Rhinocerotidae

Question 1

Describe the unique anatomy of the rhinoceros.

What is their gut type?

Do they have a gallbladder?

What features are similar to equids?

What is rhino horn made of?

Describe rhino foot anatomy?

What are some unique clinicopathologic features of rhinos?

Answer 1

• Monogastric, extensive hindgut for fermentation
• NO gallbladder
• Gutral poutches, paired ventral diverticula from pharynx later to hyoid bones
• Inguinal Mamm glands
• Placenta- diffuse, microcotyledonary, and epitheliochorial
  
  • Adenomatous hyperplasia (below photo)- found in Asian one-horned rhino assoc with placentitis/abortion
• Margo plicatus- junction of keratinized and glandular gasrtic mucosal epithelium
• Rhinos-
  • Rhino horn- compressed keratin, 2nd horn present in Ceratotherium, Diceros, and Dicerorhinus
  • Pharyngeal tonsils- epipharyngeal bursa- aggregate of mucusa glands, lymph nodules
  • Penis directed caudally, vesicular gland is multisacculate, testes are NOT descended, kidneys are lobulated
  • Weight-bearing digits (II, III, IV); fatty fibrous cushion above sole; distinct radius/ulna/tibia/fibula
  • Clin path- Higher CK values in adult males; hypophosphatemia in captive black rhinos and linked to hemolytic anemia; calcium carbonate give urine the cloudy/milky appearance- can be intensified by diet

Question 2

Describe the ideal rhino housing.

Answer 2

• Spacing between bars 0.5 m.
  
  • If have claves – may need to add chains or cables
  • If use horizontal poles – chance of climbing or horn avulsion
• Textured substrate
• Most indoor stalls are concrete but natural substrate used for outdoor enclosure
• Need access to pools and wallows
  
  • Depth of pool sufficient to allow full submersion (Indian and Sumatran)
• Can acclimate to cold temperature
  
  • Need supplemental heat to 13C when temps are 10 C (up to 55 when 50)

Question 3

Describe the feeding strategies of the five rhino species.

What should be fed to themin managed care?

Describe mineral absorption in the black rhinoceros.

What nutritional deficiencie is linked to health issues in the black rhino?

Which species are prone to iron storage disease?

What is rhino milk like (there are three papers as well)?

If a rhino calf doesn’t receive colostrum, what can you give it?

Answer 3

Nutrition (Fowler 8)

· Feeding strategies vary by species

o Browsers: Black, Sumatran

o Grazers: white

o Intermediate feeder: Indian

· Black rhino receive higher proportion of concentrates compared with other spp. and would benefit from higher proportion of browse

· In captivity – rough guideline 1-3% BW as fed no more than 1/3 calories obtained from pellets

· Grass hay – White and Indian rhino

· Grass-legume mixture or legume-browse – black and Sumatran

· Alfalfa fed browse may lead to mineral imbalance, colic, diarrhea

· Black have higher Mg and Ca absorption (compared to horses) – don’t give excessive mineral supplementation

· Vitamin E deficiency – linked to health problems (black rhino)

· Black & Sumatran – prone to iron storage disease

o Need high fiber, low iron pellets

o Minimize citrus and other produce that has vitamin C

· Rhino milk

o Less total solids compared to other hoofstock

· Neonates that don’t receive colostrum from dam can be fed bovine or equine colostrum

Question 4

Describe rhino restraint.

What can be used for physical restraint?

What size needles are needed for chemical restraint?

Where should darts be placed ideally?

Are they prone to any common complications?

Describe general drugs for standing sedation and general anesthesia?

Are any species more sensitive to opioids than others?

Answer 4

Anesthesia (Fowler 8)

• · Physical restraint – can use chutes
• · Chemical
  
  • Need long needles – min 40-60 mm
  • Ideal sites – caudal to the ear on lateral cervical, upper caudal thigh, shoulder or nuchal bump
• · Prone to developing myopathy and neuropathy
• · Standing sedation
  
  • Butorphanol +/- azaperone or alpha 2 agonist
  • Can also use low dose ultra potent
• · General anesthesia – ultrapotent opiods
  
  • Etorphine most commonly used
  • Carfenatanil – some spp causes excitable induction
  • Etorphine + thiafentanil
  • White rhino, Indian rhino – sensitive to effects of opiods compared to black rhinos
• § muscle tremors, limb paddling, hypoxia, hypercapnea and hypertension
• § can give butorphanol to antagonize respiratory effects

Question 5

Describe the ideal positioning for rhinos under anesthesia.

Which position is best for their legs? Which is best for oxygenation?

Describe the monitoring of rhinos under anesthesia.

What are some common complications? How can those be addressed?

Answer 5

Recumbency and Positioning

• Thick mats and padding – help to reduce chance of myositis and neuropathy
• Potential complication of being in sternal 🡪 irreversible muscle damage to limbs
  
  • White rhino often have muscle rigidity and paddling under opioids which can be exacerbated in lateral recumbency 🡪 place in sternal until relaxed then move to lateral
  • O2 sat higher in sternal than lateral
• Place in lateral and “pump” the legs up and down every 20 mins to aid circulation
• Optimal positioning is a balance between maintaining respiratory function (sternal) and optimal circulation to the limbs (lateral)

Monitoring

• Focus especially on respiration and blood oxygenation, plus normal vitals
• SpO2 indirect measure of oxygen saturation of hemoglobin (SaO2)
  
  • Useful for monitoring trends – if declining or < 80% intervention warranted
  • Lower O2 affinity 🡪 SaO2 likely underestimate true oxygen saturation
  • Ear (scrape off epidermis), mucosal folds, or reflectance probe
• Respiratory depression is the most significant life-threatening complication
  
  • Large size and abdominal organs compressing diaphragm
  • Hypoxemia, hypercapneia, acidosis 🡪 more prevalent in white than black rhino
  • Ensure down nostril is clear so passive regurg can occur
  • Often develop apnea when moved into different position
  • Induction: RR 10-15 brpm, Post-induction RR 4-8 brpm
  • If breathing slow, apnea, SpO2 < 80% - consider partial reversal with nalorphine, nalbuphine or butorphanol or complete reversal with naltrexone or diprenorphine. Will improve rate and depth of respiration
    
    • Doxapram – CNS excitement and exacerbates mm tremors in white rhino
  • Nasal or tracheal insufflation rapidly increased blood oxygenation, but wont correct acidosis or hypercapnia. Recommended to do.
• For every 1 degree increase in temperature, there is 10% increase in O2 consumption. Greater than 102.2F must process quickly, greater than 105.5 give antidote.
  
  • Black rhino greater level of hyperthermia related morbidity than whites
  • Often experience hyperthermia during immobilization, and again when entering crate
  • Deep infrared ear thermometer comparable to muscle temperatures to assess core body temperature.
• Palpate pulse medial auricular artery or caudal artery
  
  • HR usually 55-80 bpm
• Hypertension prevalent under etorphine anesthesia in black and white rhinos
  
  • Inc sympathetic action, vasoconstriction, hypoxemia are suspected factors

Anesthesia complications

• With opioid induced cardiopulmonary depression, many need to deliver artificial ventilation 🡪 animal moved onto side 🡪 force knee and lower leg into abdomen to force diaphragm and force air into and out of the lungs while IV opioid antagonist take effect
• Myopathies – common if excessive chase or hyperthermia. If dog sitting in crate 🡪 electric prodder on head (not hindquarters), and if this doesn’t work given IV diprenorphine or nalorphine. Can also sling it.
• Black rhino – fat nose syndrome – nostrils close up and appear edematous – potential hypersensitivity reaction.
• Propensity for dart abscess 🡪 infusion oxytetracycline into the wound

Question 6

Describe walking a rhino in the field into a crate following anesthesia.

Describe the transport of rhinos in the field - including airlifting, vehicular transport (what sedatives), and transporting orphaned calves?

How are rhinos off-loaded once they arrive to their destination?

Answer 6

Walking a Rhinoceros (blindfolded, rope on head and a hind limb)

1. White Rhinoceros
   
   1. <20-40mg azaperone, avoid alpha2 agonists
   2. 20mg torb : 1mg etorphine
   3. Reverse any drugs incrementally if unwanted recumbency occurs
2. Black Rhinoceros
   
   1. Admin 5mg butorphanol after anesthetic procedure incrementally and rock patient back and forth to stimulate
   2. Nalorphine 10-20mg IV
   3. Diprenorphine 0.25-0.4mg IV

Question 7

What are three important bacterial diseases of rhinos?

What are their clinical signs, diagnosis, and treatment like?

What lesions are associated with these diseases?

Question 8

What is an important viral disease of rhinos?

Answer 8

Encephalomyocarditis virus - transmitted from rodents

Question 9

What are some important parasites of rhinos?

Answer 9

Parasitic (F8)

• Nematodes
• Tse tse fly- transmission of trypanosome
• External – Stephanofilaria dinniki – skin ulcers in free-ranging black rhino
  
  • Transmission by a blood-sucking arthropod
  • Recent outbreak in Meru national park in white and black rhino
• Free ranging rhino ticks – Rhipicephalaus, Dermacentor, amblyoma, hyalomma

Parasites (ZP)

• Metazoa- Stephanofilaria dinniki; halicephalobus gingivalis, Schistosomiasis
• Protozoa- Piroplasmosis- tick borne; babesia or Theileria; sarcosystis neurona; Klossiella equi; neosporosis; Naegleria fowleri- necrosuppurative meningoencephalitis

Question 10

What are some toxicities reported in rhino species?

Answer 10

· Toxicity:

· 7 fatalities in black rhino due to creosote-treated wood – liver dysfunction

· White rhinos blue-green algae toxicity

· Vitamin D toxicity from improper diet

· White rhino – adverse drug reaction to firocoxib

· Food to avoid rhino: red maple, kale, onion, Brassica family, - can cause hemolysis

Question 11

Describe superficial necrolytic dermatopathy in black rhinos.

What are the typical clinical signs?

How do the lesions progress?

What is found histologically?

How are these treated?

Answer 11

Superficial necrolytic dermatopathy (ulcerative skin disease, vesicular or ulcerative dermatopathy)

• Initial signs: epidermal plaques or vesicles 🡪ulcers
  
  • Pressure points, coronary band, tail tip, ear margin
  • +/- oral or nasal ulcers
  • +/- anorexia, lethargy, weight loss, lameness
  • Decreased albumin, hct
• Most cases associated with other health problems
• Txt
  
  • Cryotherapy, steroids
  • Lesions may resolve spontaneously
  • Symptomatic treatment
  • If lesions become extensive, it may become fatal
• Skin diseases of captive black rhinos: Superficial necrolytic dermatopathy (superficial necrolytic dermatitis, necrolytic migratory erythema, vesicular and ulcerative dermatopathy, metabolic epidermal necrosis, mucosal and cutaneous ulcerative syndrome, hepatocutaneous syndrome, and ulcerative disease) - common and develop over time and variable severity, all ages, bilateral, pressure points, lateral body surfaces, coronary bands, tail tip, and ear margins along with poss oral/nasal lesions
  
  • Epidermal plaques>vesicles/pustules>erosions
  • CS: Depressed, anorexic, weight loss; hypoalbuminemia, hypocholesterolemia, and decreased HCT. Histo: layered “red, white, and blue”
  • Nodular collagen degeneration with dystrophic minteralization- rhinos
  • Eosinophilic granulomas- described in 8 captive black rhinos, hypersensitivity?

Question 12

Describe the hemolytic anemia of black rhinos.

What are some of the suspected etiologies?

What clinical signs occured with idiopathic hemorrhagic vasculopathy syndrome in black rhinos?

Answer 12

Hemolytic anemia

• Peaked in the 1990’s
• Of 47 known cases – high mortality rate has been observed
• Etiologies?
  
  • Hereditary cause – deficiency of G-6-OD, Lepto, hypophosphatemia, hypovitaminosis E
• Management
  
  • IV or oral supplementation of phosphorous
  • Vitamin E
  • Prophylactic antibiotics
  • Whole blood transfusion
• Acute intravascular hemolytic anemia (1/3 of deaths of captive black rhinos in 80s-90s (ZP)
  
  • DDX: autoimmune, equine infecious anemia, equine viral arteritis, copper tox, vitamin E def, and clostridial infection. Lepto was diagnosed in many affected ones.
  • Free rangning rhinos-anemia due to babesiosis and trypanasomiasis

Idiopathic hemorrhagic vasculopathy syndrome in seven black rhinos; JAVMA 2000

Pathological findings in idiopathic haemorrhagic vasculopathy syndrome (IHVS) of captive black rhinos

• Clinical signs
  
  • Severe limb facial and neck swelling associated with nonhemolytic anemia
  • Lethargy, respiratory stridor, laminitis, nail sloughing, aural hematoma, oral or skin ulcers
  • Acute onset of signs with no known cause
  • Recurrent episode is likely
• Fatality rate is high
  
  • Some animals recover with antibiotics, NSAID, fatty acid and phosphorous supplementation
• Cooler months (Oct-March) in animals that live Tx or Southern US

Question 13

Describe iron storage disease in rhinos.

What species are most sensitive?

What are suspected causes?

How can it be treated?

What analytes can be measured? Which appear to correlate with total body stores?

Answer 13

Iron Storage Disease (ISD)

• Significant higher tissue and serum ferritin in black and Sumatran rhino
• Levels increase over time in captivity
  
  • Ferritin levels do not appear be elevated in white or Indian rhino
• Possible causes
  
  • Vitamin E deficiency, hemolytic anemia
• Clinical signs
  
  • Hemosiderosis found in multiple organs
• Recommend low iron diets, provision of browse and phlebotomy, iron chelating agents

Serum iron analytes:

1. Transferrin is the plasma transport molecule for iron, and the percent saturation (T sat) is considered a reflection of iron absorption (but not an accurate measure of iron stores), which is related to dietary intake.
2. Total iron binding capacity (TIBC) measures the maximum amount of iron that can be transported.
3. Ferritin is an intracellular storage molecule for iron and in some species correlates well to total body iron stores.

• Iron overload- rhinos and tapirs- hepaticin is hormone regulating storage; tannins, phytates, polyphenolic compounds chelate iron. Hemosiderosis is a problem of CAPTIVE BLACK rhinos are more prone- browsers; vitamin C increases iron absorption; Lesions (hemosiderosis)- rhino-spleen, liver, and lung and intestines in some; Serum ferritin concentration is a good estimate total iron stores

Question 14

Describe the male reproductive tract of the rhino.

What accessory glands do they have?

How can these structures be evaluated?

What are some common diseases of these structures?

Answer 14

Male Reproductive Anatomy and Clinical Aspects

• Musculocavernous penis fully covered by prepucial fold
• Penis points caudally during urination and cranially during erection
• Likely cervical inseminator – horizontal flaps unfold in females vagina while process glandis locks into portio and cervical folds
• Accessory Sex Glands – prostate, seminal glands, bulbouretheral glands (ampullae not reported)
  
  • White rhino- accessory gland volume correlates w/ semen quality
• Testes w/ tightly attached epididymis located in dorsal prepucial fold
  
  • May or may not be palpable (can move up by inguinal rings)- limits conclusions by palpation
• Exam of testes and accessory glands relies on transcutaneous and transrectal ultrasound
  
  • Transrectal requires chute-trained animal or sedation
  • Transcutaneous through bars of enclosure with very little training, no sedation

Diseases of the Male Reproductive Tract:

• Penis:
  
  • Trauma d/t intramale aggression, masturbation on foreign objects
  • Edema – may result from wounds; aggressive therapy recc; body bandage penis to abdomen
  • Penile fracture – usually caused by blunt trauma to the erect penis; inability to obtain intromission thereafter
• Accessory Sex Glands:
  
  • Rarely diseased
  • Prostate cysts reported in one white rhino- can be painful, may decrease libido
• Testis:
  
  • Testicular fibrosis – common in older males; starts around 15 years; appears as bright dots in U/S which get larger and more numerous w/ time; no influence on semen quality
  • Trauma – hematomas, seromas seen as bulges/distentions of dorsal prepucial fold; see fluid pocket on U/S
  • Testicular neoplasia – Seminoma reported in black and white rhino; Hemicastration is tx of choice to avoid effects on semen quality and metastasis
  • Epididymal cysts – Reported in Sumatran and white rhino; 1-10cm fluid pockets; cause unclear; transcutaneous aspiration is treatment of choice to aid in maintaining sperm quality and allows for sperm passage
  • Trauma, neoplasia and epididymal cysts often reduce or abolish sperm quality

Question 15

Describe rhinoceros semen collection and preservation?

How is semen collected? What methods are recommended?

How is semen evaluated?

Describe the cryopreservation of rhino semen?

Answer 15

Semen Collection, Evaluation and Preservation:

• Poor semen quality does not necessarily exclude chance of fertilization
  
  • Often associated with subordinate male in group of males (social issues)
  • May be associated with lack of breeding activity
• Semen Collection:
  
  • Methods: Post-coital (from dripping female vulva), Manual stimulation, rectal massage, electroejaculation or post-mortem
    
    • Manual stim – time-consuming, requires well-trained animal and restraint chute; Inconsistent collection success and often minute volume
    • Artificial vagina – produce ejaculates but no sperm thus far
    • Electrostimulation – recommended method; Ultrasound determines position of accessory glands and probe is place above them to avoid stimulating bladder/urine contamination
    • Post-mortem – can take epididymal aspirate or entire testes and refridgerate (4C) in saline solution for cryopreservation
  • Sperm-rich fractions emitted at beginning of ejaculation, decreasing progressively
  • Sperm quality is not affected by age or season
    
    • Testicular fibrosis, common in older males, does not affect sperm quality
    • Androgen concentration is also unaffected by age or season
• Semen Assessment and Preservation:
  
  • Most relevant indicators of sperm quality = motility, morphology, viability
    
    • % motile and morphologically normal sperm in sperm-rich fraction used to rate bull semen quality
    • Motility: <75% = intermediate; <50% = poor quality
    • Quality may vary between collections; several attempts suggested
    • White rhino: Social structure and subordinate behavior have strong influence on sperm quality
  • Preservation:
    
    • Not sensitive to slow chilling
    • Facilitates sperm transport to distant locations for AI
    • Post-thaw Eval: motility, viability, morphology and acrosome integrity are most important
    • Single or double stranded sperm fragmentation recently added as critical parameter
• Cryopreservation of Sperm:
  
  • Extenders: Standard equine, skim milk-egg yolk, TEST-egg yolk medium
  • Cryoprotectants: Glycerol or dimethylsulfoxide (Me2SO)
    
    • Me2SO better in Sumatran rhino
  • Freezing methods: Liquid nitrogen traditional method
    
    • Multithermal Gradient Directional Freezing
    • Facilitates freezing large volumes, less cell damage and higher gamete survival

Question 16

Describe the female reproductive anatomy of the rhinoceros.

How are these structures evaluated?

Describe the estrous cycle of rhinos?

Acyclitiy is a problem in what rhino species?

Can estrous be induced?

Answer 16

Female Reproductive Anatomy and Clinical Aspects

Anatomy:

• Genital tract length varies – white rhino is very long (100cm), Sumatran shortest (50cm)
• Other structures: Outer and inner labia, prominent clitoris, hymen (cranial to urethral orfice; thickens w/ age; persistence = common cause of infertility in older females), cervix (thick, tortuous, can only catheterize during estrus), uterus (short body, long horns)
• Ovarian follicles rupture on surface (NOT like horse)
• Uterus and ovary not accessible for palpation
• Examination: 2-8 MHz transrectal U/S; in chute or standing sedat.
• Minimal external changes with estrous – identified by male interest or female vocalization (Indian and Sumatran)

Estrous Cycle:

• Polyestric, non-seasonal breeders in wild
• Estrous cycle length varies between spp (see table 71-2)
• 2 cycle lengths in white rhino: short (30-35d) is fertile, but long in females w/o mating history is first sign of reproductive aging and infertility (may also be observed after AI with fetus resorption)
• Sumatran rhino is an induced ovulatory**
• LH surge stimulates ovulation
• Luteinization and hemorrhagic follicles (bigger than ovulatory/Graafian follicles) are common problem (non-physiologic except in bred Sumatran – induced ovulation)

Anestrous:

• Common in white rhino and primary cause of low repro rate in captivity
  
  • 50% females in anestrous
  • Also occurs in other rhino spp
  • Young females in anestrous exhibit high ovarian activity, but never ovulate – develop hemorrhagic or atretic follicles
    
    • Over decades, leads to follicular depletion and early reproductive aging (Asymetrical reproductive aging)
    • Introducing new male or moving female to new location can help

Estrous Induction:

• Low species-specific receptor affinity of altrenogest led to early failures at induction
• Chlormadinone acetate (synthetic progestin) followed by hCG or GnRH analogue was effective in white rhino
• GnRH implants may be placed when pre-ovulatory follicle is identified to induce ovulation w/in 48 hrs

Question 17

How long is rhino gestation?

How is pregnancy diagnosed?

What is luteal insufficiency? How can it be managed?

How long does labor take?

What species has the highest rate of stillbirths?

When do you intervene in a dystocia? How?

Answer 17

Gestation and Birth

• Gestation period = 15-18 mo
• Pregnancy diagnosis:
  
  • Ultrasound – embryonic vesicle visible at 15d; confirm dx between 2-4 wks after conception
  • Fecal progesterone – 3-5 months after conception
  • Transrectal imaging limited to 1^st trimester in white and Indian rhinos due to size of fetus and thickness of fetal membranes; can use transabd in 3^rd trimester
• Luteal insufficiency – suspected cause of embryonic resorption in captivity
  
  • Treatment w/ altrenogest in 2 rhino – but receptor affinity low w/ this drug, so unknown if it was the drug that allowed rhino to carry to term
  • Comparison of progesterone levels during supplemented and un-supplemented pregnancies in one rhino showed no difference

Birth:

• Labor = 1-3hrs
• Calf stands in first hour; nurses by 2-3 hours
  
  • Some calves take up to 14hrs w/ new moms, but calves seem unaffected
• Placenta passes in 6-7 hours; female will often consume it

Dystocia:

• Dystocia and stillbirth occur in all rhino spp
• Highest stillbirth rate in Indian rhino
  
  • One study: 24% stillborn or died w/in 3 mo.
  • Craniofacial malformation (3 cases) suggests undetected neural tube defects may be associated with high stillbirth rate
  • Cause still unknown
• Lack of labor progress 4-6 hrs after fetal membranes rupture necessitates veterinary intervention
  
  • Oxytocin 100 IU
  • Reposition fetus
  • Fetotomy
  • No C-section: Difficult wound management, heavy weight of intestines, thick integument make this a bad option
    
    • All females from whom a calf was extracted fully recovered

Question 18

Describe the hand-rearing of rhinos.

What milk should be used - papers may change this.

How often should they be fed and how much?

When can they be weaned?

Answer 18

Hand Rearing:

• Milk: Equine milk exchanger or bovine skim milk = best replacement; rhino milk low in protein, solids, and very low fat, but high in sugar
• Colostrum or maternal serum should be collected pre-partum to prepare for hand-rearing
• Vitamins, gamma globulin and paraimmunity inducers should be added to milk
• Feeding schedule: 10% BW first 3 days, then 15-20%; 7-10 feeds/day; Substitute boiled rice, hay, soft fruits, veggies starting at 2 weeks
• Weaning: begins at 6 months, complete by 12-15mo; offer horse feeds or high-fiber ungulate pellet w/ hay & browse
• Post-partum aggression is common in inexperienced moms; to prepare mother, place on exhibit with small mammals, give oxytocin to stim milk production or sedate to facilitate nursing

Question 19

Describe the reproductive pathology of female rhinos.

What neoplasias are common?

Describe asymmetrical reproductive aging. What can be done to help prevent this?

Answer 19

Reproductive Pathology

• Incidence of repro disorders greater in nulliparous females and positively correlated w/ age
• Neoplasia – Esp Asian spp.
  
  • Ovarian
  • Uterine – most common in white rhino; leiomyoma is most common across spp; endometrial adenoma and adenocarcinoma also occur;
  • Cervical and vaginal most common in Indian
  • Treatment: GnRH agonist may decrease hormonal stimulation/growth and blood loss
    
    • Use caution as this may wipe out reproductive signals and males may perceive females as a territorial threat and attack or kill them
      
      • Also occurs with reproductively senescent females
• Cysts- ovarian or endometrial
• Cystic Endometrial hyperplasia – esp African spp
• Mucohydrometra
• Asymmetrical reproductive aging- reproductive disorders and ovarian exhaustion, irreversibly infertile females early during life
  
  • Reproducing female white rhinoceros in captivity may produce up to nine calves with approx. 90 estrous cycles in between
  • Incidence of reproductive disorders in parous females significantly lower- pregnancy in young animals may help to protect against development of reproductive disorders
• Infectious disease – rarely described; endometritis has been reported in Indian rhino – vaginal purulent d/c only sign (DDx: breeding trauma, abortion)

Question 20

Describe the assisted reproductive techniques used in rhinos:

Artificial insemination

Sperm sexing

Gamete rescue

Cell line culture

Answer 20

Artificial Insemination

• Successful w/ fresh and cryopreserved semen- 7 conceived, 5 born alive
• Estrous cycle in anestrous rhinos induced using chlormadinone acetate plus GnRH analogue
  
  • Presence of preovulatory follicle and accurate timing of ovulation determined by u/s
• Need rhino specific AI catheter to get through tortuous cervix

Advanced Assisted Reproduction Techniques

Sperm Sexing:

• A sorting purity of 94% for X chromosome spermatozoa was achieved
• Slow sorting rate prevents use currently, but good option for future

Gamete Rescue:

• Oocyte collection from infertile rhino has been successful in black and white rhino
  
  • Transrectal, U/S guided aspiration
• In vitro fertilization has produced an embryo
• Good option for future
• JZWM 47(4) – gamete rescue attempted in Sumatran female at Cincinnati that died of hemochromatosis; Successfully collected follicles, sperm adhered to zona pelucida, but fertilization did not occur

Cell Lines:

• Skin sampling, then in vitro culture of fibroblasts
• Cryopreserve fibroblasts
• Reprogram fibroblasts into omnipotent stem cells
• Future possibility

Question 21

A recent study described a vertical vaginal septum in rhinos.

What are proposed functions of the septum?

Can it be used to establish nulliparity?

Answer 21

Kinney, M. E., Hendrickson, D., Pennington, P., Zuba, J. R., Clancy, M. M., Howard, L. L., … & Durrant, B. (2019). Vaginoscopic identification of a vertical vaginal septum in one primiparous and three nulliparous southern white rhinoceros (ceratotherium simum simum). Journal of Zoo and Wildlife Medicine, 50(1), 274-277.

Abstract: Vaginoscopy using a 10-mm, 308 forward viewing rigid endoscope was used to evaluate the caudal reproductive tract of four subadult southern white rhinoceros (Ceratotherium simum simum). A vertical vaginal septum was documented in all four animals, including a primiparous cow that gave birth to a stillborn calf 14 months before vaginoscopy. Vaginoscopy using a 57-cm-long, 10-mm, 308 forward viewing endoscope provides adequate visualization of the caudal reproductive track in the southern white rhinoceros, and a detailed description of the vertical vaginal septum is presented. Additionally, the presence of a vertical vaginal septum in a primiparous southern white rhinoceros suggests the presence of this anatomic structure cannot be used as a proxy of nulliparity for captive southern white rhinoceros.

• Female rhinos’ reproductive tract has been heavily
  
  • Purpose of the vertical vaginal septum has not been reported
• Vaginoscopy used to visualize the caudal reproductive tract in 4 southern white rhinos
  
  • 6-9 years old
  • 1 previously given birth 14 months
  • 3 were nulliparous
• Sedated: Butorphanol (27-36 ug/kg) and medetomidine (2 ug/kg) IM
• 10-mm 30 degree forward viewing rigid endoscope in a 57-cm-long cannula w/ insufflation valve inserted into vagina
  
  • Transverse hymen manually ruptured in 2 animals to allow visualization of vertical vaginal septum
  • Commonly ruptured in rhinos during mating à persistent hymen seen with reproductive failure
• Thought to impede successful intromissionà recommend manual transection
• All animals had a vertical vaginal septum located directly on the midline, extending to the dorsal and ventral aspect of the vagina
  
  • Location, size, color, and distensibility à all same for nulliparous and primiparous rhinos
• 3 animals - septum was transected for direct visualization of the vulva opening
  
  • Histopathology (from one of the transected animals) - dense fibrous connective tissue lined superficially by squamous mucosa. Glands lined with pseudostratified columnar epithelium in submucosa. Occasional small lymphoid aggregates in the superficial submucosa/propria
• Purpose of the vertical vaginal septum 3 theories
  
  • Supports transverse hymen and maintains integrity
  • Helps stabilize/ repositions distal urethra against cervix when engaged w/ male’s penis
  • Vestigial structure or embryonic remnant
• Further study using the vaginoscopy evaluation is needed to understand the purpose of the vertical vaginal septum
  
  • Different ages, housing, reproductive cycle timing and parity
  • Understand the relationship between the cranial vaginal vault and the erect penis
  • Documentation of the septum in naturally bred rhinos that delivered alive, full term calves

Question 23

White rhino nasal sinus anatomy was recently described.

How is this sinus affected by poaching?

What are some functions of paranasal sinuses?

Answer 23

Gerard, M. P., Glyphis, Z. G., Crawford, C., Blikslager, A. T., & Marais, J. (2018). Identification of a nasoconchal paranasal sinus in the white rhinoceros (Ceratotherium simum). Journal of Zoo and Wildlife Medicine, 49(2), 444-449.

Abstract: African rhinoceros are poached for their horns using indiscriminate and aggressive methods. Rhinoceros that survive these attacks often have severe facial trauma, and treatment is limited by a lack of understanding and published information of the normal anatomy. This study was performed to investigate and describe the anatomy of the most commonly injured area of the head of the white rhinoceros (Ceratotherium simum). Two white rhinoceros cadaver heads were imaged by computed tomography and grossly dissected. A combined dorsal conchal sinus and nasal sinus (named the nasoconchal sinus) was identified and confirmed to be readily exposed by horn removal. The nasoconchal sinus communicates via a relatively large opening with the middle nasal meatus of the nasal cavity. Awareness of the combined sinus space and its single communicating pathway will assist with accurate assessment and treatment of trauma to the dorsal facial region of the white rhinoceros.

• Goal - investigate the paranasal sinus anatomy of the white rhinoceros and specifically to focus on the commonly traumatized dorsorostral region of the head under both horns
• 2 white rhino heads – CT and dissection performed
• Catheters, endoscopy and new methylene blue used to investigate communications
• The nasal opening of the dorsal conchal sinus (apertura conchonasalis, the conchonasal opening) was the sole communication between the combined NCS (nasoconchal sinus) and the nasal cavity
  
  • likely serves as an outflow pathway but may also provide a strategically placed inflow pathway for inspired air to be circulated through the sinus, possibly to enhance olfactory function
  • sinus fluid accumulation has to drain through this ostium
  • may be entry point for endoscopy of sinus
• In the white rhino, the entire dorsal nasal concha encloses a relatively vast dorsal conchal sinus, and there is no apparent communication with the frontal sinus.
• the cavernous nasal bone sinus and its dorsal conchal sinus continuation in the white rhinoceros contrasts with the long, flat, nasal bone of the horse
• Paranasal sinuses have been ascribed various roles:
  
  • weight reduction of the skull
  • absorption of trauma
  • protection of underlying structures
  • aiding facial growth
  • increasing surface area for olfaction
  • evolutionary vestigial remnants
  • improving immune defense in the respiratory system by generating nitric oxide
• Facial trauma following poaching of nasal and frontal horns will expose the nasal and dorsal conchal sinuses.
• Frontal horn removal may expose the frontal sinus, and deeper, lateral wounds will expose the maxillary sinus
• The entire nasal bone and its sinus may be lost with indiscriminate removal of both horns, along with portions of the frontal bone and maxilla, resulting in paranasal sinus disruption, nasal cavity exposure and partial loss of the nasal septum.

Question 24

A recent study evaluated Indian rhino milk.

How did nutrient composition change over time?

How did that compare to elephants?

What is the primary nutrient in rhino milk?

What is their fatty acid profile like?

What replacers may be potentially successful?

Answer 24

Gimmel, A., Hoby, S., Deillon, L., von Houwald, F., Schweizer, R., Kölln, M., … & Liesegang, A. (2018). Milk composition of indian rhinoceros (rhinoceros unicornis) and changes over lactation. Journal of Zoo and Wildlife Medicine, 49(3), 704-714.

Abstract: The objective of this study was to determine the major nutrient composition of Indian rhinoceros milk (Rhinoceros unicornis) over the first 13 months of an 18 month lactation period and to compare the results of those previous studies on rhinoceros, African elephant (Loxodonta africana) and horse milk (Equus ferus caballus). The following parameters were measured: dry matter (DM), crude ash (ASH), crude protein (CP), ether extract (EE - crude fat), nitrogen-free extract (NFE; calculated), lactose, calcium (Ca), phosphorus (P), magnesium (Mg), fatty acids (FAs), and gross energy (GE). DM, ASH, CP, and EE were determined with a proximate analysis, lactose with infrared spectroscopy and an enzymatic method, minerals with an autoanalyzer. FA with gas chromatography and GE with bomb calorimetry. Milk samples were collected from two Indian rhinoceros cows from Zoo Basel. Rhino A gave birth to her third calf on 10 September 2012; three samples were collected and analyzed (colostrum, milk 1 wk and 2 wk postpartum). Rhino B gave birth to her eighth calf on 05 October 2013; samples were collected and 15 were chosen for the analyses (from colostrum to 13 mo postpartum). The composition of rhino B’s colostrum was 13.8% DM (wet-weight basis), 4.8% ASH, 61.8% CP, 0.7% EE, 32.6% NFE, 26.7% lactose, 0.59% Ca, 0.54% P, 0.2% Mg (DM basis), and 20.3 MJ GE/kg DM. Rhino B’s sample collected 13 mo postpartum averaged 8.0% DM (wet-weight basis), 3.6% ASH, 16.3% CP, 1.8% EE, 78.3% NFE, 84.7% lactose, 0.54% Ca, 0.48% P, Mg (on DM basis), and 17.43 MJ GE/kg DM. The main FAs in rhino B’s and rhino A’s samples were C10:0, C12:0, C16:0, C18:1n9c, and C18:2n6c. Milk of the Indian rhinoceros is low in fat and protein but high in lactose, which is comparable to the milk composition of other rhinoceros species and horses, but not African elephants.

o DM, CP, EE, minerals, energy decreased over time, lactose increased.

o African elephant opposite trend: DM, CP, EE, minerals, energy increased, lactose decreased.

Conclusion:

• Indian rhinoceros mid-lactation milk has a composition with low EE (ether extract) and CP (crude protein) and a high lactose.
• The FA profile shows high proportions of medium-chain FAs.
• Milk composition of the different rhino spp is comparable to that of the horse.
• Horse milk is not an optimal substitute for rhino calves unless supplemented with a fat source that has a similar FA profile to rhino milk.

Question 25

A recent study evaluated the milk composition of the white rhino.

What was the major nutrient in the milk?

Is this similar to other rhinos?

What type of fatty acids predominate?

Answer 25

Zoo Biol. 2021 Sep;40(5):417-428.

Osthoff G, Beukes B, Steyn AC, Hugo A, Deacon F, Butler HJB, O’Neill FH, Grobler JP. MILK COMPOSITION OF WHITE RHINOCEROS OVER LACTATION AND COMPARISON WITH OTHER PERISSODACTYLA. – reviewed by ZCR

Abstract

The proximate composition of milk from fifteen free-ranging white rhinoceroses at different lactation stages is reported with detailed analysis of fatty acid composition and minerals. Lactose is the main component at 7.93 ± 0.53%, followed by 0.93 ± 0.19% protein, 1.76 ± 0.39% fat, 0.40 ± 0.18% ash, 0.05 ± 0.01% glucose, and 0.04 ± 0.02% non protein nitrogen The interindividual variation of all the components is high, showing no trend of change over lactation. The K and P content decreased over lactation. The fatty acid composition of rhinoceros milk is characteristic with a high saturated fatty acids content of 62%-84%, of which the medium chain fatty acids form the major portion. The C10:0, C12:0, C16:0, and C18:1c9 are the major fatty acids in the milk fat. The results are compared with the two other Perissodactylae families, the Equidae and Tapiridae. Differences in gross composition are small, but the milk of the Rhinocerotidae have the lowest gross energy, while the milk fats consist of the highest amounts of saturated fatty acids, while the low levels of C16:1c9 and C18:1c9 indicate the lowest mammary Δ9-desaturase activity.

Key Points

• Studies in Indian and Black rhinos – indicated low total solids content (8-16%), main components being lactose, protein and fat; high content of capric acid (18-36% of total fatty acids).
• This study presents data from 15 free-ranging white rhinos, between 0.5-18 mo lactation
• No change in dry matter (DM) over lactation
• DM, NPN, ash, protein, lactose and fat content of milk similar to previous studies, including black and Indian rhinos
• K content of milk increased over first 4 mo of lactation
• Ca:P ratio is ~3:1
• Lactose major component of the milk.
• White rhino has highest fat content of the rhinocerotidae; lowland tapir and plains zebra had highest fat content of the perissodactyls
• Saturated fatty acids were >60%

Take home message

• This paper provides a reference for rhinoceros milk for hand rearing formulas. No major changes over the course of lactation were seen, and lactose was the primary component of the milk.

References: none

Question 26

A recent study evaluated vitamin D metabolism in the black rhino.

What are the two main forms of vitamin D?

How is vitamin D activated?

What is the best form to measure?

How were rhinos affected by UV exposure and dietary supplementation?

Answer 26

Olds, J., Oltman, W., Makowski, A. J., Householder, H., & Keeley, L. L. (2018). Seasonal variation of serum 25-hydroxy-vitamin d in two captive eastern black rhinoceros (diceros bicornis michaeli) housed in a north american zoo. Journal of Zoo and Wildlife Medicine, 49(4), 943-951.

Abstract: Black rhinoceros (Diceros bicornis spp.) are critically endangered species, with less than 65 individual animals housed in captivity within Association of Zoos and Aquariums-accredited zoos within the United States, and an estimated 5,500 individual animals of all subspecies surviving in the wild. Previously published reference values for circulating vitamin D3 (25OHD3; 55.7 6 34.2 ng/ml) were based upon samples from free-ranging black rhinoceros in Africa. Recent research in human medicine has highlighted the importance of subclinical vitamin D deficiency, with links to increased risks for developing various health conditions. Serum samples collected opportunistically from two captive Eastern black rhinoceros (Diceros bicornis michaeli) housed with seasonal access outdoors in a North American zoo were tested for 25-hydroxy-vitamin D (25OHD) levels over a 3-yr period. A commercially prepared pelleted diet containing vitamin D3 was fed to both rhinos. This study correlates environmental ultraviolet (UV) index, dietary supplementation, and seasonal serum 25OHD levels to compare with known 25OHD3 levels in free-ranging African black rhinoceros. Results in these two individuals suggest that D. bicornis spp. are dependent upon sunlight or UVB for measurable circulating 25OHD, and that current vitamin D3 supplementation levels may have little effect for Diceros spp. in human care housed in northern latitudes.

• Vitamin D
  
  • Fat soluble vitamin
  • Contributes to immune system function and metabolic pathways
  • 2 main forms – ergocalciferol and cholecalciferol
  • Ergocalciferol – found in plants
  • Cholecalciferol – produced endogenously by animal skin
    
    • Requires UVB exposure
• Cholecalciferol converted to 25OHD3 in liver -> 25OHD3 modified to 1,25-dihydroxy-vitamin-D3 in kidneys under PTH stimulation
• Circulating 25OHD – best indicator of vitamin D levels
• Previous study found 25OHD levels to be higher in wild rhinos than captive rhinos
• Serum samples collected opportunistically from two captive Eastern black rhinoceros housed with seasonal access outdoors in a North American zoo were tested for 25OHD levels over a 3 year period
• Monthly blood samples collected and 25OHD levels measured via RIA
• UVB exposure was also noted for each rhino by monitoring time spent outdoors during daylight and using UV forecasts via the National Oceanic and Atmospheric Administration’s UV Index Bulletins Archives
• 25OHD low on early screening tests so an oral vitamin D3 supplement was added to diet
• 25OHD increased with increased UVB exposure but did not significantly increased with addition of dietary vitamin D3
• 25OHD decreased with increased confinement indoors and decreased UVB exposure
• Lower 25OHD levels in captive rhinos in this study suspected to be due to differences in UV intensity at the northern latitude of the zoo
• This study indicates that in black rhinos natural sunlight exposure seems to influence circulating 25OHD and dietary supplementation of vitamin D has little effect
• In humans, low 25OHD has been linked to various cancers, autoimmune disorders, abnormal cardiac function, and susceptibility to infections such as tuberculosis and sepsis
  
  • The author speculates that black rhinos may be similarly affected by hypovitaminosis D given captive black rhinos susceptibility to certain cancers and infections

Question 27

A recent study evaluated the welfare associated with rhino capture and transport.

What clinicopathologic changes were associated with capture and transport?

What allowed for smoother capture and trasnport and low lactate at the start?

Answer 27

Pohlin, F., Hofmeyr, M., Hooijberg, E. H., Blackhurst, D., Reuben, M., Cooper, D., & Meyer, L. C. (2020). Challenges to animal welfare associated with capture and long road transport in boma-adapted black (diceros bicornis) and semi-captive white (ceratotherium simum) rhinoceroses. The Journal of Wildlife Diseases, 56(2), 294-305.

Abstract: Capture and transport are part of translocation and expose animals to a variety of stressors that can lead to morbidity and mortality. We aimed to establish a better understanding of the physiologic responses to capture and transport in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceroses in Southern Africa. Fourteen adult black rhinoceroses were transported 600 km by vehicle and 32 white rhinoceroses (24 adults and 8 juveniles) were transported 1,300 km by vehicle. The black rhinoceroses had been wild-caught and boma-adapted over 6 wk prior to the translocation and were only sedated to allow for loading into the transport crates. The white rhinoceroses originated from a game farm and were chemically immobilized from a helicopter and then loaded. Paired blood samples were collected from animals at loading (capture) and after transport and evaluated for changes in clinical chemistry analytes, acute phase reactants, and oxidative stress biomarkers. The Wilcoxon rank sum test was used to compare changes in measured analytes from capture and after transport. All rhinoceroses survived capture and transport. Rhinoceroses experienced total body water loss, mobilization of energy reserves, and muscular damage. Alterations in acute phase reactants suggested that animals mounted a stress response. Oxidative stress was observed in black rhinoceroses. We identified the following challenges to animal welfare during transport: hydration status, energy balance, skeletal muscle fatigue, and stress-induced immunomodulation. Measures to mitigate these challenges, such as administration of fluids, need to be included in the planning of future translocations.

● Southern-central black rhinoceros - critically endangered IUCN

● Southern white rhinoceros - near threatened IUCN

● Current mortality rate for rhinoceros translocation in ZA and Namibia: ~5%

● All rhinoceroses survived

● Increased TP, Albumin, Na, and Cl: dehydration

○ Also white rhinos increase BUN and crea (decreased GFR) - transported longer and at higher temps than black rhinos

● Increased tBili, decreased K, Mg, P (and Ca in juvenile) - decreased intake (fasting)

○ Elevated NEFA from protein catabolism and mobilization of lipid stores

○ Increased BHB (ketone bodies) and triglycerides support negative energy balance as NEFAs are utilized for energy

● Chol decreased - response to inflammatory cytokines and acute phase reaction

● AST and CK increased - muscle exertion

● Lactate increased in black rhinos during transport and profoundly elevated in white rhinos after helicopter-capture

● No change in cortisol - possibly missing peak plasma concentrations at sampling times

● SAA increase and iron decrease (APPs) - immunomodulation in response to stress

● Elevated oxidative stress biomarkers (conjugated dienes) in black rhinos only

○ Black rhinos received NSAID, may have contributed to oxidative damage

○ White rhinos received vitamin C, E, and selenium - antioxidants may have prevented increase in lipid peroxidation products

● Challenges to animal welfare identified during transport of rhinoceroses: dehydration, negative energy balance, skeletal muscle fatigue, stress-induced immunomodulation

● Temporary confinement in bomas allowed smoother capture and transport, lower lactate at start of transport, and mitigated the need for full immobilization vs helicopter-captured

Question 28

A recent study evaluated the effects of ventilation on blood gases in white rhinos.

What are some of the most common complciations in anesthetized rhions?

Describe intubation and ventilation of rhinos?

What changes in the blood gas occurred following intubation and ventilation?

Answer 28

Jeon, M., Mama, K. R., Zuba, J. R., Lamberski, N., Oosterhuis, J. E., Clancy, M. M., … & Olea-Popelka, F. (2017). Evaluation of blood gas values in anesthetized southern white rhinoceros (Ceratotherium simum) ventilated with a novel demand ventilator in a zoological park setting. Journal of Zoo and Wildlife Medicine, 48(4), 1016-1025.

Abstract: Rhinoceros conservation efforts are essential to the survival of the species. One such effort is focused on using advanced reproductive technologies to produce viable northern white rhinoceros (Ceratotherium simum cottoni) embryos for implantation into southern white rhinoceros (Ceratotherium simum simum) surrogates. Anesthesia may be required to facilitate necessary procedures in these surrogate rhinoceros, but commonly reported side effects including hypercapnia and hypoxemia limit anesthetic recumbency time due to animal safety concerns. Although many interventions have been attempted, success in improving these physiologic parameters to date is mixed. The objective of this report is to describe arterial pH (pHa), blood gas (PaO2 and PaCO2), bicarbonate, base excess, lactate, and cardiovascular (heart rate, direct arterial blood pressure) values recorded in seven intubated and ventilated female southern white rhinoceros anesthetized for reproductive examinations in a zoological park setting. Anesthetic induction was accomplished using etorphine, medetomidine, butorphanol, and midazolam. The primary hypotheses were that PaO2 and PaCO2 would improve after intubation and mechanical ventilation. Induction and recovery observations were also summarized. Physiologic and laboratory data were analyzed using a mixed linear regression model using ranks. Statistical significance was set at P < 0.05. The PaO2 increased significantly (P < 0.001) following ventilation from a median value of 58 (range, 38–67) to 123 (range, 42–184) mm Hg. The PaCO2 significantly (P = 0.003) decreased from 63 (range, 55–73) to 52 (range, 30–75) mm Hg, with a corresponding improvement (P = 0.068) in pHa from 7.33 (7.25–7.34) to 7.37 (7.24–7.58) units. Intubation and ventilation improve respiratory parameters and may facilitate safe prolongation of anesthetic duration in white rhinoceros.

• Hypercapnia & hypoxia are major complications with anesthetized rhinos
• Efforts to alleviate respiratory compromise have included partial reversal
• Immobilization – etorphine 0.0018-0.0024 mg/kg and butorphanol (10 B:1 E) with midazolam (0.015-0.020 mg/kg) and medetomidine 20-24 mcg/kg IM
• Induction – Propofol 0.28 mg/kg, ketamine 0.17 mg/kg, or medetomidine 5.6 mcg/kg IV
• Intubation – 30 mm ID orotracheal tube with stylet & hydraulic wedge to open jaws
• Ventilation – mega-vertebrate demand ventilator @ 70 PSI to generate Fi02 of 40%
• Reversal – naltrexone 0.1-0.24 mg/kg IV and atipamezole 0.22 mg/kg – half IM half IV
• Hypoxemia is worse in adults & subadults than in calves – weight or chest wall rigidity in older animals

Take Home: Intubate and ventilate rhinos, use butorphanol (or other mixed antagonist) with etorphine

Question 29

A recent study evaluated repeated anesthesia in a rhino being managed for upper respiratory obstruction.

What anesthetic combination did they use?

Describe the positioning of rhinos under anesthesia?

What changes in physiologic and blood gas parameters were observed with this protocol?

Answer 29

Mora, I. M., Langan, J. N., Bailey, R. S., Aitken-Palmer, C., Adkesson, M. J., Tang, K. N., & Chinnadurai, S. K. (2018). Repeated anesthesia in a black rhinoceros (Diceros bicornis) to manage upper respiratory obstruction. Journal of Zoo and Wildlife Medicine, 49(4), 1041-1046.

Abstract: This report describes weekly repeated anesthesia in a 7-yr-old, 1,030 kg, female Eastern black rhinoceros (Diceros bicornis michaeli), that was immobilized six times using a combination of 2 mg etorphine (0.002 mg/kg), 5 mg medetomidine (0.005 mg/kg), 25 mg midazolam (0.024 mg/kg), and 300 mg ketamine (0.29 mg/kg)delivered intramuscularly (IM) via remote dart to facilitate long-term medical care of a bilateral, obstructive Actinomyces sp. rhinitis. The drug combination described in this study resulted in reliable, rapid recumbency of the animal within 2–8 min after initial administration via dart and produced deep anesthesia for 34–78 min without supplemental anesthetic administration. Antagonist drugs (100 mg naltrexone [0.1 mg/kg] and 25 mg atipamezole [0.024 mg/kg] IM) produced reliable and uneventful recoveries in all the procedures. During each anesthetic procedure, the animal was intubated and provided intermittent positive pressure ventilation with a megavertebrate demand ventilator. Tachycardia and hypoxia noted after induction resolved after positive pressure ventilation with oxygen. This report provides useful information on a novel anesthetic protocol used repeatedly for intensive medical management in a black rhinoceros.

• Etorphine – potent opioid commonly used in rhino procedures
  
  • Rhinos are particularly sensitive – hypertension, tachycardia, acidemia, respiratory depression with hypoxemia and hypercapnia all reported
• Alpha 2 agonists – detomidine, medetomidine – enhance sedation & analgesia, improve muscle relaxation, but can exacerbate respiratory depression and alter themoregulatory mechanisms
  
  • High doses are necessary to improve induction time
• Azaperone – butyrophenone, enhances tranquilization, shortens induction, causes peripheral vasodilation, and reduces hypertension
• Ketamine – reduces induction time and decreases supplemental drugs when used in induction
• This combination (ketamine, medetomidine, midazolam, etorphine) produces deep anesthetic plane for 34-78 minutes which allowed for intubation and ventilation
• Recumbency changed when sternal for more than 1 hour (sternal to lateral)
  
  • Sternal recumbency – better ventilation
  • Lateral recumbency – better peripheral limb circulation
  • Thick padding under the animal
• Physiologic parameters were higher in this study (HR, ETCO2, iBP) compared to standing unrestrained rhinos likely due to the anesthetic drugs
  
  • Tachycardia and low oxygenation was observed before intubation which resolved with positive pressure ventilation
  • Blood gas analysis is more sensitive than pulse oximetry which overestimates hemoglobin saturation in rhinos

Take Home: Multimodal drugs and intubation allow for safe repeated anesthetic events of the same rhino

Question 30

A recent study evaluated ferritin as a marker of iron overload disorder in the Sumatran rhino.

What is ferritin?

How did ferritin vary in Sumatran rhinos with iron storage disease?

Answer 30

Roth, T. L., Reinhart, P. R., & Kroll, J. L. (2017). Serum ferritin concentration is not a reliable biomarker of iron overload disorder progression or hemochromatosis in the Sumatran rhinoceros (Dicerorhinus sumatrensis). Journal of Zoo and Wildlife Medicine, 48(3), 645-658.

Abstract: The aim of this study was to determine if ferritin is a reliable biomarker of iron overload disorder (IOD) progression and hemochromatosis in the Sumatran rhinoceros (Dicerorhinus sumatrensis) by developing a species-specific ferritin assay and testing historically banked samples collected from rhinos that did and did not die of hemochromatosis. Ferritin extracted from Sumatran rhino liver tissue was used to generate antibodies for the Enzyme Immunoassay. Historically banked Sumatran rhino serum samples (n¼298) obtained from six rhinos in US zoos (n¼290); five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia (n¼5); and two rhinos in Sabah, Malaysia (n¼3) were analyzed for ferritin concentrations. Across all US zoo samples, serum ferritin concentrations ranged from 348 to 7,071 ng/ml, with individual means ranging from 1,267 (n¼25) to 2,604 ng/ml (n ¼ 36). The ferritin profiles were dynamic, and all rhinos exhibited spikes in ferritin above baseline during the sampling period. The rhino with the highest mean ferritin concentration did not die of hemochromatosis and exhibited only mild hemosiderosis postmortem. A reproductive female exhibited decreases and increases in serum ferritin concurrent with pregnant and nonpregnant states, respectively. Mean (6SD) serum ferritin concentration for Sumatran rhinos in Malaysia was high (4,904 6 4,828 ng/ml) compared to that for US zoo rhinos (1,835 6 495 ng/ml). However, those in Sabah had lower ferritin concentrations (1,025 6 52.7 ng/ml) compared to those in Sungai Dusun (6,456 6 4,941 ng/ml). In conclusion, Sumatran rhino serum ferritin concentrations are dynamic, and increases often are not associated with illness or hemochromatosis. Neither a specific pattern nor the individual’s overall mean ferritin concentration can be used to accurately assess IOD progression or diagnose hemochromatosis in this rhino species.

• Iron overload disorder (IOD) = excessive storage of iron in organ tissues
  
  • Affects variety of species including African black rhinos and Sumatran rhinos
  • Hemochromatosis = diseased state where organ function compromised by hemosiderin
• 20% of black rhino deaths secondary to infectious disease
  
  • Salmonella and TB have enhanced virulence in iron rich environments
• IOD possibly related to susceptibility to infectious disease and decreased longevity
• Ferritin - primary intracellular iron binding protein in organ tissues
  
  • Serum ferritin associated with body iron stores, used to diagnose IOD
  • Not specific to IOD – ferritin is an acute phase protein
    
    • Can increase secondary to inflammation 🡪 chronic infection, neoplasia
  • Ubiquitous among living organisms but immunologically species specific
• Monitoring of IOD in rhinos based on serum ferritin levels and transferrin saturation
  
  • Previous studies - serum ferritin high in Sumatran and black rhinos, increase after capture, increase with age and time in captivity
    
    • Results inconsistent in Sumatran rhinos
  • Current assay uses equine ferritin antibodies
• Retrospective study using banked Sumatran rhino serum samples obtained from six rhinos in US zoos, five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia, and two rhinos in Sabah, Malaysia
• A species-specific ferritin assay was developed, tested banked samples collected from rhinos that did and did not die of hemochromatosis
• Data from this study does not support the use of serum ferritin for monitoring IOD progression or diagnosing hemochromatosis in Sumatran rhinos
• Sumatran rhino serum ferritin concentrations are dynamic, and increases not associated with illness or hemochromatosis
  
  • Mean serum ferritin concentrations were not higher in rhinos that died of hemochromatosis compared to those that did not develop the disease
  • Significant fluctuation in ferritin concentrations between sample collections, months, and years within individuals
  • Liver iron load not correlated with mean serum ferritin concentrations or concentrations in samples taken just prior to death
• Serum ferritin in Malaysian rhinos higher than US rhinos and none of Malaysian rhinos dies of IOD
• Ferritin increases in response to tumor necrosis factor independent of iron status
  
  • Can be elevated in humans with certain cancers
  • One rhino with cancer had elevated ferritin
• Pregnancy may also affect ferritin levels
  
  • Decreased during pregnancy and increased postpartum in one rhino
• Healthy adult Sumatran rhino serum ferritin concentrations - 1,000 and 2,000 ng/ml
• The rhino specific assay provided more accurate results than the equine assay
• This study data did not support previous conclusions that serum ferritin increases with time in captivity or with age
• Diet suspected not to be the primary cause for IOD
  
  • More likely altered iron uptake regulatory mechanism
  • Decreased anemia risk and iron drain in captivity thought more likely culprit

Question 31

A recent study evaluated ferritin as a biomarker for iron storage disease in black rhinos.

What species are predisposed to iron overload disorder?

What is the difference between hemosiderosis and hemochromatosis?

What is ferritin?

What were the findings about its use as a marker for iron overload? Is this similar to other rhinos?

Answer 31

Wojtusik, J., & Roth, T. L. (2018). Investigation of factors potentially associated with serum ferritin concentrations in the black rhinoceros (Diceros bicornis) using a validated rhinoceros-specific assay. Journal of Zoo and Wildlife Medicine, 49(2), 297-306.

Abstract: Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros (Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples (n=943) collected from 36 black rhinoceros (<1-33 yr) at 14 US institutions. Mean (± SEM) serum ferritin concentration was 6,317 ± 505 ng/mL (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444+/-1, 130 ng/mL) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean value sin wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P < 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,162 ± 254 ng/mL) and adults (7,623 ± 610 ng/mL) and were not correlated with age (r = 0.143) or time in captivity (r = 0.146, wild born; r= 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 109 ng/mL; male 8,684 ± 717 ng/ml), date, month, or season of collection (P>0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.

· Iron homeostasis sustained through controlled absorption.

o Excess iron stored intracellularly within liver or spleen as hemosiderin (hemosiderosis).

o Iron overload disorder (IOD) – primates, birds, hosrses, tapir, red deer, three spp of rhinos (Af black, Sumatran, indian), results in excessive iron storage (severe hemosiderosis).

§ Excessive hemosiderin -> visceral damage (hemochromatosis term in humans).

§ Accumulation in liver, heart, endocrine organs (pancreas, pituitary, gonads). Exacerbates other dz (hemolytic anemia, mucocutaneous ulcer disorder, leukoencephalomalacia).

· Dx often postmortem – black rhinos increased hemosiderin in captive vs wild animals.

§ Correlation between stored iron measures and both age and time in captivity.

· Tx – frequent, large-volume phlebotomies, oral iron chelator.

· Tracking serum ferritin concentrations – questionable accuracy for dx IOD.

· Ferritin – acute phase globular protein complex responsible for iron storage.

· Used as indicator of total iron stores, correlated with stored iron levels in some spp.

· A ferritin enzyme immunoassay (EIA) developed for horses has traditionally been used to measure ferritin in rhinos, discrepancies in results.

· Serum ferritin concentration is not a reliable biomarker of IOD progression and dz in Sumatran rhinos. Not correlated with liver stores.

§ May be influenced by reproductive status of female, diet changes, may differ based on location, individual, or subspecies.

· Animals born in wild and brought into captivity appear to be at risk, unclear.

· Goal of study: validate rhinoceros-specific ferritin assay for measuring black rhinoceros serum ferritin and to determine if differences in ferritin concentrations are associated with age, season, sex, subspecies, and location of birth, and to evaluate the efficacy of this assay as a tool for diagnosing IOD and monitoring its progression.

· Animals and samples: 1990-2016; 36 black rhinos, <1 – 32 yrs, 14 institutions.

o Analyzed using rhinoceros ferritin EIA.

o 2 individuals during time period – 1 unknown, 1 euthanized and IOD confirmed.

o Samples were banked for other purposes and used opportunistically in this study.

· Ferritin isolation.

o Ferritin isolated from black rhino liver using methodology described for Sumatran rhinoceroses.

· Gel electrophoresis and immunoblotting.

o Black rhinoceros liver ferritin (BRF) was subjected to gel electrophoresis using purified equine spleen ferritin and Sumatran rhino ferritin positive controls.

· Ferritin enzyme immunoassay.

o Serum ferritin concentrations were measured with the rhino specific ferritin sandwich enzyme immunoassay (EIA) previously developed and validated for Sumatran rhinos (Roth et al).

· Mean serum ferritin concentration – variation among individuals was significant.

· Results suggested ferritin is not an accurate biomarker of IOD in black rhinoceros.

o Many rhinos in this study exhibited ferritin values equal to or lower than those reported for wild individuals, overall means tended to be greater. Possible all values for captive individuals are ‘abnormal’.

o High values did not indicate the animal was at risk of developing IOD

o One animal euthanized had hemosiderosis at necropsy, neither IOD or hemochromatosis diagnosed. Extreme ferritin values associated with arthritis or something else.

o Another individual had rising ferritin values before succumbing to M. tuberculosis. Hemosiderosis post mortem.

§ In one individual, ferritin was low prior to illness and signs of IOD, confirmed at necropsy. Ferritin rose but only after clinical signs, still below the species average. This animal also had pneumonia.

· Serum ferritin is often considered a reliable marker of stored iron in horses, pigs, humans.

§ However, acute phase protein – serum concentrations rise during instances of infection, inflammation, diet change, metabolic syndromes, and many other dz not specific to iron storage.

· Ferritin concentrations did not differ between age groups or with time in captivity. Not impacted by sex.

· Wild born individuals had significantly higher levels of ferritin than those born in captivity.

§ Unexpected, as ferritin values generally lower in wild versus captive individuals.

§ Possible captive born individuals may have a developmental adaptation mitigating the physiological response of ferritin, or wild born sample size was small.

§ In Sumatran rhinos, liver iron concentrations did not correlate with ferritin, and ferritin was not indicative of disease progression.

· Conclusion: Black rhinoceros serum ferritin concentrations are variable, high concentrations are not predictive of clinical illness, and low concentrations can exist in sick individuals with extensive hemosiderosis; therefore, ferritin is not a useful tool for diagnosing IOD or monitoring its progression in the African black rhinoceros.

Question 32

A recent study described infection with Mycobacterium orygis in an indian rhino.

What testing is recommended for mycobacterium?

What were the clinical signs this animal developed?

What are the antigens used for serologic testing for tuberculous mycobacteria?

Answer 32

Love, D. M., Garner, M. M., Lyashchenko, K. P., Sikar-Gang, A., Bradway, D. S., Robbe-Austerman, S., … & Ramer, J. (2020). Tuberculosis caused by Mycobacterium orygis in a greater one-horned rhinoceros (Rhinoceros unicornis): first report in the western hemisphere. Journal of Zoo and Wildlife Medicine, 50(4), 1000-1004.

Abstract: Mycobacterium orygis, a newly identified member of the Mycobacterium tuberculosis complex, has been isolated predominantly from hoofstock in eastern Africa and the Arabian Peninsula, and sporadically in cattle (Bos taurus indicus), rhesus monkeys (Macaca mulatta), humans, and a greater one-horned rhinoceros (Rhinoceros unicornis) in South Asia. In rhinoceros, tuberculosis typically presents as a chronic progressive respiratory disease. The report describes the postmortem diagnosis of tuberculosis caused by Mycobacterium orygis in a greater onehorned rhinoceros with hind limb paresis due to neural granulomatosis. Serologic assays for detection of antibodies to M. tuberculosis complex proteins before culture results allowed for appropriate herd management protocols to be initiated. Mycobacterium genus–specific polymerase chain reaction assays with direct sequencing allowed timely confirmation of the serologic results. This is the first isolation of M. orygis in the western hemisphere, showing the need for mycobacterial testing of rhinoceros before international shipments and the urgency for validated antemortem M. tuberculosis complex screening assays in rhinoceros species.

· Mycobacterium orygis most closely related to M. africanum

· Disease by M. orygis indistinguishable from classic tuberculosis, with zoonotic and zooanthroponotic transmission

· Classic signs: chronic progressive respiratory disease, typically diagnosed postmortem

o Testing

§ Tuberculin skin testing unreliable in rhinoceros

§ Serology (dual path platform- DPP) and multiantigen print immunoassays (MAPIAs) successful in many species, but not validated in rhinos

• Mycobacterial culture gold standard, but variable sensitivity and invasive sampling

· Developed intermittent gait deficit in right pelvic limb 1 year after acquisition from India

o General proprioceptive deficits, High stepping gait with limb crossing, rapid atrophy of gluteal and epaxial muscles progressed to recumbency

· Necropsy results:

o Markedly enlarged irregular abdominal lymph nodes on gross, consistent with granulomas in lymph nodes, lung, bone marrow and effacing sciatic nerve and cauda equina on histo. Rare acid fast bacteria noted in multinucleated giant cells stained with Fite’s acid fast technique (photo below, potential test question photos)

· Serum testing:

o DPP Vet TB and MAPIA testing of euthanasia blood positive, prior banked bloods then tested and variable but measurable and increasing IgG responses noted to various proteins

o ***TESTABLE– MPB83, CFP010, ESAT-6 major immunodominant antigens in members of the MTBC and generate antibody responses during TB infection in mammals. Absent in nontuberculous bacteria

• Culture and PCR consistent with M. orygis

· TAKE HOME: First report of M. orygis in greater one-horned rhinoceros in Western hemisphere

o M. orygis newly described member of Mycobacterium tuberculosis complex

o Recently reported in fatal case in free-ranging greater one-horned rhinoceros

o Unusual case presentation of hind limb paresis secondary to neural granulomatosis

Question 33

A recent case series described clinical salmonellosis in black rhino calves.

What serovars were commonly encountered?

What forms of the disease are young animals more susceptible to?

What treatments are recommended?

Answer 33

Love, D., Madrigal, R., Cerveny, S., Raines, J., Rideout, B., & Lung, N. P. (2017). Case series: Clinical salmonellosis in four black rhinoceros (Diceros bicornis) calves. Journal of Zoo and Wildlife Medicine, 48(2), 466-475.

Abstract: Although Salmonella spp. infection has been identified in captive and free-ranging rhinoceros, clinical cases in black rhinoceros (Diceros bicornis) calves have not been described. This case series describes clinical salmonellosis in four black rhinoceros calves. Two calves developed self-limiting diarrhea, recovering after treatment. The other two cases were fatal. One of the fatal cases had a short clinical course, whereas the other case was protracted, with signs reflecting multiple organ system involvement. In all cases, diagnosis was by fecal culture and/or quantitative polymerase chain reaction. A variable clinical presentation, which is typical for salmonellosis in domestic hoofstock, was a feature of these rhinoceros cases. Similarly, postmortem pathology in black rhinoceros calves was consistent with domestic neonatal ungulates with salmonellosis. Potential predisposing factors for infection were considered to be primiparity of the dam and failure of passive transfer in the calf. The case investigation included attempts to identify the source of infection, which was aided by organism serotyping. In one case, the patient’s dam and another conspecific in the facility were shown to be asymptomatic shedders of the organism strain responsible for disease in the calf. Further surveillance of captive rhinoceros Salmonella spp. carrier status is needed to inform screening recommendations for thistaxa.

• Case 1
  
  • Negative Crypto, negative giardia, positive Salmonella enterica javiana – flunixin (1.1 mg/kg PO q24h x2d) and TMS (33 mg/kg PO q24h x 7d) – recovered, nursed throughout
• Case 2
  
  • Rotavirus and coronavirus negative, Salmonella enterica Newport – amikacin 8 mg/kg IM q24h x 12d and bismuth subsalicylate 30 mL PO q24h x 18d
• Case 3
  
  • Moribund on presentation, coded, CBC – severe anemia, leukocytosis, thrombocytopenia, Chem – azotemia, severe hyponatremia, hypoproteinemia; Campylobacter & Clostridium negative, Salmonella enterica lomalinda; necropsy – ulcerative enteritis, typhylocolitis, sepsis
• Case 4
  
  • Presentation 1 - C6-T2 lesion suspected based on posture & ataxia, CBC – inflammatory leukogram, hyperfibroginemia, Anaplasma/Babesia/Bartonella/Borelia/Rickettsia/Ehrlichia/Francisella negative; EEE/VEE negative – WEE exposure (low); Clostridium & toxin negative, Salmonella enterica typhimurium – positive in feces and CSF culture
  • Presentation 2 – hemorrhagic diarrhea, severe leukopenia, IV ampicillin, metronidazole, omeprazole, flunixin, central line placement, eventual sepsis & DIC & euthanasia
• Salmonella occurs more commonly in black rhinos
• Young animals are more susceptible to enteric and septicemic forms of the disease
• Treatment – supportive care, abx controversial in adults, necessary in neonates – understand risks of potential resistance carrier status
  
  • Gastroprotectants, probiotics, transfaunation, antiendotoxic therapy, acid/base and electrolyte correction, fluid therapy, nutrition
• Culture and PCR useful for identification – PCR especially so in outbreaks
• Risk factors – primarity, failure of passive transfer

Take Home: Salmonellosis is an important disease of infant and juvenile rhinoceros. Aggressive supportive care is necessary particularly when

Question 34

A recent study evaluated the gastrointestinal parasites of the Indian rhino.

What were some of the most common parasites identified?

Answer 34

O’Connor, L., Choudhury, B., Ali, S., Bull, K., & Morgan, E. (2018). A survey of gastrointestinal parasites of wild and orphan greater one-horned rhino (Rhinoceros unicornis) in Kaziranga National Park, Assam, India. Journal of Zoo and Wildlife Medicine, 49(4), 1051-1053.

Abstract: Wild greater one-horned rhinos (Rhinoceros unicornis), orphaned juveniles in human care, and orphaned calves from Kaziranga National Park, Assam, India were surveyed coprologically for gastrointestinal parasites. Parasite infections were present in 100% of wild rhino samples, 96% of orphaned juvenile samples, and 27% of orphaned calf samples. In wild rhino, observed parasite ova were primarily of trematodes Paramphistomum sp. (100%), followed by those of strongyle nematodes (94%) and the cestode Anoplocephala sp. (56%). Orphaned juvenile and calf samples were positive only for strongyles. Total fecal parasite egg counts were recorded in wild rhino (mean 64 eggs per gram [epg], range 0–270), orphan juveniles (mean 43 epg, range 0–145), and orphan calves (mean 2 epg, range 0–10). Results suggest that parasite infection in rhinos in this setting is common, though more extensive sampling would provide further information on epidemiology and potential impacts on individual health and population viability.

• Indian Rhino population – 3,557 in 2015
• Strongyles treated with 1.5 g/300kg bolus of Fenbendazole
• Paramphistomum trematode present in all animals
• 94% had stronglyes
• 56% had Anoplocephala

Take Home: Paramphistomum trematode, Strongyles, Anoplocephala cestode common parasites of Indian Rhino

Question 35

A recent study described amebic meningoencephalomyelitis in a black rhino.

What amoeba was the cause of the disease?

What are common sources of infection of this parasite?

How can it be prevented?

Answer 35

· Naegleria fowleri – Free-living amoeba, FW, worldwide. Primary amebic meningoencephalitis in people, enters through nasal sinus. Migrates along olfactory nerve through cribriform plate. More than half cases reported in US. Fatality > 95%. Reported in cattle, sheep, and a South American tapir in US. Death within 3-7 days of symptoms. CS include anorexia, lethargy, dry cough, mucoid feces, pyrexia, nasal discharge, acute central neurologic signs (ataxia, facial paralysis, circling, weakness, blindness, seizures).

· Common infection source in cattle – stagnant canals, drinking troughs that reach high temperatures (can survive up to 133 deg F).

· Premortem dx of amebic meningoencephalomyelitis relies on ID of high polymorphonuclear leukocyte counts and amebas in CSF. MRI usually WNL. Humoral reactions usually weak. No tx. In human case – amphotericin B, fluconazole, oral rifampin resulted in successful outcome. Miltefosine (breast cancer tx; also leishmaniasis) and voriconazole have shown efficacy in vitro.

· Prevention – Elimination of warm, stagnant, FW sources.

Takeaway: N. fowleri causes primary amebic meningoencephalitis, persists in stagnant, warm FW sources. Fatal. Zoonotic.

Question 36

A recent study evaluated the prevalence of hemorrhagic septicemia in water buffalo as potential exposure to Javan rhinos.

What is the etiologic agent of hemorrhagic septicemia?

What species are commonly affected?

What did this study find?

Answer 36

Khairani, K. O., Nydam, D., Felippe, M. J., McDonough, P., Barry, J., Mahmud, R., … & Radcliffe, R. W. (2018). Surveillance for hemorrhagic septicemia in buffalo (Bubalus bubalis) as an aid to range expansion of the Javan rhinoceros (Rhinoceros sondaicus) in Ujung Kulon National Park, Indonesia. Journal of wildlife diseases, 54(1), 14-25.

Abstract: The Javan rhinoceros (Rhinoceros sondaicus) of Ujung Kulon National Park (UKNP) is the crown jewel of Indonesia’s rich natural history. The park lies on a peninsula surrounded by coastline and agriculture-dominated landscapes. The invasion of water buffalo (Bubalus bubalis) into the park carries a substantial health risk to the Javan rhinoceros and threatens plans to establish a new population outside of its only current range in UKNP. Hemorrhagic septicemia (HS), known locally as septicemia epizootica and caused by Pasteurella multocida B:2, could thwart Indonesia’s efforts to expand the range of the Javan rhinoceros. Because HS was considered eradicated from Banten Province, few preventative programs have been available to farmers. During June 2012-July 2013, biologic samples were collected from 770 water buffalo in 19 villages. Deep nasal swabs ( n=85) were taken for bacterial culture and blood samples ( n=770) were collected for serologic testing. No animals were positive on culture. The prevalence of antibody to P. multocida in this population was 1.8% (14 of 770 animals). A structured questionnaire was used to gather information about possible risk factors. Husbandry practices associated with presence of antibody in water buffalo included lack of a permanent area to house buffalo at night, low body condition score (=2), high body temperature (≥40 C), a history of clinical signs or sudden death in the previous year, and a grazing system that utilized significant forage inside the park. Antibody was not associated with sex, age, vaccination status, or season. Understanding HS disease dynamics in the buffalo adjacent to UKNP may improve the livelihoods of people and health of endangered rhinoceroses in this ecosystem.

• Javan rhinoceros are critically endangered with only 51 animals in a single population on the western tip of Java
  
  • Aiming to create a second population of Javan rhinoceros closer to humans and agriculture
• Javan rhinoceros mortalities have been associated with hemorrhagic septicemia, anthrax, and trypanosomiasis
• Hemorrhagic septicemia (HS) is caused by Pasteurella multocida B:2
  
  • Major mortality event in 1982 with continued sporadic mortalities in Javan rhinoceros
  • Transmitted from buffalo and other livestock species
  • Also affects yaks, camels, and water buffalo
  • Nasal swabs are unreliable for the detection of P. multocida due to overgrowth by resident microflora
  • Ideal culture swab = heart within a few hours of death
• After rhino HS mass mortality, HS vaccinations in livestock has created a HS-free zone surrounding current Javan rhino population, though the current vaccination program is intermittent

Methods: Studied prevalence of HS in water buffalo in 19 villages surrounding location of proposed second Javan rhinoceros population site in eastern Ujung Kulon National Park with HS serology and nasal culture

• HS case definition: severe respiratory distress with nasal discharge and frothing from the mouth, leading to recumbency and death.

Key Points:

• Three clinical cases were all positive on ELISA, but negative on culture
• Antibody prevalence in population = 1.8%
  
  • Not associated with age, sex, vaccination status, or season
  • Associated with body condition score, body temperature, no permanent holding area, grazing system inside the national park, and history of sudden death in the herd
• Analysis shows that you can identify serology positive animals by history of HS clinical signs in herd within one year and lowest BCS
  
  • Animals with sudden death in herd within one year were also at high risk for positive P. multocida antibody
• Water buffalo may be a source of HS epidemics, despite low prevalence due to carrier animals
  
  • New vaccination program initiated after the results of this study

Conclusion: Water buffalo in Indonesia are a reservoir species of hemorrhagic septicemia that may spill over to Javan rhinoceros. A low BCS and history of HS clinical signs within the past year are factors closely associated with serology positive animals.

Question 37

A recent study investigated the single dose pharmacokinetics of flunixin meglumine in the white rhinoceros.

Describe the clinical uses of flunixin.

What dose was used?

How was absorption?

How long was the dose above therapeutic concentrations?

What are some adverse effects of NSAIDs? Were any seen in this study?

Answer 37

East, B., Tell, L., Citino, S. B., Fredholm, D. V., Gamble, K., & Fajt, V. (2019). Pharmacokinetics of a single oral dose of flunixin meglumine in the white rhinoceros (Ceratotherium simum). Journal of Zoo and Wildlife Medicine, 50(2), 322-329.

Abstract: Flunixin meglumine, a nonsteroidal anti-inflammatory medication, has been used in rhinoceros species at doses extrapolated from domestic animals. There is increasing evidence to suggest significant variations exist in metabolism of drugs in exotic species. Due to the differences in drug metabolism, dose extrapolation from domestic animals may not be appropriate for exotic species. The objective of this study was to investigate the pharmacokinetics of flunixin meglumine in five white rhinoceroses (Ceratotherium simum) administered a single (1 mg/kg) oral dose of a commercial equine flunixin meglumine paste. Concentrations of flunixin and its metabolite 5-OH flunixin were analyzed, and pharmacokinetic parameters were estimated for each animal. Mean observed plasma concentrations peaked at 1,207 +- 601 ng/ml and occurred at 3 +- 1 hr. The geometric mean of the apparent elimination half-life after oral administration was 8.3 +- 1.2 hr. This data suggests that flunixin meglumine appears to be slowly metabolized or slowly absorbed in this species. No adverse clinical effects were observed during the study period. A single dose of 1 mg/kg appears safe for use in the white rhinoceros. Multidose studies are needed to determine if plasma accumulation of flunixin meglumine occurs and to evaluate safety

· Flunixin suppresses prostaglandin and thromboxane production

o Most efficacious for alleviation of equine visceral pain, effective for acute pain

· Rhinos are hindgut fermenters, so equine PK models commonly used

STUDY DESIGN: 5 adult rhinos fed flunixin paste 1mg/kg PO once, with blood samples collected up to 24hrs after drug admin, for 4 animals added 36 and 48hr samples; drug levels measured using ultra performance liquid chromatography

RESULTS: No adverse clinical effects related to flunixin administration seen; protein binding >99% in plasma. See above for relevant details.

· 1.1mg/kg flunixin PO absorbed well, peak concentration within 3 hours

o Absorption less than pigs and goats, though study dose was higher (2.2mg/kg) the concentrations were roughly the same

· Half life 7-10hrs

· Delayed absorption due to binding to hay and stomach contents may have accounted for double peak—maybe enterohepatic cycling (can be pronounced in animals with more GI residence of food)

o Secondary metabolites (5-OH flunixin) found in detectable concentrations in this study. Unknown activity in body.

· 3 animals in the study were pregnant, unknown effects of flunixin on pregnancy but may affect drug absorption

· Reported NSAID side effects include GI ulceration and hemorhage, effects on fecal microbiota in horses

· Concentrations of 200-900ng/mL effective in horses; was not an objective of this study but was over 300ng/mL for 12 hours

TAKE HOME: Flunixin is safe for use in rhinos at 1mg/kg PO, need more studies

Question 38

A recent study evaluated the pulmonayr and metabolic effects of suspending rhinos by their feet during transport.

What affects does positioning of rhinos have on their oxygenation status?

What blood gas parameters were affected by suspension?

What was their blood gas state like, regardless of positioning?

Answer 38

Radcliffe, R. W., Jago, M., Morkel, P. V., Morkel, E., du Preez, P., Beytell, P., … & Gleed, R. D. (2021). The pulmonary and metabolic effects of suspension by the feet compared with lateral recumbency in immobilized black rhinoceroses (diceros bicornis) captured by aerial darting. The Journal of Wildlife Diseases, 57(2), 357-367.

Abstract: Aerial translocation of captured black rhinoceroses (Diceros bicornis) has been accomplished by suspending them by their feet. We expected this posture would compromise respiratory gas exchange more than would lateral recumbency. Because white rhinoceroses (Ceratotherium simum) immobilized with etorphine alone are hypermetabolic, with a high rate of carbon dioxide production (VCO₂), we expected immobilized black rhinoceroses would also have a high VCO₂. Twelve (nine male, three female; median age 8 yr old [range: 4–25]; median weight 1,137 kg [range: 804–1,234] body weight) wild black rhinoceroses were immobilized by aerial darting with etorphine and azaperone. The animals were in lateral recumbency or suspended by their feet from a crane for approximately 10 min before data were collected. Each rhinoceros received both treatments sequentially, in random order. Six were in lateral recumbency first and six were suspended first. All animals were substantially hypoxemic and hypercapnic in both postures. When suspended by the feet, mean arterial oxygen pressure (P_aO₂) was 42 mm Hg, 4 mm Hg greater than in lateral recumbency (P=0.030), and arterial carbon dioxide pressure (P_aCO₂) was 52 mm Hg, 3 mm Hg less than in lateral recumbency (P=0.016). Tidal volume and minute ventilation were similar between postures. The mean VCO₂ was 2 mL/kg/min in both postures and was similar to, or marginally greater than, VCO₂ predicted allometrically. Suspension by the feet for 10 min did not impair pulmonary function more than did lateral recumbency and apparently augmented gas exchange to a small degree relative to lateral recumbency. The biological importance in these animals of numerically small increments in P_aO₂ and decrements in P_aCO₂ with suspension by the feet is unknown. Black rhinoceroses immobilized with etorphine and azaperone were not as hypermetabolic as were white rhinoceroses immobilized with etorphine.

Key Points:

• Immobilized black rhinos are suspended from their feet under a helicopter for periods of up to 30 min for translocation.
• Established protocols for capture of wild black rhinos use potent opioids i.e. etorphine.
  
  • Complications include hypoventilation, hypoxemia, hypercapnia, hypertension, and acidemia.
  • Usually partially reversed after loading for ground transportation.
  • Safe aerial suspension requires complete immobility and antagonisms is not routinely employed if air-lifting.
• Immobilized black rhinos in sternal recumbency have significantly greater median arterial oxygen pressure than in lateral, although alveolar ventilation is similar in the two postures.
• A white rhino positioned in dorsal for colic surgery experienced more profound hypoxemia vs rhinos in lateral recumbency.
• Hypothesized that immobilized black rhinos suspended by their feet from a crane would have higher arterial carbon dioxide pressure and lower PaO2 vs lateral recumbency.
• Hypermetabolic state with etorphine (high oxygen consumption and carbon dioxide production; VCO2) may contribute to hypoxemia, hypercapnia, hypertension, and acidemia when immobilized.
  
  • Hypothesized that immobilized black rhinos would have greater VCO2 than that predicted allometrically.
• Immobilized the rhinos, used collection bags attached to cuffed nostril ET tubes to collect expired gas and attached tubes to a capnography to measure end-tidal CO2. Blood collected from auricular artery toward the end of gas collection for iSTAT CG4+ and VetScan. Measured PcO2, PaCO2, arterial pH, lactate, SaO2.
• Regardless of posture, rhinos were substantially hypoxemic and hypercapnic.
• When suspended, significant differences in PaO2, SaO2, pH, PaCO2, PCO2, VD/VT, and PAO2.
  
  • PaO2, SaO2, and pH higher vs lateral. Mean PAO2 greater when suspended.
  • PcO2 lower vs lateral.
  • Only PaCO2 and PAO2 significantly affected by the order of treatment.
    
    • PaCO2 greater and PAO2 lower in the animals in lateral first, then suspended.
• Suspending immobilized black rhinos by their feet for 10 min did not impair pulmonary function vs lateral.
• All were severely hypoxemic and hypercapnic regardless of positioning.
• Suspension by the feet was associated with very slightly better respiratory gas exchange. Possibly due to improved alveolar ventilation, may have also been the reason for higher pH when suspended. Greater PaO2 with suspension likely consistent with the improved alveolar ventilation.
• Measured values for VCO2 in black rhinos considerably less vs white rhinos after etorphine.
  
  • Black rhinos may be inherently less susceptible than white to the hypermetabolic effects of etorphine.
• Likely that hypoventilation was attributable to the etorphine slowing the breathing rate.

Takeaways: All rhinos severely hypoxemic and hypercapnic regardless of positioning, suspending by feet for 10 min did not impair pulmonary function vs lateral. Slightly better gas exchange during suspension.

Question 39

A recent study evaluated the serologic evidence of Q fever in South African white rhinos.

What is the etiologic agent of Q fever?

What are teh typical clinical signs in rhinos? What about in people?

What was the prevalence of this disease? Did it change demographically?

Answer 39

SEROLOGICAL EVIDENCE OF COXIELLA BURNETII INFECTION IN THE WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN SOUTH AFRICA

Donnelly et al. JZWM 52(2), 2021

Abstract

Coxiellosis, or Query (Q) fever, a disease caused by the intracellular bacteria Coxiella burnetii, was recently described in a managed breeding herd of white rhinoceros (Ceratotherium simum) in the southeastern United States. Clinical disease often results in abortion and could represent a conservation challenge for this species. In addition to the reproductive and herd management consequences, coxiellosis is also a zoonotic disease. Infection or clinical disease in any free-ranging rhinoceros species in a national park setting has not been previously described. In this study, evidence of prior infection was measured by immunofluorescent antibody titers in 89 serum samples collected from white rhinoceros within private reserves and a national park in South Africa. Total seropositivity was 48/89 (53.9% [95% CI, 43.6–63.9%]). Animals on private reserves had a seropositivity of 21/51 (41.1% [95% CI, 27.1–55.2%]), and national park rhinoceros had a higher rate of seropositivity at 71.0% [95% CI, 55.9–86.2%] (27/38; P= 0.004). Adults had a higher seropositivity compared with subadults (P= 0.03). There was no difference in seropositivity between sexes (P > 0.05). Results demonstrate that South African white rhinoceros populations are exposed to Coxiella**, which could result in underrecognized reproductive consequences. Further studies should investigate potential implications for public health and conservation management of this species.

Key Points-

· Coxiella burnetii – intracellular bacteria responsive for Q-fever

· Outbreak associated with late-term abortions (see Bercier et al. JZWM 52(1)

· Zoonotic- fever, flu-like symptoms, pneumonia, abortion and/or stillbirth, endocarditis. Considered re-emerging with outbreaks in sheep, goats, and camels

· Diagnosis challenging- intermittent shedding, serology doesn’t indicate active infection, etc.

· Phase I/Phase II ABs produced in response to changes from smooth to rough lipopolysaccharides in the bacteria, and in humans can help differentiate acute/chronic infections

Phase I- chronic

Phase II- acute

· Both ab phases were ID’ed in the affected US white rhino herd, results suggest a temporal correlation with clinicals signs associated with infection. Coxiellosis has not been reported in any rhino species (aside from the SE outbreak) and exposure unknown

Discussion

· high seropositivity in rhino in SA- 53.9% and higher than reported in other domestic and wildlife species

· repro implications could influence recovery of a threatened species

· handle aborted tissue with caution, and include on the DDX

· Path changes associated with coxiellosis in SA white rhino not described

· This study did not ID significant associations with phase I and phase II titers across age class, sex, or management style

· Older animals more likely to be seropositive (common trend)

· Proximity to livestock appears to be risk for people, however Kruger National Park pop had sign higher exposure prevalence compared to private reserves

· Tick-borne illness in people not well described- tick burden noted in free-ranging rhinos with no intervention to manage it. Private reserves use treatments, may result in lower tick burden?

· Non-c.burnetii Coxiella species are symbiont in some ticks- possible ab cross-react and complicate interpretation of true exposure to C. burnetii

· Routes of infection in rhino currently unknown. People infected via inhalation of aerosolized bacteria or ingestion of unpasteurized milk

Question 40

A recent study evaluated the effects of supplemental etorphine on white rhinos.

What are some common side effects of etorphine in rhinos?

How did pulmonary artery pressure change with supplemental etorphine?

How did arterial oxygen change?

How did cardiac output change?

What does this mean clinically?

Answer 40

Journal of Zoo and Wildlife Medicine 49(4): 849-855, 2018

EFFECTS OF A SUPPLEMENTAL ETORPHINE DOSE ON PUMONARY ARTERY PRESSURE AND CARDIAC OUTPUT IN IMMOBILIZED, BOMA-HABITUATED WHITE RHINOCEROS (CERATOTHERIUM SIMUM): A PRELIMINARY STUDY

Jordyn M. Boesch, DVM, Dipl ACVAA, RD Gleed, BVSc, MA, Dipl ACVAA, Dipl ECVAA, DVA, MRCVS, MRCA, Peter Buss, BVSc, MMedVet, PhD, Markus Hofmeyr, BVSc, Adrian Tordiffe, BVSc, MSc, PhD, Gareth Zeiler, BVSc (Hons), MMedVet, Dipl ECVAA, Dipl ACVAA, and Leith Meyer BVSc, PhD

Abstract: The effects of etorphine on the pulmonary vascular system of white rhinoceros (Ceratotherium simum) have not been described and could play a role in the severe hypoxemia that develops after immobilization with etorphine-based drug combination. Characterization of these effects requires measurement of pulmonary vascular pressures and cardiac output (CO).To refine a technique for pulmonary arterial catheterization, five boma-habituated white rhinoceros (three females and two males weighing 1,012-1,572 kg) were immobilized by remote injection with etorphine plus azaperone followed by butorphanol. This afforded the opportunity to perform a pilot study and acquire preliminary measurements of pulmonary arterial pressure (PAP) and CO before and after supplemental etorphine given intravenously. Ultrasonographic guidance was used to insert a sheath introducer into a linguofacial branch of a jugular vein. A 160-cm-long pulmonary artery catheter with a balloon and thermistor was then passed through the introducer and positioned with its tip in the pulmonary artery. It was not long enough to permit wedging for measurement of pulmonary artery occlusion pressure. Mean PAP was 35 mm Hg (minimum, maximum 32, 47 mmHg) and increased (P = 0.031) by 83% (28, 106%) after supplemental etorphine. Thermodilution CO was 120 L/min (92, 145 L/min) and increased 27% (3, 43%)(P = 0.031). Heart rate was 100 (88, 112) beats/min and increased 20% (4, 45%)(P = 0.031) whereas arterial partial pressure of oxygen was 35 mmHg (30, 94 mmHg) and decreased 47% (20, 72%)(P = 0.031). The cardiovascular observations could result from etorphine-induced generalized sympathetic outflow, as has been reported in horses. Further studies of etorphine in isolation are needed to test this suggestion and to discern how the changes in pulmonary vascular pressures and blood flow might relate to hypoxemia in etorphine-immobilized white rhinoceros.

Key points:

· Life-threatening hypoxemia, hypercapnia, acidemia common in chemical capture of wild white rhinos

o Extreme exertion during darting may contribute, but these still occur when they are calm

· Goats given etorphine à hypoxemia, pulmonary hypertension, hypoventilation, hypercapnia

· Tissue depth in rhinos over the jugular vein makes percutaneous route impractical – use linguofacial vein instead

· 1.98 ug/kg etorphine, 0.019 mg/kg azaperone à 0.01 mg/kg butorphanol

o Supplemental etorphine dose: 0.30 ug/kg given IV 61min after initial dose

· In a rhino of this size, a pulmonary artery catheter must be longer than 160cm to wedge in a branch of the PA

· Mean PAP was ~35mmHg, similar to standing horses

· Using allometric scaling from goats, normal CO would be 55 L/min and HR would be 42 bpm

· Increase in CO, HR, systemic arterial BP observed after supplemental etorphine in rhinos agree with changes in horses

o In horses, thought that this is a consequence of upregulated sympathetic nervous activity

o White rhinos immobilized with etorphine and azaperone, plasma epinephrine correlated positively with tremor intensity and negatively PaO2 – sympathetic upregulation may be responsible for changes reported here

· Increase in PAP may be due to increased blood flow or resistance in arterial segment of the pulmonary bed, increased left ventricular preload or afterload, or another cause

· Supplemental etorphine exacerbated the hypoxemia and hypercapnia

o Before supplement: 4/5 were hypoxemic, 5/5 were hypercapnic

o After supplement: results compatible with additional functional impairment of gas exchange and decreased alveolar ventilation from etorphine

Take-home points:

· Pulmonary artery catheterization and thermodilution CO measurement are feasible in white rhinos

· Use of supplemental etorphine associated with serious adverse cardiopulmonary effects; argues against the use of etorphine to prolong immobilization in white rhinos, suggest alternative drugs

Question 41

A recent study described reproductive losses due to Q fever in managed rhinos.

What is the etiologic agent of Q fever?

What are the natural reservoirs? How is it transmitted?

What lesions were present in these cases?

What preventative measures were taken to prevent additional cases?

What serologic findings were present in these two dams?

Answer 41

Journal of Zoo and Wildlife Medicine, 2021. 52(1): 389-395

CLINICAL FINDINGS, PATHOLOGY, BIOSECURITY, AND SEROSURVEILLANCE OF COXIELLOSIS IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) ATA CONSERVATION CENTER: TWO CASES

Marjorie Bercier, DMV, DACZM, Elise E.B. LaDouceur, DVM, DACVP, and Scott B. Citino, DVM, DACZM

Abstract: A primiparous white rhinoceros (Ceratotherium simum) gave birth to a calf overnight after approximately 16 mo of gestation. The calf was found dead in the morning. Necrosuppurative placentitis with bacterial inclusions suggestive of coxiellosis was diagnosed histologically, and Coxiella burnetii was identified in fetal tissues and placenta by polymerase chain reaction and immunohistochemistry. Another primiparous female from the same herd aborted later that year after approximately 15 mo of gestation, and coxiellosis was similarly diagnosed in fetal tissues and on vaginal shedding. Estimates of exposure time, duration of vaginal shedding, and phase I and phase II antibody dynamics were determined retrospectively and prospectively for the two confirmed cases. Biosecurity measures were put in place to prevent guests, staff, and conspecific exposure to the organism. No other confirmed cases have occurred in the collection 3 yr after the initial cases. Coxiellosis outbreaks could represent an emerging threat to conservation efforts and ex situ white rhinoceros breeding programs.

Key Points:

· Q fever caused by gram negative intracellular coccobacilli, Coxiella burnetii.

o Reservoirs – domestic ruminants, wildlife, and ticks

o Transmission – aerosol or contact with birth products

· Case 1 – 7 yo primiparous white rhinoceros gave birth after 16 mo gestation. Calf found deceased the next morning with hemorrhagic effusion in chest and abdomen (premature birth).

o Severe necrosuppurative placentitis with intracytoplasmic bacterial microcolonies

o PCR and IHC of placenta confirmed Coxiella burnetii

· Case 2 – 5 yo primiparous white rhinoceros from the same herd gave birth overnight. Calf found deceased the next morning, but placenta was not found.

o Calf has atelectatic, suggesting stillbirth or abortion

o Fetal samples (spleen, LN, liver) and vaginal swab from dam were positive for C. burnetii via PCR. Vaginal shedding became negative 36d after parturition. Coxiella not detected in milk.

· Herd management – both rhinos were isolated from the rest of the herd with buffer zone between them and herd. One vehicle with dedicated keeper staff serviced them at the end of the day. PPE – coveralls, boot covers, gloves, N95, and given zoonotic training on q fever.

o Released from qt one vaginal shedding stopped at 36d.

· Banked serum samples retrospectively analyzed for acute and chronic antibodies

o Both dams had elevated phase I (chronic) and phase II (acute) titers at the time of calf delivery, however phase I titers were higher indicative of chronic infection.

§ Both were exposed after conception – exposure at any timepoint during gestation can result in clinical disease.

§ Planning to monitor antibodies of all rhinos at this facility

Take home:

First report of necrosuppurative placentitis and stillbirth secondary to Coxiella in managed rhinoceros. Both dams lacked clinical signs prior to parturition/abortion and were exposed during gestation.

Question 42

Discuss iron storage disease in rhinos.

WHat species are typically affected?

Describe iron metabolism
- How is iron absorption regulated? What about excretion?
- How is iron absorbed?
- What are the three ways the liver regulates iron?
- What is the funciton of hepcidin?
- What is the function of ferroportin?

What is the difference between primary and secondary hemosiderosis?

What is the relationship between iron overload and oxidative stress in rhinos?

What is the relationship between iron overload and infection?

How is iron overload diagnosed?

How is iron overload treated?

Answer 42

Fowler 10 Ch 90 Browsing Rhinoceros and Iron Storage Disease—An Update

Abstract key points:
- Iron overload has only been seen in rhinoceros in managed care.
- Iron accumulates over time in browsing rhinoceros species, Sumatran (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), and low amounts in Indian rhinoceros (Rhinoceros unicornis), though not in the other grazing species, white rhinoceros (Ceratotherium simum).

The role of iron and iron homeostasis
- Iron transports oxygen in hemoglobin and myoglobin
- Shifting between ferrous (Fe 2+) and ferric (Fe 3+) forms occurs at cell surfaces and contributes to oxidative damage
- Circulating red blood cells represent 80% of iron in the body.
- Iron absorption is tightly regulated. Excretion is not regulated.
- Iron is absorbed in the duodenum→stored as ferritin in the cytoplasm→loaded onto transferrin for distribution to iron-consuming organs (ie bone marrow)
- The liver regulates iron homeostasis by (1) producing hepcidin in hepatocytes, (2) storing excess iron, and (3) mobilizing iron from hepatocytes to the circulation as required.
- Hepcidin binds ferroportin, causing its endocytosis and degradation, preventing passage of iron. (Ferroportin is located at the surface of enterocytes and macrophages.)
– Hepcidin is upregulated by iron and inflammation.
– High hepcidin leads to low plasma iron, iron retention in macrophages, and reduced erythropoiesis

Iron Overload–Causes, findings, and significance
- Iron Overload Disorder–a condition in which higher amounts of iron than normal are in the circulation, iron is deposited within the body, or both.
- Hemosiderin granules stained with Prussian blue
- Can be primary (genetic) or secondary (dietary or other causes)
- Conflicting evidence of genetic cause in different rhino species
- With primary hemochromatosis, hepatocytes are the first and main target of iron loading in humans
- By contrast, reticuloendothelial cells/macrophages are the main site of storage in secondary iron overload.
- The main cell of iron-storage appears to be the macrophage in the black rhinoceros in managed care with accumulation over time, more suggestive of secondary hemosiderosis, though an element of underlying primary hemosiderosis seems possible.

Iron overload and oxidative stress
- Increased inflammation and oxidative stress are associated with iron overload.
- Oxidative stress can lead to hemolysis and fatality, reported in black rhinos
- No red blood cell enzyme deficiency has been identified in black rhinos
- Rhino RBCs seem to have lower antioxidant metabolism

Iron overload and infection
- Hyperferremia leads to increased susceptibility to infection (many pathogens need iron and acquire it from the host)
- Hosts decrease the amount of iron bound to transferrin during infection as a protective mechanism, sequestering it in macrophages.
- Patients with primary hemochromatosis appear more susceptible to certain infections.

Dietary iron
- Hard to measure dietary iron in wild diets alone as it is influenced by other factors such as tannins, vitamin C, etc
- Wild diets do seem to be lower in iron in the dry season

Diagnosis of overload
- Liver biopsy ideal but challenging
- transferrin saturation may be best for guiding antemortem diagnosis and prognosis of iron overload.
- Ferritin is an acute phase protein so can be difficult to interpret

Treatment of Overload
- Phlebotomy is recommended in primary hemochromatosis when there are changes of cirrhosis, cardiomyopathy, or diabetes mellitus in humans.
- This works because hepcidin is low and removing RBC and iron is drawn out of macrophages, erythropoiesis occurs, and excess iron is mobilized.
- Liver biopsies are collected at 5-year intervals to reassess iron status and monitor treatment.
- Secondary hemochromatosis requires chelation therapy, as these individuals are often anemic, and hepcidin levels are not generally low.

Conclusion
- While iron intake is regulated, output is not, and minimizing intake is important to reducing iron overload.
- While there may be a genetic component to iron overload in the black rhinoceros, there is a significant component of iron deposition in reticuloendothelial cells/macrophages in multiple species in managed care, more consistent with the secondary form.
- Approach to treatment of iron overload and an understanding of its effects and significance requires a better understanding of the complexity of the entity and the origin of its development.

Question 43

Describe the predictors of parturition in rhinos.

How long is gestation?

What measurements are taken of the fetus in early gestation?

What are the clinical signs of parturition?
- What signs occur 3-4 week before?
- What signs occur 3 weeks before?
- What signs occur 1-2 weeks before?
- What signs occur 2 days before?

What is the physiology of parturition induction?

What is the only accurate hormonal predictor of parturition?
- What levels are significant?

What are the three stages of labor?

What is the physiology of the second stage of labor?
- What is the classic clinical sign of this stage in this species?
- What are the two intervals to note?

What is the third stage of labor?

What are the behaviors of normal rhino calves?
- How should mom behave when first born?
- What signs necessitate an emergency response?
- When should they stand?
- When shoudl they nurse?

What are some potential complications with parturition?
- What are teh most common causes?
- What are maternal risk factors for complications?
- What are fetal risk factors for complications?

Answer 43

Pre-partum Changes and Predictors of Parturition in Rhinos

Gestational Length ~16 months
- Dependent on source gestational length varies by 7-8 weeks
- Suspected to be thought to deviate due to unnoticed mating/conception
- Parturition often occurs at night (makes it difficult to respond to emergencies)

Fetal Morphometry – reported in single cases of different rhino species
- Useful during early pregnancy but not late gestation
- Measurements during first trimester: diameter of embryonic vesicle, crown-rump length, intercostal width, orbital diameter, biparietal diameter, humerus and femur length, fetal foot size
- Limiting factors of unknown effects: maternal age, parity, individual, species variation
- Can determine proposed mating date but cannot predict parturition

Clinical Signs of Parturition
- 3-4 weeks prior: udder enlargement and genital swelling
– First early indicators for parturition
- 3 weeks prior: Colostrum produced up to 3 weeks prior to birth, significantly increases one week prior, and even more substantially 24 hours prior
– Pre-colostrum can drip from teats, leak when lying down, stain inner legs
– White rhino: increases during last week of gestation, and even more 24 hrs prior to parturition
– Can be collected, frozen and substituted for maternal Ab if hand-rearing
– Ca increase and pH decrease in pre colostrum (as in mares) may predict parturition
- 1-2 weeks prior: Genital edema and lubrication from increased cervical/vaginal mucus
– Increase in relaxin and estrogen → vaginal and cervical mucus is increased
- 2 days prior: Behavioral unrest +/- smaller fecal balls, increased urination frequency, shorter periods of recumbency
– Video analysis weeks before expected parturition can establish “normal” recumbency pattern and down times for comparison
– Night before parturition down time decreases to <2 hours
– Down times < 30 min can indicate imminent birth
– Easy to observe as a predictor of parturition

Estrogen Increase and Progesterone Decrease Prior to Parturition
- Pathophysiology: Fetal space becomes limited→ induces rise of fetal corticoids → stimulates conversion of maternal progesterone into estrogen and synthesis of PGF2a → lysis of CL → further increase in estrogen and decrease in progesterone
– Without progesterone block → allows for uterine contractions and repro secretions
- Estrogen increase is NOT a sensitive predictor of parturition
– Can increase up to 4 weeks before parturition
– Estrogen remains elevated for several weeks prior without a marked peak
- Fecal Progesterone decrease is NOT a sensitive predictor of parturition
– Progesterone metabolites excreted in feces is delayed by days
- Serum progesterone decrease IS ACCURATE predictor of parturition
– Can accurately predict parturition 1 week before and 48 hrs prior to birth
– Independent of maternal age, parity, behavioral changes and pre-colostrum secretion
– White rhino: >40 ng/ml in last trimester → <20 ng/ml in last week of gestation
– Slowly declines over last week, 48 hrs prior drops by 30%, and 24 hrs by 40%
– Dystocia suspected if P4 <5 ng/mL for 24+ hrs without progress toward stage II

Parturition
- Normally born in anterior presentation (73%), at night or early morning
- Fetus born within allantochorion membrane that mom pulls away when standing up or turning towards fetus after birth
- Ideally have two calving stalls in case need to separate mom from calf
- Three distinct labor stages
– Sometimes called “quick parturition” because most of stage I and II are not noticed

Stage I Labor (5-7 hrs): uterine contraction, dilation of cervix, fetus enters birth canal
- Difficult to determine, no single event
- Behavioral unrest (recumbency <10-20 mins), frequent urination and defecation attempts
- Can go unnoticed due to subtleness

Stage II Labor (1.5-2.5 hrs): rupture of chorioallantoic membrane, draining of fluid, birth
- Abdominal contractions (visible), flattened ears, elevated RR (>16 brpm), prolonged horizontal or curled tail position
- Pathophysiology: Fetus in birth canal → increased fetal movement due to hypoxia → increased pressure on sensory neurons in cervix/vagina → stimulates oxytocin → induced more forceful contractions → expulsion of fetus
– Expulsion takes longer if fetus in posterior position due to smaller HL not maximizing stimulation of sensory neurons
- Rupture of chorioallantoic membrane, draining of amniotic and allantoic fluid (clear), calf is born
– Chorion (fetal membrane), allantois (placenta)
– Unmistakable indicator of this phase = fluid moistening legs; even if don’t see the membrane rupture, should be able to distinguish from urination
- Interval I: rupture, fetal membranes/fetus appear in genital opening
- Interval II: first appearance of membranes/fetus, expulsion of fetus
Expulsion usually occurs <10 min (5-45 mins) after placenta rupture
– Characterized by strong labor activity, prolonged horizontal tail position, and prolonged abdominal pressing in standing or recumbent position
– Dam tends to stand/lie with back toward wall/corner to protect offspring
- Most critical stage of parturition

Stage III Labor (~4 hrs): passage of placenta
- Calf born covered with chorion membrane, placenta remains attached to uterus
- Female automatically pulls fetal membranes away when she stands up/turns toward newborn
- Should spontaneously pass placenta with forceful contractions
– Retained placenta = rare in rhinos; can administer oxytocin if needed

Perinatal Period
- Pulls away membranes, licks/suckles the calf, gently pushing the calf to initiate first breath and get in sternal position
– If no ear twitch/breath scene → suspect hypoxia → immediate measures should be taken
- Standing should occur within 1 hour (>75% of calves)
- Nursing: within 2 hrs for black, Indian; within 4 hrs for white
– Dependent on dams experience/willingness to present teats + calves abilities to find udder
– Can take up to 15 hours if inexperienced/insufficient

Dystocia and Perinatal Complications
- Stillbirth and perinatal (<3d postpartum) deaths 6-17%
- Cause usually unknown and non-infectious; one report of Q-fever (coxiella burnetti)
– Dystocia, asphyxia (most common in other species), perinatal hypoxia
– Congenital disorders (rare): cyclopia, cerebral aplasia, cystic dermoid eye, Schistosomus reflexus, deformed feet
- Potential maternal risk factors: advanced age, lack of physical fitness, maternal obesity, fetal overdevelopment
- Potential fetal risk factors: posterior positioning and prolonged expulsion
– Consider oxytocin if HL seen/suspect posterior positioning and expulsion time prolonged
– If fails consider anesthesia, IV calcium and oxytocin, and manual extraction
– Oxytocin contraindicated if malpositioned or dead rigor mortis fetus
– May require fetotomy (wait 48 hrs to do so after rigor mortis resolved)
– C-section has never been attempted in rhino
- Fetal hypoxia +/- leg paralysis: responsible for 11% of calf mortalities
– During parturition can be caused by umbilical cord compression, premature detachment of placenta, fetal malposition, prolonged expulsion of fetus
– Treatment: physical mobilization, O2, steroids, rectal glucose, caffeine
– Can return to dam to prevent hand-rearing
- Maternal problems: misdirected maternal instincts, maternal anxiety, aggression
- Infanticide reported in all rhino species; neuroleptics can be trialed if needed
- Can result in hand-rearing; may be be able to reintroduce after a few weeks

Question 44

A recent study investigated the pharmacokinetics of propofol in anesthetized white rhinoceros.

What is the scientific name of this species?

What were they anesthetized with prior to propofol administration?

What dose of propofol was used? What time frame was it given over?

What were the effects of propofol in this study?

Answer 44

Pharmacokinetics of intravenous propofol in southern white rhinoceros (Ceratotherium simum simum) after intramuscular etorphine-butorphanol-medetomidine-azaperone.
Berlin ER, Kinney ME, Howard LL, Perrin KL, Phair KA, Clancy MM, Ferris RL, Knych HK, Mama KR.
American Journal of Veterinary Research. 2023;84(4):onlinw

OBJECTIVE To determine the pharmacokinetics of a single bolus of intravenous (IV) propofol after intramuscular administration of etorphine, butorphanol, medetomidine, and azaperone in 5 southern white rhinoceros to facilitate reproductive evaluations. A specific consideration was whether propofol would facilitate timely orotracheal intubation.
ANIMALS 5 adult, female, zoo-maintained southern white rhinoceros.
PROCEDURES Rhinoceros were administered etorphine (0.002 mg/kg), butorphanol (0.02 to 0.026 mg/kg), medetomidine (0.023 to 0.025 mg/kg), and azaperone (0.014 to 0.017 mg/kg) intramuscularly (IM) prior to an IV dose of propofol (0.5 mg/kg). Physiologic parameters (heart rate, blood pressure, respiratory rate, and capnography), timed parameters (eg, time to initial effects and intubation), and quality of induction and intubation were recorded following drug administration. Venous blood was collected for analysis of plasma propofol concentrations using liquid chromatography-tandem mass spectrometry at various time points after propofol administration.
RESULTS All animals were approachable following IM drug administration, and orotracheal intubation was achieved at 9.8 ± 2.0 minutes (mean ±SD) following propofol administration. The mean clearance for propofol was 14.2 ± 7.7 ml/min/kg, the mean terminal half-life was 82.4 ± 74.4 minutes, and the maximum concentration occurred at 2.8 ± 2.9 minutes. Two of 5 rhinoceros experienced apnea after propofol administration. Initial hypertension, which improved without intervention, was observed.
CLINICAL RELEVANCE This study provides pharmacokinetic data and insight into the effects of propofol in rhinoceros anesthetized using etorphine, butorphanol, medetomidine, and azaperone. While apnea was observed in 2 rhinoceros, propofol administration allowed for rapid control of the airway and facilitated oxygen administration and ventilatory support.

Background
- Physiologic changes during recumbency due to size and drug-induced respiratory depression from potent opioids: hypercapnea, hypoxemia
- Orotracheal intubation difficult in white rhinos due to heavy head, narrow space between dental arcades, and presence of dorsal pharyngeal recess + muscle rigidity common with potent opioids

Key Points
- Tried to give propofol over 60 sec but it varied
– Animal with longest administration time had highest plasma concentrations and longest clearance
- Intubated in lateral by manual palpation with stylet and Murphy eye technique, mechanically ventilated with Zubanator (mega-vertebrate demand ventilator; 40% inspired oxygen)
- No supplemental drugs were needed before propofol, 4/5 became sternal, two got supplemental drugs mid-procedure (long after intubation)
- Jaw tone during intubation varied: moderate > mild > no jaw tone
- 2/5 had brief apnea after propofol, 1 resolved during intubation
- Induction was good&raquo_space; excellent
- Muscle relaxation was good > excellent or fair
- Recovery was ‘normal’ - extubation after 2-3 spontaneous breaths
- All 5 rhinos were initially hypertensive (MAP > 124 mmHg; consistent with etorphine use), 2 had hypercapnea, lactate decreased over time, all had mild-moderate hyperglycemia (consistent with alpha-2)
- PK: propofol concentrations declined rapidly, half-life 20-80 min, 1 outlier 210 min
– Rhinos had slower clearance than horses - similar to carprofen and enrofloxacin studies

Conclusions
- IV propofol after induction allowed adequate loss of jaw tone to allow intubation with the use of a hydraulic jack (used straps and manually opening the mouth)
- Impact of propofol on breathing was manageable in the 2/5 that became apneic - requires ability to intubate and administer IPPV

Question 45

A recent study investigated the effects of boma confinement on hematologic and biochemical values in white rhinos.

What is the scientifc name of this species.

How did rhinos respond to boma confinement?
- What were the two categorized responses?

What changes were seen with maladapted rhinos?

Answer 45

JWD 2022 58(4) 735-745
EFFECT OF BOMA CONFINEMENT ON HEMATOLOGIC AND BIOCHEMICAL VALUES IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA

ABSTRACT: Boma adaptation is an important component of rhinoceros translocations to allow transition to new diets, restricted space, and quarantine for disease screening. However, up to 20% of recently captured white rhinoceros (Ceratotherium simum) do not adjust to captivity, resulting in early release or even death. The causes and physiologic consequences of maladaptation to boma confinement are poorly understood. The aim of this investigation was to evaluate hematologic and serum biochemical changes in maladapted rhinoceros compared to animals that adapted under the same boma conditions. Ninety-six white rhinoceros were captured between 2009 and 2011 in Kruger National Park, South Africa and placed in bomas prior to translocation. Weight, complete blood count, and serum biochemical panel results were recorded when rhinoceros were placed in the boma and repeated on the day of release. In this study, the mean duration of boma confinement for maladapted white rhinoceros was 13 d (range 8–16 d) compared to 89.9 d (range 39–187 d) for adapted animals. Mean weight loss between capture and release was significantly greater in maladapted rhinoceros (224.0 versus 65.9 kgs; P,0.001). Although adapted rhinoceros had statistically significant changes in some hematologic and biochemical values, most were not considered clinically relevant. In contrast, the maladapted rhinoceros had significant changes at the time of early release from the boma, including evidence of leukocytosis with left shift, lymphopenia, eosinopenia, decreased red blood cell count and hematocrit, increased serum creatine kinase, and decreased serum calcium, phosphorus, and magnesium values. Along with loss of body condition, these findings were consistent with a stress-associated catabolic response. These changes occurred in the first 2 wk of confinement, and the results provide a foundation for evaluating adaptation in white rhinoceros. Future studies should focus on factors that improve adaptation and welfare of recently confined free-ranging white rhinoceros.

Intro
- Conservation programs for African rhinoceros depend on capture and translocation to maintain genetic diversity and restore populations
- Rhinos often placed in bomas as an intermediary step to adapt to a new diet and undergo quarantine prior to placement in a new location
- As many as 10%–20% of free ranging white rhinoceros are unable to adapt to boma confinement
- Maladaptation results in loss of translocation candidates, along with the time and associated costs and, in some cases, death of the rhinoceros.
- our study aimed to evaluate changes in hematologic and serum biochemical values in boma-adapted and maladapted white rhinoceros

M&M
- Retrospective, 96 white rhinos between 2009 and 2011
- Weight changes, CBC and chem values evaluated between adapted and maladapted rhinos

Results
- The mean length of stay for boma-adapted rhinoceros was longer than maladapted rhinos
- Postmortem of the rhino that died confirmed Salmonella gastroenteritis as the cause of death
- Adaptation scores got higher over time in the adapted rhinos and then plateaued, maladapted rhinos hit a lower plateau earlier
- the mean weight loss between capture and release of maladapted rhinoceros was statistically greater than weight loss in adapted animal
- Some changes occurred in CBC and chem values for adapted rhinos, but not considered clinically significant (comparing capture to release)
– WBC, eos and basos declined, neutrophils increased
– TP, albumin, BUN, CK and AST increased
– ALP, GGT, and Mg decreased
- Maladapted rhinos had more clinically significant changes to bloodwork (comparing capture to release)
– Increase in WBC and neutrophils, decrease in lymphocytes
– Increase in band neutrophils, though not statistically significant
– increases in BUN and CK and decreases in ALP, GGT, and Mg
- When comparing adapted and maladapted, maladapted rhinoceros had a significantly higher median WBC count, proportion of neutrophils, and a decreased proportion of eosinophils compared to adapted
– Also a decreased RBC count and hematocrit in maladapted vs. adapted
– also a significantly higher median CK and lower serum albumin, AST, BUN, calcium (Ca), phosphorus (P), and Mg values in the maladapted rhinoceros

Discussion
- There was a significant difference in the time between capture and release samples between the adapted and maladapted rhinoceros, with a relatively long period between blood collections in the adapted rhinoceros, which could have influenced these findings
- The trends in the adapted rhinoceros were suggestive of adjustment because many of the changes were not clinically relevant or were considered to show a return toward reference values
– Ie decreased WBC count was consistent with a lessening stress leukogram
- In contrast, weight loss and changes in blood values in maladapted rhinoceros were significantly greater and were consistent with a decline in condition or catabolic state, despite the shorter duration of confinement.
- There was no evidence of lymphopenia or anemia associated with boma confinement.
- The decrease in proportion of eosinophils was most probably due to the parasiticide treatment
- The relative changes in basophil and neutrophil populations were physiologically inconsequential and remained within reference ranges
- The hematologic changes in maladapted rhinoceros between capture and release were consistent with a stress response
– stress leukocytosis with left shift, lymphopenia, eosinopenia, and anemia.
– Anemia may be due to bone marrow suppression or GI ulceratiton
- The serum biochemical values that significantly changed between capture and release of maladapted rhinoceros included increased BUN and decreased Mg values
– Decreased Mg likely due to decreased food intake
– Increased BUN consistent with a catabolic state
- Although long term monitoring after release was not performed, immediate observations after release revealed that the rhinoceros started grazing almost immediately, suggesting that they might be able to recover.
- The significance of the changes during boma confinement was even more apparent when differences in blood results between adapted and maladapted rhinoceros were compared at the time of release.
- Maladapted rhinoceros had stress hemograms, with significantly decreased mean hematocrit, Ca, P, and Mg levels, and clinical evidence of catabolism supported by the elevated CK levels and rapid weight loss.
- Future studies should continue to investigate predisposing factors and effective preventive and therapeutic techniques to improve adaptation and welfare of recently captured white rhinoceros.

Question 46

A recent study described the effects of capture and immobilization on boma adaptation in free-ranging white rhinos.

What physiologic effects are seen with ultra-potent opioids?

What proportion of rhinos display maladaptation to boma confinement?

How did immobilization parameters affect maladaptation?

When did rhinos start to show signs of maladaptation?

Answer 46

JWD 58(4)
EFFECT OF CAPTURE AND IMMOBILIZATION ON BOMA ADAPTATION IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA

Abstract
Ninety-six white rhinoceros (Ceratotherium simum) were captured between February and October 2009–2011 in Kruger National Park, South Africa and placed in boma confinement before translocation. Of these, 19 rhinoceros did not adapt to the bomas and required early release (n=18) or died (n=1). The available immobilization data and physiologic parameters, including blood gas analyses, were compared between adapted and maladapted rhinoceros to determine whether predisposing causes could be identified. There were no statistical differences in age category, sex, or body weight at capture between adaptation cohorts. The recorded immobilization data, physiologic values, blood gas analytes, hematologic, or serum chemistry values were not statistically different between adapted and maladapted rhinoceros at capture, except maladapted rhinoceros had lower median serum aspartate aminotransferase, blood urea nitrogen, and phosphorus values; however, these statistically different values were not clinically important. Therefore, observable demographic or capture-related factors did not appear to predispose white rhinoceros to maladaptation to boma confinement. Further investigations into factors affecting adaptation should be performed to minimize the effect on rhinoceros health and welfare.

Key Points
- Growing need to confine white rhinos for tx or to relocate them; immobilization related complications can be compounded by transport and boma (holding facility) confinement
- Hypoxemia and hypercapnia are assoc with opioids used for capture
– Muscle rigidity, tremors, and tachycardia also possible → acidosis and hypoxemia → cardiac arrhythmias, muscle and nervous dysfunction
- Maladaptation to confinement by white rhinos occurs in up to 20%, requiring early release and possible mortality
- 96 rhinos immobilized, placed in bomas for eventual translocation
- Scored based on adaptation to bomas
- 20% of rhinos were maladapted and required early release
- Majority of animals captured were subadults > adults
- No difference in butorphanol route of administration, standing vs recumbent animals that were maladapted to bomas
- No difference in mean duration of immobilization for boma-adapted vs maladapted animals
- No difference in physiologic parameters or blood values for adapted vs maladapted animals
- Boma adaptation scores for maladptaed animals started to differ at day 8, (8-16d was the time frame where they would need to be released)
- One animal died from salmonellosis

Take home message
- Capture-related factors do not appear to influence boma adaptation in immobilized free ranging white rhinos.
- No significant differences in age, sex, body weight, immobilization variables or physiologic measurements

Question 47

A recent study assessed capillary zone electrophoresis and serum amyloid a quantification in white and black rhinos.

What are the scientific names of these species?

How did SAA varry in rhinos?
- Were black rhinos in managed care higher or lower than wild animals?
- What resulted in higher SAA in white rhinos?

How does capillary zone electrophoresis differ from traditional agarose gel electrophoresis?

How did CZE results differ from AGE results in this study?

Answer 47

JZWM 2022 53(2):319-330
Assessment Of Capillary Zone Electrophoresis And Serum Amyloid A Quantitation In Clinically Normal And Abnormal Southern White Rhinoceros (Ceratotherium Simum Simum) And Southern Black Rhinoceros (Diceros Bicornis Minor)
Meyer A, Emerson JA, Rainwater KL, et al

ABSTRACT: Capillary zone electrophoresis (CZE) and an immunoassay for serum amyloid A (SAA) were used to examine serum samples from clinically normal and abnormal southern white rhinoceros (Ceratotherium simum simum) and southern black rhinoceros (Diceros bicornis minor) under managed care. CZE resolved seven fractions as well as subfractions for α1 globulins. Reference intervals were calculated for white rhinoceros (n = 33) and found to have some differences over previously reported intervals generated using agarose gel electrophoresis (AGE) methods in sera from free-ranging animals. In addition, the coefficient of variation related to fraction quantitation was found to be overlapping or superior to that reported for AGE. No significant differences were observed in CZE measurands and total protein between clinically normal and abnormal rhinoceros. In contrast to CZE, significant differences in SAA levels (P < 0.001) were observed in samples from the white rhinoceros between clinically normal and abnormal animals. In addition, in limited sample sets with repeated measures, SAA provided prognostic value. Future studies should generate more robust reference intervals and delineate the application of both SAA quantitation and CZE in routine health assessments and in prognostication.

Key Points:
- SAA is a highly conserved major APP in many species
– A multispecies sandwich ELISA for SAA has been validated for white & black rhinos
– In the black rhinos, SAA levels were higher in animals under managed care in conjunction with elevated cytokines and insulin-to-glucose ratio
– SAA in white rhinos was significantly higher from a group with tissue injury
– In the present study, SAA levels were found to increase in varied clinical presentations in white and black rhinos
- In the present study, SAA > 20 mg/L were consistent with systemic inflammation
– Results < 7 mg/L should be interpreted as normal or possible mild inflammation
– SAA values tended to correlate with clinical signs
– SAA returned to normal when clinical signs resolved
- CZE provides increased fraction resolution vs. AGE
– AGE is a semiautomated method; proteins separated by size/charge on a gel substrate
– Fractions are then resolved using protein-binding dyes
– CZE is an automated method; protein fractionation is done via high voltage in a capillary
– The fractions are quantitated by a UV detector
- No significant differences were observed in the electrophoresis measurands between clinically normal and abnormal white and black rhinoceros
- This contrasts with injured white rhinos using AGE where changes including decreased albumin, α2, and β1 globulins were observed in acute and chronic inflammation
– These changes were associated with wounds and tissue trauma, which were often extensive and untreated

TLDR: SAA is a clinically useful major positive APP in white and black rhinos

Question 48

A recent paper described the managed of seasonal dermatitis in greater one-horned rhino.

What is the scientific name of this species?

How did these animals present?
- What findings were seen on cytology?
- What diagnostics did they perform? What were their findings?

How did they managed these cases?

Answer 48

THE USE OF INTRADERMAL SKIN TESTING AND HYPOSENSITIZATION INJECTIONS TO CONTROL SEASONAL DERMATITIS IN GREATER ONE-HORNED RHINOCEROSES (RHINOCEROS UNICORNIS)
Sarah B. Chaney, DVM, PhD, DACVP, Melissa Loewinger, DVM, Donna Doherty, Colleen M. McCann, PhD, Kenneth J. Conley, DVM, DACVP, Denise McAloose, VMD, DACVP, Andrew Rosenberg, DVM, DACVD, and John M. Sykes IV, DVM, DACZM
Journal of Zoo and Wildlife Medicine 53(2): 485–491, 2022

Abstract: Allergic dermatitis was diagnosed in a 25-yr-old female greater one-horned rhinoceros (Rhinoceros unicornis) and her 6-yr-old female offspring by skin biopsy, intradermal skin testing (IDST), and allergen-specific serum IgE testing. Dam and offspring presented with seasonal, erosive, and ulcerative dermatitis affecting the face, legs, and trunk starting at 6 and 2 yr of age, respectively. IDST was performed at the caudal pinnal base using 61 regionally specific allergens. Specific serum allergen responses were detected using Heska’s Equine ALLERCEPT Allergen Panel. Histopathology of the lesions was consistent with an allergic etiology. Injectable allergen-specific immunotherapy was initiated in both animals and within 6 to 18 mon after commencing hyposensitization clinical improvement was noted. This report documents a repeatable methodology for IDST and serological allergen testing for use in rhinoceroses. The hyposensitization protocol detailed here can help guide future treatment protocols.

Key Points:
- Described methodologies to diagnose allergic dermatitis, case management and results of hyposensitization therapy for seasonal ulcerative dermatitis
- Housed individually in indoor-outdoor areas surrounded by dense forst
- Case 1- 24 yo – worsening seasonal erosive and ulcerative dermatitis on lateral aspects of limbs, skin folds of the limbs, aural base and pinnal margins which occurred from spring to Fall
– Lesions started with erythema/depigmentation on intertriginous areas and progressed to exudative erosions/ulcerations, pruritic
– Cytology – numerous eosinophils supportive hypersensitivity reaction with secondary infection. Biopsy confirmed eosinophilic dermatitis.
– Intradermal skin testing on caudal right ear – Kentucky blue grass, mosquitos and horse fly.
– IgE – positive tiers to dust mite, storage mite and horsefly
– Hyposensitization – injections q3d and continues q10d – significantly improved with severity of ulceration and overall surface are reduced
- Case 2 – 6 yo F offspring to case 1 presented for erosive and ulcerative dermatitis on lateral aspects of all four limbs and intertriginous areas of the limbs, ventral chin, and trunk with periocular depigmentation and crusting. Seasonality from spring to fall and pruritic
– Intradermal reactions to moth, mouse, red cedar, privet and mold and IgE titers to storage mites
– Received hyposensitization injections q3d. Still receives injections q10d.
- Allergic dermatitis is diagnosis of exclusion – once diagnosed through exclusion, IDST is the gold standard to determine allergens for desensitization. Use of both IDST and IgE antibodies may strengthen the list of possible offending allergens
– Immunotherapy lessened the severity, duration and dependence on treatment for flare-ups – both rhinos now managed with topical treatments

Take-Home Message:
- Allergic dermatitis in rhinos presented with lesions beginning as erythema and fissuring between the skin folds and creases and progressed to coalescing areas of exudative and ulcerative dermatitis (ventral body, periocular or aural skin and skin folds).
- IDST may aid in selecting appropriate antigens for treatment of suspected allergic dermatopathies in rhinos