Cetaceans

Question 1

What are the two suborders of cetacea?

What are the two families of whales commonly housed in aquaria?

What are the scientific names of the following species:

Bottlenose dolphin

Killer whale

Beluga whale

Pacific white-sided dolphin

Answer 1

• Two suborders:
  
  • Mysticete - Baleen whales
    
    • Large plates of keratinized baleen
    • Feed on krill and fish by swallowing large volumes of water and forcing it out through the baleen
  • Odontocete – Toothed whales
    
    • Sperm whales, porpoises, dolphins, beluga, narwhal, beaked whales.
    • Extremely social, small or large groups (superpods)
    • Family groups in several species
    • Socialized feeding strategies
    • Nearly all piscivorous
    • Two families (delphinidae and monodontidae)
      
      • Bottlenose dolphin – Tursiops truncatus
      • Killer whale – Orcinus orca
      • Beluga whale – Delphinapterus leucas
      • Pacific white-sided dolphin – Lagenorhynchus obliquidens
      • Harbor porpoise – Phocoena phocoena
      • Many others; Amazon river dolphin (Inia geoffrensis)

Question 2

What unique anatomical adaptations do cetaceans have for life in the aquatic environment?

Answer 2

• Adaptations to life in the aquatic environment:
  
  • Lack hair, lack hind limbs, have vestigial pelvic bones
    
    • Hip bones have occasional articulations (pseudoarthrosis) located deep in muscle of the ventrolateral caudal abdominal wall. Linked to male sexual performance.
  • Forelimbs evolved into flippers
  • Tail flukes for propulsion
  • Streamlined body
  • Thick blubber layers - Insulation, nutritional reserve, aids in buoyancy
    
    • Lower bone density.
    • Long-bones of forelimbs have no medullary cavity.
    • Bone marrow can be extracted from vertebral bodies or ribs.

Question 3

Describe some of the cardiovascular adaptations cetaceans have evolved to facilitate diving, breath holding, and temperature conservation.

What is the primary blood supply to the head?

Answer 3

• Cardiovascular system:
  
  • Adaptations to facilitate diving, breath holds, temp conservation:
    
    • Large distensible veins, venous sinuses, venous valves in lungs.
    • Portal triads of the liver, venous sphincter in the common hepatic vein at junction of inferior VC below the diaphragm.
    • Network of arteries and veins between thoracic vertebral bodies (rete mirabile).
    • Periarterial vascular rete - counter current exchange systems.
    • Retes – arteries branch into number of small vessels that reconstitute into single vessel.
    • Can peripherally vasoconstrict and still perfuse the brain and other organs with warm oxygenated blood under pressure and temp extremes with depth.
    • Profound normal sinus arrhythmia.
      
      • Speeds up with each breath, slow between breaths.
  • Blood supply to cetacean head
    
    • Rete mirabile primary source of blood to the brain.
      
      • Spinal meningeal arteries enter the cranium at occipital foramen.
      • May predispose to spontaneous intravascular gas bubble formation and embolization (Caisson’s dz).
    • Internal carotids vestigial, provide blood to the ears and eyes, little to no oxygenated blood to the brain.
    • Brachiocephalic trunk and descending aorta branch off to feed intercostal and dorsal thoracic arteries.

Question 4

How does the blowhole in cetaceans work?

How is it different between mysticetes and odontocetes?

What is the goosebeak? What are the cartilages and muscles that compose it?

Describe the tracheal anatomy of cetaceans.

What adaptations prevent airway collapse while diving?

How quickly does respiratory exchange occur in cetaceans?

How does this impact them from a disease standpoint?

Answer 4

• Respiratory system:
  
  • Blowhole
    
    • External nasal opening on top of head
    • Two external openings in mysticetes, single blowhole in odontocetes. All have an underlying nasal septum in the skull (two openings for entry into resp tract).
    • Closes as associated muscles relax.
  • Ventral and lateral - right and left lateral vestibular sacs
  • Paired internal nares, paired nasal cavities extend ventrally to the nasopharynx.
  • Goosebeak
    
    • Elongated epiglottal and cricoarytenoid cartilages of larynx supported by arytenoepiglottal muscles.
    • Voluntarily displaced for eating large food items; manually for intubation
    • Held in place by muscular palatopharyngal sphincter.
    • Displacement has been associated with fatal aspiration and asphyxiation.
      
      • Short trachea
    • Trachea has a large vascular venous plexus with prominent lacunae in the subepithelium.
      
      • Critical role in fungal tracheitis.
      • Anterior to carina is right sided accessory bronchus
    • Leads to anterior right lung lobe
    • Extensive pulmonary support structures:
      
      • Complete tracheal and bronchial cartilaginous rings extend deep into bronchioles, plates and rings of cartilage that extend down to junctions of alveoli.
    • Visible as cartilage nodules throughout the parenchyma.
      
      • Lungs contain great amount of smooth muscle, elastic sphincters and visceral pleura.
    • Valves can obstruct pulmonary clearance of parasites and bacteria, lead to abscesses.
    • Lungs are non-septate and non-segmented
      
      • Type 1 and 2 pneumocytes, no type 3 (brush border cells).
      • Alveolar walls contain double rows of capillaries (unlike single in terrestrial mammals).
      • Common age-related changes – increased fibrous tissue in alveolar interstitium and pleura, likely reflecting resolution of prior chronic inflammation.
• Large tidal volumes, respiratory exchange 1-2 seconds.
  
  • Easy for particles and pathogens to move deep into respiratory tract.
  • Respiratory disease more significant cause of morbidity and mortality.

Question 5

How are baleen plates in mycicetes secured to the gingiva?

Describe the dental anatomy of delphinids, porpoises, sperm & pygmy sperm whales, beaked whatels, and narwal.

Do cetacean teeth have enamel?

Describe the multi-chambered stomach of the cetacean.

Do cetaceans have a gallbladder or caecum?

What is the ink sac of pygmy sperm whales?

Answer 5

• Gastrointestinal tract:
  
  • Oral cavity
    
    • Mysticete – multiple baleen plates
      
      • Secured by zwischensubstanz to the gingiva.
    • Odonticete – teeth vary by prey type
      
      • Delphinids – conical or spade-shaped, each tooth has one root, no incisors or molars (homodont).
      • Sperm whale – one row of conical teeth on both sides of lower jaw only.
      • Kogidae – pygmy and dwarf sperm whales only lower teeth as well.
      • Beaked whales – two teeth in lower jaw.
      • Narwhal – single erupted tooth (tusk, canine), left handed spiral, covered in cementum rather than enamel.
        
        • Canines of females can erupt as tusks (shorter).
      • Annular layers used to estimate age.
      • Sperm whale, kogidae, beaked whales lack enamel covering on teeth.
      • Others have a central pulp layer surrounded by dentin with outer layer of cementum and an enamel cap that wears with time.
      • Upper GI
        
        • Modified 3 chambered stomach
        • First chamber - Forestomach
          
          • Large, muscular, mechanical digestion
        • Second chamber - Fundic
      • Glandular, ventrolateral to first chamber on left
      • Deep red-brown folded, glandular mucosa.
        
        • Third chamber - Pyloric chamber
      • Prominent sphincter regulates ingesta into SI
        
        • Stomach ends at the duodenal ampula, followed by the small and large intestines, uniform in diameter through to the rectum.
        • All cetaceans lack a gallbladder.
        • Cecum is absent in odontecetes except river dolphins.
        • Rectum has lymphoid nodules – colonic tonsils
        • Dwarf and pygmy sperm whales (Kogia spp) have a saccular dilation of the distal colon (ink sac).
      • Expelled in dense cloud to distract predators.
      • Sperm whales – ambergris is a unique compound produced by consumption and partial digestion of squid beaks.
  • Accessory spleens (splenules)
    
    • Adjacent to the primary spleen or within mesentery.
    • May play role in EMH.
  • Stranded dolphins and whales – hyaline intracytoplasmic inclusions in hepatocytes common.
    
    • Compress and peripherally displace nucleus.
    • Inclusions randomly scattered through hepatic parenchyma.
    • Differentiated from viral inclusions by lack of associated inflammation and necrosis.

Question 6

Describe the renal anatomy of cetaceans.

Where are the testes located?

What type of penis do cetaceans have?

Where are the ovaries found on US?

What type of placentation do cetaceans have? How does this affect neonatal care?

Answer 6

• Urogenital system:
  
  • Kidneys are reniculated
    
    • Not well understood - Greater surface area may facilitate filtration of larger blood volumes
  • Male repro
    
    • Testes within the abdomen
    • Fibroelastic sigmoid-shaped penis similar to cattle
  • Female repro
    
    • Ovaries immediately caudal to kidneys
    • May be identified in most species by ultrasound
      
      • Bordered by epaxial muscles dorsally
      • Abdominal muscles ventrally
      • Narrow longitudinal window
    • Bicornuate uterus
      
      • Long horns, short body
      • Series of vaginal folds or rings
        
        • Endoscopic evaluation of the uterus difficult
    • Diffuse epitheliochorial placenta
      
      • Cetacean neonate dependent on colostrum for immediate postnatal immunity

Question 7

What is unique about cetacean skin?

What are the callosites on mysticetes?

What ectoparasites commonly infest those areas?

Answer 7

No keratin layer

• Skin:
  
  • Sensory input, streamlined
  • Callosities – normal, large patches of irregularly raised epithelial tissue especially on right (Eubalena) and bowhead (Balena) whales.
    
    • Hyperplastic, cornified skin above the eyes, along jaws and lips.
    • Becomes infested with cyamids (whale lice)
      
      • Cyamus ovalus and Cyamus gracilis on right whales (N, S)
      • Cyamus erraticus only from southern right whales
    • Similar proliferations on other baleen whales.
      
      • Gray whales – barnacles
        
        • Cryptolepas rhachianecti on calves and mothers, spends life cycle attached to the whale. Used for ID.
      • Species specific cyamids lodge within proliferative areas and spread from dams to calves.
      • Marked hyperplasia, hyperkeratosis, inflammation in superficial dermis, bacterial colonization.

Question 8

What adaptation of the cetacean skull allow for navigation in the water?

Describe cetacean hearing.

Answer 8

• Skull is asymmetrical
  
  • Melon
    
    • Serves as an “acoustic lens”
    • Sound production
      
      • Baleen whales produce powerful low frequency sounds that can travel hundreds of miles
      • Toothed whales high frequency sounds, useful for echolocation/navigation.
      • Clicking sounds transmitted through the melon, sound waves bounce off objects and returning signals received through mandible (pan) and conducted to the inner ear.
      • Noise is significant in captivity!
• Ears
  
  • Rudimentary ear canal
  • Bones are dense with ligamentous attachments to the adjoining skull, tympanoperiotic air sinuses homologous to guttural pouches in horses.
  • Can collect ear wax at necropsy for hormonal and chemical analysis/contaminant exposure.

Question 9

Cetacean housing requirements are established by what legislation?

What governing body inspects facilities?

What are the two types of systems used to house cetaceans?

What are the coliform counts expected to be in marine mammal housing?

How can coliforms be reduced?

How does group social dynamics play into welfare evaluation?

Answer 9

• Housing requirements and water quality
  
  • Established by the Animal Welfare Act
    
    • United States Department of Agriculture
    • Routine inspections and enforcement conducted by Animal Plant Health Inspection Service (APHIS)
  • Housing requirements
    
    • Depth, volume, surface area, “minimum horizontal distance” i.e. length across pool
    • Space requirements also include number of animals per facility and species specific requirements
  • Water quality
    
    • Critical component of overall health and well-being
    • Two primary forms of water management
      
      • Open systems
      • Closed systems
    • Open systems
      
      • Lagoon or sea based
      • Quality of water dependent on environment
    • Closed systems
      
      • Designed pools usually filled with synthetic sea water
      • Hybrid semi closed systems rely on access to natural seawater that is pumped into a land based facility
      • Mechanical filtration prior to and after reaching the animal habitat
      • Closed systems most intensive
        
        • Water is continuously recycled
        • May experience shift in concentration because of incidental FW additions i.e. rain or run off
          
          • Salinity, acid base changes
        • Marine cetacean water systems sal 27-32 ppt
      • APHIS regulates amount of coliform bacterial in marine mammal systems*
        
        • Considered indicator bacteria
          
          • Numbers reflect efficacy of water disinfection
        • Requires samples be collected weekly and most probable number of coliform bacteria not exceed 1000 per 100 mL of water.
          
          • May be result of 3 samples over 48 hrs
        • Chlorine-based oxidants and ozone highly effective water disinfectants
        • Purely biologic filtration systems in attempt to reduce risks assoc with chlorine oxidant exposure show promise
  • Social grouping and behavioral compatibility important
    
    • Significant factor in health and wellness

Question 10

Discuss the nutrition and feeding of cetaceans in managed care.

What is their general diet?

How much is fed?

How is quality of fish maintained?

What vitamins are oxidized as a result of freezing the fish?

How does sustainability play a role in selection of food fish?

How does removing the slime layer of the fish affect cetaceans?

Describe the effects of histamine in thawing fish?

Answer 10

• Feeding and nutrition:
  
  • All cetacean species in zoological parks are piscivorous
    
    • Fish species most commonly fed:
      
      • Herring (~50%), capelin, squid, mackerel, smelt
      • In general, minimum of three spp of food items should be available to ensure varied, high-quality diet that is balanced in volume and nutrients and not prone to interruptions in supply.
    • Quantity fed depends on spp
    • Generally, 2-5% body weight per day for adult delphinids/monodontids
    • Fish quality is paramount!
      
      • Individually quick frozen fish (IQF)
        
        • Higher quality and integrity when thawed
      • Fish should remain frozen below -2-4 deg C until immediately prior to being fed
      • Do not to use frozen fish that have been stored more than 12 months.
      • Air thawing of frozen fish no more than 24h prior to feeding recommended. Dedicated refrigerators. Thawing in water not recommended, promotes bacterial growth
      • Feeding whole fish recommended
        
        • Loss of nutritional value with loss of organ meats/fats otherwise
      • Spoilage can lead to loss of nutritional value and depending on species, scromboid poisoning (tuna and mackerel)
      • Body weight in captivity should be monitored twice weekly to assess errors in feeding plans
      • Proper daily supplementation with multi-vitamins specifically for piscivorous marine mammals
    • Freezing food fish results in oxidation of vitamins B9, C, fat sol vit A, D, E, K.
      
      • Thiamine is critical
      • Thiamine deficiency manifests as depression, body tremors, CNS signs i.e. seizures
      • Nutritional complication of frozen fish-based diets
        
        • Freeze thaw cycles promote activity of thiaminases in fish
    • Considerations for sustainability:
      
      • Impact on zoological community on fisheries
      • Efforts to identify other sustainable fish species for feeding
      • Additionally, fish analogs of high grade sustainable fish meal supplemented with vitamins in gel forms
        
        • May also improve storage, provide nutritional flexibility
  • Removal of slime layer of fish has been associated with reduced incidence of septicemic form of erysipelas and severity of nonsepticemic form in cetaceans.
  • Any change in appetite or acceptance of food should immediately be reported to the veterinarian.
• Histamine
  
  • Can rapidly form in fish tissues at warm temps.
  • Tissue histamine levels can be used to evaluate integrity of the cold chain and good hygienic practices for handling fish.
  • Elevated levels can trigger toxicity, but susceptibility varies between marine mammal spp and individuals.
  • Maximum allowable peroxide values and histamine of 20 meq/kg of fish fat and up to 100 mg/kg, respectively are often employed in cetaceans. Empirical, not validated.

Question 11

What routine diagnostics can be accomplished with operant conditioning in cetaceans?

Describe the physical restraint of cetaceans. What tools are needed?

Answer 11

• Restraint and handling:
  
  • Behavioral training techniques
  • Operant conditioning
    
    • Facilitation of clinical procedures
      
      • Physical examination
      • Body weight measurement
      • Routine diagnostics
        
        • Blood collection
        • Gastric fluid collection
        • Samples of urine, feces
        • “Chuff” samples
        • Endoscopy
        • Ultrasound
  • Physical restraint
    
    • Slings or stretchers with holes for pectoral flippers
    • Pools that can be drained or false-bottom pools with bottoms that lift
    • While out of water, protect from contact injuries, overheating, drying out
      
      • Use foam padding, keep out of sunlight
      • Keep moist or wet
      • Monitor for distress, when in doubt, place back in water

Question 12

Describe the monitoring of cetaceans undergoing anesehtsia.

What devices can be used, how are catheters placed, what values are concerning?

Answer 12

• Chemical restraint
  
  • Sedation or general anesthesia (more details below, West chapter)
  • Comprehensive approach to anesthesia monitoring
  • Anatomic and physiologic challenges
    
    • Monitoring
      
      • PICC (peripherally inserted central IV catheter) may be placed in common branchiocephalic vein and hepatic vein with US guidance
      • Challenges with maintaining a clean catheter and vascular access site
      • Place ECG on before induction to get a baseline.
        
        • HR < 60 BPM concerning under anesthesia.
      • ETCO2 monitoring – even if not intubated (sedation), can hold above the blowhole.
      • Blood gas measurements
        
        • Pure arterial or venous samples difficult.
        • True venous samples may be collected from brachiocephalic and hepatic veins.
        • ECG
      • Yaw, T. J., Kraus, M. S., Ginsburg, A., Clayton, L. A., Hadfield, C. A., & Gelzer, A. R. (2018). Comparison of a smartphone-based electrocardiogram device with a standard six-lead electrocardiogram in the Atlantic bottlenose dolphin (Tursiops truncatus). Journal of zoo and wildlife medicine, 49(3), 689-695.
        
        • Pulse ox can be used on the tongue
        • CVP can be measured with PICC lines
        • Body temp flexible thermometer probe rectally
      • Vascular heat exchange systems for internal gonads may interfere with thermometer readings if the probe is too deep.
        
        • Skin must be kept moist whenever out of water; eye lube.

Question 13

Describe the intubation and ventilation of cetaceans.

Describe tracheal bifurcation in cetaceans how that affects intubation?

Describe the approach in smaller cetaceans.

How do cetaceans naturally breathe? How can that be mimicked with ventilation?

Answer 13

• Intubation & Ventilation
  
  • Redirect elongated larynx rostral by manually manipulation
  • Trachea is short in cetaceans. Caution not to advance the ET tube past the separate right accessory bronchus.
  • Mechanical ventilation while under general anesthesia is necessary
    
    • Cetaceans have a specialized respiratory cycle with short, rapid exchange phases followed by long inspiratory apneustic plateaus
    • Ventilators that allow for an apneustic plateau (prolonged inspiratory hold) in cycle important
    • Fewer, deeper breaths; RR 2-4/min on vent.
  • Oral approach may not be possible in smaller odontocetes.
    
    • Tube can be inserted through right or left nasal cavity.
    • Use of bronchoscope for visualization of goosebeak and stylet.
      
      • Possible adverse effect of abnormal blowhole function.

Question 14

How is general anessthesia typically induced in cetaceans?

How does their unique uni-hemispheric sleep pattern affect them under anesthesia?

What sedation drugs are commonly used?

What analgesics are commonly used? Any potential adverse effects?

Describe recovery of cetaceans following anesthesia.

Answer 14

• General anesthesia:
  
  • Propofol smooth, reliable IV.
  • Maintained with isoflurane or sevoflurane.
  • Profound respiratory sinus arrhythmia is normal.
  • Loss of swimming motion in tail flukes may be the most reliable indicator that a surgical plane of anesthesia has been reached.
    
    • Uni-hemispheric sleep
      
      • Slow waves appear very rarely/briefly in both hemispheres simultaneously.
      • Short periods of EEG slow waves in both hemispheres when awake, but slow waves in both hemispheres not seen during breaths.
      • Under anesthesia, slow waves on EEG of both brain hemispheres, will not breath – have to breathe for them.
• Consideration of dose scaling and basal metabolic rate must be made.
  
  • Larger cetaceans may require smaller dosages of drug on mg/kg basis.
• Analgesia
  
  • Several MM acute negative side effects with use of NSAIDs.
• Sedation
  
  • Benzodiazepines and opioids are most commonly used.
    
    • Diazepam and midazolam.
    • Diazepam and tramadol – enhanced sedation and some analgesia.
    • Midazolam generally safe in bottlenose dolphins.
      
      • Profound sedation and respiratory and cardiac depression in Pac bottlenose dolphins!
      • Flumazenil may be given orally or IV in an emergency.
  • Opioids.
    
    • Butorphanol.
    • Meperidine (Demerol).
• Parenteral anesthetics
  
  • Thiopental, pentothal, propofol have been used.
  • Ketamine and medetomidine in one animal.
  • Medetomidine alone – significant respiratory and CVP depression, light sedation.
  • Propofol is of choice
• Inhalant anesthetics
  
  • Isoflurane is currently the inhalational anesthetic of choice.
  • Sevoflurane should be considered.
• Local anesthesia
  
  • 2% xylocaine - Dental blocks
• Recovery
  
  • Requires replacement of the goosebeak.
• There should be a high index of suspicion that sick animals may have respiratory compromise (pneumonia is common).

Question 15

Describe the regular monitoring of cetacian health from both an animal care and veterinary perspective.

Answer 15

• Diagnostic techniques and preventive medicine
  
  • Cetaceans mask signs of clinical illness
  • Changes in breathing, body posture, other illness cues subtle
  • Urination/defecation difficult to appreciate
  • Any small indicators of disease including partial or complete inappetance should be thoroughly examined
  • Robust and routine preventive health program is critical
  • Routine preventive care is mainstay of wellness programming for cetaceans in zoos and oceanaria
    
    • Regular health assessment increases likelihood of early disease detection – weekly, monthly, quarterly, or annually depending on spp.
    • Daily behavioral assessments, routine wellness checks, nutritional assessments, social and environmental evaluations, vaccination, parasite prophylaxis.

Question 16

Describe the visual and physical examination of cetaceans.

List some differentials for buoyancy issues.

Answer 16

• Regular health assessments should include:
  
  • Visual examination
    
    • Social behavior, activity, swimming patterns, buoyancy, skin, eyes, posture.
    • Other cetaceans may become attentive to a pod member that is sick. The animal that stops eating may not be the sick one.
    • Interaction between animal and trainer.
    • Buoyancy
      
      • Best evaluated at rest during normal inspiratory breath hold.
      • Space-occupying masses, fluid accumulation possible causes of decreased buoyancy.
      • Increased buoyancy usually from abnormal gas accumulation in GIT, abdomen or thorax.
      • A near catatonic sinking to the bottom of the pool may indicate estrous in female Tursiops.
      • Vision impairment may result in listing to one side.
      • Normal newborns or early postpartum calves will often list if temporarily abandoned by the mother.
      • Imaging (US/centesis/rads).
      • Physical examination
        
        • HR, body temp
        • Respiratory rate and character

Question 17

What are the venipuncture sites in cetaceans?

Answer 17

• CBC, serum chemistry
  
  • Phlebotomy sites include periarterial vascular retes PAVR on dorsal and ventral midlines of the fluke blades
    
    • Understanding that it is almost always a mixed venous/arterial sample.
    • IV meds can be administered here, but size and microanatomy of the vessel, accuracy of needle stick, and dynamic nature of peripheral blood vessels can limit success.
  • Peduncle PAVR
    
    • Ventral aspect of terminal vertebral bodies
    • Landmarks ventral peduncle ridge proximal to confluence of right and left fluke PAVR
    • Bigger vessel for IV injections.
  • Dorsal fin PAVR
    
    • Challenging, useful for killer whales or fractious animals, or for when under anesthesia.
    • Blood draining from this site goes directly to the gonads.
  • Hemal arch/caudal vascular bundle
    
    • Runs longitudinally along the ventral midline of the caudal vertebral bodies within an arch formed by the chevron bones
    • Low pressure, mostly venous system
    • Can be catheterized for IVF or drug administration
    • Rete can influence rates of absorption of drugs.
  • Common brachiocephalic vein CBV
    
    • For purely venous blood samples, to measure venous BP
    • More successful with ultrasound guidance
• Hepatic vein
  
  • Local anesthesia, US guidance, PICC can be advanced into caudal vena cava.
  • Risk of bleeding.
  • Gelfoam is recommended following removal of catheter.

Question 18

Describe the affects of inflammation on routine bloodwork in cetaceans.

What are the most reliable analytes as indicators of inflammation?

How is hemaglobin and PCV affected?

What is the best method for measuring fibrinogen?

What is the ESR?

How does iron change?

How does ALP work as a prognostic indicator?

Describe the interpretation of the white cell count and neutrophil recruitment.

What values are used to asses liver health?

Answer 18

• Analytes proven to be most helpful as indicators of inflammation are reticulocyte count, white blood cell count, differential count, ESR, plasma fibrinogen, serum albumin, serum globulin, ALP, serum iron.
  
  • Hb and PCV decrease 7-10% with inflammation.
    
    • Distinguish from blood loss anemia based on reticulocyte count (regenerative or not).
  • Plasma fibrinogen – most reliable indicator of inflammatory dz in cetaceans.
    
    • Photo-optical test is best.
    • Early inflammation and response to tx.
      
      • Mirrors levels of IL6 (takes days to measure, less useful clinically).
    • Elevations of as little as 20% above high normal levels important, will usually be 50% elevated or more with significant inflammation.
  • ESR – presence and severity of inflammation.
    
    • Prone to fluctuation, use in conjunction with PF.
    • Dehydration can increase viscosity of blood, slows sedimentation of RBCs but will not affect concentration of fibrinogen.
  • Serum iron – decreases acutely in septic animals.
    
    • Sequestering iron in liver in form not available to pathogenic bacteria.
      
      • Supplementation not indicated.
      • Hepatocellular damage often assoc with high serum iron.
    • Can drop 20% or less within 24h.
    • Ferritin may be alternate.
    • Serum iron may be first analyte to start normalizing with tx, but can also fluctuate and may not be reliable.
  • Reticulocyte counts – often low with chronic infection.
    
    • Chronic slow blood loss or decreased regeneration.
    • Chronic low-grade pneumonia may lead to increased inflammatory parameters and low-grate NR anemia.
      
      • Can differentiate from gastric ulceration.
  • Serum albumin – decreases with bacterial infection.
    
    • 10-20% decline expected with inflammation, globulins may remain unchanged.
    • Hereditary bisalbuminemia reported in two groups of bottlenose dolphins.
  • ALP – low in inflammation.
    
    • Usually normally higher than terrestrial spp.
    • Drops dramatically with illness, considered reliable prognostic indicator.
      
      • ALP < 50 in killer whale very serious dz, < 25 going to die.
  • Total WBC count – life threatening pneumonia can be associated with unremarkable CBC.
  • Differential blood cell count – neutrophils mature and segment faster than terrestrial mammals.
    
    • Bands may be zero even with inflammation and active neutrophil recruitment.
    • Bands redefined as any neutrophil with chromatin spanning the lobules.
      
      • 2-5% in normal individuals.
    • Fluctuate and can be different even with site of venipuncture.
  • Serum transaminases – increased ALT indicative of hepatocellular leakage or damage.
    
    • Acute liver dz – spike.
    • Chronic – wax and wane.
    • GGT, ALP, LDH, ferritin iron, TG, cholesterol and bile acids should also be used.
    • Modification of the clotting pathways based on absence of factor XII aka Hageman factor (loss of intrinsic arm of clotting cascade).
  • Can impede collection of serum.
  • For minimal hemolysis, clotting profiles, whole blood sedimentation times, and plasma fibrinogen:
    
    • Orange top (thrombin) tube induces clotting
    • Light blue (sodium citrate) tube coag assays, fibrinogen
    • Potassium EDTA tube for hematology and ESR
    • For genomic assays – green top (lithium heparin) tube
• Following rehydration, many inflammatory parameters increase – WBC count, ESR, fibrinogen with corresponding dc in Hb, iron, albumin.
  
  • Reperfusion injury may trigger systemic inflammation.
• Inflammation associated with pregnancy.
  
  • Implantation, endometrial development, parturition and abortion are mediated by hormones, interleukins, cytokines.
    
    • Main causes of inflammation – IL6 and CRP, produce cervical ripening and softening.

Question 19

Describe the collection of urine, stool, and milk in cetaceans.

How can fecal occult blood testing be done?

Answer 19

• Urinalysis
  
  • Can be obtained from trained animals
  • Can also obtain urine with U catheter
  • Gastric samples, fecal samples, chuff samples easily collected
  • Cytology or microbial assessment (may be poorly representative of disease)
  • Urine from mature males will invariably contain spermatozoa.
• Stool analysis
  
  • Tube inserted into rectum into descending colon. Do not apply suction.
  • Cytology and culture.
  • Fecal occult blood will always be positive if done on stool from cetaceans fed whole fish.
    
    • If eating entirely washed fish filets for 2-3 days, normal stool will convert to occult blood negative (clinically useful).
    • Use clinically normal animal as a control for this.
• Milk analysis
  
  • Clinically normal cetacean milk can contain white blood cells.
• Upper respiratory tract evaluation
  
  • Bronchoscopy and BAL are the most appropriate procedures for determination of etiologic agents in the lower respiratory tract.

Question 20

Describe the imaging of cetaceans

Answer 20

• Ultrasound
  
  • Routine screening of abdomen and thorax, reproductive evaluation
  • Evaluation of peripheral lymph nodes
• Martony, M. E., Ivančić, M., Gomez, F. M., Meegan, J. M., Nollens, H. H., Schmitt, T. L., … & Smith, C. R. (2017). Establishing marginal lymph node ultrasonographic characteristics in healthy bottlenose dolphins (Tursiops truncatus). Journal of Zoo and Wildlife Medicine, 48(4), 961-971.
• FNA and biopsies can be performed
• Radiographs
  
  • Skull, spine, thorax, abdomen
    
    • Lack of visceral fat limits abdominal rad assessments
• CT extremely valuable
• Endoscopic evaluation – gastroscopy, bronchoscopy, colonoscopy

Question 21

What vaccines should be considered for cetaceans?

Are there any risks associated with adminstration?

Answer 21

• Vaccines
  
  • Erysipelas rhusiopathie – ER BAC PLUS vaccine is regularly used in zoos.
    
    • Commercial swine vaccine, immunogenic to bottlenose dolphins.
    • Administer IM, shallow pool for 20 min – 1 hr after to observe for hypersensitivity reaction.
    • May premedicate with diphenhydramine.
    • Annual vaccination, 3 week booster especially in young.
    • Risk of transient adverse reactions toward the vx did increase with number of vaccines administered.

Question 22

Describe parasite prophlyaxis in cetaceans.

What drugs have been safely used?

What drugs should be avoided?

Answer 22

• Parasite prophylaxis
  
  • Freezing of fish can kill IM tissue stages of parasites inc aptivity
  • Internal parasite prevention and control should be used for cetaceans with access to live fish.
  • Wild-caught/rescued cetaceans – Crassicauda spp, Nasitrema spp, Karyoikeus spp.
  • Routine monitoring of feces and deworming.
    
    • Fenbendazole, ivermectin, praziquantel have been safe and effective.
    • Cetaceans are sensitive to CNS side effects of levamisole, avoid.

Question 23

Describe normal breeding behavior in cetaceans.

How can cycling be monitored in cetaceans?

What assisted reproductive technologies have been used?

How is estrus synchronized?

How is artificial insemination timed?

Answer 23

• Breeding behavior and conception
  
  • Copulation at or near surface, brief
  • Females will lie at surface in lateral, exhibit shaking or side-to-side head movement.
  • May be monitored with urinary hormone measurement, ovarian US, observation in pre-ovulatory period to predict and detect ovulation.
• Assisted reproductive technologies
  
  • ARTs – semen cryopreservation, estrus synchronization, artificial insemination.
  • Estrus synchronization with a progestin (usually altrenogest).
  • Ovulation 20-30 days after withdrawal of tx.
  • For AI, detection of LH surge via urinary hormone monitoring TID and inseminating 28-35 hours after LH surge must be performed.
  • Semen deposited in uterus with sterile catheter through flexible endoscope.
  • Ability to manage sex ratio in bottlenose dolphins.

Question 24

How is pregnancy diagnosed in cetaceans?

What is the duration of gestation in bottlenose dolphins? What about killer whales?

What hematologic and biochemical changes are associated with pregnancy?

Answer 24

• Pregnancy diagnosis.
  
  • Mean duration in bottlenose dolphins 376 days.
  • Killer whales 536 days.
  • Pregnancy best dx with ultrasound.
    
    • Fetus by end of first trimester.
    • Serum progesterone peaks weeks 9-12 after conception.
    • Weekly blood samples recommended to confirm rise in progesterone.
      
      • Pregnancy is confirmed on the basis of degree of progesterone persistence, not increase.
• Hematologic and chemistry changes with pregnancy
  
  • Mild inflammatory state including reduced HCT, hemoglobin, RBC, decreased serum transaminases, increased fibrinogen and 60-minute erythrocyte sedimentation rate, increased serum iron.
  • Monthly US monitoring of developing fetus is recommended.

Question 25

Describe the stages of labor in cetaceans.

Answer 25

• Calving:
  
  • Stages of labor
    
    • 1 – difficult to appreciate.
    • 2 – presentation of tail fluke tips or fetus being visible within vulvar opening, ends with the successful delivery of calf (< 2 hrs).
    • Oxytocin may be given.
    • Fluke-first presentation most common.

Question 26

What contraceptive options exist for cetaceans?

Answer 26

• Contraception:
  
  • Separation of males and females.
  • Vigilant monitoring of estrus cycles with US and urinary hormone assessment.
  • Regu-mate (altrenogest) may prevent ovulation in bottlenose dolphins, killer whales, belugas, pacific white-sided dolphins.
    
    • Pregnancy may still occur with dolphins and killer whales.
  • Deslorelin implants for about 1 year, prevention of estrus and ovulation in bottlenose dolphins.
  • Leuprolide acetate every 28 days, reduces testosterone, produces azoospermia in bottlenose dolphins.
  • Testosterone will increase for first 14 days after tx.
  • Megesterol acetate is not reliable in male dolphins, pregnancies have occurred in females. Glucocorticoid activity may affect adrenal function.
  • Houser, D. S., Champagne, C. D., Jensen, E. D., Smith, C. R., Cotte, L. S., Meegan, J. M., … & Wasser, S. K. (2017). Effects of oral megestrol acetate administration on the hypothalamic-pituitary-adrenal axis of male bottlenose dolphins (Tursiops truncatus). Journal of the American Veterinary Medical Association, 251(2), 217-223.

Question 27

Describe the administration of medications in cetaceans.

Are there any medications that are absolutely contraindicated?

How are oral medication affected by being placed in fish?

Describe IM and IV administration.

What challenges exist with ophthalmic medications?

Answer 27

• Medical intervention:
  
  • If there is clinical evidence of illness, assume it is serious.
  • Medications:
    
    • Fatal adverse reactions – sulfamethoxazole, phenothiazines, haloperidol, levamisole.
    • Empiric first line abx typically amoxicillin/clavamox.
      
      • Esp due to susceptibility to fatal Erisepelas.
  • Routes of administration:
    
    • Oral is preferred.
      
      • Feeding medication in fish.
      • If anorexic, stomach tube for oral meds and fluids.
    • Many medications require the enteric coating to pass the acidic stomach and be absorbed in the small intestine, should be loaded into food fish immediately prior to administration.
      
      • Put meds in 2-3 fish in case a conspecific eats the medicated fish, will not get entire dose.
      • Rapid GI transit times (few hours).
    • IM injection.
      
      • Limit IM injection volume to maximum 20 mL per site.
        
        • Avoid ischemic necrosis at site of injection.
        • Avoid thoracic cavity.
        • Inject off midline, slightly anterior to or parallel to the dorsal fin.
    • IV injection.
      
      • Fluke vessels.
      • Indwelling catheter in lateral peduncle vein, difficult to maintain. Limited success.
    • Topical treatment.
      
      • Corneal lesion.
        
        • May not penetrate tear plug or reach the corneal epithelium.
        • Can admix with mucolytic acetylcysteine to allow penetration of the tear plug.
        • Palpebral conjunctiva is highly vascularized, subconjunctival injections can cause profuse hemorrhage.

Question 28

How much water do cetaceans consume a day?

What are markers of dehydration?

How can fluids be administered?

Answer 28

• Fluid therapy:
  
  • Dolphins consume an average of 47 ml/kg water per day (in fish, some from seawater).
    
    • Amount of available fluid absorbed is unknown.
    • Ill individuals may consume excessive seawater, leading to sodium toxicity and death.
    • Other markers of dehydration – PCV, TP, BUN to Cr ratio, serum Na and Cl levels.
    • CRT in back of the throat.
    • Usually respond well to oral fluids.
      
      • Stomach tube (2-4L) well tolerated by bottlenose dolphins and belugas respectively.
      • SQ fluid space behind the scapula.

Question 29

Describe the management of inappetance and weight loss in cetaceans.

What medications may be helpful in stimulating appetite? What concerns are there with their use?

How can energy demands be lessened for anorexic cetaceans?

How can nutrition be supplemented?

Answer 29

• Managing inappetance:
  
  • Prednisone, dexamethasone most effective appetite stimulants in cetaceans through a mechanism of iatrogenic insulin resistance.
  • Masks clinical signs.
  • Should not be administered prior to diagnostics.
  • Taper more gradually than domestic animals.
  • Diazepam may help with anxiety but not appetite stimulation.
• Managing weight loss:
  
  • Increase temp of water gradually to counter body heat dissipation and lower caloric needs.
  • Tube feeding whole fish blended in oral electrolyte solutions.
  • Larger food fish can be used to assist feed.
  • Tube feed with cetacean neonatal milk replacer, even in adults.

Question 30

Describe the care of stranded cetaceans.

What are some poor prognostic indicators?

What are some standard treatments prior to transporting to a rehab facility?

Answer 30

• Immediate care of stranded cetaceans:
  
  • Prognostic indicators.
    
    • K+ higher than 5-6
    • ALP lower than 30
    • NA higher than 180
    • Injuries to the head, high respiratory rate > 3-4 seconds
    • Open thoracic or abdominal wounds
  • Assume they are dehydrated.
  • Tube small volume of oral electrolyte solution.
  • Can mix with up to 50% dextrose.
  • If hypothermic, weak and rapidly declining, hypoglycemia is likely.
  • Glucose, HCT, pH, lactate, Na will guide initial care after arrival to rehab center.
    
    • Can give bolus of LRS during transport.
    • Diazepam to relieve anxiety unless severely depressed.
  • Stranding related myopathy – contracture of the epaxial or hypaxial muscles at level of peduncle.
    
    • Oral diazepam, selenium, vit E, anti-inflammatory agent may further air in preventing stranding myopathy.

Question 31

What surgical procedures are commonly performed in cetaceans?

What are some complications with cetacean surgery?

Answer 31

• Surgery:
  
  • Dentistry, wound management, abscess treatment, superficial biopsy, liver biopsy, endoscopic procedures, jaw fracture repairs.
  • Purulent infections deep to blubber will dissect along the interface with the muscle.
  • Tamura, J., Yanagisawa, M., Endo, Y., Ueda, K., Koga, H., Izumisawa, Y., & Yamashita, K. (2017). Anesthetic management of an indo-pacific bottlenose dolphin (tursiops aduncus) requiring surgical debridement of a tail abscess. Journal of Zoo and Wildlife Medicine, 48(1), 200-203.
  • Rosenberg, J. F., Haulena, M., Bailey, J. E., Hendrickson, D. A., Ivančić, M., & Raverty, S. A. (2017). Emergency anesthesia and exploratory laparotomy in a compromised Pacific white-sided dolphin (Lagenorhynchus obliquidens). Journal of Zoo and Wildlife Medicine, 48(2), 581-585.

Question 32

What analgesics have been studied in cetaceans?

Answer 32

• Pain management:
  
  • Meloxicam PK study
    
    • Simeone, C.A., H.H. Nollens, J.M. Meegan et al. 2014. Pharmacokinetics of single dose oral meloxicam in bottlenose dolphins (Tursiops truncatus). J Zoo Wildl Med 45: 594–599.
    • Drug elimination is prolonged, levels detectable for up to 7 days.
  • Tramadol can be used alone or in combination with NSAIDs.
    
    • Elimination may also be prolonged.

Question 33

Describe the classic lesions of poxvirus affecting cetaceans?

What type of virus is this?

What do these lesions typically occur?

Answer 33

• Pox virus
  
  • DNA virus, tattoo lesions
  • Non-proliferative skin lesions present for extended periods of time; hyper-pigmented, may have dark pinpoint surrounded by pale margin
    
    • Wax and wane over months to years
  • Both toothed and baleen whales, no pattern in species-specificity
  • Flippers and flukes generally less affected
  • Commonly next to or directly associated with rake marks
  • Within stratum intermedium – small, round, eosinophilic intracytoplasmic viral inclusion bodies displace nucleus.

Question 34

What is the tropism for papillomaviruses in cetaceans?

What are the typical lesions?

Answer 34

• Papillomavirus
  
  • Proliferative mucosal and cutaneous lesions
  • At least 5 variants
  • Serologic positivity is high in wild dolphins and common in captive dolphins
  • ELISA reactivity higher in males
  • In wild ~11yo, captive ~30yo
  • Common in bottlenose dolphins, likely horizontal transmission with oral papilloma development early in life.
  • Tropism for vulval and vaginal mucosa, penile mucosa, oral and esophageal mucosa and frenulum of tongue.
  • Far less common – skin lesions that are warty or plaque-like.

Question 35

Describe the affects of cetacean herpesviruses.

What two groups of herpesviruses have been documented?

What are their clinical signs?

Answer 35

• Herpesvirus
  
  • Gammaherpesvirus
    
    • Skin lesions, genital lesions, nephritis, encephalitis, disseminated infection.
    • Host immunosuppression, latent infections exacerbated by debilitation.
    • Specific causation not determined.
    • Possibly associated with oral SCC, unclear.
  • Alphaherpes virus
    
    • Much less frequent
    • Incidental and asymptomatic to systemic necrosis in multiple organ systems

Question 36

What type of virus is cetacean morbilivirus?

What are the clinical signs?

What strains exist?

How is this disease transmitted?

What are the histologic findings, including the inclusion bodies?

How else can this disease be diagnosed?

What is the atypical chronic form?

What biosecurity considerations should be taken with this virus?

Answer 36

• Dolphin/Cetacean morbillivirus
  
  • Paramyxovirus (RNA Virus)
  • Clinical Signs
    
    • Debilitation, pneumonia, encephalitis, immunosuppression & secondary infections
  • Multiple strains – Porpoise (PMV), Dolphin (DMV), Pilot whale (PWMV), Beaked Whale (BWMV)
  • Horizontal transmission by inhalation of aerosols shed by infected individuals.
    
    • Placental and lactation transmission also occurs.
  • Histologic Fingins
    
    • Intracytoplasmic and intranuclear, eosinophilic inclusion bodies can be found.
  • Diagnosis
    
    • IHC useful when histo ID is difficult (decomposition, heavy secondary infection)
    • Virus isolation gold standard. RT-PCR followed by sequencing also useful. Serology useful for epidemiological surveillance.
  • Atypical chronic CeMV – profound lymph depletion, secondary infections, without typical MV lesions has been seen in stranded animals
  • Mortality worldwide but mostly Atlantic Ocean.
  • Biosecurity
    
    • Consideration for rehab facilities with resident cetaceans in close proximity or within same water system.
    • Stranding personnel should not work with naïve cetaceans without prior decontamination.

Question 37

Describe brucellosis in dolphins?

What is the primary organism?

How is this disease transmitted?

What are the typical clinical signs?

Answer 37

• Brucella
  
  • B. ceti – vertebral osteomyelitis, abortions in bottlenose dolphins.
  • Zoonotic
  • Caused bovine abortion by experimental challenge.
  • Replicates in host macrophages and trophoblasts, causes chronic dz.
  • Animals tend to overcome infection and act as carriers/potential shedders.
  • Cultured from 70% of stranded dolphin in Costa Rica.
  • No antibodies found in 70 cetaceans sampled in Italy.
  • Transmission likely involves three pathways.
    
    • Brucellae are non-motile, labile in environment, unlikely to persist outside host.
    • Physical contact during intercourse.
    • Exhaled breath samples.
    • Exposure to expelled fluids and placental tissues during delivery or nursing.
    • Isolated from reproductive tracts, milk, vertical transmission documented with in utero pneumonia in a fetus.
    • Has been recovered from fish mucus and cycles in nematodes and fish.
      
      • Possibility of vector transmission.
      • Halocercus and Pseudalius spp lungworms.
  • Lesions in CNS, reproductive, and skeletal systems.
    
    • Females – endometritis, placentitis, abortions.
      
      • Multifocal placental necrosis, edema, bacteria.
      • Gram stain and IHC.
      • Isolated from mammary glands of sperm whales and dolphins.
    • Males – epididymitis, orchitis.
    • Testicular enlargement, abscesses, granulomas.
  • Based on case series:
    
    • Most common lesions in CNS.
    • CSF increased in volume and cellularity, acquired hydrocephalus common sequela.
    • Meningoencephalomyelitis, nonsuppurative inflammation.
    • Expansion of inflamed tissues in choroid plexus, leptomeninges, subependymal neuropil (periventriculitis) obstruct CSF flow, cause secondary hydrocephalus.
    • Predilection – cerebellum, brainstem, spinal cord, medulla oblongata.
  • In bones and joints – discospondylitis, fibrinopurulent osteoarthritis, degenerative joint changes.
  • Blubber abscesses, pneumonia, vegetative valvular endocarditis with intralesional Brucella spp positive macrophages.

Question 38

Describe the course of infection in cetaceans with Erysipelothris rhusiopathiae?

What are the two primary forms of the disease?

Where can this bacteria be found?

What is the recommended treatment?

Answer 38

• Erysipelothrix rhusiopathie
  
  • Gram-positive rod
  • Peracute septicemia and mortality or dermatologic diamond skin disease.
    
    • Acute – sudden death, ascites, mottled liver, multifocal necrotizing lymphadenitis and hepatitis. Intralesional gram positive bacilli.
    • Milder cutaneous form – beluga whales, bottlenosed dolphins in captivity. Rhomboid shaped patch of skin discoloration, ulcerative dermatitis, loss of appetite, inflammatory leukogram.
  • Bacteria found in mucous layer of feed fish, growth may be promoted in association with poor food handling.
  • Presumptive route of exposure is oral or through rake marks or ulcers in skin.
  • Dx by blood culture unless peracute form.
  • Sensitive to wide variety of abx – fluoroquinolones, potentiated beta lactams. Vaccination programs.

Question 39

What are the two primary acid-fast organisms affecting cetaceans?

How are they differentiated?

What are the clinical signs?

Answer 39

• Mycobacteria spp
  
  • M. abscessus, M. chelonea, M. marinum
  • Acid-fast, rod-shaped, intracellular
  • Respiratory and cutaneous forms in bottlenose dolphins, serum antibodies in free-ranging Atlantic bottlenose.
  • Panniculitis, pleuritic, pneumonia, lymphadenitis.
  • Environmental exposure.
    
    • Cannot pass through mucosa or integument without percutaneous inoculation or secondary contamination of skin wounds.
  • TB causing mycobacteria have not been reported in cetaceans.
• Nocardia
  
  • Gram-positive rod, high protality with multi-organ disease in Atlantic bottlenose dolphin, beluga whale, killer whales.
  • Most common presentation is systemic form, lungs, thoracic LN. Pyogranulomatous inflammation.
  • Cutaneous nocardia abscess seen in beluga whales.
  • Responds to early, aggressive antimicrobial treatment
    
    • Trimethoprim sulfadiazine may reduce risk of bone marrow suppression.
  • Cytology – branching, rod-shaped bacteria, gram-positive, positive with modified acid fast and Grocott’s methenamine silver special stains.
  • Juveniles more commonly affected.

Question 40

Describe staphylococcal and streptococcal infections in cetaceans.

What lesions are common?

Answer 40

• Staphylococcus spp
  
  • Staph aureus
    
    • Sepsis in a killer whale
    • Granulomatous inflammation in harbor poises
      
      • Myocarditis, leptomeningitis, abscesses in LN and skeletal muscle
      • Has been associated with tooth root abscessation and suppurative pneumonia.
      • Likely opportunistic.
• Streptococcus spp
  
  • Strep phocae can be cultured from dead pinnipeds, blowhole debris and fecal material of asymptomatic cetaceans.
    
    • Coinfection/sepsis with morbillivirus.
  • 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.
  • Strep iniae
    
    • Commensal and fish pathogen
    • Exposure from contaminated prey or feed
    • Geralized sepsis and localized infections – pneumonia, pleuritic, pyothorax, dermatitis, myositis, panniculitis.
      
      • Mix of acute necrotizing and chronic pyogranulomatous inflammation.
  • Likely opportunistic.

Question 41

Describe the following fungal infections in cetaceans: aspergillosis, candidiasis, and coccidiomycosis.

What are the typical clinical signs?

Are there any predisposing factors?

What are the lesions?

Answer 41

• Aspergillosis – pneumonia, tracheal lesions. High mortality.
  
  • Often associated with morbillivirus co-infection in cetaceans.
  • Well delineated, grey nodules in parenchyma with hemorrhagic margins or large cavitations composed of fungal hyphae.
  • Tracheal mortality reported in four bottlenose dolphins in captivity.
  • Severe mycotic otitis in a juvenile harbor porpoise.
• Candida – oral, urinary, esophageal, regional.
  
  • May result from long-term antibiotic therapy, aggressive abx treatment, spontaneous.
  • Prophylactic oral nystatin two or three times daily during antibiotic tx has been used for prevention.
  • High resistance to itraconazole and fluconazole. Voriconazole some efficacy.
• Coccidiomycosis
  
  • Sporadic cases in bottlenose dolphins, common dolphins (frequent in CSL and southern sea otters).
    
    • Milliary nodules, centeal caseous necrosis throughout lung and perihilar LN.
    • Intralesional fungal arthroconidia and immature and mature spherules.
    • Necrotizing encephalitis.

Question 42

Describe the effects of cetacean cryptococcosis.

What are the two main organisms?

What are their distributions?

What are the reported lesions?

Answer 42

• Cryptococcosis
  
  • C. neoformans, C. gattii.
    
    • Yeast with prominent mucinous capsules, tropism for lung and LN.
    • Entrance via resp tract, subsequent hematogenous dissemination.
    • C. neoformans worldwide, C. gattii tropical and subtropical regions (eucalyptus trees).
    • In PNW, C. gattii primary fungal pathogen recovered from stranded cetaceans, esp harbor porpoises.
      
      • Generalized emaciation, pulmonary consolidation, generalized lymphadenopathy.
      • Pulmonary lesions, pneumonia.
      • Multisystemic fungemia has been reported.
      • In contrast to terrestrial spp, adrenal gland involvement tends to be localized to the medulla.
      • Cell walls can be highlighted with methenamine silver and Fontana-Masson stains.
      • Capsule stains red with mucicarmine and blue with Alcian blue stains.

Question 43

What causes lobomycosis in cetaceans?

What is the current name of this organism?

What are the typical clinical signs?

Is this zoonotic?

Answer 43

• Lacaziosis (causes lobomycosis in humans).
  
  • Chronic granulomatous skin disease caused by Lacazia loboi (yeast-like).
    
    • Potentially zoonotic.
    • Repeated surgical excision with wide margins.
    • Recent molecular studies have identified Paracoccidioides brasiliensis (paracoccidiodomycosis ceti) as the causative organism.
    • Dolphin cases typically from Gulf of Mexico to SA.
    • Gross lesions along torso but may occur anywhere. Focally extensive to disseminated, smooth, cutaneous swellings that progress to papules, nodules, plaques, verrucae, and ulcerations on occasion.
    • PST/GMS positive yeast single to multiple budding cells or chains connected by tubular isthmus.
    • Usually limited to the skin.

Question 44

Describe the clinical course of zygomycosis and entomophthoramycosis in cetaceans.

What are the primary etiologic agents of each?

How does their distribution differ?

How do the clinical signs differ?

Answer 44

• Zygomycosis
  
  • Typically lungs and LN affected, dx – rads, CT, bronchoscopy, BAL, US guided aspirate.
  • Mucorales and Entomophthorales orders.
  • Mucormycosis – Mucor spp, Rhizopus spp, etc.
    
    • Worldwide, associated with soil and detritus.
    • Inhalation, ingestion, or percutaneous inoculation of spores.
    • Angioinvasive. Disseminated.
• Entomophthoramycosis.
  
  • Tropical regions.
  • Basidiobolus spp, conidiobolus spp.
  • Infections are chronic, indolent.
  • Inhalation or direct inoculation of spores.
  • Tend to not invade blood vessels.
  • Rarely disseminate from primary site.
  • Skin and subcutaneous infections.

Question 45

Describe toxoplasmosis in cetaceans.

Are there certain species or geographic areas that appear to be affected?

What lesions are present?

Answer 45

• Toxoplasma
  
  • Serological survey St. Lawrence estuary – 27% beluga whales seroprevalence.
  • Vertical transmission in a Risso’s dolphin. ID in fetal tissues, placenta.
  • Nonsuppurative meningoencephalitis, disseminated parasitemia, necrotizing placentitis, abortion in cetaceans.
  • Hector’s and Maui dolphins, may suggest excess marine contamination from terrestrial run-off.

Question 46

Describe the effects of Sarcocystis neurona infection in cetaceans.

Is there a geographic area of concern?

What are the lesions?

Are there any particularly affected species?

Answer 46

• Sarcocystis
  
  • Sarcocystis neurona
    
    • PNW
    • Marked, nonsuppurative and necrotizing meningoencephalitis, perivascular lymphoplasmacytic cuffing, scattered gliosis, intralesional protozoal schizonts and dispersed merozoites in a pacific white-sided dolphin.
    • Harbor porpoise with pneumonia, intravascular accumulations of protozoa, encephalitis, myocarditis, tonsillitis.
    • Cysts in skeletal muscle and heart incidentally.

Question 47

What is the gastric nematode of cetaceans?

What lesions are typically observed with infestation?

Answer 47

• Anisakis simplex – gastric nematode
  
  • Associated with crater-like ulcerations
  • Nonglandular forestomach bottlenose dolphins and harbor porpoises
  • May be in any of the three gastric compartments, intestinal lumen.
  • Gastritis.
  • Perforation uncommon.
  • Present within muscles of many marine fish.

Question 48

What are the two most common cetacean trematodes?

Where are they typically find?

What are the associated lesions?

Answer 48

• Trematodes
  
  • Braunina cordiformis – digenic trematode of small delphinids.
    
    • Common within a mat of mucus attached to the second chamber of the stomach.
    • Occasionally will attach to mucosa of gastric pylorus and duodenal ampula.
    • Produces small focus of chronic gastritis at attachment site.
    • Heavy parasitism can be intense, over 50% gastric mucosa.
  • Nasitrema globicephalae
    
    • Inhabit pterygoid sinuses and tympanic cavities of odontocetes.
    • Most likely through consumption of infected fish.
    • Aberrant migration can result in neuritis of 8^th CN, otitis media, marked meningoencephalitis.
    • Migration tracts in gray and white matter of brain.

Question 49

What are the amphipod parasites of whales?

Where are they typicallly located?

How are they transmitted?

Answer 49

• Ectoparasites
  
  • Cyamids
    
    • External amphipod parasites, baleen whales and odontocetes (less frequent).
    • Aka whale lice.
    • Associated with callosities, genital folds, ventral pleats, nostrils.
    • Periphery of eyes, around skin wounds.
    • Each whale species generally has its own spp of cyamid parasite.
    • Infection transferred by contact.
      
      • From dams to calves during birth, nursing, contact.
      • In sperm whales, sex-specific.
      • Cyamus catodontis exclusively on males, Neocyamus physeteris only on females and calves.

Question 50

Describe iron overload disease in cetaceans.

What is a secondary syndrome that can occur as a result?

How is it diagnosed?

How is it managed?

Answer 50

• Iron overload disease:
• Captive bottlenose dolphins, high circulating levels of serum iron (> 600-700 mcg/dL), ref 100-300; and elevated transferrin saturation.
• Histo – accumulation of iron in hypertrophied Kupffer cells, diffuse cytoplasmic iron accumulations within hepatocytes.
  
  • Positive stain with Prussian blue.
  • In absence of hemochromatosis (necrosis, hepatitis, fibrosis), dx of hemosiderosis is appropriate (rare).
  • Associated with diabetes and metabolic syndrome.
  • Chronic inflammation, iron sequestration secondary to sepsis, excessive dietary iron, emaciation, inborn errors of metabolism, maladaptive type syndrome may contribute, not well understood.
• Hepatitis/hepatopathy syndrome – Atlantic bottlenose dolphins
  
  • Anorexia, inflammatory blood profile with rise in LDH, ALT, AST, GGT.
  • Dramatic increases in serum iron.
  • Waxes and wanes, not responsive to abx.
  • Variable response to steroid and hepatoprotective tx.
  • Dx – percutaneous hepatic biopsy.
  • Iron overload dz has been treated, serial phlebotomies 1-3 L whole blood weekly for 20-30 weeks.
  • Relation between iron metabolism and hepatic disease syndrome still unknown.

Question 51

Gastric ulceration is common in cetaceans.

What are some of the causes?

Answer 51

• Gastric ulceration
  
  • Parasitic infections often associated with crateriform ulcers
  • Helicobacter cetorum ID in numerus delphinid spp with gastritis
    
    • Ulceration in pylorus can perforate and result in fatal peritonitis.
    • Spiral to fusiform bacteria stain with Warthin Starry stain.
    • Role of helicobacter spp not yet fully resolved.
    • Ulcerations with consumption of high levels of histamine in fish.
  • Elevated histamine from freeze/thaw cycles.
    
    • Unlikely this occurs in free-ranging dolphins.
  • Stressors

Question 52

Describe hepatic lipidosis in cetaceans.

Is it associated with any particularly diseases?

What demographic groups may present with this disease?

Answer 52

• Hepatic lipidosis
  
  • Common; considered physiologic in very young animals.
  • Has been associated with insulin resistance and metabolic syndrome in Atlantic bottlenose dolphins.
  • Stranded animals starving or nursing calves with a dietary intake high in carbohydrates will accumulate excessive triglycerides in hepatocytes.

Question 53

Describe amyloidosis in cetaceans. Are there particular places for deposition?

What species is susceptible to lysosomal storage disease?

Answer 53

• Amyloidosis
  
  • Likely AA amyloid, inflammatory process.
  • Corticomedullary regions in the kidneys, around acini of palatal salivary gland and thyroid gland.
• Lysosomal storage disease
  
  • Reported in multiple beluga whales, associated with reduced cognitive function (adults).
    
    • Neuron expansion, filling with lysosomal lamellar bodies.

Question 54

Describe urolithiasis in cetaceans.

What are the most common stone types in bottlenose dolphins? What about pygmy sperm whales?

What predisposing factors exist?

What sequelae may occur?

Answer 54

• Urolithiasis
  
  • Ammonium urate stones, bottlenose dolphins.
  • More common in managed dolphins. High dietary load of acid and purines may result in increases in urinary supersaturation of soluble ammonium urate salt. Metabolic etiology.
  • Nephroliths associated with clinical hematuria and nephritis, renal atrophy. May become obstructive (ureters, hydronephrosis).
  • Hypocitraturia may be predisposing.
    
    • Mechanism unknown.
  • Diagnosis
    
    • Changes in BUN/Cr may not manifest for many years with nephroliths.
    • AU stones radiolucent.
    • CT, renal US may ID stones.
    • Two reports of obstructive urolithiasis.
    • Ddx for any dolphin with acute abdominal pain.
    • Dramatic and acute azotemia.
  • Urolithiasis of penile urethra reported in adult pygmy sperm whale (struvite).
    
    • Klebsiella oxytoca on culture. Bacterial infection etiology.
    • Vaginal struvite urolithiasis also reported in a common dolphin

Question 55

Describe neoplasia in cetaceans.

What is the most common neoplasia in belugas? Is one population overly affected?

What is the most common neoplasia in bottlenose dolphins?

What etiologic agent is it associated with?

Describe its behavior.

Answer 55

• Neoplasia
  
  • Gastrointestinal neoplasia in belugas
    
    • St. Lawrence estuary
    • Among highest % neoplasia in wild cetaceans in world.
    • Gastrointestinal carcinoma
    • Also metastatic mammary carcinomas, thyroid carcinomas.
    • Environmental exposure, persistence and bioaccumulation of carcinogens such as polyaromatic hydrocarbons.
  • Urogenital papillomas in free-ranging and captive dolphins
  • Oral SCC in bottlenose dolphins in captivity
    
    • Closely associated with genital papillomas
    • Only oral masses have been ID to transform into carcinoma or SCC.
      
      • Papillomas – raised, well-circumscribed, pale, irregular masses or plaques along oral mucosa and tongue.
      • Tend to recur after excision, may progress to carcinoma o SCC in situ.
        
        • Irregular, poorly demarcated ulcerative areas.
    • Oral SCC slowly progressive but highly aggressive.
      
      • Metastasis to tonsils, thoracic LN, prescap LN and lung.
      • Aggressive nature of these lesions reported to enhance during pregnancy.
      • Possibly associated with exposure of oral cavity to UV light when feeding in aquariums.
      • Papillomaviruses and herpesviruses assoc with the papillomas and SCC in cetacean spp.

Question 56

Describe the mechanisms and affects of the following toxins from harmful algal blooms in cetaceans:

Saxitoxins

Brevitoxins

Domoic acis

What lesions occur?

Answer 56

• Toxins
  
  • Harmful algal blooms (HABs)
  • Predominantly free swimming unicellular dinoflagellates
    
    • Dinoflagellates can sexually reproduce, encyst for varying periods of time, then reemerge under appropriate environmental cues and produce toxins.
  • May result in mass strandings.
    
    • Saxitoxins
      
      • Neurotoxic, effect voltage sensitive sodium channels
      • Impaired nerve impulse propagation and paralysis
      • May impede foraging and avoidance of marine vessel traffic.
    • Karenia brevis
      
      • Gulf of Mexico, Atl coast of FL, Caribbean
      • Toxins release on rupture of cell membrane
      • Prolonged opening of voltage gated sodium channels in neurons – brevitoxisosis
      • Ingestion of heavily contaminated menhaden and aerosol exposure in strandings of bottlenose dolphins
      • Pulmonary and nasopharyngeal edema and hemorrhage, multisystem congestion.
      • IHC shows brevitoxin in pulmonary alveolar lymphocytes, histiocytes, pulmonary LN.
    • Domoic acid
      
      • Direct causal link to CS and pathology not resolved in Cetaceans.
      • Extrapolation from CSL – acute pyriform lobe necrosis, myocardial degeneration, pulmonary edema, pneumonia, GI hemorrhage, repro failure. Chronic hippocampal atrophy and emaciation.
      • Degenerative cardiomyopathy with pericardial serous effusion.
      • Histo – multifocal myocardial cytoplasmic vacuolation, edema. Chronically replacement of myocardial fibers by adipocytes.

Question 57

Describe the effects of oil spills on cetaceans.

Answer 57

• Oil spills
  
  • Contact with skin, eyes, oral cavity, blowhole, ingestion of contaminated prey.
  • Deep Water Horizon Spill – adrenocortical atrophy, pneumonia, reproductive loss significant findings.

Question 58

Describe the effects of persistant organopollutants on cetaceans.

Answer 58

• Persistant organopollutants (POPs)
  
  • Due to aerosolization and widespread dispersal, environmental persistence and longevity of many cetacean spp.
  • Impacts immune function, endocrine disruption.
  • Reduction in splenic and lymphoid populations with increasing POP levels.

Question 59

Why do cetaceans accumulate heavy metals?

What metals are commonly elevated?

Answer 59

• Heavy metals
  
  • Cetaceans have protein complexes (metallothioneins) and bind copper, zinc, mercury, selenium.
  • Sequester metals in liver and skin.
  • Case series South Australian bottlenose dolphins – increased liver, cadmium, copper and zinc associated with renal pathology.
    
    • Distention of Bowman’s space and peoreinuria and bone lesions consistent with osteoporosis of ribs ID with elevated liver cadmium levels.

Question 60

What are some of the congenital conditions affecting cetaceans?

Answer 60

• Congenital/Genetic
  
  • Aberrant white colorations reported in cetaceans, presumed genetic.
    
    • One case of white killer whale – congenital Chediak-Higashi syndrome.
  • Cardiac anomalies
    
    • VSD, ASD, PDA, right ventricular hypertrophy, subvalvular pulmonic stenosis.
  • Thyroid glands
    
    • Lobulation increases normally with age
    • Average follicle diameter larger in wild dolphins
    • Congenital hyperplastic goiter associated with perinatal captive bottlenose dolphin deaths.
  • Skeletal malformations
    
    • Prognathism, brachygnathism, ‘double-faced’ monster, scoliosis.
    • Incomplete fusion of dorsal processes of cervical vertebrae.
    • Spina bifida in humpback and killer whales reported.

Question 61

Describe cardiac disease in cetaceans.

What lesions are commonly seen?

What is Kogia myopathy?

Answer 61

• Cardiac disease – common
  
  • Myocardial degeneration and fibrosis, endocardiosis, valvular fibrosis, mitral leaflet thickening, left ventricular hypertrophy.
  • Cardiomyopathy first described in pygmy and dwarf sperm whales. Aka Kogia myopathy.
    
    • Males over-represented in both species.
    • Unknown etiopathogenesis. Could be stress related.
    • Probably chronic, progressive, advancing to DCM.
    • Enlarged heart with pale, dilated right ventricle and thin interventricular septum.
    • HCM and DCM.
    • Pulmonary and hepatic congestion secondarily common.
• Atherosclerosis

Question 62

Describe adrenal gland hyperplasia in cetaceans.

What is it associated with?

Answer 62

• Adrenal gland hyperplasia
  
  • Increased thickening associated with either diffuse or nodular cortical, medullary, or extracapsular hyperplasia and cortical cyst formation.
  • Zona fasciculate and glomerulosa primarily, but all three zones can be involved.
  • Not associated with pathology.
    
    • Contrast with adrenocortical atrophy – animals exposed to petroleum products, profound effects associated with hypoadrenocorticism, possibly predisposing or exacerbating secondary bronchopneumonia in affected animals.

Question 63

How does decompression sickness develop in cetaceans?

What lesions are observed?

How does that change chronically?

Answer 63

• Decompression sickness
  
  • Beaked whales, common dolphins, Risso’s dolphins, harbor porpoises.
  • Change in the dive profile contributes to gas bubble formation, subsequent embolization, impaired vascular perfusion, ischemia, infarction.
  • Tactical nasal sonar and net entanglement result in abrupt ascent from depth to the surface.
  • Bubbles seen throughout vasculature, hemorrhage in acoustic fat, pleura, epicardium.
  • Acute phase – fat emboli in pulmonary vasculature, epidural rete, subcapsular sinuses LN, renal vasculature.
    
    • If survives, may resolve through vascular resorption and exhalation or offloading of gases.
  • Chronic phase – fibrous encapsulation of pseudocysts with granulomatous infiltrates and Langhan’s type multinucleated giant cells.

Question 64

Describe the effects of barotrauma on cetaceans.

How do they get the trauma?

What are the respiratory lesions?

What are the auditory lesions?

Answer 64

• Barotrauma
  
  • Blast and sonar related
  • Detonation of explosive results in pressure changes propagated radially.
    
    • Impacts animal, leads to implosive pressure differentials that result in up to 9x increase in pressure.
    • Underwater explosions more lethal, shock wave propagation up to three times farther than on land.
  • Respiratory, alimentary, circulatory, and auditory systems involved.
  • Hemorrhage may be in trachea and lungs with pulmonary hematomas, edema, emphysemia worse on side of impact.
  • Histo – frank hemorrhage in bronchoalveolar spaces, rafts of detached and aspirated ciliary respiratory epithelia.
  • May have hemorrhage, perforation of GIT.
  • Ear pathology – peribullar hemorrhage and fractured tympanic bullae.
    
    • If ears are harvested and perfused within 18-24h after death, ultrastructural evaluation of stereocilia and immunofluorescence studies of afferent nerves may provide evidence of barotrauma.
    • Examine acoustic fats, auditory canals, eyes, nasal sinuses.

Question 65

Describe the lesions associated with boat strike in cetaceans.

Answer 65

• Ship strike
  
  • Speeds of 14-15 knots or more associated.
  • Blunt force or sharp trauma.
  • Massive hematomas.
  • Focally extensive bulging of fibroelastic sheath.
  • Organ herniation.
  • Histo – muscle samples remote to site of impact may have hyalinized segmental myodeneration attributed to agonal catecholamine surge.
• Propeller injuries

Question 66

Describe the management of respiraotry disease in a cetacean.

What are your differentials?

What diagnostics and treatments would you employ?

Answer 66

• Respiratory disease
  
  • Pneumonia one of the most common causes of illness in cetaceans
  • Chronic pneumonia usually clinically silent, may have some increases in inflammatory prameters
  • Dx – US, rads, CT, culture and sensitivity with BAL and aspirates of pleural effusion.
    
    • Culture of blow swab and exhalates will yield mixed bacteria and be unreliable.
  • Tx – broad-spectrum abx – oral meds, aminoglycoside of fluoroquinolone aerosol therapy.
  • Monitor blood inflammatory parameters often.
    
    • Refractory to tx, consider pseudomonas aeruginosa, nontuberculous mycobacteria, fungal infection, nocardiosis.
      
      • Tx – injectable amikacin, multimodal drug tx
      • Fungal serology
      • PCR of BAL for mycobacterium and nocardia
      • Nocardiosis can be managed with trimethoprim sulfadiazone (bacterial folate inhibitor).
      • Azole therapy (fungal dz can result from prolonged abx therapy).

Question 67

Describe the management of GI disease in cetaceans.

What is the normal pH of the cetacean stomach?

What is a potential side effect of H2 blockers in cetaceans?

How can you diagnose lower GI disease?

What medications may be recommended?

Answer 67

• GI disease
  
  • Emesis and defecation issues can easily be missed without direct observation.
  • Normal postprandial gastric pH of 1.5.
  • Dx – gastric fluid cytology, gastroscopy, fecal cytology.
  • Endoscoic removal of FB.
  • H2 blockers, proton pump inhibitors mainstay of tx for ulcerative gastritis.
  • If pH of stomah is too high, bones will not demineralize and will accumulate in the stomach until they produce gastric upset and vomiting.
    
    • Monitor feces of cetaceans on H2 blockers for presence of undigested bone spicules.
    • Sucralfate preferentially binds fish skin over gastric mucosa.
    • Monitor progression of gastritis using fasted gastric fluid samples or via repeated gastroscopy.
  • Lower GI dz – fecal cytology, measurement of folate and cobalamin.
    
    • Tang, K. N., Nollens, H. H., Robeck, T. R., & Schmitt, T. L. (2018). Serum cobalamin and folate concentrations as indicators of gastrointestinal disease in killer whales (orcinus orca). Journal of zoo and wildlife medicine, 49(3), 564-572.
  • Antibiotics, oral hydration, temporarily lowering total daily food intake.
  • Butylscopolamine may provide some relief of abdominal discomfort.
  • Simethicone.
  • Nystatin for yeast overgrowth on fecal.
  • Metronidazole can cause anorexia. CNS side effects possible.

Question 68

Ophthalmic disease is common in cetaceans.

What are some causes?

What are some potentially effective treatments?

Answer 68

• Ocular disease
  
  • Corneal dz is the primary ophthalmic problem in dolphin.
    
    • Trauma, edema, ulceration, perforations.
    • Good water quality with low residual oxidants is paramount for prevention and tx of corneal injuries.
    • Shade and dark pool colors may alleviate a UV component.
    • Systemic and topical abx.
    • Oral fluoroquinolones – excellent penetration into eye.
    • Oral tetracyclines similar.
      
      • Prolonged tx may be required.
    • PRP drops unrewarding. Subconj administered PRP, adipose derived stem cells, and abx may be more useful.
    • Raising ambient water temp to 26.7 deg C may allow for faster, more comlete corneal hearing.
    • Cataracts rarely reported, usually left untreated.
    • Impaired vision can be supplemented with echolocation.

Question 69

What are some potential causes of hepatic disease in cetaceans?

How can these diseases be managed?

Answer 69

• Liver disease
  
  • Exposure to toxins, response to medications, active infections, metabolic conditions.
  • Exposure to petroleum products from oil spills, PCBs, dioxins from runoff, chronic mercury associated with increased liver enzymes.
  • Viral hepatitis – one case of adenoviral hepatitis in belugas.
    
    • Acute spikes in liver enzymes.
    • Approx. 30% of stranded wild, free-ranging bottlenose dolphins have moderate to severe hepatic fibrosis, most unknown etiology.
  • Lipid deposition, iron deposition, hepatitis, fibrosis.
    
    • Underlying nutritional and metabolic drivers.
  • Dx – liver enzymes, GGT, ferritin, iron, TG, cholesterol, inflammatory indicators.
  • Tx – fluid therapy, appetite stimulants, nutritional support, abx (doxy, rifampin).
    
    • Phlebotomy, dietary changes for iron overload and fatty liver disease.

Question 70

What is the most common renal disease of cetaceans?

What are some contributing factors?

How is this diagnosed?

What are some common findings on bloodwork?

How is this treated?

Answer 70

• Nephrolithiasis in bottlenose dolphins (ammonium acid urate).
  
  • Contributors may include dietary fish types high in purines, nutrient or metabolic states leading to more acidic or concentrated urine, larger meal sizes that cause fluctuations in postprandial urinary ammonia.
  • Hypocitraturia and older age are risk factors.
  • Dx – renal sonography, CT, US.
  • US visualization of collecting duct is indicative of advanced disease (> 20 nephroliths).
  • Anemia, high BUN, high Cr, low estimated GFR.
  • Advanced cases may also have urinary erythrocytes, occult blood, lower pH.
  • Obstruction – acute anorexia, progressive azotemia, high Cr, electrolyte abnormalities.
  • Tx – oral electrolyte solutions. AAU stones can be tx with potassium citrate but efficacy is unknown.
    
    • Modified diets using fish with lower purines and lower acidity.
    • Laser lithotripsy.

Question 71

Describe metabolic syndrome in cetaceans.

What is it associated with?

What metabolites should be measured?

Are there any protective fatty acids?

What other treatments should be considered?

Answer 71

• Metabolic syndrome
  
  • Wild and managed bottlenose dolphins.
  • Mild to advanced elevations in insulin and lipids.
    
    • Associated with fatty liver and iron overload.
    • Progresses with age if untreated.
    • Dx – feed one third of daily diet in morning and collect 2 hour postprandial blood samples for the measurement of ferritin, insulin, glucose, and TG levels.
      
      • Early – may have mild increase in serum ferritin.
      • Advanced – ferritin may exceed 10,000 ng/mL; indicator of amount of iron stored, appears to contribute to insulin resistance in dolphins.
        
        • Elevated postprandial TG and insulin may be present.
        • Glucose may be elevated, least remarkable change with metabolic syndrome.
    • Dietary fatty acids i.e. heptadecanoic acid have been ID in prevention.
      
      • C17:0
      • Fish with this include mullet, mackerel, herring.
      • Capelin and squid not detectable.
      • Therapeutic target of greater than 0.4% serum total C17:0 associated with lower ferritin within 1 month and normalization of TG, insulin, glucose within 6 months.
      • Phlebotomy to tx iron overload may also alleviate metabolic syndrome.
        
        • Consistent with human literature.

Phlebotomy tx insulin resistance.

Question 72

A recent study evaluated the vaccination of bottlenose dolphins against Erysipelas.

Erysipelothrix rhusiopathiae is what type of pathogen?

Where is it commonly isolated?

What are teh typical disease forms? What are the three forms in cetaceans?

How have previous vaccination efforts been?

What did this sutdy find? How many vaccines are necessary?

Answer 72

Lacave, G., Cui, Y., Salbany, A., Flanagan, C., Grande, F., & Cox, E. (2019). Erysipelas vaccination protocols in dolphins Tursiops truncatus evaluated by antibody responses over twenty continuous years. Diseases of Aquatic Organisms, 134(3), 237-255.

Abstract: Erysipelas is an infection caused by Erysipelothrix rhusiopathiae that affects many different species around the world, including cetaceans. The acute septicemic form can rapidly cause death in bottlenose dolphins Tursiops truncatus. The ultimate goals of this long-term study were the development and identification of the most effective vaccination protocol against clinical erysipelas in T. truncatus using a commercially available swine vaccine, and to determine whether there is a need for a semi-annual vaccination versus an annual vaccination. The present study concentrated on the immunization of a dolphin population (7 wild-born and 22 captive-born individuals) with 2 swine vaccines, the European ‘Eurovac Ery®’ vaccine and the American ‘ER Bac Plus®’ vaccine, and immunological profile results over a 20-yr time period. The general protocol was a primo-vaccination (between 3 and 7 mo of age for calves) with or without a booster 1 mo post primo-vaccination and either annual or semi-annual vaccination thereafter. Sera were collected prior to vaccination, 2 wk post-vaccination and monthly. A dolphin-specific ELISA was developed to analyze the erysipelas-specific antibody response of vaccinated animals. The final ELISA results (n = 1362 samples from 29 animals at pre- and post-vaccination time) suggest that (1) there is a significant difference in antibody levels at the start of the vaccination between older and younger animals; (2) at least 3 vaccinations are necessary to obtain antibody levels above the levels at pre-vaccination; (3) thereafter, annual vaccinations seem sufficient to keep antibody levels above the levels at pre-vaccination; and (4) both vaccines induced similar responses. No case of erysipelas infection was observed in this population during the study.

• Erysipelothrix rhusiopathiae
  
  • Affects fish, amphibians, reptiles, birds, and mammals
  • Facultative pathogen – facultative anaerobe, intracellular, non-motile, non acid-fast, non-sporulating, gram-positive rod-shaped bacterium
  • Can be isolated in soil, contaminated food and water, can be carried by a pigs (not all are sick), can be disinfected but when organic material is difficult cleaning must occur first
  • Disease forms – acute septicemia, endocarditis, arthritis, skin lesions
  • Zoonotic
• Eyrsipelas in cetaceans
  
  • Many reports of wild and managed cetaceans
  • Occurs through ingestion of contaminated fish – organism gains access to bloodstream via breach in GI mucosa usually
    
    • Other sources have been reported as well as conjunctival, respiratory, parental, and cutaneous spread
  • Disease forms – acute septicemic (death) or diamond skin disease caused by peripheral arterial thrombosis. Less commonly a vesicular glossitis has been reported

Previous vaccination efforts

• Modified live vaccines produced disease and subsequent death
• Older inactivated killed vaccines produced anaphylaxis

Take home message

• Newer vaccines produce less reactions but three initial vaccines followed by annual boosters is needed to maintain sufficient antibodies

Question 73

A recent case report described the anesthetic management of a dolphin needing surgical debridement of a tail abscess.

What drugs were used?

What monitoring was used?

How was recovery facilitated?

Answer 73

Tamura, J., Yanagisawa, M., Endo, Y., Ueda, K., Koga, H., Izumisawa, Y., & Yamashita, K. (2017). Anesthetic management of an indo-pacific bottlenose dolphin (tursiops aduncus) requiring surgical debridement of a tail abscess. Journal of Zoo and Wildlife Medicine, 48(1), 200-203.

Abstract: This report describes the anesthetic management of a 14-yr-old, 160-kg, female Indo-Pacific bottlenose dolphin (Tursiops aduncus) that underwent surgical debridement for a refractory subcutaneous abscess twice within a 6-mo interval. The animal was otherwise in good physical condition at each anesthetic procedure. Following premedication with intramuscular midazolam and butorphanol, anesthesia was induced with propofol and maintained with sevoflurane by intubation. During surgery ventilation was controlled. Blood pressure was indirectly estimated using either oscillometric or pulse oximetry. Presumed hypotension was managed by adjusting the sevoflurane concentration and infusion of dopamine. During recovery, the dolphin regained adequate spontaneous respiration following intravenous administration of flumazenil and doxapram. The dolphin was extubated at 85 min and 53 min after the first and second surgeries, respectively. Successful weaning from the ventilator and initiation of spontaneous respiration was the most important complication encountered. Establishment of a reliable blood pressure measurement technique is critical to success for anesthesia in this species.

• 14yo F IndoPac bottlenose dolphin presented for sx debridement of a refractory SQ abscess with osteitis secondary to S. aureus infection, involved transverse process 4^th and 5^th caudal vertebrae.
• Anesthetized twice within 6 mos.
• First surgery – PM midazolam, intubated, induced with propofol, sevoflurane intermittent PPV with either manual compression of bag or ventilator.
• Monitored with ECG, RR, MAP (indirect with pulse-ox), SpO2, ETCO2.
• Lidocaine/bupivacaine local infiltration.
• Period of apnea/cyanosis following extubation, was intubated again and ventilated. Administered flumazenil and doxapram. Began spontaneously breathing.
• Second surgery – similar protocol. Premed with midaz and butorphanol.
  
  • Arterial cath on palmar aspect of flipper attempted for direct art pressures.
  • Vessel was found superficial to the underlying carpal bone.
  • Presumed hypotension was treated with dopamine.
  • Reversed with flumazenil and extubated.
• Discussion points:
  
  • Amount of propofol differed greatly between procedures.
  • Sevo MAC was observed to be similar to dogs premedicated with butorphanol ~2.3 %.
  • Direct arterial pressure measurement failed in this case.
    
    • After administration of dopamine, increased MABP and BP pulse along with increased HR were observed.
• MABP and BPpulse may indicate estimated BP levels or trends.
• Successful weaning from the ventilator and initiation of spontaneous respiration was the most important complication encountered in this case.
  
  • Considered that admin of doxapram facilitated smooth weaning from the vent.
  • Additionally, restablishment of reliable BP measurements is critical for safe ax management of dolphins

Question 74

A recent case report described the anesthesia, surgery, and post-operative management of a pacific white-sided dolphin.

Describe the anesthetics used.

How was the surgical site closed?

What was found on necropsy?

Answer 74

Rosenberg, J. F., Haulena, M., Bailey, J. E., Hendrickson, D. A., Ivančić, M., & Raverty, S. A. (2017). Emergency anesthesia and exploratory laparotomy in a compromised Pacific white-sided dolphin (Lagenorhynchus obliquidens). Journal of Zoo and Wildlife Medicine, 48(2), 581-585.

Abstract: Anesthesia and surgery in cetaceans have inherent risks and have rarely been utilized as viable treatment options. This report represents the first known multidisciplinary team approach to emergency laparotomy in a compromised, 22-yr-old, female Pacific white-sided dolphin (Lagenorhynchus obliquidens). The inciting clinical signs and ancillary diagnostics were consistent with a mechanical ileus. Although no torsion or obstruction was apparent during surgery, severe enteritis and peritonitis were noted. Postoperatively, the animal was maintained on aggressive medical management with continuous supportive care until succumbing 3 days later with clinical pathology indicative of terminal sepsis and profound inflammation. Postmortem findings included generalized vascular stasis and segmental intestinal volvulus with infarction. To the authors’ knowledge, this is one of the most-complex surgical and anesthetic procedures performed in a cetacean. Though the outcome was unsuccessful, this case represents the aquatic veterinary community’s collective advances in the ability to treat cetaceans under human care.

• 22yo adult F PWSD, Vancouver Aquarium.
• Acute onset anorexia, lethargy, disregard for behavioral cues, decreased fecal output.
• CBC, chemistry, fibrinogen, serum iron, ESR, progesterone.
  
  • Leukopenia, dehydration.
• Cytology of vagina, rectum, and blowhole unremarkable.
• Rx metronidazole, ciprofloxacin, prednisone, ranitidine, maropitant, diazepam.
  
  • Leukopenia, azotemia ongoing.
  • Toxic change, mild left shift.
  • Rx itraconazole, nystatin.
• AUS – fluid-distended forestomach and severe fluid distension of small bowel loops.
• AXR – numerous dorsally displaced, gas-distended small bowel loops containing horizontal fluid lines.
• Enema – expulsion of necrotic debris, frank hemorrhage.
• Ddx – intestinal torsion, severe enteritis.
• Placed IVC, left lateral caudal subcutaneous vein.
  
  • IV fluid bolus, ceftriaxone.
• Subsequent repeated US revealed bicavitary effusion.
  
  • Abdominocentesis – modified transudate.
  • Concern for mechanical ileus, pursued emergency exploratory laparotomy.
• Ax – propofol, sevoflurane.
  
  • Surgical exploration through ventrolateral flank incision.
  • Orad sections of small intestine severely hemorrhagic, dark in coloration, distended with fluid.
  • Serosal surface of affected intestine petechial and ecchymotic hemorrhage.
• Four-layer closure performed.
• Post-op care:
  
  • Progressive inflammatory leukogram with evidence of sepsis.
  • Rx – metronidazole, metoclopramide, ceftriaxone, tramadol, meloxicam.
  • Died 3 days post-op.
  • Nx – cause of death distal ileum intestinal volvulus and segmental infarction with septic shock.
  • Suggested obstruction acquired following surgery.
  • Culture of bicavitary effusions showed e. coli and clostridium perfringens alpha toxin.

Question 75

A recent study evaluated the prevalence of Steptococcus phocae among wild phocids, otariids, harbor porpoises, and sea otters.

What were the two most common sites for bacterial infeciton.

Where does the bacteria come from?

Coinfections occur with what other pathogens?

Answer 75

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 conditions. 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.

• Strep phocae – isolated first during epidemic in harbor seals, associated with healthy and dz animals.
  
  • Commensal, tropism for oropharymx and URT, no published evidence supporting these sites as preferred.
  • CS – pneumonia, sinusitis, emaciation, abortion, pyometra, lymphadenopathy.
    
    • Also assoc with urogenital neoplasia in Steller sea lions and ski nabscesses in southern sea otters.
• Lung tissue culture-positive for S. phocae most frequently (25% total positive tissue cultures).
• Most common isolate found in mixed culture was nonhemolytic E. coli.
• Bronchopneumonia, meningoencephalitis, and cellulitis most consistent with S. phocae-associated lesion.
  
  • Bronchopneumonia most consistent lesion.
  • Lesions in the repro tract were not a consistent finding in this study.
• Mixed microbial infection, largest percentage with S. phocae assoc sepsis, mostly CSL with lepto and harbor seals with PHV-1.
• Strep phocae historically assoc with resp, repro, and integument dz.
• In this study, reported bronchopheumonia, sepsis, meningitis and encephalitis, omphalitis, cellulitis, and abscesses.
  
  • Suppurative meningoencephalitis was the second most commonly observed pathologic manifestation of infection.
  • Majority of positive cultures were from lung tissue.
    
    • Pure culture predominating from spleen and LN was strong supportive evidence for sole in antemortem morbidity.
  • Skin isolation and repro lesions were less prevalent in this study.
    
    • Bacterium may be a commensal in the female urogenital tract.
  • Cases with positive bacterial cultures form spleen, LN, or both likely represented true infections vs PM translocation or contamination.
  • Concurrent pure culture form spleen and LN raised suspicion for significant role of Hg in antemortem morbidity.
  • Infections secondary to debilitation in young animals more likely associated with inadequate colostral transfer and possible malnutrition.
  • In older animals, associated with severe parasitic dz and Se:Hg imbalance

Question 76

A recent study evaluated postmortem findings in cetaceans stranded in an Italian sanctuary.

What infectious diseases were found as causes of strandings in that are?

What anthropogenic causes of mortality were observed?

For natural causes of death, animal in poor body condition were attributed to what pathogens? What about those in good body condition?

Answer 76

Giorda, F., Ballardini, M., Di Guardo, G., Pintore, M. D., Grattarola, C., Iulini, B., … & Dondo, A. (2017). Postmortem findings in cetaceans found stranded in the Pelagos sanctuary, Italy, 2007–14. Journal of wildlife diseases, 53(4), 795-803.

Abstract: Between 2007 and 2014, 83 cetaceans were found stranded along the Ligurian coast of Italy, in the Pelagos Sanctuary, the largest marine protected area in the Mediterranean basin. Forty-nine (59%) were submitted to complete or partial necropsy, depending on the conservation status of the carcass. Based on gross and histological pathology and ancillary testing, the cause of death was determined and categorized as anthropogenic or natural (i.e., nonanthropogenic) in origin for 33 animals (67%) and of undetermined origin in the remaining 16 (33%). Natural causes of death, accompanied by either poor or good nutritional status, were attributed to 29 animals (59%), whereas four (8%) were diagnosed with an anthropogenic cause of death, consisting of interaction with fishing activities. Infectious and noninfectious disease was the most common cause of death, involving 29 cetaceans (59%). These data are valuable for understanding health and mortality trends in cetacean populations and can provide information for establishing policies for cetacean conservation and management in such an important protected area of the Mediterranean basin.

• Dolphin morbillivirus (DMV) has caused at least 4 epidemics in Mediterranean over last 25 years.
• Toxoplasma gondii has been attributed a primarily etiologic role in abortion, lethal systemic dz, and nonsuppurative encephalitis.
• Brucella first reported in MM in 1994, causes placentitis, abortion, encephalitis.
  
  • Striped dolphin calves beached most often in spring and summer.
  • Anthropogenic causes of death – all animals in this category died due to interactions with fishing activities.
    
    • Accidental capture, sudden death secondary to serious injury inflicted by fishermen.
      
      • Number of individuals in this category lower than previously reported rates in this area, may be due to reduction in interactions with fishing gear/driftnet fishing no longer used in the Ligurian Sea.
    • Or, only animals lacking severe pathological conditions were included in this category.
    • High percentage of calves – juvenile dolphins are thought to be at higher risk for human-induced injury.
  • Natural causes of death – pathological disorders leading cause of mortality (60%).
    
    • Poor nutritional status – related to parasitic infections.
      
      • DMV and T. gondii coinfections.
        
        • High prevalence of T. gondii suggests role as a secondary pathogen co-occurring with other immunosuppressive agents.
    • Good nutritional status – encephalitis frequently observed.
      
      • DMV, HV, T. gondii, Brucella ceti are the main etiologic agents described in cases of NS meningoencephalitis in stranded cetaceans.
        
        • Neither HV nor Brucella detected in these cases.
  • Pneumonias were also found in many cases, some verminous, some associated with septicemia.

Question 77

A recent study evaluated the neuropathology of stranded cetaceas in Italy.

What were the most common findings?

What are the four morbilliviruses that affect marine mammals?

What are teh three strains of Cetacean morbillivirus?

What other pathogens were identified?

Answer 77

Pintore, M. D., Mignone, W., Di Guardo, G., Mazzariol, S., Ballardini, M., Florio, C. L., … & Serracca, L. (2018). Neuropathologic findings in cetaceans stranded in italy (2002–14). Journal of wildlife diseases, 54(2), 295-303.

Abstract: We summarized the neuropathologic findings in 60 cetaceans stranded along the Italian coastline from 2002 to 2014. The following neuropathologic changes were detected in 45% (27/60) of animals: nonsuppurative meningo-encephalitides (30%, 18/60), nonspecific lesions (12%, 7/60), suppurative encephalitis (2%, 1/60), and neoplasm (2%, 1/60). No histologic lesions were found in 47% (28/60) of the specimens. Five (8%, 5/60) samples were unsuitable for analysis. Analysis with PCR detected Brucella spp., morbillivirus, and Toxoplasma gondii infection in one, six, and seven individuals, respectively. Immunohistochemical analysis confirmed positivity for morbillivirus and for T. gondii infection in three cases each. No evidence of the scrapie-associated prion protein PrPSc was detected. Our findings underscore the importance of an adequate surveillance system for monitoring aquatic mammal pathologies and for protecting both animal and human health.

• Morbilliviruses – at least 4 spp pathogenic in MM.
  
  • Cetacean morbillivirus – CeMv
    
    • Three main strains – porpoise morbillivirus, dolphin morbillivirus, pilot whale morbillivirus, along with newly characterized CeMV clade members from Hawaii and southern hemisphere.
  • Phocine distemper virus – PDV
  • Canine distemper virus – CDV
  • Monk seal morbillivirus
• Toxoplasma gondii – protozoan implicated as cause of systemic dz in several MM species, opportunistic.
  
  • May be concurrent with morbillivirus infection.

Most frequent finding was nonsuppurative meningoencephalitis in 30% of brains.

• Microscopic hallmarks included vasaculitis, perivascular cuffing, and meningitis.
• All three major neurotropic pathogens investigated (DMV, T. gondii, Brucella) were detected, in contrast to previous study where only morbillivirus was detected.
• Two cases of coinfection morbillivirus and t. gondii.
  
  • Parasitic cysts may be present without histologic lesions.

Question 78

A recent study evaluated hypermucoviscous Klebsiella from stranded marine mammals.

What type of pathogen is Klebsiella? Where can it be isolated from?

What are typical clinical signs in marine mammals? How does the hypermucoviscous form differ?

How prevalent was this pathogen in stranded marine mammals?

What plasmids and capsular types were evaluated in this study?

What are recommended treatments?

Answer 78

Whitaker, D. M., Reichley, S. R., Griffin, M. J., Prager, K., Richey, C. A., Kenelty, K. V., … & Johnson, S. (2018). Hypermucoviscous klebsiella pneumoniae isolates from stranded and wild-caught marine mammals of the us pacific coast: prevalence, phenotype, and genotype. Journal of wildlife diseases, 54(4), 659-670.

Abstract: Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs (n=11) and of archival HMV and non-HMV isolates from stranded marine mammals (n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p-rmpA, whereas 92% (23 of 25) were PCR positive for p-rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 10² colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

• Klebsiella pneumoniae – Gram negative, facultative anaerobic bacterium in family Enterobacteriaceae.
  
  • Mammalian mucosal surfaces and in environment.
  • MM – assoc with polyarthritis, meningoecephalitis, peritonitis.
  • Infects multiple hosts, rapidly evolving antimicrobial resistance patterns.
  • Hypermucoviscous KP (HMV) – hypervirulent form.
    
    • Ability to cause life-threatening, community acquired infection in healthy individuals, causes metastatic infections, greater virulence in animal models.
      
      • Assoc with pleuritic, suppurative pneumonia in CSL with septicemia and meningitis in NZ sea lion pups.
• Prevalence 1.2% among 336 stranded MM tested..
  
  • No isolates from PHS, NES, or NFS, likely due to small number of animals.
• HMV KP is significant cause of death for NZ SL pups historically.
  
  • Baseline prevalence reported here in CSLs was lower than previously reported prevalence of the isolate for NZSLs during those epidemics.
• Prevalence of this organism in stranded and wild-caught CSLs was compared, NSF.
• All but one HMV isolate possessed plasmid forms of both rmpA and rmp A2 (similar to humans).
  
  • This study suggests the rmpA gene has potential as a molecular marker for HMV KP and molecular dx method for this isolate.
• Several capsular types identified assoc with hypermucoviscosity in addition to types K1 and K2, which are the ones most freq assoc with HMVKP in humans.
  
  • In this study, most isolates were type K2, but two isolates were negative for K1 and K2, suggesting other capsular types may be assoc with HMV phenotype in MM.
• In humans, they use wcaG gene virulence factor as a potential marker.
  
  • This was not a likely reliable indicator of HMVPK type in the species tested in this study.
• When MICs for non-HMV isolates was compared to those of HMV KP isolates, non-HMV isolates had higher MICs for many antimicrobial agents.
  
  • Potential for acquisition of antimicrobial resistance.
  • Most of the hypervirulent strains are susceptible to many common antibiotics.
    
    • Based on MIC in this study, Ceftiofur may be an appropriate initial choice for treatment of suspected HMVKP infections in CSLs along US Pac coast.
    • MICs for HMV wild-caught CSL isolates were significantly higher than those of HMV stranded CSL isolates for several abx (ceftiofur, amoxicillin, tetracycline, gentamicin, florfenicol, penicillin).
• Detection in MM stool samples by cultivation methods seems to be a reliable method for detecting the presence of this bacteria in CSL.
  
  • Does not grow well on MacConkey agar typically used for other Enterobacteriaceae.

Question 79

A recent study evaluated the prevalence of cryptosporidium & giardia in marine mammals in northern European waters.

How did they test for the parasites?

How prevalent were the parasites?

Do marine mammals play a role in maintaining the aquatic sylvatic cycle of these pathogens?

Answer 79

Brief Communication:

Grilo, M. L., Gomes, L., Wohlsein, P., de Carvalho, L. M., Siebert, U., & Lehnert, K. (2018). Cryptosporidium species and Giardia species prevalence in marine mammal species present in the German North and Baltic Seas. Journal of zoo and wildlife medicine, 49(4), 1002-1006.

Abstract: Marine mammals are known to harbor Cryptosporidium spp. and Giardia spp., especially species or genotypes commonly associated with humans. In animals of the North and Baltic Seas, this information is missing. In this study, fecal samples (n = 97) from harbor porpoises (Phocoena phocoena), harbor seals (Phoca vitulina), and grey seals (Halichoerus grypus) present in German waters were examined via microscopic examination (by using a modified Ziehl-Neelsen technique [MZN]) to estimate the prevalence of these parasites. Additionally, intestinal tissue samples (n = 1,049) from specimens of 10 marine mammal species were submitted for histopathologic examination (HP). A low prevalence was detected (2.1 and 1.3%, for MZN and HP, respectively), associated with Cryptosporidium spp. Giardia spp. was not observed. Current results suggest a low prevalence of these parasites in marine mammals present in Germany. However, methods with higher sensitivity, such as molecular methods, are crucial to confirm the prevalence, establish origin routes, and determine epidemiologic factors associated with the low prevalence observed.

• Cryptosporidium and Giardia ubiquitous enteric protozoans, worldwide.
  
  • Humans, domestic animals, and wildlife contribute to dispersal from land to aquatic environment.
  • Public health concern related to recreational water use and food safety.
• Several marine mammal species harbor Cryptosporidium, Giardia, or both.

Goal: Evaluate prevalence of Crypto and Giardia in marine mammals in the German North and Baltic Seas, and characterize lesions associated with the parasites.

• Fecals collected during health monitoring surveillance programs.
  
  • Harbor porpoises, harbor seals, gray seals.
  • For detection of Crypto and Giardia, fecal smears stained by modified Ziehl-Neelsen (MZN).
  • ID based on typical staining and morphology of Crypto oocysts and Giardia cysts.
    
    • Crypto – small, rounded to oval, red or rose stained, with four sporozoites inside. Resembles reddish half moon.
    • Giardia – blue greyish to transparent oval structures and can be identified due to the four nuclei.
    • Intensity of infection accessed with a semi-quantitative method on the basis of the number of oocysts or cysts per field.
• Histo records of intestinal tissue samples also analyzed.
  
  • Harbor seals, harbor porpoises, grey seals, ringed seals, short-beaked common dolphins, common mink whales, orca.
• MZN – highest prevalence newborn or immature animals, mainly harbor seals.
  
  • Infections normally associated with cestode and acanthocephalan spp.
• Histo – enteric protozoans detected via HP examination only in harbor seals originating from the North Sea.
  
  • Neonates or immature animals.
  • Coparasitism with Diphyllobothrium spp and Corynosoma spp in half of the animals.
  • In confirmed cases of crypto, protozoan organisms observed on the surface of the crypt epithelial cells.
    
    • Additional findings – lymphoid depletion, single-cell necrosis of the Peyer patch, presence of crypt abscesses with degeneration of the epithelium.
• Screening failed to detect Giardia spp infection in any of the examined animals.
• Factors suggested to influence the prevalence of protozoans in aquatic animals – proximity to human sewage or agricultural outflow and association with raining seasons.
• These results suggest it is unlikely that these marine mammal populations play a role in maintenance of an aquatic sylvatic cycle of crypto. North and Baltic Sea coasts of Germany appear to be low-prevalence areas of enteric protozoans for marine mammals.

Question 80

A recent paper evaluated ultrasonography of the marginal lymph node in bottlenose dolphins.

What does the marginal lymph node drain?

What are the landmarks for probe placement?

What does the node look like sonographically?

How did the nodes differ by age groups?

Answer 80

Martony, M. E., Ivančić, M., Gomez, F. M., Meegan, J. M., Nollens, H. H., Schmitt, T. L., … & Smith, C. R. (2017). Establishing marginal lymph node ultrasonographic characteristics in healthy bottlenose dolphins (Tursiops truncatus). Journal of Zoo and Wildlife Medicine, 48(4), 961-971.

Abstract: Pulmonary disease has been well documented in wild and managed dolphin populations. The marginal lymph nodes of the dolphin thorax provide lymphatic drainage to the lungs and can indicate pulmonary disease. This study standardized a technique for rapid, efficient, and thorough ultrasonographic evaluation of the marginal lymph nodes in bottlenose dolphins (Tursiops truncatus). Thoracic ultrasonography was performed on 29 clinically healthy adult bottlenose dolphins. Reference intervals for lymph node dimensions and ultrasonographic characteristics of marginal lymph nodes were determined from four transducer orientations: longitudinal, transverse, oblique, and an orientation optimized to the ultrasonographer’s eye. The relationship between lymph node dimensions and dolphin age, sex, length, weight, origin, and management setting (pool versus ocean enclosure) were also evaluated. The mean marginal lymph nodes measured 5.26 cm in length (SD = 1.10 cm, minimum = 3.04 cm, maximum = 7.61 cm, reference interval [10th to 90th percentiles per node dimension] 3.78–6.55 cm) and 3.72 cm in depth (SD = 0.59 cm, minimum = 2.64, maximum = 5.38 cm, reference interval 2.98–4.50 cm). Sex, dolphin length, weight, and management setting had no effect on lymph node dimensions. Dolphins >30 yr of age had longer node lengths than dolphins 5–10 yr old. Node dimensions did differ between dolphins from various origins. Most commonly, the lymph node was found to be hyperechoic relative to surrounding soft tissues (98%) and to have irregular caudal borders (84%), ill-defined deep borders (83%), flat superficial border (67%), triangular or rounded triangle shape (59%), irregular cranial border (55%), and moderate heterogeneity (34%). The data reported in this study serve as a baseline reference that may contribute to earlier detection of pleural and pulmonary disease of managed and wild cetacean populations.

• Pulmonary disease is common in dolphins. Ultrasound is most practical first line method to image lungs, but has limitations. Only visceral surface can be imaged in normal lungs.
• Dolphins have exceptions to lymph node organization:
  
  • Marginal lymph nodes, diaphragmatic lymph nodes, anal tonsil complex, no appendix
    
    • Marginal LNs drain lung lymphatics and are readily imaged
    • Large, solitary, bilateral, always present
    • External: Generally caudoventral to pectoral flipper insertion. Internal: Caudo lateral to heart, on ventral border of lung where lung intersects with diaphragm.
• Image below: Green = heart (appreciate lung tissue overlying its caudal margins that sits between heart and marginal LN (Yellow)). This causes gas artifact that makes LN border indistinct in some views. Blue = margin of diaphragm.
• 4 views taken. Longitudinal, transverse, oblique, and optimal view (modified oblique).
  
  • Maximal length and depth most often found in oblique and optimal views (Eliminated rib shadowing). Longitudinal and transverse images underrepresented true dimensions.
• Prevalent u/s characteristics: hyperechoic to surrounding tissues, irregular caudal borders, ill-defined deep borders, flat superficial border, triangle/rounded triangular in shape, irregular cranial border, moderate heterogenicity. No difference between left and right sides.
• Key differences in other domestic species: irregular borders are often associated with infiltrative pathology. Mixed echotextures are associated with pathology. Nodes are typically oval. Nodes are typically iso or hypoechoiec
  
  • Node length was not associated with length, sex, weight, or management setting (pool vs. ocean pen). But was positively correlated with age.
• Dolphin with Pacific origin (vs. Atlantic) had statistically smaller nodes. More difficult to image.
  
  • Pacific dolphins reported to have larger lung volumes. Suspect larger lung volume underrepresents node size due to increased gas artifact.
• Single case example of dolphin with pulmonary coccidiomycosis. Diffusely enlarged nodes compared to reference animals, hypoechoic (not seen in any healthy animals)
• Marginal LN scans rapid and effective technique
• Healthy dolphins have characteristics of this node different from conventional assessment of healthy nodes in other domestic animals.
• Pacific origin dolphins have nodes that appear smaller on u/s

Takeaway: Marginal LN evaluation may be beneficial for diagnosis and monitoring thoracic/pulmonary disease. Dolphins > 30y had larger LN lengths in healthy individuals.

Question 81

A recent study evaluated cobalamin and folate as indicators of GI disease in killer whales.

What are typical clinical signs of GI disease in cetaceans?

Deficiencies in cobalamin and folate are indicative of malabsorption at which sites?

Which vitamin was reduced in clinically ill whales? By how much?

What was a common pathogen in these cases? How can that affect these vitamin concentrations?

Answer 81

Tang, K. N., Nollens, H. H., Robeck, T. R., & Schmitt, T. L. (2018). Serum cobalamin and folate concentrations as indicators of gastrointestinal disease in killer whales (orcinus orca). Journal of zoo and wildlife medicine, 49(3), 564-572.

Abstract: Cobalamin and folate are water-soluble vitamins that are useful indicators of chronic gastrointestinal (GI) function in humans and some animal species. Serum cobalamin and folate concentrations in an ex situ population of killer whales (Orcinus orca) were measured and factors that may affect their serum concentrations were identified. Serum samples (n = 104) were analyzed from killer whales (n = 10) both while clinically healthy and during periods of clinical GI disease as defined by clinical signs and fecal cytology. To characterize serum cobalamin and folate concentrations in clinically healthy animals, a mixed-model regression was used, with cobalamin and folate both significantly affected by weight (cobalamin: P < 0.0001, folate: P = 0.006) and season (cobalamin: P < 0.0001, folate: P < 0.0001). The marginal mean concentrations for cobalamin and folate across weight and season were 742 ± 53.6 ng/L and 30.2 ± 2.6 μg/L, respectively. The predicted 95% confidence intervals (CI) for these analytes were then compared with samples collected during periods of GI disease. Across individuals, 22% (2/9) of the folate and 80% (8/10) of the cobalamin samples from the animals with GI disease fell outside the 95% CI for the population. When comparing samples within an individual, a similar pattern presented, with 100% of cobalamin of the observed abnormal samples reduced compared to healthy animal concentration variability. The same was not true for folate. These results suggest that serum concentrations of cobalamin and folate may be useful minimally invasive markers to identify GI disease in killer whales, especially when values are compared within an individual.

• Clinical signs of GI dz in cetaceans – vague, decreased appetite, adnominal splinting, lethargy, tenesmus, bottom-resting.
  
  • Feces is normally characteristically loose.
  • Observation and sampling of diarrhea is difficult.
• Cobalamin and folate are dietary water-soluble vitamins absorbed in the small intestine, shown to be important markers for GI, pancreatic, and hepatic dz.
  
  • Essential cofactors for multiple enzymes, including isomerases, methyltransferases, dehalogenases.
  • Deficiencies sequelae of GI dz when the disease is associated with malabsorption or with overgrowth of bacteria capable of synthesizing or consuming the vitamins.
  • Cobalamin deficiency associated with poor clinical outcome.
    
    • Indicates malabsorption in the ileum.
  • Folate deficiency indicates malabsorption in the jejunum.
  • Species differences:
    
    • Intrinsic factor (required for transport of cobalamin from duodenum to ileum and absorption in the ileum) produced in different organs in humans (gastric mucosa), dogs (stomach, pancreas) and cats (exocrine pancreas).
• • Cobalamin ranged between 309 – 1540 ng/L, folate concentrations ranged 8.9-55.6 mcg/L.
    
    • Using samples that were previously frozen and thawed found to not affect results.
    • Effect of GI dz on cobalamin and folate:
      
      • Five events with clinical signs and abnormal fecal, four with clinical signs without fecals, one with normal fecal and clinical signs, one with no clinical signs and an abnormal fecal.
      • Most common bacteria in abnormal fecal cytology was Clostridium spp.
      • For cobalamin, all abnormal samples reduced vs healthy animal concentrations.
        
        • Not true for folate.
        • In the animal with mesenteric torsion, both were elevated.
• • Cobalamin concentrations were 80% reduced when compared within an individual to its own healthy measurements.
    * Except in the acute mesenteric torsion case.
    * Possible increases occurred due to pathologically increased transit time, alterations in the mucosal barrier, and small intestinal bacterial overgrowth.
    * Possible causes of decreased serum cobalamin concentrations include congenital receptor defects, decrease mucosal absorption, and excess competition from the intestinal biome.
    * Clostridial spp known to consume cobalamin, most commonly found bacteria in abnormal fecals in these studies.
    * Decreased cobalamin concentrations cannot be definitively attributed to either bacterial overgrowth or malabsorption.
    * Holotranscobalamin (metabolically active portion of cobalamin) has been considered more sensitive and specific for true cobalamin deficiency, but this has not been evaluated in veterinary medicine.
    * Fecal cytology may help differentiate and corroborate cobalamin results.

Question 82

A recent study compared a smartphone-based ECG to that standard six lead ECG in bottlenose dolphins.

How do cetacean ECGs differ from other mammal species? What species is this similar to?

How is the anatomy of their Purkinje fibers different?

What were some limitations with this technique?

Answer 82

Yaw, T. J., Kraus, M. S., Ginsburg, A., Clayton, L. A., Hadfield, C. A., & Gelzer, A. R. (2018). Comparison of a smartphone-based electrocardiogram device with a standard six-lead electrocardiogram in the Atlantic bottlenose dolphin (Tursiops truncatus). Journal of zoo and wildlife medicine, 49(3), 689-695.

Abstract: A bipolar, single-lead electrocardiogram (ECG) device is capable of recording ECGs with an integrated smartphone application. To determine the utility of this device, phone-based ECGs (pECG) were compared with standard six-lead ECGs (sECG) in four female Atlantic bottlenose dolphins (Tursiops truncatus) at the National Aquarium. Study animals were trained to haul out onto a dry deck in ventral recumbency and allow simultaneous 30-sec ECG acquisition using the two devices. The pECG device was held against the thoracic wall caudal to the left axilla. The sECGs were recorded in the frontal plane. Instantaneous heart rates were obtained from identical QRS complexes on both ECGs. Three boarded cardiologists independently evaluated the rhythm and the polarity of the QRS depolarization for each recording and the results were compared. The mean heart rate was 80 beats/min (range 62–92 beats/min) and 80 beats/min (range 60–92 beats/min) for the pECG and sECGs, respectively. All four dolphins displayed sinus respiratory arrhythmia, and one animal had occasional atrial premature contractions. Rhythm diagnosis and QRS polarity were identical for the pECG and sECG. Dolphin vocalizations created artifacts on the pECG that were not present on the sECG, so pECGs had to be acquired without vocalization. The pECG appears to be an accurate and useful method to monitor the heart rate in Atlantic bottlenose dolphins. This system is inexpensive and portable, making it valuable for health examinations, transport monitoring, and stranding responses.

• ECG results have been published for bottlenose dolphin (Tursiops truncatus), beluga whale (Delphinapterus leucas), humpback whale (Megaptera novaeangliae), harbor porpoise (Phocoena phocoena) and orca (Orcinus orca).
  
  • Android ECG device transforms electrical signals of the ECG generated by the sensor assembly to a frequency-modulated inaudible ultrasonic sound signal received by the phone’s microphone and displayed as a single-lead ECG.
  • Direct thorax lead placement requires no cables, reduces equipment.
• Results
  
  • Mean HR 80 BPM on both ECGs.
  • All dolphins displayed sinus respiratory arrhythmia and one animal had occasional atrial premature complexes.
  • 100% agreement in HR and rhythm between the two ECG systems, assessed by cardiologists.
  • Vocalizations produced artifacts on the smartphone-based device, not on the standard six-lead ECG.
• Discussion
  
  • Presence of negative polarity (deep S wave instead of an R wave) observed in dolphins is similar to ventricular activation patterns in horses, cows, sheep, pigs; differs from carnivores and primates.
  • Cetaceans are closely related to ungulates, cytoarchitecture of the Purkinje fiber network is classified as group 1.
    
    • Penetrates from the endocardium to epicardium in both the subendocardial and intramural Purkinje networks participate in rapid depolarization throughout the ventricular myocardium.
      
      • As a result, most of the ventricular mass is activated simultaneously and QRS morphology does not correlate with ventricular mass or heart size in these species.
  • ECGs obtained on the phone can be easily uploaded via PDF and sent to cardiologists for review.
  • Main limitation is the signal overlap created by vocalizations. Also need to be in close proximity to the animal. Also only provides a single-lead recording.

Question 83

A recent study evaluated auditory evoked potentials in small cetaceans.

What are some causes of hearing loss in cetaceans?

What are auditory evoked potentials?

What were the findings in stranded dolphins?

What about managed dolphins?

Answer 83

Strobel, M. M., Houser, D. S., Moore, K. T., Davis, M. R., Clough, P. L., Staggs, L. A., … & Walsh, M. T. (2017). Auditory evoked potentials and behavioral considerations with hearing loss in small cetaceans: Application as a standard diagnostic test in health assessment. Journal of Zoo and Wildlife Medicine, 48(4), 979-986.

Abstract: The primary sense in odontocetes is hearing and a large portion of the odontocete brain is devoted to the auditory processing of echolocation signals. Hearing deficits in odontocetes potentially compromise the ability to forage, navigate, socialize, and evade predators. This presents a challenge to survival and reproduction in wild odontocetes and can affect the general welfare of odontocetes under human care. Currently, little empirical information on how odontocete behavior is affected by hearing loss exists. This study investigated hearing deficits in several species of stranded dolphins and age-related hearing deficits in dolphins kept under human care through auditory evoked potential (AEP) testing and evaluated whether individual behavior correlated with hearing impairment. Behavioral questionnaires for participating animals were completed by individuals with extensive knowledge of the animals’ history and behavior. A chi-square analysis determined whether animals with hearing impairment demonstrated behaviors that differed significantly from those considered normal. All tested individuals under human care over 35 years of age had some degree of hearing loss, as did a large percentage of previously stranded animals. Individuals with hearing loss exhibited a range of behavioral changes, including delays in learning new behaviors, accepting novel enrichment, and habituating to new environments. Some individuals with profound hearing loss also displayed a change in vocalization rate in various situations. Findings within previously stranded animals suggest AEP studies should be conducted in all stranded individuals entering rehabilitation. It is further recommended that dolphins living under human care undergo hearing tests as part of their normal health assessments, with emphasis on aging individuals and animals that exhibit delayed learning, respond poorly to audible cues, or show atypical vocalization behavior.

• Hearing loss:
  
  • Trauma: cerebellum, vestibulocochlear nerve, ear
  • Conductive hearing loss: reduced ability for sound to be transmitted to inner ear
  • Sensorineural loss: inability of inner ear, vestibulocochlear nerve and brain to process incoming sounds
• Auditory evoked potentials (AEP): brain voltages produced in response to hearing sound
• Stranded dolphins: 8/13 had hearing loss, 7 = severe to profound loss Consistent w/ conductive or sensorineural hearing loss over range of hearing.
• 6/8 w/ hearing loss reported to learn behaviors more slowly. Hearing loss animals may take longer to acclimate to new enrichment or new pools/pool mates and have increased vocalizations and stereotypic behavior. Hearing loss rough toothed dolphins may sleep in center of group or log more
• 4/5 normal learned behaviors average or faster
• Managed care dolphins: all 8 over 35 yo had hearing loss
• High frequency sensorineural consistent with presbycusis (hearing loss with age), likely can still echolocate. Magnitude of loss not strongly associated with age. Those with hearing loss may be less responsive to whistle signals

Takeaway: Older dolphins in managed care experience hearing loss. Captive dolphins should undergo hearing tests as part of their normal health assessments, with emphasis on aging individuals and animals that exhibit delayed learning, respond poorly to audible cues, or show atypical vocalization behavior.

Question 84

A recent study evaluated the hematology and biochemistry of rehabilitated rough-toothed dolphina (Steno bredanensis).

How did values differ between healthy and stranded dolphins?

What analytes may have prognostic value?

Answer 84

Manire, C. A., Reiber, C. M., Gaspar, C., Rhinehart, H. L., Byrd, L., Sweeney, J., & West, K. L. (2018). Blood chemistry and hematology values in healthy and rehabilitated rough-toothed dolphins (Steno bredanensis). Journal of wildlife diseases, 54(1), 1-13.

Abstract: Rehabilitation efforts for live stranded marine mammals are guided by diagnostic measures of blood chemistry and hematology parameters obtained from each individual undergoing treatment. Despite the widespread use of blood parameters, reference values are not available in the literature from healthy rough-toothed dolphins (Steno bredanensis) with which to infer the health status of an animal. We examined serum or plasma chemistry and hematology data from 17 rough-toothed dolphins either housed at Dolphin Quest French Polynesia or during their rehabilitation at the Dolphin and Whale Hospital in Sarasota, Florida, US between 1994 and 2005. Blood parameters were compared among healthy animals, rehabilitation animals that were eventually released, and rehabilitation animals that died. This study indicated significant differences in many blood parameters for the poorly known rough-toothed dolphin that are likely to vary between healthy and sick animals. These included aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, bicarbonate, and globulins, which were greater in sick dolphins, and alkaline phosphatase and total protein which were greater in healthy individuals. Total white blood cell counts were lower in healthy animals as were the absolute numbers of neutrophils, monocytes, and eosinophils. Analysis of first blood sample levels for glucose, sodium, and erythrocyte sedimentation rate may have value for triage and prognostic evaluation.

Background: RTD found in warm pelagic waters throughout the world. Majority of stranded dolphins (of all spp) most commonly affected by diseases such as bronchopneumonia, fibrosis, GI disease, pancreatitis, hepatitis, encephalitis. No baseline bloodwork established for this species.

• Healthy: ESR, WBC count, neutrophils, eosinophils, monocytes, total bilirubin, AST, ALT, globulin level, glucose, LDH, and ESR were significantly lower. Albumin, BUN and ALP significantly higher.
• TCO2 was significantly higher in all dolphins that died, but this is the exact opposite finding of another paper evaluating stranded common dolphins.
• Initial liver enzyme values were not prognostic (ALP did not differ between surviving sick and fatally sick), but later samples could be prognostic. ALP shown to decrease with infection in bottlenose and pacific white-sided dolphins.
• Previous studies showed low PCV associated with death, this study found RBC highest in fatally sick group. Alb was lowest in fatally sick animals and could be prognostic.
• Glucose, ESR, and sodium may have prognostic value (if considered together)
• Lower ESR rate correlated well with healthy studies (lower rate, healthier dolphin), and high rates may be prognostic at presentation However, there were animals that had high glucose, low sodium, and low ESR that survived.
• WBCs did not differentiate healthy vs sick at presentation.
• Creatinine not different among groups. Per authors severe kidney disease not common in strandings.

Takeaway: Glucose, liver enzymes (esp ALP), WBC and ESR have values as (potential) indicators of health status and in predicting survival in stranded rough toothed dolphins.

Question 85

A recent study evaluated the effects of oral megestrol acetate on various hormones in bottlenose dolphins.

What is megestrol acetate?

What is it used for in people and in zoological collections? How has it been used in dolphins?

What adverse reactions have been reported (previously and in this study)?

What was the effect of megestrol acetate on the following hormones: ACTH, cortisol, epinephrine, norepinephrine, T3, T4?

Answer 85

Houser, D. S., Champagne, C. D., Jensen, E. D., Smith, C. R., Cotte, L. S., Meegan, J. M., … & Wasser, S. K. (2017). Effects of oral megestrol acetate administration on the hypothalamic-pituitary-adrenal axis of male bottlenose dolphins (Tursiops truncatus). Journal of the American Veterinary Medical Association, 251(2), 217-223.

OBJECTIVE To evaluate the impact of oral megestrol acetate (MA) administration on adrenal function in male bottlenose dolphins (Tursiops truncatus).

DESIGN Serial cross-sectional study.

ANIMALS 8 adult male dolphins, all of which were receiving MA at various daily doses (range, 0 to 60 mg, PO) for the control of reproductive behavior.

PROCEDURES Blood samples were collected every 2 weeks for 1 year from dolphins trained to voluntarily provide them. Cortisol, ACTH, and other hormone concentrations were measured in serum or plasma via radioimmunoassay or ELISA. Fecal samples, also provided by dolphins voluntarily, were assayed for glucocorticoid metabolite concentrations. Effects of daily MA dose on hormone concentrations were evaluated.

RESULTS Daily MA doses as low as 10 mg strongly suppressed cortisol secretion in nearly all dolphins, and except for a single measurement, no dolphin had measurable serum concentrations at doses ≥ 20 mg. Variations in serum cortisol concentration were unrelated to season but were directly related to ACTH concentrations, suggesting primary effects upstream of the adrenal gland. Cessation of MA administration resulted in almost immediate restoration of measurable serum cortisol concentrations, although concentrations continued to rise in a few dolphins over the following weeks to months.

CONCLUSIONS AND CLINICAL RELEVANCE Caution should be exercised when administering MA to control reproductive behavior in male dolphins. Because the hypothalamic-pituitary-adrenal axis appeared to be sensitive to even small doses of MA in dolphins, duration of treatment may be the most critical consideration.

Background:

• Megestrol acetate (MA) = progesterone derivative, used in humans for appetite stimulation and antineoplastic (e.g. hormone assoc. cancers such as ovarian).
• Adverse effects of MA in humans is due to glucocorticoid-like activity impacting the HPA axis via agonistic activity at GC receptors. Unclear if MA acts at hypothalamus or pituitary.
• MA markedly suppresses ACTH and cortisol secretion (is assoc. with Cushing’s and diabetes in humans).
• MA used in zoos for reproductive management, and for control of male-male aggression.
• Has been used in male bottlenose to suppress reproductive behaviors such as refusal to eat, increased sexual aggression. Doses for this purpose range 10-120 mg.
• Goal: determine the effects of oral MA at various doses on secretion of ACTH, cortisol, thyroid hormones, and epinephrine/norepi.

Results:

• ACTH significantly lower on MA, ACTH levels recovered after stopping MA.
  
  • ACTH suppression achieved with doses as low at 10 mg in some animals.
• Relationship between MA and cortisol was similar to ACTH. Fecal glucocoritcoids reflected serum cortisol levels. Baseline cortisol ~20 umol/L, on MA was <2.5umol/L
• Epi levels were significantly lower on MA, Norepi also lower, but to a lesser extent.
• No clinically significant effect of MA on T3 and T4

Discussion:

• Variations in cortisol and ACTH were directly related to MA. Effect of MA on HPA consistent with previous reports in humans. However, dolphin HPA appeared to be more sensitive to MA. Dolphin doses as low at 10 mg significantly reduced ACTH.
  
  • This suggests MA works predominantly at the level of the hypothalamus-pituitary interaction.
• Decreased epi and norepi suggest influence of MA on both cortical and medullary regions of adrenals
• MA does not appear to interact with the hypothalamic pituitary thyroid axis in dolphins.
• Negligible plasma ACTH and decreased cortisol consistent with secondary adrenal insufficiency
• One dolphin developed hypopigmentation after long-term tx. Melanocyte stimulating hormone and ACTH originate in pituitary and share common precursor (proopiomelanocortin). MA compromised production of tropic hormones at the level of the pituitary gland. This has been reported in humans.
• Recovery from MA admin was variable among dolphins. Some rebounded immediately, others in wks-months.
• Caution should be used when stopping MA administration, authors recommend weaning off MA.
• MA may affect ability to mount glucocorticoid response to stressors.

Conclusion: While MA is beneficial for managing repro behaviors in dolphins, there are adverse effects such as secondary adrenal insufficiency, that appear to be dependent on dose and duration of tx. Dolphin ACTH secretion appears to be sensitive to low doses of MA. Caution should be used when administering MA to dolphins.

Easy to confuse progestin contraceptives:

• Melengestrol acetate (MGA) – comes as implant, liquid, feed.
• Megestrol acetate (Ovaban, Megace) (MA) - oral
• Medroxyprogesterone acetate (MAP, Depo-Provera) – depot injection lasting 2-3 months
• Mechanism of action: Continuous exposure to progestins mimics luteal activity or pregnancy à inhibits LH/FSH (decreases cervical mucus, increases viscosity, decreased oviductal transport, inhibits implantation).
• Side effects include uterine and mammary pathology, polyphagia/weight gain, diabetes-like symptoms, PU/PD, androgenic activity. Is safe for lactating female.

Question 86

A recent paper classifiied bottlenose dolphin adenovirus 2 (BdAdV-2) as a host-jumping virus.

Discuss the taxonomy of cetaceans and what hosts this virus may have come from.

Viruses with more recent host jumping events are rich with what nucleotides?

What are some other viruses that have jumped hosts into cetaceans?

What are the typical clinical signs in marine mammals of adenovirus infection?

What are some other cetacean adenoviruses?

Answer 86

Standorf, K., Cortés-Hinojosa, G., Venn-Watson, S., Rivera, R., Archer, L. L., & Wellehan Jr, J. F. (2018). Phylogenetic analysis of the genome of an enteritis-associated bottlenose dolphin mastadenovirus supports a clade infecting the Cetartiodactyla. Journal of wildlife diseases, 54(1), 112-121.

ABSTRACT: Adenoviruses are nonenveloped, double-stranded DNA viruses, known to infect members of all tetrapod classes, with a similarity between phylogenies of hosts and viruses observed. We characterized bottlenose dolphin adenovirus 2 (BdAdV-2) found in a bottlenose dolphin (Tursiops truncatus) with enteritis. Virions were seen by negative staining electron microscopy of feces. Initial sequences obtained using conserved PCR primers were expanded using primer walking techniques, and the complete coding sequence was obtained. Phylogenetic analyses were consistent with coevolution of this virus and its bottlenose dolphin host, placing BdAdV-2 into a monophyletic group with other mastadenoviruses of Cetartiodactyla. When considering the low guanine/cytosine (G/C) content of BdAdV-2 with the phylogenetic data, this virus may represent a host-jumping event from another member of Cetartiodactyla. Analysis of partial polymerase indicated that bottlenose dolphin adenovirus 1, previously identified in Spain, and BdAdV-2 are sister taxa with harbor porpoise adenovirus 1, forming a cetacean clade. Bottlenose dolphin adenovirus 2 includes a highly divergent fiber gene. Two genes homologous to the dUTPase superfamily are also present which could play a role in enabling viral replication in nondividing cells. We used sequence data to develop a probe hybridization quantitative PCR assay specific to BdAdV-2 with a limit of detection of 10 copies.

• Adenoviruses – all tetropods, nonenveloped DNA viruses that reploicate within host nuclei.
  
  • Mastadenovirus only in amammals.
  • Aviadenovirus only in birds.
  • Atadenovirus likely originated in squamates, also found in birds, ruminants, marsupials.
  • Siadenovirus unknown origin.
  • Ichtadenovirus in white sturgeon.
  • Testadenovirus in turtles.
• Summary of placental mammal superorders:
  
  • Afrotheria – elephants, manatees, aardvarks, etc.
  • Xenarthral – armadillos, sloths, anteaters.
  • Boreoeutheria
    
    • Laurasiatheria – carnivores, Cetartiodactyla (whales, even-toed ungulates), Perissodactyla (odd-toed ungulates), insectivores, bats.
      
      • Cetartiodactyla – Camelids, suids, hippos, cetceans, ruminants.
        
        • Cetruminantia – cetaceans, ruminants, hippos.
    • Euarchontoglires – rabbits, rodents, primates.
• Host-jumping
  
  • Genomes rich in adenine/thymidine assoc with more recent host-jumping events.
  • Closely related host spp make it easier for jumping events to occur.
• CS – typically respiratory, hepatitis.
• Adenovirus in MM:
  
  • First established in cetaceans in sei, bowhead, and beluga whales.
  • Also found in mesenteric LN of harbor porpoise.
  • Bottlenose dolphins in Spain – anorexia, diarrhea, vomiting, BdAdV-1.
    
    • Here they describe a second novel bottlenose dolphin adenovirus from dolphin with diarrhea and develop a PCR.

16 genes common between all 5 recognized genera of adenoviruses.

• Significant differences in fiber gene.
• Fiber protein is first viral component to interact with host cells through receptors, may be involved in initial stages of infection.
• BdAdV-2 does not display any homology to known fiber sequences.
  
  • Likely possesses a fiber protein with an unrecognizably distinct sequence including an immunoglobulin domain.
• Most mastadenoviruses infecting Cetartiodactyla including BdAdV2 appear monophyletic.
  
  • Likely that the origin of this virus is another member of the Cetartiodactyla (not bottlenose dolphins).
  • Relationship between adenoviruses found in cetaceans with those found in ruminants may present strong implications for management of agricultural runoff and other open-water contaminants.
  • Other examples of cetacean viruses that likely originated in bovids – Tursiops truncates parainfluenza virus 1, bovine enterovirus.
• BdAdV-2 appears more closely related to harbor porpoise adenovirus 1 than to BdAdV-1, most consistent with host jumping between cetacean spp.

A sensitive and specific qPCR was developed for rapid diagnostic evaluation of potentially infected individuals.

Take home: second novel bottlenose dolphin adenovirus – BdAdV-2 – may represent host-jumping event

Question 87

A recent study investigated the prevalence of pseudaliid lungworms in cetaceans.

What are some genera of these worms?

What are typical clinical signs in cetaceans? Where do infestations occur?

How does transmission occur?

How prevalent were these worms in stranded striped dolphins?

Answer 87

Pool, R., Chandradeva, N., Gkafas, G., Raga, J. A., Fernández, M., & Aznar, F. J. (2020). Transmission and Predictors of Burden of Lungworms of the Striped Dolphin (Stenella coeruleoalba) in the Western Mediterranean. Journal of Wildlife Diseases, 56(1), 186-191.

Abstract: Pseudaliid lungworms infect the lungs and sinuses of cetaceans. Information on the life cycle and epidemiology of pseudaliids is very scarce and mostly concerns species that infect coastal or inshore cetaceans. Available evidence indicates that some pseudaliids are vertically transmitted to the host, whereas others are acquired via infected prey. We documented pseudaliid infections in an oceanic cetacean, the striped dolphin (Stenella coeruleoalba) in the western Mediterranean, and investigated the possibilities of vertical vs. horizontal transmission and the potential influence of host body size, sex, and season on infection levels. We found two species of lungworm in 87 dolphins that stranded along the Spanish Mediterranean coast between 1987 and 2018. One or two larvae of Stenurus ovatus were found in three adult dolphins. Larger numbers of larvae and adults of Skrjabinalius guevarai were collected in 51 dolphins, including unweaned calves. These observations suggested that Skrjabinalius guevarai could be vertically transmitted. The abundance of Skrjabinalius guevarai increased significantly with host size, which suggested that it could be trophically transmitted, as well, with larger hosts consuming more infected prey. Infection levels peaked in spring, outside of the calving season, which is likely a reflection of a seasonal shift in dolphin diet. In summary, results indicate that Skrjabinalius guevarai was capable of both vertical and horizontal transmission, but future research should be directed at clarifying the potential mechanics behind transmission and intermediate hosts.

• Pseudaliidae – Evolutionairly distinct family of metastrongyloid lungworms, almost exclusively infect cetaceans.
  
  • Lungs and cranial sinuses.
  • May result in lesion sin the cranial sinus, verminous pneumonia.
  • Vertical and horizontal routes of transmission have been proposed.
  • Halocercus – Has been found in fetuses and suckling calves. Poss vertical transmission.
  • Pseudalis inflexus found in wild dab, prey of harbor porpoises, support horizontal transmission.

MM: 87 striped dolphins stranded along the Med coast of Spain, analyzed lungs for worms via light microscopy, estimated total worm burdens. Evaluated between age classes. Investigated relative importance of vert and horiz transmission by comparing burdens between neonate (feeds on milk) suckling (milk and prey) and weaned (prey).

• All infections of Stenurus ovatus (n=4) were composed of larvae only.
• Skrjabinalius guevarai found in 59% (n=87). One neonate – strongly suggested vert transmission. Transplacental vs consumption of infected milk or contact with contaminated spray during breathing or feeding.
  
  • Neither prevalence nor abundance differed significantly among age classes.
  • Lungworm infection somewhat higher in males, highest in spring followed by winter, summer, and autumn.
    
    • May be related to calving season.

Takeaway: Pseudaliid lungworms infect sinuses and lungs of cetaceans, lifecycle unknown, suspected vertical and horizontal transmission, may be more common in Spring and coincide with calving season.

Question 88

A recent study evaluated the effect of inflammation on immunomediator gene transcription in beluga whales.

What are some pro-inflammatory immunomediators? What are some anti-inflammatory ones?

How did the beluga with the nonhealing wound and the one with gastritis compare to control whales?

Answer 88

IMMUNOMEDIATOR GENE TRANSCRIPTION PROFILING IN BELUGA WHALE (DELPHINAPTERUS LEUCAS) CLINICAL CASES.

Hofstetter, A.R., Van Bonn, W. and Sacco, R.E.

Journal of Zoo and Wildlife Medicine, 2020;51(2):334-349.

There is an unmet need for specific diagnostics of immune perturbations and inflammation in beluga whale (Delphinapterus leucas) clinical care. Quantitative real-time polymerase chain reaction (qPCR) has been used to measure immunomediator gene transcription in beluga whales. The study hypothesis was that a qPCR-based immunomediator assay would supplement routine clinical data with specific and sensitive information on immune status. Two beluga whale clinical cases provided an opportunity to test this hypothesis: a whale with a skin laceration and a whale with gastrointestinal inflammation. Mitogen-stimulated immunomediator gene transcription (MSIGT) was compared between the cases and healthy contact whales. In both case studies, mitogens increased transcription of IL1B, PTGS2 (Cox-2), TNF, HIF1A, and IL2 but decreased IL10 transcription in peripheral blood mononuclear cells (PBMC) from the abnormal whale over the control. Correlations were identified between most immunomediators tested and one or more standard blood clinical values. Considering all 15 immunomediators tested, the whale with gastrointestinal inflammation had a more unique MSIGT signature than the whale with a laceration. These results support further elucidation of beluga whale PBMC cytokine profiles for use as immune biomarkers.

Background

• qPCR to assess potential of mononuclear cell potential for gene transcription of cytokines in cetacean blood after mitogen stimulation
  
  • mitogen stimulated immunomediator gene transcription (MSIGT)
• Assay panel of 15 immunomediators:
  
  • Pro-inflammatory: IL-1B, IL-12p40, IL-18, IFN-y, TNF-a
  • Anti-inflammatory: IL-4, IL-10, IL-13, TGF-B
  • IL-2: growth factor
  • IL-6: pleiotropic effects
  • PTGS2: encodes Cox-2, inflammatory downstream of IL-1
  • TLR-4: pathogen pattern recognition receptor
  • HIF-1a: transcription factor regulating oxygen hemostasis
    
    • CXCR4: target of HIF-1a, chemokine receptor for immune cell trafficking, vasculogenesis, angiogenesis

Key Points

• 4 beluga whales: 2 sick each with a control
  
  • Whale A (4 yo F, normal in captivity) compared to whale B (35 yo F wild-born, nonhealing wound)
    
    • Two time points: 1 during healing, 1 after complete resolution
  • Whale C (14 yo M, normal in captivity) compared to whale D (7 yo M, captive, GI strangulation from congenital anomalous vessel, peritonitis and died)
    
    • One time point during illness
• Maximum MSIGT observed at 5hr with similar or decreased levels at 18 hr
  
  • IL18, TGFB1, CXCR4, and TLR4 rarely differed from unstimulated cells more than 2 fold
  • Con A (mitogen) was better for IL12B vs PHA (mitogen) was better from TNF and IL6
  • Greatest MSIGT: IFNG, IL13, IL6, IL1B, TNF, IL12B
  • Least or decreased MSIGT: IL2, CXCR4, HIF1A, IL10, IL18, TGFB1, TLR4
  • IL4 and PTGS2 varied based on aquarium
• MSIGT profile of whale B during treatment differed from after resolution and from control whale A
  
  • Increased IL1B, PTGS2, TNF, HIF1A, CXCR4, IL2, and IL13
    
    • IL13 anti-inflammatory and CXCR4 for angiogenesis were unique to this whale, may reflect wound healing response
  • Decreased IL10
  • After resolution: IL2 was increased and IL13 was decreased from whale A samples
• MSIGT profile of whale D differed from control whale C
  
  • Increased: IL1B, PTGS2, TNF, IL12B, IL6, HIF1A, IL2
  • Decreased IL10
  • No change in IL18 vs decreased in whale C
  • Whale D differed from all other whales: highest IL6, IL12B, PTGS2 and lowest IFNG, only one to have slight increase for TLR4
• Similar trends between the two affected whales vs controls:
  
  • Increased IL1B, PTGS2, TNF, HIF1A, and IL2
  • Decreased IL10 MSIGT
• All immunomediator genes correlated with at least one blood value except TFGB1
  
  • PTGS2 positive correlation with MCH, negative correlation with hematocrit
  • IL10 positive correlation with serum iron
  • IL12B positively correlated with MCH
  • IL13 positively correlated with monocyte %

Conclusions

• Mitogen-stimulated immunomediator (cytokine) gene transcription of peripheral blood mononuclear cells detected by qPCR of beluga whale blood may be an indicator of inflammatory response
• Maximal cytokine transcription occurred after 3 hr incubation with mitogens
  
  • 18hr incubation had similar or slightly reduced levels as 5hr incubation
  • IL1B, PTGS2, TNF, HIF1A, IL2 (increased) and IL10 (decreased) varied from normal controls in both cases and most correlated with serum iron and A/G ratio
  • IL18, TGFB1, CXCR4, and TLR4 rarely differed from unstimulated cells more than 2 fold

Question 89

A recent study evaluated the correlation of traditional inflammatory markers in cetaceans with acute phase proteins.

What are some classic markers of inflammatory disease in cetacans?

What are acute phase proteins?

How did serum amyloid A and haptoglobin compare to other inflammatory markers?

How did the APP differ between free-ranging and managed dolphins?

How did they differ between healthy and unhealthy dolphins?

Answer 89

SERUM ACUTE-PHASE PROTEINS IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) AND CORRELATION WITH COMMONLY UTILIZED INFLAMMATORY INDICES

JZWM 2020 51(3) 657-662

Abstract:

Acute-phase proteins (APP) are the foundation to the innate immune response and valuable biomarkers that increase with inflammation, infection, neoplasia, stress, and trauma.2,4,16 Little is known about the acute-phase response in cetaceans and if these proteins can be used for health monitoring in individuals and free-ranging populations. The purpose of this study was to characterize serum concentrations of haptoglobin (Hp) and serum amyloid A (SAA), as well as electrophoretic profiles of common bottlenose dolphins (Tursiops truncatus) in free-ranging (n = 33) and professional care (n = 27) settings. Results were correlated to commonly utilized inflammatory indices including erythrocyte sedimentation rate (ESR), fibrinogen, total white blood cell count (WBC), and absolute neutrophil count. SAA levels, measured with a dolphin-specific enzyme linked immunosorbent assay (ELISA), were significantly higher (P = 0.05) in free-ranging dolphins (mean = 4.26; SE = 1.12) when compared with those under professional care (mean = 1.82; SE = 0.45). For dolphins under professional care, a statistically significant correlation was identified between ESR and Hp (P < 0.001; r = 0.69), ESR and SAA (P < 0.001; r = 0.67), fibrinogen and Hp (P = 0.001; r = 0.58), and fibrinogen and SAA (P = 0.002; r = 0.56). In addition, there was a significant correlation between WBC and SAA (P = 0.01; r = 0.38) and absolute neutrophil count and SAA (P = 0.04; r = 0.32). There were no significant correlations between study variables observed in free-ranging dolphins. The variable correlation of APPs with commonly utilized inflammatory indices demonstrates that these proteins are independent measures of inflammation with unique sensitivity, specificity, and timeline of expression. The results of this study contribute to improved health monitoring of dolphins and have the potential to assist in identification of compromised health.

Summary:

· evaluation of inflammation in cetaceans – WBC count, absolute neutrophil count, erythrocyte sedimentation rate (ESR), fibrinogen

· Acute phase proteins (APP)

o innate immune response

o biomarkers - increases with inflammation, infection, neoplasia, stress, trauma

o acute-phase response - dynamic process involving systemic and metabolic changes providing an early nonspecific defense mechanism against insult before specific immunity achieved

o has been used as prognostic indicator in some cases

· objectives:

o characterize plasma concentrations of two positive APP, haptoglobin (Hp) and serum amyloid A (SAA), and electrophoretic profiles of bottlenose dolphins under professional care and free-ranging populations

o assess correlations with traditional inflammatory markers utilized in cetacean medicine (ESR, fibrinogen, WBC, and absolute neutrophil count)

· M + M

o Blood collected from captive and free ranging bottlenose dolphins

§ dolphin-specific ELISA for SAA

§ Hp measurements using Tridelta colorimetric method

§ electrophoresis measurements using Helena SPIFE Split Beta gels

§ CBC, ESR, and fibrinogen also measured

· Results/discussion

o free-ranging dolphins had significantly higher concentrations of 10/13 measured inflammatory markers compared with healthy captive dolphins 🡪 SAA

§ no significant difference in Hp (different expression timeline than SAA)

o unhealthy captive dolphins had significantly higher levels of multiple inflammatory indicators but not APP 🡪 alpha-1 globulins, globulins, absolute neutrophils

§ no significant elevation in SAA or Hp (positive APP)

§ no significant difference in albumin (negative APP)

o captive dolphins:

• § positive correlations between*
• · ESR and Hp*
• · ESR and SAA*
• · fibrinogen and Hp*
• · fibrinogen and SAA*

§ significant correlation between:

· WBC and Hp

· WBC and SAA

· absolute neutrophil count and Hp

· absolute neutrophil count and SAA

o previous study - SAA concentrations higher during third trimester and postpartum period in pregnant bottlenose dolphins

Question 90

A recent study evaluated the changes in respiratory function in dolphins stranded versus those in the water.

How did tidal volume and respiratory flow differ between the groups?

What are some stranded associated health effects of cetaceans?

Describe the respiratory cycle of cetaceans.

How do dolphins compensate while on land?

Answer 90

Journal of Zoo and Wildlife Medicine, 2021. 52(1): 49-56

RESPIRATORY CHANGES IN STRANDED BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS)

Andreas Fahlman, PhD, Micah Brodsky, VMD, Julie Rocho-Levine, BSc, Daniel Garcia-Parraga, LicVet, Dipl ECZM(ZHM), Dipl ECAAH(N-P), Marina Ivancic´, DVM, DACVR, Carlos Camarena, Leonardo Ibarra, MVZ, and Joan Rocabert, MSc

Abstract: Lung function (breath duration, respiratory flow [V], and tidal volume [VT]), and end-expiratory O2 were measured in 19 adult bottlenose dolphins (Tursiops spp.) while at rest in water or beached for up to 10 min. The results show that inspiratory VT, expiratory VT, or inspiratory V did not differ on land or in water. The average expiratory V for all dolphins on land decreased by 16%, and the expiratory and total breath durations increased by 5% and 4%, respectively, compared with in water. There were temporal changes observed during beaching, where expired and inspired VT and inspired V decreased by 13%, 16%, and 9%, respectively, after 10 min on land. These data suggest that dolphins compensate for the effect of gravity by adjusting respiration to maintain alveolar ventilation and gas exchange, but during extended durations, the increased work of breathing may impede ventilation and gas exchange. Continuous monitoring of lung function and gas exchange may help prevent long-term damage during out-of-water medical procedures, optimize animal transport conditions, and improve survival during stranding events.

Key Points:

• In cetaceans, when on land, the weight of the body pushes down on the highly compliant thorax which may have cardiorespiratory effects
  
  • Stranding associated health effects - hyperthermia, dehydration, suffocation, hypoxia, abrasions, sunburn, cardiomyopathy, skeletal muscle myopathy
  • Respiratory tract disease identified in as many as 20% of stranded dolphins
• Respiratory cycle - begins with expiration followed by immediate inspiration and respiratory pause. The elastic recoil of the chest is responsible for emptying the lung. Inhalation is an active process
• Lung function testing may be an effective diagnostic tool in stranding situations, during transport or medical intervention
  
  • Objective – respiratory flow (V), and end-expired O2 concentration were measured while inactive in water or up to 10 minutes on land.
    
    • Placement of pneumotachometer over the blowhole (trained behavior) while in water or when slid out on dry area next to the habitat.
• There were no differences in the overall average inspiratory VT, expiratory VT, end expired O2, or inspiratory V between land and water, however significant temporal changes occurred
• Average expiratory V for all dolphins on land decreased, and the expiratory and total breath durations increased compared with in water
• Expired and inspired VT and inspired V decreased after 10 min on land, but there were no changes in end-expired O2 concentration - despite reduced tidal volume, dolphins compensated their alveolar ventilation to prevent hypoxia.
  
  • The tidal volume decreased progressively with time
  • Reduced inspired tidal volume caused by reduction in inspired respiratory flow because of gravity

Take home:

There are progressive reductions over time in respiratory capacity during beaching in bottlenose dolphins. However, there were no changes in expired O2 levels during the 10-minute time limit of this study, thus the dolphins were able to compensate for these reductions through their respiratory functional reserve.

Question 91

A recent study evaluated the use of human recombinant granulocyte colony-stimulating factor (filgrastim aka neupogen) in neutrophenic cetaceans.

What are some causes of neutropenia in cetaceans?

What is the function of granulocyte colony-stimulating factor?

How did the neutropenic cetaceans that were administered this product do?

Were they species, dose, or route dependent responses?

How long did neutrophil counts remain elevated?

Any adverse effects?

Answer 91

EFFICACY OF HUMAN RECOMBINANT GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF, FILGRASTIM; NEUPOGEN®) IN NEUTROPENIC CETACEANS

Journal of Zoo and Wildlife Medicine 52(3): 1042–1053, 2021

Abstract: Neutrophils are one of the initial cell lines of protection against pathogens, and when their concentrations in the blood are low, animals are highly susceptible to infections. Neutropenia has been reported in cetaceans secondary to administration of systemic sulfa antibiotics or antifungal medications and severe, overwhelming infection. Filgrastim was administered to treat neutropenia over a 15-y period in 11 cetaceans comprising four species—beluga (Delphinapterus leucas, n ¼ 1), bottlenose dolphin (Tursiops truncatus, n ¼ 4), killer whale (Orcinus orca, n ¼ 5), and short-finned pilot whale (Globicephala macrorhynchus, n ¼ 1)] ranging in age from 1 wk to .24 y. Seven study animals received multiple doses (2–6). All animals responded to at least one dose (1–7 lg/kg) of parenteral filgrastim characterized by an increase in peripheral immature (band) neutrophils, segmented neutrophils, or both. In most cases (9/11), neutrophil counts increased within 48 h of a single dose. Duration of response varied but was at least 2 wk in eight of the 11 animals and 5–9 d in the remaining animals. No adverse reactions were observed in any cases.

Intro

• Neutropenia has been reported in cetaceans secondary to systemic sulfa antibiotics,2 ketoconazole, and micafungin use and to severe infection
• In adult mammals, granulopoiesis is facilitated by several mediators, including granulocyte colony-stimulating factor (G-CSF), a glycoprotein cytokine
• Filgrastim (Neupogent, Amgen Inc, Thousand Oaks, CA 91320, USA), is a human recombinant G-CSF effective in increasing peripheral neutrophil counts in numerous mammalian species
• This case series describes the use of filgrastim to treat neutropenia in 11 cetaceans over a 15-y period

Case reports

• Beluga
  
  • 1 week old calf with FPT, treated with 2 doses of neupogen, neutropenia resolved (but calf ultimately died)
• Bottlenose dolphins
  
  • Tt1-adult female, mild signs of illness along with neutropenia (cause of illness undetermined), treated with two doses of neupogen, marked increases in neutrophils within 24 hrs, hemogram remained normal 1 month after tx
  • Tt2-subadult female being treated for severe gastroenteritis, developed neutropenia, treated with 1 dose of neupogen—neutropenia resolved within 48h but returned, neupogen administered at a higher dose and neutropenia resolved, clinical illness resolved
  • Tt3-adult female, chronic inflammatory disease, developed neutropenia-neupogen SID for 5 days—WBC count did not increase after the first dose, day 6 bands increased but were still low, by day 13 tWBC and neutrophil count were normal
  • Tt4-subadult male, gastritis, dermatitis, systemic inflammation and progressive neutropenia. Treated with one dose of neupogen (also on abx, PPI, and antifungals) – resolution of neutropenia within 48 hr, maintained for up to 49 days after
• Killer whales
  
  • Oo1 – adult male, lethargy and inflammatory blood profile progressing to neutropenia. One dose of neupogen along with abx, antifungals, analgesics, glucocorticoids and gastroprotectants – tWBC increased to normal after 48hr
  • Oo2 – adult female, panleukopenia after pneumonia diagnosis – treated with one dose of neupogen, seg neutrophils and tWBC normalized after 48 hr, neutropenia recurred five months later and was treated again, worked again within 48 hr
  • Oo3-adult male, chronic pneumonia, developed panleukopenia – neupogen given once daily for 6 days, twbc remained static, but band and seg counts increased mildly after the first dose and normalized after the fourth dose
  • Oo4- adult female chronic occult inflammation, developed pancytopenia, treated with neupogen once, twbc and neutrophil counts started to increase after 24 h and were normal by 5 days post treatment
  • Oo5 – juvenile male, nonspecific clinical signs with nonregenerative anemia and mild neutropenia – one dose of neupogen and abx, within 48 h tWBC, seg and band counts were normal
• Pilot whale
  
  • Gm1-subadult female, occult inflammation, developed progressive neutropenia, two doses of neupogen and a third dose 21 days after the second, within 72h of the first 2 doses and 96h of the third dose tWBC and seg counts had risen

Summary

• All cetaceans in this report responded to filgrastim, and responses appeared to be neither species dependent nor dependent upon the route (SC, IM, or IV), order, or number of filgrastim doses.
• In 82% of cases (9/11), response occurred within 24–48 h of a single dose of filgrastim
• In 91% of cases (10/11) the response was characterized by an initial moderate to robust increase in neutrophil progenitor cells in conjunction with, or followed by, an increase in segmented neutrophils
• For most cases in this report (73%; 8/11), neutrophil counts remained normal or elevated for at least 2 wk after the last filgrastim dose was administered.
• No immediate or long-term adverse reactions to filgrastim were seen.

Discussion

• The 11 cetaceans included in this report had variable signalments, clinical presentations, treatments, and filgrastim administration regimes, yet all of them responded to filgrastim.
• In some species (cattle, human and nonhuman primates) response has been reported to be dose dependent, whereas in others (cats) it has not.9 A clear dose-dependent pattern was not seen in the cetaceans of this report
• Neutrophil counts remained normal or elevated for at least 2 wk after the last filgrastim dose was administered to the majority of cetaceans in this report (73%; 8/11).
• No adverse reactions to filgrastim were seen in the cetaceans of this report or other publications

All animal survived except the FPT neonate

Question 92

A recent study evaluated the social behavior of orcas in managed care.

What are some affiliative behaviors observed?

What about conciliatory behavior?

Answer 92

Zoo Biology. 2019; 38:323–333.

Social interaction analysis in captive orcas (Orcinus orca).

Paula Sánchez–Hernández | Anastasia Krasheninnikova2 | Javier Almunia |Miguel Molina–Borja

Abstract: The management of socially complex species in captivity is challenging. Research on their social behavior improves our understanding of interactions in captive animals and captive‐group management. We conducted a detailed analysis of social relationships shown by the orcas kept at Loro Parque zoo and their tendency to reconcile after aggressive episodes. Affiliative interactions were the most frequent social activities compared to agonistic or sexual interactions. Within affiliative behaviors, we documented the pattern “gentle tongue bite”, where an animal touches the other’s tongue with his teeth but does not bite it. Affiliative interactions between a specific pair of orcas occurred significantly more often than expected by chance, and together with low levels of agonistic interactions, indicated particular affinity between some individuals. The most frequently observed low-level agonistic relationship was that of the two older males (Tekoa–Keto); however, they also showed frequent sexual and affiliative interactions. Sexual-like behaviors (pursuit, mount, and penis between males) were found in both sexes. Finally, the observed corrected conciliatory tendency (31.57%) was within the range described for other primate and cetacean species. This study provides a systematic way to assess social interactions as well as conflict management strategies in cetaceans housed in zoos and zoo-like facilities and may help to improve animal welfare and management of animals in controlled environments.

Take Home: Orcas have complex social behaviors including the ability to reconcile after agonistic interactions.

Question 93

A recent study evaluated the effect of swim with dolphin programs on visitors.

How is dolphin behavior affected by these programs?

Do the number of participants affect their behavior?

What effect do these programs have on participants?

Answer 93

Zoo Biology 40: 551-562, 2021

VISITOR ATTACHMENT TO DOLPHINS DURING AN INTERACTION PROGRAMME, ARE THERE IMPLICATIONS TO DOLPHIN BEHAVIOR?

Thomas Welsh, Samantha Ward – Reviewed by BCJ

Abstract

Millions of people visit zoos and aquariums globally each year, with a smaller number choosing to participate in animal interaction programmes which allows visitors closer contact with individual animals. These are reportedly having mixed effects in increasing conservation‐related behaviors. Human‐animal interactions (HAIs) during these programmes are generally positive experiences for the human participants, however are there behavioral implications for the animals involved? The Bottlenose Dolphin (Tursiops truncatus) is the most widely used cetacean for dolphin interactions, known as “swim with dolphin” (SWD) programmes. This study investigated visitor attachment to the dolphins they interacted with, whilst assessing behavioral implications of the dolphins. A total of 41 visitors to a Spanish dolphinarium, who participated in a SWD were surveyed using a modified version of the Lexington Attachment to Pets Scale. Alongside this, 15-min continuous focal samples monitored three female dolphins (D1, D2, and D3) aged 22–40, split into pre (n = 96), during (n = 96) and post (n = 96) SWD. 80% of visitors reported a sense of attachment to the dolphin they interacted with. An exploratory factor analysis extracted three factors from the survey, these were “relationships,” “emotional attachment,” and “non-attachment.” A Friedmans two-way analysis of variance produced significant results for some behavior categories for each individual, including locomotory (D1: F2 = 9.556, p < .01), rest (D2: F2 = 14, p < .01, D3: F2 = 10.889, p < .01) and individual play (D1: F2 = 11.677, p < .01 D2: F2 = 6.353, p < .05) however, pairwise comparison showed no differences pre-post SWD. In this context it can be implied that participating in the SWD was neither enriching nor aversive for the individual animals, although due to the small sample size further research is required. As visitors reported a sense of attachment post HAI, this can have applications in improving conservation education during SWD. This study has provided scope for further research into methods that facilities can use to utilize the emotional attachment developed to individual animals to facilitate learning about conservation issues.

Key Points: After one interaction, 80% of respondents reported a sense of attachment with the dolphin that they had interacted with

· Females indicated a stronger sense of attachment

· Repeat visitors are more likely to participate in conservation-related behaviors

· Increasing visitor numbers did not have adverse effect on behavior

Take-Home Message: Animal encounters such as SWD have a place in zoos to elicit a positive emotional response from visitors which, with further research can be used as a factor to improve proconservation mindedness

References:

· Collins, C., Corkery, I., Haigh, A., McKeown, S., Quirke, T., & O’Riordan, R. (2017). The effects of environmental and visitor variables on the behavior of free‐ranging ring‐tailed lemurs (lemur catta) in captivity. Zoo Biology, 36(4), 250–260. https://doi.org/10.1002/zoo.21370

Question 94

A recent study described recurrent pleural effusion in a Bottlenose dolphin.

How did this animal present?

How was it managed?

What serum markers corresponded with volume of pleural effusion?

What was the suspected etiology of the effusion?

Answer 94

Chronic nonchylous lymphatic pleural effusion in a bottlenose dolphin (Tursiops truncatus)
Russell et al
Abstract
CASE DESCRIPTION
A 19-year-old male bottlenose dolphin (Tursiops truncatus) presented with inappetence and avoidant behavior.
CLINICAL FINDINGS
Ultrasound revealed a large-volume left-sided pleural effusion, which was consistent with chronic nonchylous lymphatic
effusion and mild chronic hemorrhage by cytology. Computed tomography identified ipsilateral rib fractures,
atelectasis, nodular pleuritis, marginal lymph node enlargement, and suspected dilation of the thoracic duct and internal
thoracic veins. Fifteen lipids were significantly higher in serum of the dolphin as compared with controls (n = 3) using
nontargeted lipidomics.
TREATMENT AND OUTCOME
A series of thoracentesis procedures were performed. Follow-up CT demonstrated marked reduction in pleural effusion
with persistence of thoracic duct dilation and mass-like areas of pleural thickening. Ultrasonographic resolution of
pleural effusion occurred 14 months after presentation; however, recrudescence was noted 5 months later. Over a total
of 24 months, 21.52 L of pleural effusion was removed. Despite the presence of pleural effusion, the patient was clinically
stable during this time and quality of life was considered good on the basis of continuous animal welfare evaluations.
Humane euthanasia was elected following acute clinical decline 27 months after initial diagnosis. Necropsy confirmed
severe pleural effusion, chronic severe pleural fibrosis with chronic hemorrhage, and mediastinal fibrosis with entrapped
lymph nodes and thymic tissue.
CLINICAL RELEVANCE
Pleuritis and effusion were suspected sequelae of previous rib fractures. To our knowledge, this is the first report of
nonchylous lymphatic pleural effusion with repeated pleural drainage and diagnostic imaging for clinical management in a
bottlenose dolphin.

Key Points:
- Throughout treatment, SAA concentration was the only abnormal serum marker and concentrations were
positively associated with amount of pleural fluid seen ultrasonographically
- Each procedure – diazepam Po then IV midazolam
- Drainage site selection was based on maximum depth of fluid on ultrasound (approx 20 cm), approximately between the 7th to 9th rib spaces, as for domestic mammals.
- Drainage was performed 7 times over a 24-month period, by use of the technique described, with a total volume
of 21.52 L removed.
- Necropsy revealed healed mid-diaphyseal
fractures of the fourth and fifth ribs
- It is unclear why recrudescence occurred, but it is speculated that the severity of pleural fibrosis resulted in impaired lymphatic and
venous return.

Question 95

What are the common health conditions of stranded small cetacean?
- What disease processes have been linked to mass strandings?
- What respiratory conditions are common?
- What neurological conditions are common?
- What are some of the common traumatic lesions?

What are some of the problems from being stranded on land?

What are some poor prognostic indicators for stranded cetaceans?

What are the definitions of success for post-release?

Answer 95

Stranded Small Cetaceans

Common Health Conditions
* Systematic examinations of live and dead stranded marine mammals provide morbidity and mortality data to inform local clinical case management as well as global ocean health matrices and conservation policies
* Disease is more common in single stranded than mass stranded cetaceans, but mass mortalities have been linked to harmful algal blooms, primarily in North America, and Morbillivirus outbreaks in both hemispheres
* Pneumonia- common in stranded cetaceans
– may be recognized in the field through physical examination, pulmonary ultrasonography, or portable radiography, with bacterial, viral, fungal, parasitic, or mixed etiologies
* Neurological conditions including various encephalitides, age-related neurodegeneration, vestibulocochlear neuritis, and pterygoid sinusitis with middle and inner ear involvement
* Mild to moderate gastrointestinal parasite burdens are common but are rarely of clinical significance
* Trauma from rough substrates (e.g., rocks, oyster shells) or predation (e.g., from gulls, canids) cause lacerations and abrasions, most of which would not significantly compromise an animal clinically but may elevate stress
– Ocular and blowhole bird predation may be debilitating in some cases
* Clinical problems from being on land include:
– Visceral compression, poor end-organ perfusion, and vascular shunting, poor respiratory exchange due to pulmonary edema, thoracic compression, and exhaustion of respiratory muscles; dehydration if stranding is prolonged; capture myopathy including exertional myopathies, stress cardiomyopathy, and myoglobinuric nephrosis; and shock
- An initial shock assessment looking at overall responsiveness, palpebral reflex, mucous membrane color and capillary refill time, percent dehydration, and heart and respiratory rates and rhythms indicates if emergency treatments are warranted

Prognostic Indicators
- Prognostic indicators from physical examination and blood analyses were determined for stranded short-beaked common dolphins (Delphinus delphis) based on postrelease monitoring efforts
- Dolphins that died were thinner, more anemic, acidemic, and dehydrated than those that survived
- Also had evidence of liver disease (elevated gammaglutamyl transpeptidase and alanine aminotransferase) and capture myopathy (elevated aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and lactate)

Post-release success
- Targeted focal follows of healthy satellite tagged single stranded short-beaked common dolphins found that they were capable of reintegrating into social groups after being relocated and released alone
- Success criteria for released, tagged small odontocetes have ranged from 3- to 6-week minimums
- Differences in species (specifically, dorsal fin thickness impacting tag retention), environment, as well as tag design and programming will affect the duration of satellite tag transmissions

Question 96

Describe the management of stranded large whales.

What is the leading cause of mortality for the North Atlantic Right Whale?

Describe the physical examination of a large stranded whale?
- Where can blood be collected from?
- When is euthanasia indicated?

In the rare cases a large stranded whale appears healthy enough for rescue and release, how do you go about doing that?

Answer 96

Stranded Large Whales

Mortality Data and Conservation
- Necropsy data confirm that many large whales are victims of anthropogenic trauma
- Entanglement in fishing line and vessel strikes are the leading causes of death for the critically endangered North Atlantic right whale
- Large whales also strand with evidence of both infectious and noninfectious diseases

Live Stranded Large Whales
- Stranding-induced conditions that were previously outlined for small cetaceans are exacerbated by whales’ large size and the resulting gravitational effects
- Operating around large whales presents tremendous risks to humans; helmets and other personal protective equipment should be worn at all times and every effort made not to place people in jeopardy

Physical examination of large whales will be limited due to their size and human safety concerns
- The examination should start visually from a distance, noting degree of submersion, overall alertness of the animal, behavior, skin condition (e.g., blistering, sloughing), body condition, injury (e.g., propeller or entanglement wounds, entanglements, shark bites), musculoskeletal abnormalities (e.g., concavity of epaxial muscles, scoliosis of the spine), and respiratory rate, rhythm, and quality
- Trends in respiration rate and quality should be continuously monitored and documented
- If safe to approach the animal, evaluate palpebral reflexes, jaw tone, adduction of pectoral flipper in response to manual abduction, and reflexive closure of the blowhole in response to manual probing
- Blood may be drawn from the vascular bundle between the radius and ulna
- Euthanasia or palliative care are the most appropriate for cases that are thin, emaciated, minimally responsive, those with rapid, shallow, or weak respirations, or those with no viable rescue options available

Priorities of supportive care include maintaining an airway, monitoring breathing, moderating body temperature, and providing protection from the sun, wind, and scavengers.
- In rare cases when stranded whales are apparently healthy, and options exist, a rescue may be attempted
- Methods appropriate for large whales include flotation, trenching/dredging, and towing; often a combination is necessary
- Whales should not be towed backwards by attaching a line or strap around the peduncle to avoid trauma to the spinal cord, vertebral column, and potentially the pectoral flippers
- Forward towing using a rapid release axillary bridle, allowing the whale to swim off gear-free, is likely the best towing approach

Question 97

Describe the euthanasia of large cetaceans.

What conditions would warrant euthanasia in a large whale?
- What is the time frame from the time of stranding before odds of succdssful release diminish rapidly?

What are some of the challenges of euthanizing large whales?
- How do you reliably estimate body weight?
- What are some risks associated with barbiturate administration?
- What sedation protocols are recommended?
- How do you euthanize a large whale?
- What injection sites can be considered for euthanizing a large whale?
- What size needle is needed for IC injection? And what are the landmarks?

What are some of the typical responses of large whales during euthanasia?

What physical methods of euthanasia can be considered?

What considerations for the public need to be considered?

Answer 97

Large Whale Euthanasia

“Stranded”: animal is found dead or live on land, is found in shallow water or otherwise out of normal habitat or in need of medical attention
- Human safety is top priority in any stranded whale response
- Hazards: drowning, blunt or crushing trauma from flukes or pectoral fins, foot or leg entrapment under animal, zoonotic disease, drug exposure, needle sticks, knife cuts, ballistics or explosives, slippery footing, high tension lines, heavy equipment, exhaustion, hyperthermia and hypothermia, death

Euthanasia
- Conditions supporting euthanasia may include the following: emaciation, serious disabling locomotor injuries, wounds that involve a large percentage of surface area or that have full penetration into the thoracic or abdominal body cavity, blistering and scavenger damage to an extensive surface area, or critical areas: eyes, blowhole; significant hemorrhage from any orifice, loss of reflexes at anus, genital opening, blowhole, tongue, eyelids or eyes, other signs of neurologic abnormalities, marked prolonged hypothermia or hyperthermia, extended length of time beached (over 24 to 48 hours)
- Gravitational effects= respiratory and circulatory collapse
- Refloating could cause more stress to terminal animal
- Allowing a stranded whale to expire naturally may be reasonable and should always be considered- however, pending on the animal it may be ideal to end the suffering

Beyond 3-4 tides after stranding- odds for successful either assisted or self rescue begin to diminish rapidly
“Humane Death”: minimized pain, distress and anxiety prior to loss of consciousness and results in rapid unconsciousness followed by cardiac or respiratory arrest

Large whales pose unique challenges= drug volumes involved, drug administration, costs, potential for relay toxicity, and difficulty applying physical methods effectively
- Reasonable accurate estimate of body weight is essential for appropriate dosing - app WhaleScale- along with a tape measure to help with length
- Administering preeuthanasia sedation and analgesic drugs decreases risk to personnel working closely- reduces pain/distress, and may sometimes suffice to effect euthanasia themselves in severely debilitated animals
- NOTE: barbiturates pose a risk for relay toxicity to scavengers and are environmentally persistent- therefore if proper disposal is not possible their use is precluded

Original Protocol
- Pre- euthanasia: midazolam, acepromazine, xylazine - now butorphanol is added)
- Intracardiac saturated KCl solution
- Xylazine and alpha-2= associated with excitement in cetaceans
- Lidocaine local block at the IC injection site is not required if pre-euthanasia drugs have been administered to proper effect but is simple and can provide additional layer of analgesia
- Long needles needed to get through skin and blubber = Custom 31 to 55 cm x 16 to 18 G needles
- Intravascular access can sometimes be achieved in pectoral fin vessels (between radius and ulna)
- Vascular access at fluke vessels or caudal peduncle is HAZARDOUS
- IC injections: long robust needles, and pressurized plastic canister for administration of high volume
- Access point: low on the body wall via right or left axillary spaces
- Ventrally from parasternal approach

Once completed- insert hand behind lips or into blowhole for sensation (this will not be tolerated normally- clamp shut)
- Death- terminal muscle activity “last swim”, arching and guttural exhalation
- Warn bystanders of these movements
- Death can also be confirmed by ECG or lack of cardiac excursions via the IC needle left in place after injection
- May be best to trim IM injection sites for separate disposal to reduce risk to scavengers

Physical Methods: HAVE to quickly destroy brain or brain stem to be considered humane
- Shaped explosive charges caudal to the blowhole directing a shock wave into the brainstem are highly effective for euthanizing cetaceans over 7 m length
- This is effective but takes time to set up- administration of sedation could be beneficial
- Keep thorough records of animals condition prior to euthanasia
- Public education vs visual barriers

Question 98

Describe the necropsy of large cetaceans.

Why is time of the essence in these cases?

How many people are needed to conduct the necropsy? How should teams be divided?

What windows should be made for best organ access?

How should external lesions be examined?

How is deep trauma determined?

Answer 98

Large Whale Necropsy
- Once euthanized the carcass starts to heat up instead of cool down due to insulative blubber layer- making a “pressure cooker”
- Cause of Death necropsies are traditional focus; BUT a large whale necropsy provides an opportunity to gather data on life history and health of the species
- Note: most countries had specific permits to collect and possess tissues from endangered whales- there are legal requirements!
- Split into teams: prosecting team and sampling team
- Prosecting: quickly gets carcass open- efficiently removing large amount of tissue
- Sampling: collecting tissue samples

Examples:
- 5 m neonate whale= 4-5 people team
- 10 m humpback whale= backhoe loader (with skilled operator) and 4-5 people team
- 15 m NARW= full on construction operation with at least one excavator and loader and 10-20 people
- Can take up to 2 days for sampling and documentation
- Beneficial to create “windows” in the carcass to get access to organs
- Thoracic wall (caudal to flipper): thoracic organs and multiple lymph nodes to be reached, removed and sample
- Cranial abdomen window: stomachs, pancreas and spleen
- Caudal abdomen: colon, urinary bladder, and reproductive tissues
- Make sure to examine and document external lesions!
- Entangling lines: mouth, flippers, and insertion of flukes
- Sharp wounds= propeller or bow of a vessel (but examine to see if pre-, peri- or post mortem
- Make sure to describe and number all lesions
- Blubber can mask deep trauma- so in order to fully document- may need to remove the entire blubber layer from the body in one to two longitudinal strips

Sampling Station
- Set up away from necropsy
- Once documenting a lesion or organ- remove a sample and transfer to sampling station
- All protocols may be filled and logged in and sample/data sheets may be kept clean
- Collect complete suite of tissues in 10% neutral buffered formalin, cryovials, whirl paks, and/or vials
- Write up the gross necropsy observations, document samples collected,
- High quality whale mortality data accumulate slowly and require long-term commitment to their successful use

Question 99

A recent study validated an ELISA to measure DHEA in narwhals.

What is the scientific name of the species?

Stress Hormones:
- What is dehydroepiandrosterone?
- How is its role different than cortisol?
- What ratio can be used to evaluate chronic stress?
- DHEA tends to be higher in what species and during what life stages?

What did this study find regading DHEA and the ELISA to measure them in Narwhals?

Answer 99

VALIDATION OF ENZYME-LINKED IMMUNOSORBENT ASSAY TECHNIQUES TO MEASURE SERUM DEHYDROEPIANDROSTERONE (DHEA) AND DHEA-S IN NARWHALS (MONODON MONOCEROS).
Béland K, Lair S, Guay M, Juette T, Bédard C, Black SR, Marcoux M, Watt CA, Hussey NE, Desmarchelier M.
Journal of Zoo and Wildlife Medicine. 2023;54(1):119-30.

Narwhals (Monodon monoceros) are increasingly exposed to anthropogenic disturbances that may increase their stress levels with unknown consequences for the overall population dynamics. The validation and measurement of chronic stress biomarkers could contribute toward improved understanding and conservation efforts for this species. Dehydroepiandrosterone (DHEA) and its sulfated metabolite DHEA-S are collectively referred to as DHEA(S). Serum DHEA(S) concentrations combined in ratios with cortisol [cortisol/DHEA(S)] have been shown to be promising indicators of chronic stress in humans, domestic animals, and wildlife. During field tagging in 2017 and 2018 in Baffin Bay, Nunavut, Canada, 14 wild narwhals were sampled at the beginning and end of the capture-tagging procedures. Serum DHEA(S) were measured with commercially available competitive enzyme-linked immunosorbent assays (ELISA) developed for humans. A partial validation of the ELISA assays was performed by the determination of the intra-assay coefficient of variation, confirmation of the DHEA(S) dilutional linearity, and the calculation of the percentage of recovery. Mean values (nanograms per milliliter ± standard error of the mean) of narwhal serum cortisol, DHEA(S), and cortisol/DHEA(S) ratios, at the beginning and at the end of handling, respectively, are reported (cortisol = 30.74 ± 4.87 and 41.83 ± 4.83; DHEA = 1.01 ± 0.52 and 0.99 ± 0.50; DHEA-S = 8.72 ± 1.68 and 7.70 ± 1.02; cortisol/DHEA = 75.43 ± 24.35 and 84.41 ± 11.76, and cortisol/DHEA-S = 4.16 ± 1.07 and 6.14 ± 1.00). Serum cortisol and cortisol/DHEA-S were statistically higher at the end of the capture (P= 0.024 and P= 0.035, respectively). Moreover, serum cortisol at the end of handling was positively correlated to total body length (P = 0.042) and tended to be higher in males (P = 0.086). These assays proved easy to perform, rapid, and suitable for measuring serum DHEA(S) of narwhals and that calculated cortisol/DHEA(S) are potential biomarkers for chronic stress in narwhals and possibly other cetaceans.

Background
- General climatic warming trends in the Arctic, reducing sea ice coverage, presence of predatory killer whales, growing tourism industry with increasing cruise ship traffic, shipping of natural resources that increase shipping traffic and underwater noise
- Cortisol shown to be elevated in narwhal blubber with chronic stress
– Usually lower in cetaceans than stressed pinnipeds and terrestrial mammals
- Dehydroepiandrosterone (DHEA) and sulfated metabolite (DHEA-S) - androgen hormone precursor, anabolic hormones with protective and regenerative role
– Cortisol is catabolic hormone
– Ratio of serum cortisol:DHEA(S) reported to represent catabolic/anabolic balance
- Cortisol:DHEA(S) ratios generally higher in chronically stressed animals, potential to discriminate acute from chronic stress
– Chronic stress is a dysregulation in hypothalamic-pituitary-adrenal axis leading to chronically decreased DHEA(S)
- DHEA in killer whales was much higher than harbor and gray seals and humans
– DHEA tends to increase during reproduction and gestation in killer whales but decreases with advanced age

Key Points
- No overt health concerns
- Serum DHEA was significantly positively correlated after 12 and 24 mo of freezing
- Cortisol and cortisol:DHEA-S were significantly higher at the end of the handling period
– DHEA(S) and cortisol:DHEA were not significantly different
- Serum cortisol was higher in longer individuals, tended to be higher in males but not stat sig
- Values comparable to free-ranging beluga whales, lower than killer whales
- Results suggest serum DHEA(S) are less influenced by acute stress, similar to harbor and gray seals

Conclusions
- Commercially available ELISAs were partially validated for DHEA and DHEA-S in narwhals
- DHEA(S) and the cortisol:DHEA(S) ratios require further investigation to assess their relevance as chronic stress biomarkers in narwhal and other marine mammals

Question 100

A recent case series described cervical lymphadenitis causes by Streptococcus phocae in bottlenose dolphins.

What are the characteristics of infection with Strep phocae
- what types of disease are caused by this organism?

How did the cases present in this paper?
- What findings were common on clincial pathology?
- How were they diagnosed and treated?
- What are the landmarks for finding the superficial cervical lymph node on US?

Answer 100

JZWM 2023 54(1) 192-201 SUPERFICIAL CERVICAL LYMPHADENITIS ASSOCIATED WITH STREPTOCOCCUS PHOCAE IN FIVE COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS): A CASE SERIES

Abstract
Between 2009 and 2018, five common bottlenose dolphins (Tursiops truncatus) at the US Navy Marine Mammal Program presented with superficial cervical lymphadenitis. Clinical findings included ultrasonographic evidence of cervical lymph node enlargement, severe leukocytosis, elevated erythrocyte sedimentation rates, and reduced serum iron. Three of the dolphins presented with clinicopathologic changes without presence of clinical signs, and the other two cases additionally presented with partial to complete anorexia, lethargy, and refusal to participate in training sessions. Ultrasound-guided fine needle aspiration or biopsy of the affected lymph nodes yielded Streptococcus phocae by PCR in all cases, and the organism was cultured in one of five cases. Animals were treated with a combination of enteral, parenteral, intralesional antimicrobial, or a combination of those therapies and supportive care. Time to resolution of clinical disease ranged between 62 and 188 days. To the authors’ knowledge, this is the first report of Streptococcus phocae cervical lymphadenitis in cetaceans. Streptococcus phocae lymphadenitis should be a differential for cervical lymphadenopathy in this species, especially when associated with pronounced systemic inflammation and a history of potential exposure.

Key Points
- Strep phocae is a beta-hemolytic streptococcal species, primary and opportunistic pathogen
- Respiratory disease, abortion, pyometra, abscess formation, septicemia, meningitis and cellulitis
- Cause of clinical dz, most likely primary pathogen, affecting superficial cervical lymph nodes of bottlenose dolphins at the Navy
- 3 cases in 1 yr, then 2 cases 10 yr later; all in open ocean pens.
- Presented with neutrophilia, inflammatory biomarker response
- Novel presentation of S phocae in bottlenose dolphins
- c/s - lethargy/anorexia, inflammatory leukogram +/- cervical swelling. Identified on US but not always apparent on US at the time of presentation
- All successfully managed with abx, no drainage
- Authors recommend cephalosporine and Beta-lactams as empirical first choice abx in cases of S phocae lymphadenitis in cetaceans; C&S when possible
- ⅗ cases had recent or concurrent illness
- For US of superficial cervical LN: cranially - EAM, caudally-scapula

Take home message
- First report of cervical lymphadenitis caused by Strep phocae in cetaceans.
- Nonspecific evidence of systemic inflammation + US evidence of cervical swelling
- Prolonged treatment with antimicrobials was curative in all cases

Question 101

A recent study investigated cardiac biomarkers in marine mammals.

What species were evaluated?

How were samples categorized?

What was the only group with elevated cTnI levels?

Was NTproBNP a useful cardiac biomarker according to this study?

Answer 101

JZWM 2022 53(2) 373-382
INVESTIGATION OF THE USE OF SERUM BIOMARKERS FOR THE DETECTION OF CARDIAC DISEASE IN MARINE MAMMALS

Abstract: Cardiac disease has been extensively documented in marine mammals; however, it remains difficult to diagnose antemortem. Assays measuring cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are used as sensitive and specific biomarkers of cardiac disease in many species, but have not been widely investigated in marine mammals. This study aimed to provide a set of reference values for cTnI and NT-proBNP in belugas (BW) (Delphinapterus leucas), sea otters (SO) (Enhydra lutris), Steller sea lions (SSL) (Eumetopias jubatus), and California sea lions (CSL) (Zalophus californianus) with and without cardiac disease, and to determine if these biomarkers are useful indicators of cardiac disease in these species. First, existing immunoassays for cTnI and NT-proBNP were successfully validated utilizing species-specific heart lysate spiked serum. Cohorts were defined by histopathology as animals with no evidence of cardiac disease (‘‘control’’), with confirmed cardiac disease (‘‘disease’’), and with concurrent renal and cardiac disease (‘‘renal’’) for which serum samples were then analyzed. Serum concentration ranges for cTnI (ng/ml) and NT-proBNP (pmol/L) were determined for control and disease cohorts. There was significantly higher cTnI (P = 0.003) and NT-proBNP (P = 0.004) concentrations in the CSL disease cohort, as well as positive trends in BW, SO, and SSL disease cohorts that did not reach statistical significance. NT-proBNP concentrations were significantly higher in the CSL renal cohort compared with the control (P , 0.001) and disease (P = 0.007) cohorts. These results suggest that cTnI and NT-proBNP may be clinically useful in the antemortem diagnosis of cardiac disease in CSL, and warrant further investigation in BW, SO, and SL.

Intro
- Relatively few non-invasive antemortem diagnostics exist in marine mammals
- The main objective of this study was to determine if cTnI and NT-proBNP assays are useful in the antemortem diagnosis of cardiac disease in belugas (BW) (Delphinapterus leucas), sea otters (SO), Steller sea lions (SSL) (Eumetopias jubatus), and California sea lions (CSL).

M&M
- Banked serum tested from healthy animals, those with confirmed cardiac disease (disease group) and those with concurrent renal and cardiac disease (renal group)
- Animals with cardiac disease were also categorized based on chronicity and severity

Results
- Assays validated
- Ranges and medians established for each species
- No significant association with sex or age class on cTnI or NTproBNP concentrations in any of the four species
- Concentrations of cTnI were significantly higher in the disease CSL cohort when compared with the control
- Although not statistically significant, broader ranges and higher maximum concentrations of cTnI were observed in disease BW, SO, and SSL, and higher median concentrations of cTnI were observed in disease SO and SSL compared with controls
Median NT-proBNP concentrations were significantly higher in the disease CSL cohort compared with controls
- There was no significant difference in NT-proBNP concentrations in disease vs control SSL.
- NT-proBNP concentrations remained below the LLOQ for all disease and control BW and SO in this study
- Statistically significant differences in cTnI and NT-proBNP according to disease chronicity or severity compared with the control were only seen in CSL
- The concentration of cTnI was significantly higher in CSL with acute disease and moderate disease compared with the control
- Higher acute disease is expected as cardiomyocyte injury in most species results in a rapid early increase in circulating troponins
- Concentrations of NT-proBNP were significantly higher in CSL with both acute and chronic disease compared to control
- significantly higher NTproBNP was seen only in the mild disease cohort when compared with the control
- No significant differences between disease groups (acute vs chronic or mild, moderate, severe)
- Concentrations of NT-proBNP were significantly higher in CSL in the renal cohort compared with the control, but remained higher in the cardiac cohort indicating that renal disease did not drive the biomarker response
- NT-proBNP can increase with renal disease alone in some species, so caution should still be used when interpreting this in azotemic animals
- There was no significant difference in cTnI concentrations for the CSL renal cohort compared with the control.

Takehome: cTnI and NTproBNPmay have particular utility in detecting cardiac disease in CSLs. NTproBNP may be a useful adjunct in diagnosing renal dysfunction in CSLs, but additional studies are warranted. Further research needed in the other species evaluated