Marinkovich, M., Wack, R. F., Field, C. L., Whoriskey, S. T., Kass, P. H., & Gjeltema, J. (2019). Evaluation of serial blood lactate and the use of a point-of-care lactate meter in live-stranded pinnipeds. *Journal of Zoo and Wildlife Medicine*, *50*(1), 137-146. ABSTRACT: Live-stranded pinnipeds often present to rehabilitation centers systemically debilitated with dehydration and poor perfusion. **In many terrestrial mammals, blood lactate elevation has been correlated with global tissue hypoxia and decreasing circulating blood volume.** Serial blood lactate measurements in companion animals and humans have been used to **guide fluid resuscitation therapy and evaluate prognosis.** The primary objective of this study was to **evaluate the prognostic value of serial blood lactate levels in live-stranded pinnipeds** in a rehabilitation setting. The secondary objectives were to **evaluate the use of a point-of-care lactate meter, and potassium oxalate-sodium fluoride (gray-top) tubes for storing samples** for lactate analysis in pinniped patients. Fifty-five live-stranded pinnipeds (30 northern elephant seals [*Mirounga angustirostris*], 21 Pacific harbor seals [*Phoca vitulina richardsi*], and four California sea lions [*Zalophus californianus*]) that presented to a rehabilitation center were manually restrained for blood collection and lactate measurement using a point-of-care analyzer (Lactate Plus) prior to fluid or other medical therapy. Lactate measurements were repeated 72h post admission and prior to euthanasia or release. Plasma samples from gray-top tubes were frozen for 30-100 days at -80degC, then thawed and evaluated using both the LactatePlus and bench-top (ABL 815) analyzers. **The point-of-care analyzer was successful in measuring lactate in pinnipeds. Gray-top tubes were effective at preserving lactate levels in frozen plasma samples for up to 100 days. Released animals had significantly greater lactate clearance at 72 hr (P = 0.039) than animals that died or were euthanized.** Therefore, lactate clearance, determined by serial blood lactate measurements, may useful for evaluating prognosis in live-stranded pinnipeds. Initial lactate and lactate values prior to euthanasia or release were not significantly associated with outcome. Given these findings, clinicians should interpret isolated lactate values in pinnipeds with caution. * Serial lactate measurements highly predictive of outcome in humans hospitalized in ICU. * Studies evaluating lactate performed in zoological species. * Moose (*Alces alces*), rhinoceros (*Ceratotherium simum*), captive flamingos (*Phoenicopterus ruber*), free-ranging avian species, sea turtles (*Caretta caretta*), three spp of fish. * Sea turtles – lactate decreased over time following presentation and fluid therapy. * Fish lactate levels post-surgery were higher in individuals that did not survive. Primary objective of the study reported here was to **evaluate the relationship between lactate clearance using serial blood lactate values (before and after resuscitation therapy) and survival in live-stranded pinnipeds in a rehabilitation setting.** Secondary objectives **to evaluate use of a portable, poolside, lactate meter and potassium oxalate-sodium fluoride (gray-top) tubes as a means of measuring and preserving pinniped lactate** levels, respectively. * Point-of-care serial lactate evaluations * **Pacific harbor seals, northern elephant seals, California sea lions** within 600 mile range of stranding presented to TMMC. * On presentation, PE, mentation assessment, blood collection, age, spp, sex recorded. Categorized based on diagnosis. * **Venous blood sample collection prior to tx, restrained less than 5 minutes, \< 3 attempts for venipuncture. Placed in gray-top potassium oxalate-sodium fluoride tubes.** * **Run with Lactate Plus meter.** * **All patients received parenteral fluids for at least 3 days and enteral tube feedings.** * **Venous blood sampling repeated at 72 hours as well as within 24 hours of euthanasia or being cleared for release.** Lactate Plus and potassium oxalate-sodium fluoride tube evaluation * 43 frozen plasma samples from all three spp thawed, run on Lactate Plus and established bench top lactate analyzer (Radiometer ABL 815) at UC Davis. * **Pre-freezing and post-thaw LactatePlus values compared to determine the effectiveness of gray-top tubes for preserving samples.** * Post thaw lactate plus values compared with bench top analyzer * Percent bias for each evaluation study was then determined using the difference of means from paired samples. * Serial lactate evaluation * April to June 2016, initial lactate measurements for 55 individuals * 30 NES, 4 CSL, 21 HS. * 44 study animals had a second measurement at 72h post presentation, 34 measurements before euthanasia or release * **Released animals had significantly greater lactate clearance at 72 hours than animals that died or were euthanized.** * All other parameters evaluated including presenting lactate, lactate prior to euthanasia or release, sex, age class, mentation and cause of stranding not significantly associated with outcome. LactatePlus and potassium oxalate-sodium fluoride tube evaluation * Plasma samples, 43 tubes frozen 30-100 days thawed and evaluated for meter and tube evaluation studies. * **Pre-freezing LactatePlus values positively correlated with post-thaw LactatePlus values.** * Bland altman plot of potassium oxalate soium fluoride tube evaluation study showed a mild proportional bias (11%). * **Post-thaw LactatePlus positively correlated with post-thaw bench top lactate values.** \> 72h blood lactate clearance assoc with a positive outcome (release) in live stranded pinnipeds. * Consistent with similar studies in humans and companion animals. * **Initial lactate and lactate values prior to euthanasia or release not significantly associated with outcome.** * **Initial lactate levels lower overall in dehydrated pinnipeds compared with ill companion animals.** * Pinnipeds may be able to process and clear lactate better than terrestrial animals, related to diving and hypoxia tolerance. * **Lactate levels in surviving animals prior to release were higher than their initial lactate.** * Potential impact of restraint (required more restraint when feeling better). * **The present study demonstrated that the point of care LactatePlus analyzer was effective in measuring lactate in pinnipeds.** Instantaneous results without bulky laboratory equipment. * **Potassium oxalate-sodium fluoride tubes effective at preserving lactate in frozen plasma for up to 100 days.** Gold standard for blood lactate preservation in humans.

Answer 49

Gundlach, N. H., Schmicke, M., Ludes-Wehrmeister, E., Ulrich, S. A., Araujo, M. G., & Siebert, U. (2018). New approach to stress research in phocids—potential of dehydroepiandrosterone and cortisol/dehydroepiandrosterone ratio as markers for stress in harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus). *Journal of Zoo and Wildlife Medicine*, *49*(3), 556-563. Abstract: Cortisol is known to reflect the level of the stress response. However, measuring the cortisol concentration only once fails to provide sufficient information about the duration of the stress exposure. Moreover, handling is an acute stressor and increases cortisol secretion especially in wildlife species. Yet, in phocids reliable indicators are missing that reflect potential chronic effects of stress. The adrenal-derived steroid hormone **dehydroepiandrosterone (DHEA)** has been described as biomarker for the assessment of the stress status but has not been applied to marine mammal stress research. Therefore, DHEA, the sulfated precursor DHEAS and the cortisol/DHEA ratio were determined in serum of different seals. One group consisted of harbor ( Phoca vitulina) and gray seals ( Halichoerus grypus) that were habituated to human handling. The other two groups included healthy and free-ranging seals suffering from a disease. Blood samples were taken from 11 habituated (six males, five females), 17 wild\_healthy (13 males, four females), and nine wild\_diseased seals (four males, five females). No differences in serum cortisol levels could be detected between wild\_healthy and wild\_diseased seals. On the contrary, wild\_diseased seals showed the lowest DHEA concentration compared to the other two groups. Wild\_diseased seals also revealed the highest cortisol/DHEA ratio compared to the habituated\_zoo (2,074.7 ± 351.4 vs. 121.5 ± 26.8, P \< 0.001) and wild\_healthy seals (827.9 ± 214.3, P \< 0.01). DHEA and the cortisol/DHEA ratio may reflect differences in the functionality of the hypothalamic-pituitary-adrenal axis and therefore represent valuable tools for the assessment of stress-related effects in seals. * Cortisol easily influenced by handling/ sampling situations and are therefore not a good indicator of chronic stress * Dehydroepiandrosterone (DHEA) used to assess chronic stress in humans -\> chronic hyperresponsiveness of hypothalamic-pituitary-adrenal (HPA) axis causes shift towards cortisol production resulting in quantitative reduction of DHEA * Compared cortisol, DHEA, DHEAS, cortisol/ DHEA ratio, and insulin-like growth factor I (IGF-1) in 1) healthy habituated/ captive seals, 2) healthy wild seals, 3) diseased wild seals. * Blood collected from captive seals done under voluntary control, wild seals done under manual restraint or directly post-mortem for some of the diseased seals. * Captive seals had significantly lower cortisol concentrations compared to both wild groups; no significant difference between healthy and diseased wild seals -\> suggests cortisol highly influenced by situational collection factors * **DHEA was significantly lower in the diseased wild seals compared to the healthy wild seals (and captive seals)-\> indicates more reliable indicator of HPA axis activity in phocids** * **Wild diseased seals had highest cortisol/ DHEA ratio as well as highest neutrophil/ lymphocyte ratio** * **IGF-I decreased in wild diseased seals, indicating likely a good parameter for assessment of energy balance in phocids**

Answer 50

MYCOBACTERIAL DISEASE AND SUBSEQUENT DIAGNOSTIC INVESTIGATIONS IN A GROUP OF CAPTIVE PINNIPEDS IN NEW ZEALAND Chatterton et al. JZWM 2020;51(1):177-187 *Abstract:* This case series includes a single case of disseminated tuberculous disease due to *Mycobacterium pinnipedii* in a New Zealand fur seal (*Arctocephalus forsteri*), which was being cared for by a zoo in New Zealand. The remaining five pinnipeds in the colony underwent extensive mycobacterial disease surveillance over the following 4 yr, involving a total of 26 anesthetic procedures and numerous diagnostic tests that included comparative intradermal tuberculin skin tests, mycobacterial antibody serology, respiratory and gastric lavages, and computed tomography (CT) scans. An additional case of chronic sinusitis due to *Mycobacterium marinum* and *Pseudomonas aeruginosa* was identified in a California sea lion (*Zalophus californianus*). **Results from CT and the respiratory lavages were the most helpful antemortem diagnostic tests for active mycobacterial disease in this case series.** Of the remaining four animals, two were euthanatized and two remain alive, and none of them had evidence of active mycobacterial disease. Further mycobacterial disease surveillance in staff and animals was performed, and no other case was identified. There are no validated mycobacterial surveillance tests available for pinnipeds and so it remains unknown whether the two surviving pinnipeds are truly negative or whether they have latent mycobacterial infection that could develop into active mycobacterial disease in the future. For this reason, increased levels of biosecurity and quarantine remain permanently in place for the pinniped colony. * *Mycobacterium pinnipedii* - aerobic, nonmotile, slow growing acid fast bacillus * Most closely related to *M. bovis* * Clinical disease reported in wild and captive pinnipeds in the southern hemisphere and in domestic cattle in new zealand. * Zoonotic disease reported * *Mycobacterium marinum* free-living nontuberculous mycobacterium is omnipresent in freshwater and marine environments. * Opportunistic pathogen - usually of aquatic ectotherms * Has caused cutaneous granulomas in marine mammals and humans with occasional disseminated disease in marine mammals. DISCUSSION: * *M. pinnipedii* * CS - can be asymptomatic or nonspecific (lethargy weight loss and anorexia) * Reported in wild pinnipeds → current cases suspected to have had latent infection on admission * cITST (intradermal test) detects cell-mediated immune response - lowe sensitivity and specificity for M-TB * *M. marinum* * Association with sinusitis not previously reported * Suspect opportunistic * Zoonotic → self limiting skin lesions * Testing M-TB * Mycobacterial culture is gold standard with 100% specificity * M-TB PCR has similar specificity and increased sensitivity * AFB stain - lower sp/sn * BAL vs Tracheal wash - BAL may only sample a small section of the lung thus tracheal wah may be better * surveillance of active tuberculosis - tracheobronchial lavage to dedicated TB laboratory for AFB, M-TB PCR, and mycobacterial culture * Antibody detection * Confirms prior exposure but not optical for latent or active infection determination * DPP Vet TB not confirmed in pinnipeds * Cannot differentiate between M. pinnipedii and M marinum * Results in the current case were intermittently positive in non positive animals. * Low specificity * CT - can help r/o active microbial disease * RECOMMENDED TESTING: * 2 tracheobronchial lavage (AFB, M-TB PCR, and mycobacterial culture) 8 wks apart + 1 CT scan * Case highlights need for validated testing for latent TB in pinnipeds and risks of admitting wild pinnipeds to zoological collections * TAKE AWAY: Difficult to detect latent infections with m. pinnipedi due to unvalidated tests. M-TB PCR and culture are gold standard and can be performed on tracheobroncial lavage, however these will only detect active infections. cITST has low sensitivity and specificity in pinnipeds as does antibody testing - thus the authors recommend CT and tracheobronchial laval serially for surveillance of disease in pinnipeds.

Answer 51

Kennedy, S. N., Castellini, J. M., Hayden, A. B., Fadely, B. S., Burkanov, V. N., Dajles, A., ... & Rea, L. D. (2019). **REGIONAL AND AGE-RELATED VARIATIONS IN HAPTOGLOBIN CONCENTRATIONS IN STELLER SEA LIONS (EUMETOPIAS JUBATUS) FROM ALASKA, USA.** *Journal of wildlife diseases*, *55*(1), 91-104. ABSTRACT: Varying concentrations of the highly conserved acute phase response protein, haptoglobin, can indicate changes to the health and disease status of mammals, including the **Steller sea lion (SSL**; Eumetopias jubatus). To better understand factors relating to acute phase response in SSLs, circulating **haptoglobin concentrations (Hp) were quantified in plasma collected from 1,272 individuals sampled near rookeries and haulouts off the coast of Alaska, US**. We compared Hp in SSLs between sexes and among different age classes (young pups, young-of-the-year, yearlings, subadults, and adults) sampled within distinct regions in Alaska (Aleutian Islands, Gulf of Alaska, Southeast Alaska). Regional and age- related differences were observed, particularly in younger SSLs. No sex-related differences were detected. **We identified weakly significant relationships between Hp and hematology measurements including white blood cell counts and hematocrit in young pups from the Aleutian Islands and Southeast Alaska**. No relationship between Hp and body condition was found. **Lastly, a nonlinear relationship of plasma Hp and whole blood total mercury concentrations (THg) was observed in SSLs from the endangered western distinct population segment in Alaska**. These results demonstrated that regional variation in Hp, especially in younger SSLs, may reflect regional differences in health and circulating THg. * **Haptoglobin - protein biomarker of inflammation in mammals** * **Upregulated during the primary inflammatory response & concentrations vary w/ physical / environmental stressors** * **Binds free reactive hemoglobin released from damaged red blood cells & prevents damaging redox reactions→ minimizing oxidative stress**

Answer 52

Malberg, S., Gregersen, H. A., Henrich, M., & Herden, C. (2017). Epitheliotropic intestinal T-cell lymphoma in a harbor seal (Phoca vitulina). *Journal of Zoo and Wildlife Medicine*, *48*(2), 568-572. Abstract: A 14-yr- old, male harbor seal (Phoca vitulina) presented with depression, anorexia, and weight loss. Clinical examination revealed no specific abnormalities. Diagnostic imaging displayed an enlarged mass in the dorso-cranial abdomen. The animal was euthanized due to progressive worsening of symptoms. Grossly, severe emaciation and a massive enlargement of one mesenteric lymph node were found. Microscopically, a neoplastic round cell population with morphologic characteristics of mature lymphocytes infiltrated the mesenteric lymph node as well as the jejunal mucosa and exhibited a marked invasion of the mucosal epithelial layer. Immunohistochemically, the majority of cells expressed CD3, but not CD79a, indicating a T-cell origin of the tumor cells. Microscopic as well as immunohistochemical findings enabled the diagnosis of an epitheliotropic intestinal T-cell lymphoma, a neoplasm that has yet not been described in a harbor seal. * 14 yr male harbor seal presented for several weeks lethargy and hyporexia * weight loss and mild intermittent diarrhea * CBC/chem - mild anemia, moderate hypoproteinemia and hypoglobulinema, moderate increase BUN * Fecal – negative * Rads, AUS, and CT - mass in dorso-cranial abdomen * Euthanasia and necropsy * smallcell mucosal T-cell lymphoma with epitheliotropism, affecting jejunum and mesenteric lymph node * incidental mononuclear interstitial nephritis * microscopic and immunohistochemical features of neoplastic cells match criteria for feline small-cell enteropathy-associated T-cell lymphoma Lymphoma is rare in pinnipeds * Possible causes – viral, environmental pollution, especially organochlorines * Intestinal lymphoma can occur spontaneously in harbor seals, based on this report First description of epitheliotropic intestinal T-cell lymphoma in a harbor seal * Signs, lab results, imaging, location, and morphologic features comparable to canine and feline intestinal lymphoma as well as to a Californian sea lion case

Answer 53

Whitmer, E. R., Borremans, B., Duignan, P. J., Johnson, S. P., Lloyd-Smith, J. O., McClain, A. M., ... & Prager, K. C. (2021). Classification and regression tree analysis for predicting prognosis in wildlife rehabilitation: a case study of leptospirosis in california sea lions (zalophus californianus). *Journal of Zoo and Wildlife Medicine*, *52*(1), 38-48. **Abstract:** 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. · Leptospira interrogans serovar Pomona is endemic to CSL o Yearly outbreaks in the fall with larger epizootics every 3-5 years o CS – PUPD, lethargy, hyporexia, melena, abdominal pain o CP – azotemia, hyperphosphatemia, hypernatremia, variable leukocytosis o Nephritis, pneumonia, GI ulceration commonly reported o Case fatality rate in rehabilitation is 71% o Treatment reported – CCFA on arrival, oxytetracycline if not eating, doxy if eating, for 14 days, SC fluids o Large outbreaks can stretch facility limits – euthanasia criteria are needed to best allocate resources and prevent animal suffering · Poor prognostic indicators – BUN \> 280 mg/dL, not voluntarily eating within first 3 days, Sodium \>178 mmol/L, Creatinine \>3.5 mg/dL (\>7.9 for sure) · CART analysis sometimes criticized for data mining without clinical relevance – they tried to exclude redundant or nonclinical parameters – similar findings with logistic regression **Take home**: BUN, appetite, sodium, and creatinine can be used to assess the prognosis of CSL shortly after admission for leptospirosis treatment **References:** · Deming AC, Colegrove KM, Duignan PD, Hall AJ, Wellehan JFX, Gulland FMD. Prevalence of urogenital carcinoma in stranded California sea lions

Answer 54

Whitmer, E. R., Trumbull, E. J., Harris, H. S., Whoriskey, S. T., & Field, C. L. (2021). **Use of potassium chloride for low-residue euthanasia of anesthetized California sea lions (Zalophus californianus) and northern elephant seals (Mirounga angustirostris) with life-threatening injury or disease.** *Journal of the American Veterinary Medical Association*, *259*(2), 197-201. Objective: To demonstrate the efficacy of potassium chloride (KCl) for low-residue euthanasia of anesthetized pinnipeds in field settings for which carcass retrieval for disposal is not feasible. Animals: Stranded, free-ranging California sea lions (CSLs; *Zalophus californianus*; n = 17) and northern elephant seals (NESs; *Mirounga angustirostris*; 6) with life-threatening injury or disease between May and August 2020. Procedures: Each animal was anesthetized and then received a lethal dose of KCl solution administered by IV or intracardiac injection. The effective KCl dose; durations to cardiac arrest, respiratory arrest, and pupil dilation; and presence or absence of agonal breaths, muscle fasciculations, or skeletal movements were recorded. Results: Mean effective dose of KCl was 207.4 mg/kg (94.3 mg/lb) for the 17 CSLs and 209.1 mg/kg (95.0 mg/lb) for 5 of 6 NESs (1 outlier NES was excluded). The range in duration from the beginning of KCl injection to cardiac arrest was 0 to 6 minutes, to pupil dilation was 0 to 5 minutes, and to respiratory arrest was 0 to 5 minutes. Muscle fasciculations, skeletal movements, and agonal breaths were observed in both species during and after KCl administration. Conclusions and Clinical Relevance: The use of KCl provides an effective, low-residue method of euthanasia in anesthetized CSLs and NESs. Our recommended dose for these species is 250 mg KCl/kg (113.6 mg KCl/lb) delivered by intracardiac injection. Compared with euthanasia by barbiturate overdose, the use of KCl reduces the potential for secondary intoxication of scavengers and is appropriate in field scenarios in which the carcass cannot be retrieved for disposal. Key Points: * For instances in which carcasses will be left in situ after euthanasia, euthanasia by anesthetic overdose may result in drug residues in tissues and pose secondary intoxication risk to scavengers. * **High serum potassium suppresses myocardial electrical condition, leading to arrhythmias and cardiac arrest.** * **KCl is inexpensive, readily acquired and transported, not controlled, and does not pose an intoxication risk to scavengers.** * KCl solution prepared with 300 g KCl powder and room termperature tap water to generate 1 L of solution, resulting in approximately 300 mg/mL or 4 mEq K+/mL. * CSL IV access through subclavian vein, NES IV access through epidural sinus. * Pupil dilation was used as an approximate indicator of brain death. * Presence or absence of muscle fasciculations, muscle contractions resulting in skeletal movements, or agonal breaths was recorded. **Fasciculations observed in 4 of 17 CSLs, 4 of 6 NES. Not observed in CSLs that received inhalant anesthetic prior to the euthanasia. Large contractions and limb rigidity occurred in 1 NES and 6 CSLs.** * No postmortem artifacts attributed to KCl administration were found in any of the animals on gross necropsy, consistent with other studies that show negligible or absent artifacts with KCl. * KCl dose administered ranged widely for both species. Clinically useful dose of 250 mg KCl/kg suggested. This is higher than the lethal dose recommended across taxa. * **Skeletal muscle contractions have been reported during and after administration of KCl in other anesthetized spp.** In this study, physical signs were considered within the range of accepted adverse effects for euthanasia given that all animals was at an appropriate plane of anesthesia. * Additional considerations for field settings – human safety, environmental conditions, supplies and equipment. Removal of IM injection sites if other drugs used. In cold ambient temperatures, precipitate may form in the KCl solution and obstruct drug delivery. Takewaways: IC or IV administration of KCl could be effectively used in CSL and NES for euthanasia. Reduces potential for scavenger intoxication and is appropriate for situations where a carcass must be left. **Death by lethal injection of KCl in conscious or sedated vertebrates is unacceptable (GA required first).**

Answer 55

OPTIMIZING A PLATELET-RICH PLASMA CONCENTRATION PROTOCOL FOR SOUTH AMERICAN SEA LIONS (OTARIA FLAVESCENS) Journal of Zoo and Wildlife Medicine 52(3): 956–965, 2021 Abstract: Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. **This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols**. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. **Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations**. **The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min.** **The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min**. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol. Intro · Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology · Platelet rich plasma (PRP) has been used successfully to that end in multiple species—obtained by centrifuging whole blood--but no protocols exist for sea lions o Autogenous source of growth factors that promote healing o Also suppresses cytokine release, limiting inflammation while stimulating tissue regeneration · According to the American Red Cross, human PRP must contain a platelet concentration greater than a twofold increase compared with that of whole blood · In this study, both single and double centrifugation protocols are evaluated for platelet isolation, as former studies proved a double centrifugation protocol to be the most efficient way to concentrate platelets in autologous blood · The main objective of this study was to define a simple, reproducible, and efficient centrifugation protocol to obtain the highest-quality PRP from the South American sea lion (Otaria flavescens) M&M · 7 healthy adult south american sea lions (1 male, 6 females) · Voluntary blood draw · Citrated blood samples · 12 different centrifugation protocols (different rpm, different length of centrifugation, 1 and 2 step protocols) · TEM was used to evaluate platelet morphology Results and discussion · tatistically significant differences (P , 0.01) were found between the total platelet count in the PRP obtained by centrifuging whole blood for 3 min and the PRP obtained by centrifuging blood for 6 min, with the concentration achieved by the 3-min centrifugation significantly higher · In addition, when analyzing the centrifugation speeds, significant differences were found between 500 and 1,500 rpm and 900 and 1,500 rpm. The difference between 700 and 1,500 rpm was not statistically significant, but there was a clear tendency for lower centrifugation speeds to produce higher absolute platelet counts compared with higher centrifugation speeds · The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. · The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min (4.73 increase in platelets) · TEM confirmed that platelets were not damaged with these methods · **Low speeds and short centrifugation times resulted in a higher platelet concentration in PRP**

Answer 56

Journal of Zoo and Wildlife Medicine 52(3): 1054–1060, 2021 **THE USE OF EPHEDRINE TO TREAT ANESTHESIA-ASSOCIATED HYPOTENSION IN PINNIPEDS** Emily J. Trumbull, DVM, Fabian Okonski, MD, Cara L. Field, DVM, PhD, DACZM, Dave Casper, DVM, Abby M. McClain, DVM, Sarah Pattison, RVT, Emily R. Whitmer, DVM, Sophie Whoriskey, DVM, and Shawn P. Johnson, DVM, MPVM **ABSTRACT:** Hypotension is a common adverse effect of general anesthesia that has historically been difficult to measure in pinniped species due to technical challenges. **A retrospective case review found seven pinniped cases that demonstrated anesthesia-associated hypotension diagnosed by direct blood pressure measurements during general anesthesia at The Marine Mammal Center (Sausalito, CA) between 2017 and 2019**. **Cases included five California sea lions (CSL: *Zalophus californianus*), one Hawaiian monk seal (HMS: *Neomonachus schauinslandi),* and one northern elephant seal (NES: *Mirounga angustirostris*).** Patients were induced using injectable opioids, benzodiazepines, and anesthetics including propofol and alfaxalone. Excluding the HMS, all patients required supplemental isoflurane with a mask to achieve an anesthetic plane allowing for intubation. Each patient was maintained with inhalant isoflurane in oxygen for the duration of the anesthetic event. Each patient was concurrently administered continuous IV fluids and four patients received fluid boluses prior to administration of ephedrine. **All hypotensive anesthetized patients were treated with IV ephedrine (0.05–0.2 mg/kg).** The average initial systolic (SAP) and mean (MAP) arterial blood pressures for the CSL prior to ephedrine administration were 71 +/- 14 mmHg and 48 +/- 12 mmHg respectively. The average SAP and MAP for the CSL increased to 119 +/- 32 mmHg and 90 +/- 34 mmHg respectively within 5 m of ephedrine administration. The NES initial blood pressure measurement was 59/43 (50) (SAP/diastolic [MAP]) mmHg and increased to 80/51 (62) mmHg within 5 m. The initial HMS blood pressure was 79/68 (73) mmHg and increased to 99/78 (85) mmHg within 5 m following ephedrine administration. All patients recovered from anesthesia. **These results support the efficacy of IV ephedrine for the treatment of anesthesia-associated hypotension in pinnipeds.** **Study Design**: Case series (n = 7): five California sea lions, one Hawaiian monk seal, and one northern elephant seal **Goal:** Reviews the diagnosis and management of suspected anesthesia-associated hypotension in three pinniped species **Key Points:** * Noninvasive indirect blood pressure cannot be reliably measured due to technical challenges of using oscillometric cuffs on pinniped extremities * _The placement of arterial catheters in pinnipeds can be achieved in the median artery in otariids and the superficial plantar metatarsal artery in phocids using ultrasound guidance_ * Currently there are no published reference data for baseline blood pressure of conscious pinnipeds * Free-ranging CSLs undergoing rehabilitation have a higher reported perianesthetic mortality rate compared with other species * The exact cause of perianesthetic death in pinnipeds is not always identified, but we speculate that hypotension contributes to poor anesthetic outcomes * _Treatment options for anesthetic-induced hypotension includes reducing anesthetic depth; fluid therapy; blood products; and anticholinergic, vasopressor, and inotrope drugs_ * Anticholinergics such as atropine or glycopyrrolate can be used to decrease vagally mediated bradycardia, however in some species such as rabbits, the response is limited * _The response of pinnipeds to anticholinergics also seems to be minimal and therefore is not utilized by the authors_ * In hypotensive human patients under general anesthesia, the use of inotropes in addition to fluids is associated with lower morbidity and mortality compared with fluid administration alone * Previously described treatment of hypotension in pinnipeds includes dobutamine (0.2–2.0 mcg/kg/m IV) and dobutamine combined with ephedrine (0.05 mg/ kg IV) * **Dobutamine is a synthetic catecholamine that acts as a positive inotrope increasing forward cardiac flow by acting predominantly on beta-1 cardiac receptors** * **Ephedrine is a sympathomimetic amine that acts directly on alpha and beta cardiac receptors, as well as indirectly by decreasing the re-uptake of norepinephrine at the synaptic junction** * **These combined actions increase systemic vascular resistance and cardiac output, and therefore raise blood pressure** * _Maintaining normothermia can be a challenge for debilitated pinniped patients under prolonged general anesthesia despite utilizing multiple modes of thermal support_ * Two patients had a prolonged recovery to which a low body temperature may have contributed * _Sternal recumbency appears to allow for greater lung excursion and improved peripheral perfusion and therefore was the preferred position for pinnipeds during these procedures_ * Pinnipeds commonly exhibit hypercapnia or apnea during general anesthesia, especially during longer procedures or during deep planes of anesthesia, and therefore are regularly mechanically ventilated * _Ephedrine is typically administered by bolus dosing due to its longer duration of action compared with other inotropes and vasopressors_ * _The pinniped cases reviewed here suggest ephedrine can result in sustained normotension following a single bolus dose of 0.1-0.2 mg/kg IV_ * This dose range is similar to published effective doses in humans (0.05–0.1 mg/kg IV) as well as canine and equine patients (0.2 mg/kg IV) * The duration of effects were more similar to horses, which have demonstrated normotension for 90 m post infusion, unlike canine and human patients where effects generally only last for a few minutes * Both phocid patients in this case series required multiple doses of ephedrine, though it is unknown if this was necessarily due to the lower doses they received (0.05–0.07 mg/kg IV), differing vascular access points, or other species variation * Tachyphylaxis, or a diminished response due to depleted synaptic norepinephrine, can occur with patients with low catecholamine stores or in patients receiving multiple doses of ephedrine * Tachyphylaxis was not considered to have occurred in the current small sample of pinniped cases due to each dose administered resulting in increased blood pressure **TLDR:** * Hypotension was documented in a single otariid species and two phocid species * Hypotension occurred despite differing anesthetic protocols and across varying age ranges * The use of ephedrine in this limited group of pinniped patients demonstrates its potential as a single agent to treat anesthetic-associated hypotension * The effective single dose of ephedrine among the seven treated patients presented here was 0.1–0.2 mg/kg IV

Answer 57

OPTIMIZATION OF FECAL FLOTATIONS IN MARINE PARASITOLOGY AND DETERMINATION OF THE SPECIFIC GRAVITY OF HELMINTH EGGS IN PINNIPEDS. Rodgers JS, Schaffer PA, Field CL, Ballweber LR, McGrew AK. Journal of Zoo and Wildlife Medicine. 2021;52(3):949-955. Recent studies have sought to optimize the fecal flotation procedure to improve the detection of helminth eggs in terrestrial domestic species. It is unclear, however, whether these efforts in optimization are applicable to parasite species of marine environments, and verification of veterinary diagnostic procedures is clinically important. It was hypothesized that the eggs belonging to the parasites of pinnipeds would have different specific gravities (SpG) than those belonging to terrestrial hosts. **Fecal samples were collected from each of 25 stranded pinnipeds representing three species (Zalophus californianus (22), Phoca vitulina (2), Mirounga angustirostris(1)), and modified double centrifugal flotations** were performed on 1-g samples. Among the 22 California sea lions sampled, trematode, ascarid, and cestode eggs were detected in 17/22 (77%), 10/22 (45%), and 4/22 (18%) individuals, respectively. Sugar-gradient modified centrifugation flotations were then conducted on a subset of 10 samples from California sea lions to evaluate the distribution of eggs in fractions representing varying SpG. **Higher numbers of ascarid eggs were found in fractions representing a lower SpG (1.00–1.15), whereas trematode eggs belonging to the genus Zalophotrema were found in significantly higher numbers in the fraction representing 1.25 (P , 0.05)**. In conclusion, the SpG of trematode, ascarid, and cestode eggs from pinnipeds appears to be similar to those from terrestrial hosts, but numerous factors may affect their ability to be detected using traditional diagnostic approaches. Further exploration into the nature of the variability noted may lead to improved diagnostics in marine parasitology. **Background** **Key Points** * CSL Float: Trematodes prevalence 64% (*Apophallus zalophi*, *Zalophotrema hepaticum),* Cestodes 14% (*Diphyllobothrium),* Nematodes 36% (*Anasakis* or *Contracaecum)* * Sediment: Trematodes 50%, cestodes 14%, ascarids 14% * No acanthocephalans identified on float or sediment * Protozoans: *Giardia* sp. not detected, *Cryptosporidium* sp. in one sample * No association with helminth egg counts and sex or age cohorts * Harbor seals: no trematodes or cestodes on float but one case *Apophallus,* one case *Zalophotrema* on sediment; ascarids one case positive on float but not sediment, on ecase positive on sediment but not float * Elephant seal: *Diphylobothrium* and *Zalophotrema hepaticum* detected * High variability of specific gravity of nematode, trematode, and cestode eggs * Significantly more *Zalophotrema* eggs at SpG 1.25 * Significantly more ascarid eggs at SpG 1.05, similar to terrestrial **Conclusions** * SpG of _\>_ 1.25 is recommended for detection of pinniped trematodes, ascarids, and cestodes * Consider using a detergent to reduce debris and egg deformation from high SpG * Important to examine both float and sediment, especially when EPG is required

Answer 58

Journal of Zoo and Wildlife Medicine 52(3): 872–879, 2021 **PHARMACOKINETICS OF SUBCUTANEOUS ALPHA LIPOIC ACID, A PROPOSED THERAPEUTIC AID FOR DOMOIC ACID INTOXICATION IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS)** Cara L. Field, DVM, PhD, DACZM, Sophie T. Whoriskey, DVM, Xianguo Zhao, PhD, and Mark G. Papich, DVM, MS, DACVCP **Abstract**: **Domoic acid (DA) is a potent neurotoxin produced by certain marine algae that can cause neurologic and cardiac dysfunction by activating glutamate receptors. Glutamate receptor overexcitation results in continuous cellular activation, oxidative damage, and cell death. DA toxicosis causes disorientation and seizures, and antiseizure medications are the primary treatment**. _Alpha lipoic acid (ALA), a powerful antioxidant and glutathione precursor widely used in humans and dogs, can cross the blood-brain barrier to provide antioxidant availability to brain tissue_. Hundreds of stranded California sea lions (CSL; Zalophus californianus) are diagnosed annually with DA toxicosis and thus are an appropriate animal in which to establish ALA dosing recommendations for treatment. The objective of this study was to determine the population pharmacokinetics of a single 10- or 20-mg/kg dose of ALA administered subcutaneously into the interscapular region to healthy rehabilitated CSL. Blood was collected at two time points between 15 min and 24 h after administration. Serum ALA concentrations were measured by liquid chromatography-mass spectrometry, and parameters were evaluated using a nonlinear mixed effects model. **ALA was rapidly absorbed for each dose, peaking within 20 to 30 minutes, and t1/2 of 40 and 32 min (10 and 20 mg/kg, respectively), followed by an initial steep distribution phase and prolonged elimination phase. Peak concentration (CMAX) was 1,243 ng/ml (10-mg/ml dose) and 5,010 ng/ml (20-mg/ml dose). Serum from 13 CSLd with DA toxicosis treated with 10 mg/kg ALA for 1 to 9 d had measurable levels, and ALA was also measurable in cerebrospinal fluid from two treated CSLs. Therapeutic effects are noted with a CMAX of 4,000 to 5,000 ng/ml in humans; thus in CSLs, 20 mg/kg administered subcutaneously once daily may be sufficient to achieve a therapeutic level in this species.** Determination of efficacy and optimal dosing interval and duration require additional investigation. · Domoic acid (DA) o Produced by marine diatoms (Pseudo-nitzchia) and bioaccumulates in the food chain o CS in sea lions – disorientation, seizures, coma, nausea, vomiting, diarrhea o Known to cause neurologic dysfunction, cardiotoxicity, and GI upset when ingested o Binds to Kainite & alpha-amino-5-methyl-3-hydroxyisoxazolone-4-propionate (AMPA) subclasses of ionotropic glutamate receptors resulting in excitotoxic effects § Glutamate binding normally has a rapid desensitizing response, however DA results in slow or nondesensitizing overexcitation that leads to oxidative damage and cell death o DA is rapidly excreted – most of the damage is done by the secondary excitotoxic effects o Common pathologies include hippocampal atrophy, long-term cognitive impairment, and cardiomyopathy · Alpha lipoic acid is a precursor of the antioxidant glutathione. It is both amphiphilic so it easy crosses the BBB. · This study resulted in sufficient levels to be therapeutic in humans · Future studies to be performed on its efficacy in treating the secondary excitotoxic effects of DA toxicity **Take Home**: ALA given subcutaneously achieves levels that may be sufficient to reduce the secondary excitotoxic effects of DA in sea lions

Answer 59

Journal of Wildlife Diseases, 57(4), 2021, pp. 808–819 [**ANTIBODIES AGAINST INFLUENZA VIRUS TYPES A AND B IN CANADIAN SEALS**](https://doi.org/10.7589/jwd-d-20-00175) Lena N. Measures and Ron A. M. Fouchier **ABSTRACT:** Influenza viruses have been reported from marine mammals worldwide, particularly in pinnipeds, and have caused mass mortalities of seals in North America and Europe. Because influenza viruses in marine mammals can be zoonotic, our objective was to examine Canadian phocids for exposure to influenza A and B viruses in order to understand health risks to wild populations as well as to humans who consume or handle these animals. **Blood was collected from 394 seals in eastern Canada from 1994 to 2005. Sera were screened for exposure to influenza viruses in three resident species of seals: harbour, *Phoca vitulina* (n=66); grey, *Halichoerus grypus* (n=82); ringed, *Phoca hispida* (n=2); and two migrant species: harp, *Pagophilus groenlandica* (n=206) and hooded*, Cystophora cristata* (n=38).** **Included were samples from captive grey (n=1) and harbour seals (n=8) at two aquaria.** Sera were prescreened using indirect enzyme-linked immunosorbent assay (ELISA), and antibodies against influenza A virus were confirmed using a commercial competitive ELISA (IDEXX Europe B.V.). **A subset of influenza A virus positive sera was used to determine common virus subtypes recognized by sera using reference strains. All positive sera in the indirect ELISA reacted with influenza A virus subtypes H3, H4, and H10 using a hemagglutination inhibition assay. Sera from harbour, grey, harp, and hooded seals had antibodies against influenza A and influenza B viruses (some cross-reactivity occurred). Overall, 33% (128/385) of wild seals were seropositive to influenza viruses, with the highest seroprevalence in harp (42%) followed by harbour (33%), grey (23%), and hooded (11%) seals.** Antibodies were detected in both sexes and most age classes of wild seals. **Two of eight captive harbour seals were seropositive to influenza B virus and four had cross-reactions to influenza A and B viruses.** This study reports antibodies against influenza A and B viruses in four seal species from the same geographic area in eastern Canada **Study Design**: Retrospective cross sectional serosurvey **Goal:** Determine exposure to influenza viruses in ringed, harbour, grey, hooded, and harp seals in Canadian waters **Key Points** * **Mass mortalities of pinnipeds due to pneumonia associated with influenza virus infection have been reported** * **Epizootics, primarily involving harbour seals have been caused by Influenza A subtypes H3, H4, and H10 in the eastern US and northeastern Europe** * **Dense aggregations of seals, unusual environmental conditions, and secondary infections may play a role** * **Influenza A virus infection in seals can cause respiratory distress, nasal discharge, and swollen neck and thorax** * **Histopathology reveals diffuse hemorrhagic pneumonia with necrotizing bronchitis and bronchiolitis as well as hemorrhagic alveolitis and subcutaneous emphysema of the thorax and neck** * Influenza A viruses are widespread in wild aquatic birds, particularly Anseriformes and Charadriiformes, which are the natural reservoirs * Influenza A viruses are classified based on their variable surface antigens, hemagglutinin (HA) and neuraminidase (NA) proteins * Influenza A viruses reported in marine mammals are of avian origin * Influenza B and C viruses are human influenza viruses with no known wildlife reservoir and are not observed in aquatic birds * Influenza B virus identified in harbour, gray, Caspian, and South American fur seals were circulating in adjacent human populations before identification in seals * Influenza D viruses are found in livestock * Generally good agreement between the three assays used (indirect ELISA, commercial competitive ELISA, and HI assay) * Seroprevalence varied annually in all four seropositive species of seals, particularly in harp seals * **No effect of age or sex with seroprevalence of influenza A virus except for harp seals** * **Seroprevalence was greater in adult harp seals than in juveniles or pups and declined with increasing age among adults** * **It is unknown how long seals retain antibodies against influenza viruses and whether antibodies are protective against subsequent infection** **TLDR:** * Four species of Canadian seals within the St. Lawrence ecosystem were exposed to influenza A and B viruses * **Seroprevalence of influenza A virus was relatively high in harp (42%) and harbour seals (33%), lower in grey (23%) and hooded seals (11%), and seronegative in ringed seals**

Answer 60

Journal of Zoo and Wildlife Medicine 52(2): 827–837, 2021 **TOTAL EAR CANAL ABLATION AND LATERAL BULLA OSTEOTOMY (TECA-LBO) IN ATLANTIC HARBOR SEALS (PHOCA VITULINA CONCOLOR) FOR SUCCESSFUL SURGICAL MANAGEMENT OF OTITIS MEDIA** Zachary C. Ready, DVM, Jennifer E. Flower, DVM, MS, Dipl ACZM, Joshua E. Collins, DVM, Dipl ACVS, Edward Kochin, DVM, Dipl ACVS, and Charles Rogers Williams, VMD – Reviewed by MSM **Abstract:** Chronic, severe otitis media was diagnosed in four Atlantic harbor seals (Phoca vitulina concolor), three of which were stranded animals undergoing rehabilitation. All seals presented with unilateral purulent aural discharge that would intermittently recur despite prolonged topical and systemic antimicrobial therapy. Aerobic culture from aural discharge isolated multidrug-resistant organisms in all seals, including Pseudomonas aeruginosa, Staphylococcus pseudintermedius, Klebsiella pneumoniae, and/or Enterococcus faecalis. Computed tomography was used in three cases to conﬁrm otitis media and positive contrast ear canalography was used in one case to conﬁrm tympanic membrane rupture. **Given the persistent nature of otitis, surgical intervention in the form of a total ear canal ablation and lateral bulla osteotomy (TECA-LBO) was indicated.** Surgery was successful in achieving complete clinical resolution of otitis in all seals. **Postoperative complications included temporary unilateral paralysis of the left nare (2/4) and a transient left ptosis (1/4). Partial to complete surgical site dehiscence occurred in all cases; however, complete healing was achieved by second intention in 60 d or less.** One rehabilitated seal was ﬁtted with a satellite tag that conﬁrmed normal swimming and diving patterns post release. In harbor seals, TECA-LBO can be performed safely to treat persistent cases of otitis media and should be considered in cases of chronic otitis that are not responsive to medical management. · Chronic otitis externa can progress to otitis media (middle ear, tympanic bulla, typnanic membrane, auditory ossicles by drainage through the ruptured or eroded typnamic membrane o Etiologies include dermatologic conditions, parasites, foreign bodies, neoplasia, secondary bacterial & fungal infections o Refractory cases are often managed with the TECA to remove disease tissue. The TECA-LBO is a salvage procedure to excise all the affected tissue o TECA-LBO has been performed in rabbits, chinchillas, American bison, alpaca, and Bongo · These cases were anesthetized with midazolam (0.15 mg/kg), butorphanol (0.1 mg/kg) and propofol (3.8 mg/kg) · A 14-ga red rubber catheter was used to cannulate the ear canal to make manipulation and dissection easier. Dissection was accomplished with electrocautery and Metzenbaum scissors staying close to cartilages. Bulla was exposed with periosteal elevators and a surgical burr was used to remove the lateral wall of the bulla. Mucoperiosteal lining was removed in a single piece with gentle traction. · Dehiscence was reported in all cases but the addition of modified lambert cruciates reduced its severity as did the use on nonabsorbable suture and delaying suture removal beyond 14 days – blubber is poorly vascularized and takes some time to heal · Unique phocid anatomy o Lack external pinnae and vertical ear canal that has a normal lumen that is easily occluded by stenosis. o Canal is surrounded by a venous plexus that distends during diving as a proposed mechanism of pressure equilibration and ear protection during diving o Seals allowed to dive in deeper pools during rehabilitation had a decreased incidence of otitis **Take Home Message**: TECA-LBO can be performed in end stage otitis cases in phocid seals

Answer 61

**EFFECTS OF A GNRH VACCINE (IMPROVAC®) ON PATAGONIAN (OTARIA BYRONIA) AND CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).** Martinez-Giménez J, Ferrero-Vicente L, Feltrer-Rambaud Y. Journal of Zoo and Wildlife Medicine. 2021;52(2):721-725. Improvac® is a gonadotropin-releasing hormone vaccine developed to reduce “boar taint” in the meat of male domestic pig. The use of Improvac for contraception of zoo and free-living animals has been increasing in recent years. This study reports the use, efficacy, and side effects of Improvac on five male sea lions. **Administration of two injections of 600 µg of Improvac (gonadotropin releasing factor analogue–protein conjugate) 4–5 wk apart were delivered to two Patagonian and three California sea lions (12-15 yo) to reduce testosterone-related aggression, anorexia, and lethargy that occur during the breeding season. Behavior and physical changes were recorded for all individuals, and blood samples were taken from one Patagonian sea lion to measure plasma testosterone concentrations over time.** Observations revealed a descension of the testes into the scrotum, orchitis, lameness, anorexia, and lethargy in all individuals for the first 3–5 d after the first administration of the vaccine. Plasma testosterone concentrations rose after the first dose of the vaccine and remained elevated for 1 mo, decreasing after the second injection to undetectable levels. Improvac administration can cause a peak of testosterone and breeding behavior just after the first inoculation, as previously described in swine and elephants, but has not been documented in pinnipeds. None of the treated animals in this study showed breeding behaviors during their normal breeding season (July–September). **Background** * Pinnipeds - seasonal breeders * CSL, PatagonianSL testes only descend during breeding season * Improvac: GnRH vaccine, stimulates anti-GnRH antibodies; 2 doses 4 wk apart, booster q6mo **Key Points** * Given 14 wks prior to breeding season * **6 hr post vacc: signs of breeding behavior (testes descended, orchitis, apathy, floating, anorexia)** * **No signs of breeding behavior or physical changes during 6 mo after 2nd dose** * **Testosterone levels peaked at time of 2nd injection then fell to baseline and remained for 6 mo** * Inflammation at injection site, severe lameness 3-5d post injection * Dexamethasone post 1st injection and at time of 2nd injection improved pain/lameness **Conclusions** * GnRH vaccine, Improvac, can be used to control breeding season behavior in CSL, PSL * **Lameness was a major adverse reaction in all 5 animals lasting 3-5 days and improving with steroid treatment** * Signs of anorexia, lethargy, orchitis are natural during breeding season, consistent with initial elevation in testosterone, not an adverse effect of vaccination

Answer 62

**Serum estradiol and progesterone profiles during estrus, pseudopregnancy, and active gestation in Steller sea lions** Sattler and Polasek ZooBio 2017 **Abstract** While the proximate driver behind the decline of the Western stock of Steller sea lions (Eumetopias jubatus, \>80% since 1970s) is likely multifactorial, the population reduction may have been powered by a decrease in fecundity. A harvest of Steller sea lions in the 1970s and 80s revealed a 30% reduction in the proportion of pregnant females from early (October–November) to late gestation (April–May). Identification and quantification of these reproductive failures are difficult when we lack species-specific data on endocrinology associated with discrete stages of the reproductive cycle (i.e., estrus, implantation, and gestation). We tracked changes in serum estradiol and progesterone in three adult female Steller sea lions from 2011 to 2015. In all years and most females, **a discrete increase in estradiol was observed during the breeding season (June–August), indicative of estrus. Estradiol concentrations from October to May in a pregnant female compared to her corresponding values when non-pregnant did not consistently differ through gestation.** ***An elevation in progesterone was observed in all females and all years beginning approximately in June and lasting through November.*** This likely results from progesterone production by the corpus luteum in both pregnant and pseudopregnant females. **Serum progesterone shows promise as a diagnostic tool to identify pregnancy during months 3–5 (December–February) of the 8-month active gestation following embryonic implantation.** This study provides ranges of key hormones during estrus, embryonic diapause/pseudopregnancy, and gestation in pregnant and non-pregnant females for studying reproduction in Steller sea lions. **Key Points** * Background: * Western stock listed as endangered in 1997 * Currently overall stable or increasing but there are strong regional differences * Fecundity and pup production are suggested as potential sources for the continued lack of recovery * Female Steller sea lions become reproductively mature at 4.6 years of age, pup in mid-May to mid-July, and spontaneously ovulate 11 days later * Following copulation, if an egg is fertilized, females undergo a 4-month embryonic diapause and delayed implantation, followed by an 8 month active gestation * Non pregnant female otariids exhibit an obligate pseudopregnancy after ovulation for approx. 4 months, followed by an 8-month period before their next ovulation Goals of the study: * Quantify annual estradiol and progesterone hormone ranges for pregnant and non-pregnant adult females * Quantify changes in estradiol levels associated with copulation events following parturition * Determine if hormone levels differ between pregnant and non-pregnant females Methods: Sampled 3 adult female Eastern stock Steller sea lions at ASLC * From September through April, females were housed independently from breeding males * May-August – nonpregnant females were housed with the male and removed only for sample collections * Pregnant females were housed separate from the male until 7-16 days post-parturition * Three pregnancies occurred in this study * Samples were collected every other month from April 2011- Feb 2015. Weekly collections in June or July * Results/Discussion * Could identify a fetal heartbeat as early as December but reliably in January * Estradiol concentrations during the breeding season were highly variable for all females * A distinct spike was observed during the breeding season for all females * External visible signs of estrus included mucoid vaginal discharge and swollen and darkly pigmented vulvar area * High individual variability in estradiol concentrations was observed over the course of the study period * Copulation occurred during the 7-16 days post-parturition window * Mean progesterone concentrations in non-pregnant animals during the breeding season were approximately double those observed throughout the non-breeding seasons * Progesterone was elevated in all females beginning approx. in June and lasting through November * Elevated progesterone was present irrespective of pregnancy status * Annual trends in progesterone fluctuated largely among females and with reproductive status * Progesterone concentrations differentiated between a pregnant vs nonpregnant female beginning in December through May * **Take Home** * **Observed a correlation between increased estradiol and copulation** * **Serum progesterone shows promise as a diagnostic tool to identify pregnancy during the 3-5 months window (Dec-February) of the 8-month active gestation. Following this period, progesterone in pregnant animals decreases as parturition nears and thus distinguishing pregnant animals from non-pregnant during months 6-8 active gestation (March-May) using progesterone may not be possible.**

Answer 63

**Discrimination between bycatch and other causes of cetacean and pinniped stranding** Dis Aquat Org 127: 83–95, 2018 - Yara Bernaldo de Quirós et al. (WHOI/IFAW) ABSTRACT: **The challenge of identifying cause of death in discarded bycaught marine mammals stems from a combination of the non-specific nature of the lesions of drowning, the complex physiologic adaptations unique to breath-holding marine mammals, lack of case histories, and the diverse nature of fishing gear**. While no pathognomonic lesions are recognized, signs of acute external entanglement, bulging or reddened eyes, recently ingested gastric contents, pulmonary changes, and decompression-associated gas bubbles have been identified in the condition of **peracute underwater entrapment (PUE) syndrome** in previous studies of marine mammals. **We reviewed the gross necropsy and histopathology reports of 36 cetaceans and pinnipeds including 20 directly observed bycaught and 16 live stranded animals that were euthanized between 2005 and 2011 for lesions consistent with PUE.** **We identified 5 criteria which present at significantly higher rates in bycaught marine mammals: external signs of acute entanglement, red or bulging eyes, recently ingested gastric contents, multi-organ congestion, and disseminated gas bubbles detected grossly during the necropsy and histologically.** **In contrast, froth in the trachea or primary bronchi, and lung changes (i.e. wet, heavy, froth, edema, congestion, and hemorrhage) were poor indicators of PUE.** This is the first study that provides insight into the different published parameters for PUE in bycatch. For regions frequently confronted by stranded marine mammals with non-specific lesions, this could potentially aid in the investigation and quantification of marine fisheries interactions. Basics: * Bycaught marine mammals that die peracutely are those which become entangled in fishing gear such as gillnets and trawls where the proximate cause of death is forced submersion * **Peracute underwater entrapment (PUE)** - term applied to mortalities of marine mammals caused by entanglement and forced submersion and can entail complex determinations of ultimate cause of death * Gas bubbles in PUE marine mammals is a consequence of post mortem offgassing from hauling out deceased animals with nitrogen saturated tissues * Macroscopic gas bubbles in bycaught animals is higher than in stranded marine mammals * Gas bubbles were mainly composed of nitrogen, reinforcing the decompression hypothesis * DDX for gas bubbles: Decompression-like sickness linked to anthropogenic sound or infection by *Clostridium perfringens* **Mechanisms that mar mam use to compensate for long periods of breath-holding at depths (alveolar collapse, tracheobronchial compression, and blood redistribution) may inhibit inspiration during forced submersion, leading to cardiac inhibition and severe laryngospasm, hypoxia, and convulsions** * **CO2 narcosis has also been suggested; but, studies in shallow-diving terrestrial species (beaver and otter) have shown that toxic levels of CO2 do not accumulate before the onset of hypoxia** Details: * **5 criteria for PUE that were higher in bycaught animals :External signs of acute entanglement, red or bulging eyes, recently ingested gastric contents, disseminated congestion, and disseminated gas bubbles** * **Gas bubbles were found more frequently in lymph nodes and livers** of bycaught animals. * Prevalence of gas bubbles (75% for bycatch; 50% stranded) were lower than previous studies * Gas bubbles more widely distributed and numerous in bycaught vs stranded animals, suggesting distribution and number of bubbles is an important discriminating PUE criterion in fresh animals * **Evidence of acute entanglement (line marks, net marks, etc) was most specific lesion for PUE** * **Froth in the respiratory tract and lung changes are poor indicators of PUE** * Barbiturate euthanasia may have caused congestion, hemorrhage, and edema in lungs in stranded animals * Presence of chyle in lymphatic ducts should be scored as a more reliable predictor of recent feeding * Taxon appears to have no effect on the presence of the lesions identified in this study

Answer 64

**Visitor effects on a zoo population of California sea lions (*Zalophus californianus*) and harbor seals (*Phoca vitulina)*** Amber J. de Vere Zoo Bio 2018 **Abstract** The effects of visitor presence on zoo and aquarium animals have become increasingly well studied, using measures such as behavioral responses and exhibit usage. Many taxa remain underrepresented in this literature; this is the case for marine mammals, despite widespread public concern for their welfare in managed care settings. The current study therefore used behavioral activity budgets and exhibit usage to assess the responses of California sea lions (Zalophus californianus) and harbor seals (Phoca vitulina) to visitors at the Seal Cove exhibit at Six Flags Discovery Kingdom, Vallejo CA. Data was collected via focal follow video recordings over the summer season of 2016, and analyzed using MANCOVAs, discriminant analyses, and modified Spread of Participation Indices. The sea lions showed no significant changes in behavior when visitors were present, but did show greater preference for the water bordering visitor viewing areas during these times. Two sea lions gave birth during the study period, and showed greater preference for land areas both adjacent to and out of sight of visitors when nursing compared to while pregnant. In contrast, the harbor seals showed significant behavioral changes in the presence of visitors, including increased vigilance and feeding. This was associated with increased preferential use of water areas adjacent to the visitor viewing area. Visitors were able to purchase fish to throw to the animals, which likely contributed to the differences observed. Overall, this study found little evidence for negative visitor impacts on two pinniped species in a zoo setting. **Key Points:** * Aim: To provide an assessment of visitor effects on pinnipeds in a zoo setting * Two species were examined- harbor seals (Phoca vitulina) and California sea lions (Zalophus californianus) * Both behavior and enclosure use were assessed * Data collection * Observations were made over 3 months during the facility’s (Six Flags Discovery Kingdom) summer season using a video camera * The study population contained six harbor seals and four California sea lions. Two sea lions were born during the study period and were included. All animals were captive born with the exception of 1 sea lion who was a wild rescue. * Results * Harbor seals spent more time submerged when visitors were present, and less time engaged in social interactions * In contrast, the presence of visitors did not have a significant effect on the behavior of the CSLs with differences seen only for the females before and after giving birth * CSLs showed greater variability between individuals in regards to exhibit usage * * Visitors significantly affected the behavior of harbor seals but not CSls * Assessing the preferences of zoo animals is vital for not only determining whether environmental features have negative impacts on welfare, but also for pre-emptively providing animals with known preferred options. * For instance, the preference of the harbor seals for aquatic areas over land suggests that it is important for this species to be provided with sufficient water space

Answer 65

**JZWM 2020 51(3): 598–605 RETROSPECTIVE STUDY OF SURGICAL TREATMENT OF REFRACTIVE OSTEOMYELITIS AND INFECTIOUS ARTHRITIS IN THE FLIPPERS OF SEALS IN THE NETHERLANDS** Amputation surgery in pinniped rehabilitation centers is a feasible procedure when animals are presented with open fractures, osteomyelitis, and/or infectious arthritis of the flippers that appear to be refractory to medical treatment. From 2011 to 2017, the Sealcentre Pieterburen in The Netherlands admitted 3,775 seals for rehabilitation. Of these, 37 individuals presented clinical and radiologic signs of bone abnormalities indicative of osteomyelitis or infectious arthritis refractory to medical treatment. Seven cases resulted in euthanasia, and 30 cases underwent amputation surgery. The surgical procedure involved amputation of part of a flipper (24; two animals twice) or of a complete flipper (eight). All procedures were done under general anesthesia except one that was performed with local anesthesia, and all 30 animals were released. In two cases, the osteomyelitis presented with the rare Totenlade phenomenon, a sequestrum surrounded by new periosteal bone formation. The purpose of this retrospective study was to evaluate the outcome of the operative treatment of osteomyelitis and infectious arthritis in the flippers of harbor (Phoca vitulina) and grey seals (Halichoerus grypus) during this 6-yr period. - **Osteomyelitis and infectious arthritis are the most coon complication of open fractures and bite wounds.** - Main indication for amputation in seals is due to refractory osteomyelitis or infectious arthritis of the flipper M&M - 3775 animals, 144 with radiographs. 44 showed abnormalities in the flippers - 7 animals improved with time and medical treatment and did not need surgery - 4 euthanized due to osteomyelitis of a front flipper - 33 -- \> exploratory surgery o 3 = surgery was not possible -- \> euthanized o 30 = amputation, 2 had a second surgery due to infection at later stage § 32 surgeries · 26 involved hind flippers, 6 front flipper - Housed individually and dry docked for 7-10 days o Short swimming times gradually increased - 100 days average = released - Medical treatment: anti-inflammatories, analgesics and antibiotics o Carprofen tramadol, clindamycin, enro, clavvamox, gentamycin - 50% cases = culture - Radiographs repeated 2 weeks after medical treatment, if improvement seen, no further treatment. If worsening, surgery - Amputation: 1) complete amputation of flipper at level of tibia/fibula. 2) amputation of a ray or part of it with amputation level from the tarsus or carpus up to the metatarsal=phalangeal or metacarpal joint. 3) amputation of a digit of part of it - Description of surgery is mentioned Results - See abstract - Totenlade (sequestrum) seen in 2 grey seals Discussion - Marine mammals incorporate the proximal limb bones within the fusiform body, protruding only tarsus and phalanges - **Flippers of seals have minimal weight bearing compared to sea lions and polar bears** o **Functionality is mostly for steering and making turns under water** **- Hind flippers used in propulsion under water** **- Amputation of the hind flipper did not lead to problems in swimming but may lead to severe complications if the front flipper is removed.** **o Removal of the from flipper would deprive from proper locomotion. Not recommended**

Answer 66

Journal of Zoo and Wildlife Medicine52(4): 1149–1158, 2021 **CLINICAL AND MICROBIOLOGICAL CHARACTERIZATION OF LYMPH NODE ABSCESSATION IN PUP AND YEARLINGCALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) UNDERGOING REHABILITATION IN A MARINE MAMMALCENTER** Zachary C. Ready, DVM, Emily R. Whitmer, DVM, Sarah E. Wright, DVM, Carlos Rios, MLS(ASCP), Pa ́draig J. Duignan, MSc, DVM, PhD, and Cara L. Field, DVM, PhD, Dipl ACZM **Abstract**: 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 phoca e(n=10) and Staphylococcus spp. (n¼9). 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.** **Key Points**: * Previous LN abscess studies in pinnipeds * E coli & beta hemolytic strep identified as most common in a CSL study in the 90s * Gram negative isolates were 72% of cultured organisms from CSL, PHS, NES abscesses also in the 90s * Abscess formation occurs when bacteria enter the node and cannot be cleared * Difficulties in treating include the thick capsule and exudate that makes penetration of antibiotic difficult * This study * E coli was most prevalent followed by strep phocae * Surgical treatment was superior to natural fistulation or not opening the node (lowest mortality) * Treatment with at least one antibiotic was superior to one antibiotic or no antibiotics * Bloodwork showed findings consistent with malnutrition and inflammatory disease – mature neutrophilia, hypoalbuminemia * Mortality rate of these cases similar to others presenting to TMMC **Take Home**: Drain LN abscess, flush regularly, and treat with broad spectrum antibiotics

Answer 67

Journal of the American Veterinary Medical Association 259(10): 1196-1205. 2021. **Clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis** Sophie T. Whoriskey dvm Pádraig J. Duignan dvm, phd Abby M. McClain dvm Mauricio Seguel dvm, phd Frances M. D. Gulland vetmb, phd Shawn P. Johnson dvm, mpvm Cara L. Field dvm, phd – Reviewed by MSM **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. **Key Points:** * Sarcocystis neurona * 2-stage life-cycle. Virginia opossum is the only definitive hosts, but numerous intermediate hosts have been documented * Ingestion of contaminated feces or water and result in intermediate host infection * Sea otters and harbor seals develop meningoencephalitis, similar to horses, leading to strandings, depressed mentation, tremors, seizures, and coma. * Sarcocystosis is also a frequently reported cause of hepatic disease in marine mammals * Sarcocystis sporocysts can survive in fresh water for months – duration in sea water is unknown, but Toxoplasma gondii cysts can survive for up to 3 months * This study * An IgG titer \> 1:320 was considered positive * Previous retrospective found that 2-13% of stranded CSLs had titers to sarcosystis neurona * Anesthesia – midazolam (0.2-0.3 mg/kg), butorphanol (0.1-0.3 mg/kg) +/- medetomidine (0.01-0.03 mg/kg) for muscle biopsy from supraspinatus, sternocephalicus muscle * Biopsies showed lymphoplasmacytic and histiocytic myositis with concurrent muscle necrosis and regeneration. Sarcocystis like cysts were often present. * Treatment – ponazuril, clindamycin, or TMS * Clinical signs – generalized muscle atrophy leading to prominence of several bony structures; dysphagia, regurgitation, and dyspnea due to megaesophagus in more severe cases * Clin Path – elevated ALT and CK were more common * Other treatments to consider – decoquinate * Annual number of CSLs appears to be increasing **Take Home:** Sarcocystic neurona induced rhabdomyositis and megaesophagus are important differentials for emaciated adult CSLs presenting stranded. **References**: CRC protozoal chapter

Answer 68

Journal of Wildlife Diseases, 56(4): 863-872, 2020 HELMINTH FAUNA OF ICE SEALS IN THE ALASKAN BERING AND CHUKCHI SEAS, 2006–15 Heather S. Walden, Anna L. Bryan, Antoinette McIntosh, Pam Tuomi, Anne Hoover- Miller, Raphaela Stimmelmayr, and Lori Quakenbush (Reviewed by AJC) ABSTRACT: Climate warming may affect the distribution of helminth parasites, allowing endemic species to increase in prevalence and new species to appear. We analyzed tissues from 141 ice associated seals collected in the Alaskan (US) Bering and Chukchi seas during 2006–15 for internal helminth parasites and compared results with past studies. Specimens were collected from: ringed seals (Pusa hispida), bearded seals (Erignathus barbatus), spotted seals (Phoca largha), and ribbon seals (Histriophoca fasciata). Helminths were present in 94% (133/141) of the seals sampled. Nematodes were most prevalent in bearded (97%, 72/74) and spotted seals (93%, 13/14). Cestodes were most prevalent in bearded seals (82%, 61/74) and absent in ribbon seals, trematodes were only found in bearded (64%, 47/74) and ringed (5%, 2/44) seals, and acanthocephalans were mostly found in ringed (61%, 27/44) and spotted (64%, 9/14) seals. Although no helminths were new to the Bering-Chukchi Seas region, this study found a previously unreported host record for the lungworm Parafilaroides (Filaroides) gymnurus in a ribbon seal. We also found the lungworm Otostrongylus circumlitus in a ribbon seal and P. (F.) gymnurus in bearded seals, representing location records previously unreported from the Bering-Chukchi Seas region (although they have been reported from the Sea of Okhotsk). We found the cestode genus Pyramicocephalus in bearded seals (3%, 2/74) at a lower prevalence than was reported previously for Pyramicocephalus phocarum (44–100%) in the Bering-Chukchi Seas region. We found no species of the acanthocephalan genus Bolbosoma, although the genus was previously identified in ringed, spotted, and ribbon seals. This study yielded no new helminths and no increases in the prevalence of endemic parasites in these seal species. Key Points: · Helminths infect seal prey as larvae and are ingested by seals – individuals in poor health/immunosuppressed tend to have greater number and diversity of helminths. · Evaluation of helminths in ringed, bearded, spotted, and ribbon seals from Alaska over 2006-2015 when harvested. · Helminths present in 94% of seals sampled – most nonpathogenic o Nematode (roundworms) – lungworms and heartworms have potential to cause morbidity and mortality § **Four seals had adult lungworms or larvae (Parafilaroidea gymnurus, Otostrongylus circumlitus, Otostrongylus sp) - all had secondary lung disease (pneumonia, fibrosis and congestion)** **§ Acanthocheilonema spirocauda (heartworm) noted in bearded seal and spotted seal** **· Heartworms found in right ventricle and pulmonary artery à proliferative endarteritis and may completely occlude pulmonary artery** **· Thought to be transmitted by seal lice (Echinophthirius horridus)** o **Trematodes (Orthosplanchnus articus or Orthosplanchnus sp) can cause thickening of the bile ducts in bearded seals.** **§ Two adult bearded seals had evidence of trematodes in the gallbladder**. **o Bearded seals – generalists feeding on variety of fish and inverts which may explain why they had the highest diversity of helminths.** · No new helminths to the region nor change in numbers/diversity of parasites despite predicted changes associated with climate change. Take home: · Overview of multiple helminth species affecting phocid species. Majority of helminths nonpathogenic (exceptions – lungworm, heartworm, some trematodes). No new helminths nor changes in numbers/diversity of parasites noted. References: None

Answer 69

*J Zoo Wildl Med* 2021 52(4): 1217–1223 [**MEASUREMENT OF INTRAOCULAR PRESSURE USING REBOUND TONOMETRY IN ANESTHETIZED PERUVIAN FUR SEALS (*ARCTOCEPHALUS AUSTRALIS*) FROM PUNTA SAN JUAN, PERU**](https://doi.org/10.1638/2020-0222) Kane, Lauren P., Adkesson, Michael J., Sheldon, Julie D., Allender, Matthew C., Jankowski, Gwen, et al. **ABSTRACT:** Ocular disease in pinnipeds under human care is well described, and intraocular pressure (IOP) can be impacted by a variety of ophthalmic conditions. Species-specific reference parameters from clinically normal animals are instrumental for understanding how ophthalmic diseases may impact ocular pressures. _IOP measurements were obtained using rebound tonometry from free-ranging Peruvian fur seals (*Arctocephalus australis* unnamed subspecies) at Punta San Juan, Peru, over a 6-yr period (2010–2016). Retrospective data obtained from 108 (81 adults and 27 neonates comprising 69 females and 39 males) anesthetized fur seals with normal anterior segment ophthalmic examinations was included in the analysis_. Differences in IOP from each eye were compared to categorical variables (age, year, sex, restraint) using an independent-samples *t* test. All univariate results with a significance of *P* \< 0.05 were included in multivariate analysis. Of the 13 general linear models evaluated, the top two for both the right and the left eye included age class when all variables were evaluated simultaneously. _Neonates had significantly lower IOP values than adults in both the right eye (17.5 mm Hg; 95% confidence interval [CI]: 14.0–21.1 mm Hg compared to 33.5 mm Hg; 95% CI: 31.0–36.1 mm Hg, respectively) and the left eye (18.4 mm Hg; 95% CI: 14.4–22.5 mm Hg compared to 32.3 mm Hg; 95% CI: 29.3–35.3 mm Hg, respectively). Anesthesia method was not statistically significant (*P* \> 0.05). This is the first report of normal IOP measurements for any fur seal species._ Described data can be used to improve diagnosis and management of ocular alterations in pinnipeds **Goal:** Characterize normal IOP using rebound tonometry in anesthetized free-ranging Peruvian fur seals with regard to the impact of sex, age, year, and restraint method **Key Points:** * Evaluating the posterior segment can be challenging in pinnipeds due to lack of adequate mydriasis * A large globe, spherical lens, and muscular iris aid in low-luminosity vision underwater, while a region of flattened cornea allows nearly equal refraction in water and air * Pinniped corneas are thicker than those of terrestrial mammals, protecting the eye from underwater cooling and mechanical damage * IOP via manometry is gold standard; rebound tonometry provides a reliable estimation * Topical anesthetic is not required compared to applanation tonometry since the rebound tonometer probe has only minimal and brief contact with the cornea * The TonoVet has three manufacturer-recommended settings: * ‘‘d’’ for dogs and cats * ‘‘h’’ for horses * ‘‘p’’ for species that lack a calibration curve * Previous studies have shown the ‘‘p’’ setting underestimates IOP and has inaccurate readings as compared to the ‘‘d’’ setting **Free-ranging adult South American sea lions and California sea lions under human care without ocular disease had similar IOP measurements (~25-40 mmHg)** **Like this study, Humboldt penguins have lower mean IOP as chicks compared to adults, suggesting a possible association in animals adapted for deep marine diving** **Anesthetic effects on IOP are variable with some agents increasing IOP (alfaxalone), decreasing IOP (isoflurane), having no impact on IOP (medetomidine and midazolam), or transiently increasing and decreasing IOP (butorphanol)** * This study did not reveal a significant difference in IOP based on anesthetic drugs **TLDR:** * **Free-ranging Peruvian fur seal IOP: ~14-22 mmHg (neonate), ~30-35 mmHg (adult)** * **Neonates had lower IOP measurements than adults** * **Adult females had unilaterally higher IOP than males, but this may not be clinically relevant**

Answer 70

**Histologic lesions in placentas of northern fur seals (Callorhinus ursinus) from a population with high placental prevalence of coxiella burnetii**. Conway R, Duncan C, Foster RA, Kersh GJ, Raverty S, Gelatt T, Frank C. The Journal of Wildlife Diseases. 2022;58(2):333-340 Coxiella burnetii is an intracellular bacterial pathogen that can be associated with significant reproductive disease or acute mortality in livestock and wildlife. **A novel marine mammal–associated strain of C. burnetii has been identified in pinnipeds of the northwestern Pacific Ocean**. Little is known about C. burnetii infection in regard to reproductive success or population status. Our objective was to characterize the severity and extent of histologic lesions in 117 opportunistically collected placentas from presumed-normal northern fur seals (Callorhinus ursinus) in July 2011 on St. Paul Island, Alaska, US, where a high placental prevalence of C. burnetii had been reported. Sections were examined by histology and immunohistochemistry and impression smears with modified acid-fast stain. The nature and frequency of histologic changes were compared with target COM1 PCR-confirmed C. burnetii positive and negative placentas. Overall, histologic changes were similar to placental lesions described in aborting ruminants; however, changes were variable within and between placentas. **Vasculitis and occasional intracellular bacteria were seen only in C. burnetii PCR-positive placentas. Dystrophic mineralization, edema, and inflammation were seen in PCR-positive and negative placentas, although they were statistically more common in PCR-positive placentas. Results suggest that C. burnetti and associated pathologic changes are multifocal and variable in placentas from these presumably live-born pups**. Therefore, multiple sections of tissue from different placental areas should be examined microscopically, and screened by PCR, to ensure accurate diagnosis as the genomes per gram of placenta may not necessarily represent the severity of placental disease. These limitations should inform field biologists, diagnosticians, and pathologists how best to screen and sample for pathogens and histopathology in marine mammal placental samples. Background * Decline in northern fur seals on St. Paul Island, Alaska * Longitudinal mortality study shows high rate of perinatal mortality, increasing over time * Coxiella burnetii detected by PCR in 75% of northern fur seal placentas from live births since 2010 - suspect marine mammal-specific adapted strain * Sheep and goats - reproductive failure with intro of an asymptomatic carrier, abortion storms * Dairy cattle - asymptomatic infection, persistent shedding in milk, sporadic abortions * Histo - variable inflammation, necrosis, myriad intracytoplasmic bacteria in Steller sea lions and Pacific harbor seals Key Points * 76% positive for C. burnetii by COM1 PCR * None had gross lesions, no bacteria on impression smear cytology * Histo: intracytoplasmic bacteria in 4% localized in hemophagocytic zone and placental labyrinth * Inflammation and necrosis associated with bacteria * Confirmed C. burnetii on IHC * IHC did not reveal bacteria in any PCR positive placenta that were not seen on histo * All placenta with vasculitis were positive on PCR, histo looked like infarcts, edema next to vessels -- 1 case also had Brucella spp. (IHC and PCR) -- 1 case of Acanthocheilonema odendhali microfilaria in labyrinth * Inflammation, coagulative necrosis, and dystrophic mineralization often present in labyrinth and more common in labyrinth of PCR positive placenta Conclusions * Presence of labyrinth inflammation, necrosis, and vasculitis and intralesional C. burnetii were increased in PCR-positive placenta of northern fur seals * Histo sections should be collected from multiple locations of placenta including labyrinth and marginal hemophagocytic area due to multifocal nature of disease * A single section of fresh tissue appears sufficiently for PCR detection, IHC did not reveal bacteria that were not readily apparent on histo, impression smear cytology was not a sensitive method for detection.

Answer 71

**ISOLATION, CHARACTERIZATION, AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA ISOLATED FROM SEA LION (ZALOPHUS CALIFORNIANUS) PUPS IN NORTHWESTERN MEXICO** Zavala-Norzagaray et al Abstract Bacterial infections have been documented in marine mammals for decades, and some are considered emerging pathogens with zoonotic potential. **The aerobic oral (n=16) and rectal (n=17) bacterial microbiota and their antimicrobial resistance were characterized for 17 apparently healthy California sea lion pups (Zalophus californianus) captured with a hoop net in Farallon Island, Sinaloa, Mexico, in 2016**. Bacteriologic cultures, Analytical Profile Index, and PCR were used to identify bacterial species. The Escherichia coli phylogenetic groups were identified by PCR, Salmonella serotypes were identified, and resistance to antibiotics was evaluated. Overall, 39 bacterial species were isolated, including E. coli and Salmonella spp. (35.9% each) and Pseudomonas aeruginosa (28.2%). For E. coli, UNKNOWN phylogroup was the most prevalent (57.7%), followed by the A phylogroup (37.1%). Most Salmonella serotypes were identified as Newport (92.8%); serotype Saintpaul was also identified (7.2%). Sea lions with bacterial co-colonization included 24.2%, from which two bacterial species were isolated, and 3% with three species. **Overall, 59% of bacteria were resistant to at least one antibiotic tested, and 25.6% were extensively drug resistant**. Bacteria were highly resistant to ampicillin and cefotaxime. This study demonstrates the importance of characterizing the microbiome of sea lions, and the potential effect of pathogens with antimicrobial resistance on wildlife conservation and public health. Key Points: * sea lions may serve as reservoirs of Salmonella spp. and shed this zoonotic bacterium in haul-out sites along the California coast * The colonization of sea lions by Salmonella spp. could be related to fecal contamination from seabird reservoirs and human feces in waste-water discharges from boats * It has been estimated that around 30% of sea lion mortality is related to bacterial infections The objective of the study was to document the oral and rectal bacterial flora in sea lion pups from Farallon de San Ignacio island reproductive rookery in the Gulf of California, Mexico, and determine their antimicrobial susceptibility. - Farallon de San Ignacio island, Mexico - 1.5–2-mo-old sea lion pups were captured with hoop nets - Antibiotic susceptibility testing of pathogenic isolates was performed by the Kirby-Bauer disk diffusion method Three combinations of two bacterial species were identified: 1) E. coli and P. aeruginosa, 2) E. coli and Salmonella spp., and 3) Salmonella spp. and P. aeruginosa One sea lion had all 3 (and some had none) Overall, bacteria demonstrated resistance most commonly to cefotaxime (38.5%); ampicillin (30.7%); nalidixic acid, sulfamethoxazole-trimethoprim, and tetracycline (25.6%, each) - All bacteria were sensitive to ciprofloxacin. - A total of 59% of bacteria were resistant to at least one antimicrobial, and 25.6% were XDR. - Pseudomonas aeruginosa was the most resistant by antimicrobial class > E. coli less resistant > Salmonella least resistant - The three bacteria species that we isolated from sea lions may be opportunistic pathogens for sea lions, especially in stress situations such as during rehabilitation - The high prevalence of Salmonella Newport indicates that it may be endemic in this specific sea lion population. Take-home point questions: What three bacterial species were isolated from sea lion pups in northwest Mexico? - **E .coli, Salmonella (esp Salmonella Newport – maybe endemic), and Pseudomonas aeruginosa** How many of the isolates showed AMR? - **Over half (59%) were resistant to at least one antibiotic and a quarter were multidrug resistant ** Which bacterial species showed the most and which showed the least AMR? - **Pseudomonas> E. coli> Salmonella** Which antibiotic were all isolates susceptible to? - **Ciprofloxacin ** Which antibiotics were most commonly resistant? - **Cefotaxime > ampicillin > TMS, tetracycline, nalidixic acid **

Answer 72

**EVALUATION OF TRACE ELEMENT CONCENTRATIONS IN THE SERUM AND VIBRISSAE OF PERUVIAN PINNIPEDS (ARCTOCEPHALUS AUSTRALIS AND OTARIA BYRONIA)** Kooyomijian, Giarikos, Adkesson, Hirons Abstract Concentrations of 15 trace elements (aluminum, arsenic, cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury, nickel, selenium, tin, vanadium, and zinc) were determined in vibrissae (whiskers) and serum of two sympatric pinniped species, the Peruvian fur seal population (PFS; Arctocephalus australis Peruvian subpopulation) and South American sea lion (SASL; Otaria byronia) at Punta San Juan, Peru during 2011–19 sampling events. Element concentrations were 2–20 times higher in vibrissae than in serum. Vibrissae and serum concentrations of several elements, including aluminum, arsenic, and lead, suggest that environmental contaminants may affect the health of pinnipeds at Punta San Juan. Although toxicity thresholds are unknown in pinnipeds, high concentrations of some elements (especially aluminum, arsenic, and lead) may have adverse impacts on their health such as immunosuppression and impaired reproduction. Arsenic was the only element that increased in mean vibrissae concentration throughout the study period. Female SASL vibrissae contained a mean arsenic concentration three times higher than the male SASL vibrissae mean arsenic concentration, and twice as high as the arsenic mean for all PFS vibrissae. The mean male SASL vibrissae cadmium concentration was five times higher than the vibrissae cadmium mean for both PFS males and females and nearly three times higher than the vibrissae cadmium mean for SASL females. Serum concentrations of aluminum, arsenic, copper, and manganese were significantly higher during moderate to extreme El Niño years compared to La Niña years. With stronger and more frequent El Niño-Southern Oscillation events predicted in the future, it is vital to understand how these trace elements may affect pinniped population health. Key Points: - Every 5-7 years, ENSO events cause pinniped deaths due to the overall disruption of the ecosystem. Results/ discussion: * Keratinous vibrissae elemental concentrations were 2–20 times higher than serum concentrations. * Female pinnipeds can offload trace elements during gestation and lactation * Male pinnipeds do not offload elements through these routes, so they may accumulate higher elemental concentrations. * Aluminum serum concentrations in PFS and SASL were higher than toxic levels in human serum, implying that Peruvian pinnipeds may be at risk of Al toxicity * Acidic waste from mining activities may increase Al and other metal bioavailability in the environment, ground water, and surface water * Blood lead levels were also above human toxicity thresholds * Arsenic vibrissae concentrations increased over the study period (2011-2019) * Serum Al, As, Cu, and Mn concentrations increased during El Nino years. Take-home points: **They found toxic concentrations of Al, Pb, and increasing concentrations of As in vibrissae and serum relative to human thresholds** Practice Questions: Were concentrations higher in serum or whiskers? **Vibrissae were 2-20x higher than serum ** Which elements exceeded toxic thresholds? **Aluminum and lead (blood levels)** Which element increased over the study period? **Arsenic ** Which elements increased in El Nino years? **Serum aluminum, arsenic, copper, and manganese concentrations** Was there a difference between males and females? **Female South American sea lions had 3x higher arsenic in vibrissae Male south American sea lions had higher vibrissae cadmium.**

Answer 73

JZWM 2023;53(4):705-713 **Retrospective Review Of Neurologic Disease In Stranded Atlantic Harbor Seals (Phoca vitulina concolor) Along The New England Coast** Wright SE, Díaz-Delgado J, Rivard MR, Flower JE, Sirpenski G, Tuttle AD ABSTRACT: Harbor seals (Phoca vitulina) are a common species admitted to marine mammal rehabilitation facilities. As important indicators of marine ecosystem health, monitoring trends of disease in harbor seal populations is critical. However, few studies have evaluated neurologic disease in this species. T**he general objective of this study was to retrospectively review and delineate neurologic disease in free-ranging Atlantic harbor seals (P. vitulina concolor) that stranded along the New England (United States) coast and entered a rehabilitation facility between 2006 and 2019**. Any Atlantic harbor seal that stranded live along the New England coast during the study period and was diagnosed with neurologic disease on either antemortem or postmortem evaluation was included; medical records and pathologic reports were reviewed. From 211 records, 24 animals met the inclusion criteria. **Prevalence of neurologic disease was 11% in the study population and six major categories of neurologic disease were identified including: inflammatory (54%), idiopathic (33%), trauma (4%), congenital (4%), and degenerative (4%). Of the seals diagnosed with neurologic disease, 13 (54%) seals died during rehabilitation, 10 (42%) seals were euthanized, and 1 (4%) seal survived to release**. Unique cases seen included a seal with Dandy-Walker-like malformation and another seal with histopathologic findings compatible with neuroaxonal dystrophy, a degenerative process that has not been previously reported in marine mammals. This study contributes to the overall knowledge of the health of free-ranging Atlantic harbor seals and may aid clinicians in characterizing neurologic conditions that may be present in seals undergoing rehabilitation. Background: - Harbor seals inhabit temperate coastal habitats throughout North America, Europe, and Asia - There are five subspecies of Harbor seal recognized: Eastern Atlantic, Western Atlantic, Ungava, Pacific, & Insular Key Points: * Prevalence of neurologic disease over the 13-yr period was 11% -- Most animals stranded in 2018 -- An UME took place from 2018 to 2020 across the coast of New England -- The etiologic agent was suspected to be PDV * CNS inflammatory disease (e.g., meningoencephalitis) = most common disease process -- Suppurative inflammation -> bacterial etiologies -- Lymphohistiocytic or lymphoplasmacytic inflammation -> viral or protozoal infections * Neuroaxonal dystrophy = inherited degenerative disorder characterized by swelling of the axons' distal segment within the CNS * Dandy-Walker-like malformation = rare congenital malformation of the cerebellum involving cystic dilatation of the fourth ventricle, enlarged posterior fossa, and cerebellar hypoplasia TLDR: Neurologic disease diagnosed in small percentage of harbor seals; survival was low in animals diagnosed with neurologic disease

Answer 74

**BARIUM SULFATE GELATIN AS A NOVEL APPROACH TO FACILITATE GASTROINTESTINAL TRACT POSITIVE CONTRAST STUDIES IN A CAPTIVE HARBOR SEAL (PHOCA VITULINA) AND CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) WITHOUT RESTRAINT**. Barrett CE, Cutler D, Anderson CE, Clauss TM, Dennison SE. Journal of Zoo and Wildlife Medicine. 2023;53(4):838-843. Gastrointestinal tract contrast medium studies are a valuable diagnostic modality to evaluate gastrointestinal anatomy, motility, and pathology. Four positive contrast medium studies were performed twice on a harbor seal (Phoca vitulina) and once each on two California sea lions (Zalophus californianus) to evaluate for gastrointestinal abnormalities by using barium-impregnated gelatin. Because marine mammals in human care routinely receive plain gelatin as a component of their diet and as secondary reinforcement, feeding a barium gelatin mitigates the need for tube feeding or restraint. Operant conditioning was used to position the pinnipeds for voluntary radiographs. The barium gelatin permitted adequate evaluation of gastric emptying and intestinal transit times and good evaluation of the structure of the intestinal tract. Full evaluation of gastric anatomy was limited due to the gelatin blocks not conforming to the stomach in their solid form and the barium quickly exiting the stomach as barium gelatin blocks disintegrated and because orthogonal views could not always be acquired. Even with these limitations, barium gelatin resulted in diagnostically valuable contrast imaging in a stress-free patient setting with reduced risk of aspiration and eliminated the effects that anesthesia can have on gastric motility as well as other anesthesia-associated risks. Key Points * 4 successful positive contract studies in pinnipeds using voluntary ingestion of barium-impregnated gel. * 60% barium for total dose of 1.3 ml/kg in ratio 1:2 or 1:4 barium:gelatin by volume * Orthogonal views especially from 15-30 minutes to assess stoamch * Limitations: doesn’t coat the stomach like liquid so can’t do full anatomical gastric evaluation (rapidly enters intestines once gelatin blocks dissolve)

Answer 75

**SEROLOGIC RESPONSE AND ADVERSE EFFECTS OF RECOMBINANT CANINE DISTEMPER VACCINATION IN THREE AQUARIUM-HOUSED WALRUSES (ODOBENUS ROSMARUS)** Pujol et al Abstract Fatalities have been associated with phocine and canine distemper viruses in marine mammals, including pinnipeds. No data are available regarding distemper disease or vaccination in walruses. This study evaluates seroconversion and clinical adverse effects following administration of a canarypox-vectored recombinant distemper vaccination (two 1-ml doses, 3 wk apart) in three adult aquarium-housed walruses. Serum antibodies to distemper were measured using seroneutralization on blood samples collected under operant conditioning prior to and for 12 mon after vaccination or until titers were < 32. All walruses seroconverted. Medium positive titers (64–128) were detected for 4 to 9.5 mon in two of three individuals. Interindividual variability was noted, with one individual displaying only low positive titers. Major swelling at the site of injection and lameness for a week following injection occurred in all three walruses. Further studies on dosing amount and interval are needed to make vaccine recommendations in this species. Key Points: * Morbilliviruses, including canine distemper virus (CDV) and phocine distemper virus (PDV), have caused mortality events in free- ranging marine mammals, especially in phocid species. -- In 1988, more than 18,000 grey seals (Halichoerus grypus) and harbor seals (Phoca vitulina) died in northern Europe * cross protection has been demonstrated in harbor seals between different morbilliviruses * a vaccination program against CDV have been implemented in free- ranging endangered Hawaiian monk seals (Neomonachus schauinslandi) * One study about Atlantic walruses in Nunavut, Canada, showed that 16% of walruses had antibodies to CDV and 50% had detectable antibodies to PDV * PDV has been circulating in the Atlantic walrus population since the 1970s and could be endemic * A prospective study was performed in three walruses housed at the Aquarium du Que ́ bec * All walruses were vaccinated subcutaneously with operant conditioning in the gluteal region with two 1-ml doses of canarypox-vectored distemper vaccine (Ferret PureVax) 3 weeks apart * After vaccination, all walruses seroconverted – high variability between individuals -- One walrus had a protective titer for 1 yr, the others shorter < 6 mo * the magnitude of the immune response was inversely related to the body weight of walruses

Pinnipeds Flashcards

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