Amphibians Flashcards

Question

What are some benefits and uses of minimally invasive surgery in amphibians? What are some potential risks? Descrie the approach for laparoscopic exam and biopsy. Describe endoscopic orchiectomy.

Answer 1

F9 Ch 54 Minimally invasive surgery of amphibians * Laparoscopy in amphibians may permit the visualization of almost all the coelomic organs from a single point of entry * Indications: Gender ID, retrieval of FB from upper GI, assessment of intracoelomic lesions, diagnostic tool for nonspecific presentations, biopsies of tissues and organs, reproductive endosurgery. * Size may be a contraindication, as are compromised individuals with greater anesthetic risk * General anesthesia & analgesia is requisite for MIS * Risks from damaging the ventral abdominal * Laparoscopic Examination and Endoscopic Biopsy * Dorsal recumbence is recommended, unless sp. is laterally compressed * Insufflation can be used at a lower rate that sm. Mammals * CO2 insufflation w/ pressures of 0.5–2 mm Hg with a flow rate not exceeding 0.5 L/min * Surgical prep: * Cleansing the surgical site with sterile gauze soaked in 0.75% chlorhexidine solution and left on the surgical site at least 10 minutes prior to surgery * Coelom permits the visualization of liver, gallbladder, heart, lungs, digestive tract, gonads, kidneys, bladder, and fat body from a single entry point * Monofilament nylon is the best choice of suture in amphibian skin * On recovery, the animal is transferred to a warm, anesthetic-free bath and is rinsed copiously with fresh, well-oxygenated water * Endoscopic Orchiectomy * Incision with an 11 blade to insert atraumatic 5mm forceps * Cauterize surrounding vessels and epididymis with diode laser * Deflate animal post procedure * Close with 1-2 interrupted sutures

Answer 2

* MBD aka Nutritional Secondary Hyperparathyroidism * Most common form of MBD in amphibians. * Usually results from dietary Ca deficiency, vitamin D3 deficiency, or combinations. * Most of the commonly fed prey items have an inverse Ca:P ratio and low total calcum. * Intake of Ca during larval stage critical for normal skeletal development and calcium homeostasis of adults. * Consider environmental water composition when evaluating altered intake ratios of Ca:P. * Fluorosis may contribute, but the animals from that report also lacked adequate UVB exposure. * Ca absorption depend son available UVB, dietary vit D3, Ca, healthy GIT, kidneys, integument, MSK system. * Cuttlebone or other Ca source should be provided for tadpoles of many spp. * CS – Typically starts with spastic tetany after stenuous movement i.e. leaping, improves during rest. Coelomic distension from GI stasis, gas buildup may be seen. * Spinal deformities, angulation of long bones, bowed mandibles (osteomalacia). * Lateral processes of the vertebrae and urostyle become radiolucent early in the course. * Long bones become radiolucent after the ghosting of the lateral processes of the vertebrae and urostyle. * Tetany, spastic movement, muscle fasciculation, bloat, limb paralysis, ridigity considered a medical emergency. * Tx: 100 mg/kg Ca gluconate ICe, IM, IV, SQ SID-BID or more until signs resolve. * Then continue weekly injections until eating, then switch to heavy oral supplementation with Ca carbonate. * Oral or injectable vit D3 may facilitate Ca utilization. * Baths of 2-5% Ca gluconate and 2-3 IU/mL vit D3 may be helpful in some spp but should not be the single course of correction. * Tx requires several months for resolution of rad changes. * Supportive nutritional care with critical care diets, etc PRN. * Calcitonin therapy reported in reptiles NOT effective in amphibians – major structural differences between amphibian calcitonin and salmon calcitonin used for tx osteoporosis in humans.

Answer 3

* Hypovitaminosis A (Anurans; not reported in caudates) * Short tongue syndrome. * Squamous metaplasia of the mucous glands of the tongue, does not stick to prey. * Submit entire tongue on necropsy for examination of a longitudinal section (not cross section). * Bladder, kidneys, eyelids should also be assessed histologically. * Lesions commonly found in oral mucosa, esophagus, cloaca, and urinary bladder. * Frozen liver submitted for vit A analysis – Confirmatory diagnosis requires determination of hepatic retinol. * Unable to synthesize carotenoids, leads to vision loss, epithelial hyperplasia, squamous metaplasia, keratinization of mucosal epithelium. * Captive-reared insects lack significant amounts of pre-formed vit A and the plant-based carotenoids found in wild sinecs that are converted to active vit A. * Squamous metaplasia within the kidney or urinary bladder may result in hydrocoelom. Conjunctival swelling has been reported. * Impaired immune system, secondary infection by Bd and other pathogens. * Insects generally low in vitamin A. * Supplements lose potency if stored in areas with high humidity, warm temps, or both. * Multivitamins should be used within 6 months of opening. * Human-grade vit A supplements can be diluted with propylene glycol to appropriate concentrations, may be irritating to some amphibians.

Answer 4

* Obesity * Amphibians regularly offered rodents. * Most stored fat is in coelomic fat pads -\> grossly distended abdomen and normal limbs and tail. * Gonadal fat bodies located cranial to gonads and kidneys, numerous finger-like structures in anurans. * Other common sites of adipose deposition: * Epicardium, tail (plethodontid salamanders). * Inguinal region (toads, Bufonidae). * Bone marrow.

Answer 5

* Gastric overload or impaction * Result of eaving a single overly large prey or consuming too many prey items in a short period of time. * Amphbians stimulated to eat when seeing movement, can ingest smaller cage mates. * Swallowing overly large meals may affect respiratory ability. * Bacterial decay can outpace enzymatic digestion, leads to bloat and endotoxemia, can lead to perforation. * Undigested hair and bones can cause impactions. * Gastric washes with hypotonic saline may remove ingesta if acute. * Endoscopic retrieval. * Gastroenterotomy. * Consider pre and post op abx.

Answer 6

* Corneal lipid deposision aka lipid keratopathy aka corneal lipidosis. * Most commonly reported ocular disease in amphibians. * Presence of cholesterol clefts. * Associated with captive diets that differ in cholesterol content or other lipid composition compared to diets of free-ranging animals. * Hazy white discoloration at limbus with extension over time to completely cover the cornea with thick white nodules and plaques. * May present as a vertical line across the cornea. * Most also have hypercholesterolemia. * Females may be at increased risk. * Associated with pinkie mice and cultured crickets, which have different FA profiles vs wild prey. * Tx unrewarding, can debulk lesions and manipulate the diet. * Use of wild-caught insects vs monoculture crickets, feeding small amphibians or reptiles instead of mice. * Basking spots should be offered to any amphibian with corneal lipidosis, inappropriate thermoregulation in some spp may play a role in development.

Answer 7

* Renal oxalosis * Waxy monkey frogs that consumed crickets fed an oxalate-containning plant. * Tadpoles of wild anurans feeding on oxalate containing plants. * Leads to renal dysfunction and calculi, hycrodoclom or SQ edema, white foci within kidneys. * Gout * Unkonwn etiology, probably results from combo of dehydration, renal failure, inappropriate diets, toxicities. * Most likely to occur in those amphibians that excrete uric acid. * Waxy tree frogs (Phyllomedusa sauvagii). * Ammonium urate urolithiasis * Formation of large urinary bladder stones reported in waxy monkey frogs. * Contributing factors – Unique uricotelic metabolism of this spp, high-protein captive diets, dehydration. * Metastatic mineralization reported for captive frogs with dietary hypervitaminosis D.

Answer 8

* Gastric and cloacal prolapse. * Usually indicates a serious disorder when noted in salamanders. * Cloacal prolapse is commonly seen. * Predisposing factors – Dehydration, hypocalcemia, hypoglycemia, malnutrition, GI GB, cystic calculi, gastroenteritis, hyperthermia, trauma, parasitism. * Keep tissue moist and protected, genetly try to replace, abx if indicated. Tx underlying cause.

Answer 9

* Toxicities (Mader Ch 89 Amphib Medicine): * Keeping spring water that is aerated is an inexpensive solution for clinics seeing occasional amphibians. * Chlorine (Cl2) is easily removed. * Sodium thiosulfate, sodium hydroxymethanesulfonate (AmQuel), and/or carbon filters. * Chloramine (NH2Cl) – Combo of chlorine and ammonia. * Difficult to remove. * Sodium thiosulfate can be used to break apart the chlorine and ammonia and remove the chlorine, but ammonia remains. * Zeolite or biological filter needed to remove the ammonia. * Sodium hydroxymethanesulfonate effectively splits the chlorine ammonia bone and also neutralizes the resulting ammonia. * Avoid use of plastic containers that have held anything other than water. * Many common disinfectants are toxic to amphibians: * Povidone iodine, chlorhexidine, quaternary ammonium compounds, chlorine, ammonia. * Stoskopf reported an incident of iodine intoxication of poison dart frogs, resulted in agitation and abnormal posture before death. * Suspected to have diffused out of a plastic enclosure despite rinsing prior. * Ammonia \> 0.2 ppm suspicious for toxicosis. * Levels \> 1 ppm verification of toxicity. * Complete water change recommended. * Sodium hydroxymethanesulfonate may be used to temporarily bind ammonia if water change is not practical. * Chlorine intoxication is likely if a major water change has occurred within the past 24h. * Dangerous levels \> 0.5 ppm. * Bath of 100 mg/mL sodium thiosulfate can be used to treat confirmed chlorine toxicosis. * Heavy metals i.e. lead, copper, Zn may be in plumbing. * Salt toxicosis may occur with marine aquariums kept near an amphibian enclosure. * Keep amphib enclosures away from marine tanks and label containers distinctly to avoid confusion. * Presticides. * If needed for treatment around amphibians, insect growth regulators are preferred over toxicants. * Nicotine. * Secondhand tobacco smoke is irritating. * Keep in smoke-free rooms.

Answer 10

* Miscellaneous (Path book): * Limb malformations * Trematode parasite Ribeiroia ondatrae * Polymelia or amelia n free-ranging anurans. * Supernumerary limbs or missing limbs. * Enhanced by environmental eutrophication. * Disruption of the embryonic limb bud by encysting metacercaria. * Spindly leg syndrome * Thin underdeveloped forelimbs from time of metamorphosis. * Cause is unknown – Genetic vs malnutrition, water quality, enclosure temps, etc. * Animal generally dies, euthanasia is recommended

Answer 11

* Coelomic effusion and lymphedema are common nonspecific clinical presentations. * Commonly caused by chronic renal disease. * Polycystic nephropathy occurs in captive anurans from Panama. * Hypocalcemia with secondary lymph heart failure. * Hypoproteinemia. * GIT disease. * Heart failure. * Renal toxicity. * Congenital anomalies of lymph hearts. * African clawed frogs – Ovarian hyperstimulation syndrome following lab tx with human chorionic gonadotropin. * Infectious causes of edema – Ranavirus infection, sepsis, mycobacteriosis, metacercariae of echinostome trematodes.

Answer 12

* Neoplasia: * Lucke’s renal adenocarcinoma. * Free-ranging and captive northern leopard frogs. * Ranid herpesvirus 1. * Chromatophoromas, melanophoromas, iridophoromas.

Answer 13

* Iridoviridae. * Large, icosahedral, DNA viruses, enveloped or nonenveloped. * Worldwide, significant morbidity and mortality. * Geographic and Host Distribution. * Frov Virus 3 (FV3). * First reported in leopard frogs. * Outbreaks now throughout the world. * Various ranaviruses account for epizootics: * FV3, ambystomma tigrinum virus (ATV), soft-shelled turtle iridovirus (STIV), Bohle iridovirus (BIV). * FV3 is the only iridovirus in turtles in NA and most commonly reported for anurans. * ATV – salamanders, W US. * BIV – main ranavirus in Australia. * ALl ranaviruses * Frog Virus 3 – FV3 (anurans) * Tadpole edema virus - TEV * Ambystoma tigrinum virus – ATV (salamanders) * Bohle iridovirus – BIV (anurans) * Midwife toad virus (anurans) * All ranaviruses have a wide species distribution, significant differences in species susceptibility even within same isolate. * All anurans, caudates, chelonians considered susceptible. * Some uniquely sensitive i.e. tiger salamanders to ATV, wood frogs to FV3, eastern box turtles to FV3. * NA – larval amphibians considered more susceptible. * EU – adult mortality more commonly reported. * Adult chelonians more commonly develop FV3-like lesions. * All age classes should be considered susceptible to the disease. * Juvenile/larval age classes may be at increased risk in some species.

Answer 14

* Pathogenesis. * Transmission – contact with infected individuals, water, fomites. * Chelonians cohoused with infected amphibians or exposed to shared water with infected fish induced disease in 20-30%. * FV3-like DNA detected in mosquitoes during an eastern box turtle mortality event, may be another route of transmission. * Proposed amphibian reservoir spp include those that develop over 1 year (bullfrog), have neotenic development (tiger salamanders), or have aquatic adult life stages (red-spotted newt). * Caudate salamanders likely both the reservoir and hosts of FV3 infection in Canada. * EBT that recovered from a ranavirus infection experimentally reinoculated a year later demonstrated less than 20% mortality. * Surviving animals continued to shed, potential reservoir. * Clinical Signs and Pathology. * Systemic disease, epithelial necrosis. * Skin ulcerations or systemic hemorrhaged in absence of skin lesions. * Salamanders – abnormal swimming patterns, subcutaneous edema. * Frogs – loss of pigmentation, lordosis, epithelial sloughing, petechiation. * Larval amphibians – erythema at tail base, ventrum, legs, swelling of multiple areas of body. * Multisystemic infections – Hematopoietic tissues, endothelial cells, epithleial cells. * Erythema, petechial and ecchymotic hemorrhages. * Tiger salamanders and Chinese giant salamanders – Raised cutaneous plaques or polyps. Tadpole edema virus, adults asymptomatic. * Ecchymosis and petechiation of the skin are common. * Chelonians – nasal, ocular, oral discharge, oral plaques. * Epithelial lesions less consistent than fish and amphibians. * Pathologic findings similar between adults and juveniles of both amphibians and reptiles. * Systemic organ failure due to cellular necrosis in the spleen, liver, kidney, and intestine. * Hemorrhagic syndromes common in anurans in the UK and in salamanders in western NA. * Tiger salamanders – polypoid lesions over most of body. * Nonspecific changes – lymphocytosis, lymphoid depletion, vacuolation of hepatocytes and renal tubular cells. * Basophilic viral inclusions inconsistently observed. * Erythrocytes, leukocytes, epithelial cells, meninges, gills, neuroepithelium, nasal tissues, adipose, trachea, muscle, osteoclasts. * Histologic lesions can include: * Necrosis of hematopoietic tissues. * Multicentric hemorrhage with vasculitis and endothelial necrosis. * Necrosis of epithelial cells – Kidney, liver, skin, etc. * Characteristic basophilic intracytoplasmic inclusion bodies frequently observed. * May be present within glandular cells of the stomach.

Answer 15

* Diagnosis. * PCR commonly used, but limited and does not confirm active infection. * Dx most successful on fresh or frozen tissues. Recovery from fixed tissues is possible. * Evaluation of antemoretem toe clip and postmortem liver samples both effective methods for detection. * Specificity and sensitivity of PCR – tail clips underestimate prevalence, agreement increases as time after exposure increases. * Reptiles – necropsy tissues commonly tested. * Whole blood and oral swabs sensitive and specific in agreement with tissue sampling. * It is recommended that a dual testing strategy of both whole blood and oral swabs is needed for greatest testing success. * qPCR detects fewer viral copies than conventional PCR. * Immunohistochemistry – useful for quick confirmation of pathogen in formalin-fixed tissue. * Serologic assays. * May result in false positives. * Treatment and Prevention. * Therapeutic intervention in wild settings often impractical. * Acyclovir – guanine analog antiviral drug, active against herpesvirus due to presence of the thymidine kinase TK enzyme, which rapidly activates acyclovir to the monophosphate form. * Several isolates of iridoviruses have been shown to have TK genes or functional TK enzymes. * No significant differences in survival based on dose in one study. * Other mechanisms – temp alteration, disinfection. * Viral loads highest at 10 deg C vs 26 deg C. * Chlorhexidine 0.75%, sodium hypochlorite 3%, potassium peroxymonosulfate 1% effective at inactivating ranavirus after 1 minute exposure. * Potassium permanganate (100%) after 60 minutes, chlorhexidine 0.25% and sodium hypochlorite less than 3% ineffective.

Answer 16

* Mortality Events. * Significant impact on local populations. * Up to 90% mortality. * Animals in higher-density population shad higher mortality rates and died more quickly. * Extinction events were unlikely because as mortality reduced density below a critical threshold, remaining animals were able to recovery. * Turtles – less well described. * An outbreak in a zoo demonstrated 40% mortality, detection of copathogens (herpes, mycoplasma). Turtles with copathogens had a nonsignificantly lower mortality than individuals only detected with ranavirus. * In captive animals, clinical course of disease may be short with minimal signs. * Several reports in which surveys of reptiles and amphibian populations failed to demonstrate molecular evidence or exposure to ranavirus. * Conservation. * Global trade in amphibians significant factor in disease epidemiology and threatens conservation. * Bait salamander used by fishermen – significant threat to releasing ranavirus into native populations. * Infect fish without killing them, potentially leading to viral persistence and spread of infections. * Humans have both directly and indirectly increased ranaviral dz in amphibians, reasonable to consider similar case for reptiles.

Answer 17

* Ranid herpesvirus 1 (RaHV-1) * Cause of Lucke’s renal adenocarcinoma. * Cooler winter temps \<7C permissive for replication. * Proliferation of neoplastic cells minimal during that time. * In contrast, when temps are warmer the calid phase begins with increased proliferation of neoplastic cells and great reduction in viral replication. * Mets during calid phase may go to the liver. * Viral shedding occurs in spring, during spawning, offspring become infected. * CS – Emaciation, hydrocoelom, hydrops related to renal failure. * Coeliocentesis may yield neoplastic cells. * Cowdry type A intranuclear inclusion bodies typical of herpesvirus may be seen during the algid phase (winter months). * Ranid herpesvirus 3 – Europe, gray to white coalescing skin lesions/vesicular.

Answer 18

* Red leg syndrome aka bacterial dermatosepticemia * Historically Aeromonas hydrophila * Other gram-negatives i.e. Pseudomonas aeruginosa, Elizabethkingia meningoseptica (Flavobacterium meiningosepticum), Klebsiella pneumonia, Crhyseabacterium indologenes. * Gram-positives i.e. Streptococcus inae. * CS – Cutaneous hyperemia, hemorrhage distributed on limbs, digits, ventral abdomen. * Ddx ranavirus! * May also see bloating from GI gas, hydrocoelom, hydrops, sudden death. * Typically multiple animals affected. * Culture of blood and internal organs of dead amphibians recommended.

Answer 19

* Mycobacteriosis * Mycobacterium marinum. * M. chenolae, M. fortuitum, M. xenopi, M. ranae, M. ulcerans, M. liflandii. * Chronic subclinical infections become lethal with immunosuppression. * Adult animals. * Nodules, abscesses, effusion, coelomitis. * Demonstration of acid-fast bacteria with inflammatory lesions. * Fite’s stain more reliable for aquatic mycobacteria on histo. * If Acid-fast bacteria detected, samples should be submitted for culture or PCR and identification. * Speciation important since some spp have more zoonotic potential. * Infection occurs through direct contact with infected cohorts, consumption of infected organisms, exposure to infected water. * Can remain subclinical for long periods of time. * CS typically includes weight loss despite a good appetite. * Immediate euthanasia is recommended because tx is ineffective, and the diseases are likely to be zoonotic. * Removal and euthanasia of infected individuals is required to control the spread throughout the colony and prevent infection of human handlers.

Answer 20

* Brucellaceae * Gram-negative facultatively intracellular bacteria * Abscesses, spinal arthropathy with enlarged intervertebral joints, pyogranulomatous inflammation. * B. inopinata-like organisms readily isolated.

Answer 21

* Chlamydia (Chlamydophila) pneumoniae * African clawed frogs * Chlamydia psittacii * C. pneumoniae. * Amphibians may be reservoir hosts. * CS – cutaneous hyperemia, excessive shedding of skin, SQ edema, hepatosplenomegaly. * Dx PCR. * Large intracytoplasmic granular basophilic inclusions – Common in spleen or liver. * TEM – Characteristic elementary and reticulate bodies. * Tx – Doxycycline drug of choice, although tetracycline may be effective.

Answer 22

* Saprolegnia * Primarily aquatic amphibians, caused by 20+ spp of fungi. * Opportunistic, following skin trauma or low environmental temps, high environmental organic loads, or poor water quality i.e. high ammonia. * Cottonlike, covers underlying cutaneous ulcers. * Acute infections characterized by light-colored fungal mats, more chronic infections may turn green with algae or brown as debris is trapped in the fungal strands. * Secondary bacterial infections may occur. * Incidence markedly decreases at water temps over 68F (19C). * Wet mounts of cotton mat reveals fungal hyphae. * Thin-walled, nonseptate, usually nonbranching hyphae. * Stain poorly with PAS and well with GMS. * Postmortem overgrowth should be considred. * Tx – Salt, itraconazole, or benzalkonium baths. * Topical miconazole, dilute benzalkonium chloride, malachite green for isolated lesions.

Answer 23

* Chromomycosis * Light-tan or gray to dark raised nodules. * Signs of debilitation, weight loss. * Presumptive dx based on finding pigmented fungi in wet mounts. * Tendency to become systemic, unlike saprolegniasis. * Chromomycotic granulomatous lesions throughout viscera. * Tx unrewarding. * Euthanasia is recommended for infected amphibians due to zoonotic potential.

Answer 24

* Fusarium spp, zygomycetes (Basidiobolus ranarum, Mucor spp, Rhizopus spp). * Similar gross findings to chytrid and chromoblastomycosis, ranaviriosis, red leg. * Fusarium hyphae typically slender with parallel walls, frequent separation. * Mucor hyphae are wide and infrequently septate. * Mucor amphibiorum, disseminated mycosis in Australian anurans.

Answer 25

* Microsporidiosis. * Pleistophora myotrophica – Myositis, high mortality in Eu common toads. * White discoloration and streaking of pectoral, abdominal, femoral musculature. * Tx with chloramphenicol sodium succinate ICe in combo with topical oxytet and polymyxin B appeared effective.

Answer 26

* Etiological agents – Batrachochytrium dendrobatidis (Bd), Batrachochytridium salamandrivorans (Bs). * Both are intracellular pathogens of keratinocytes. * Life cycle is simple: * Intracellular fungal bodies (thalli) mature to form zoosporangia containing infective flagellated zoospores. * Bd – OIE reportable * Broad host range, infects 520+ spp. * Anurans, caudates, and caecilians. * Panamanian golden frogs highly susceptible. * Global distribution linked to international movement of amphibs for research and pet trade. Asia proposed origin. * Grows on sterile bird feathers and arthropod exoskeletons, keratinous paw scales of waterfowl, GIT of crayfish. * Crayfish = Potential nonamphibian vector for Bd. * Motile zoospores can swim and infect cohorts. * Bd feeds on keratin, anurans with keratinized skin usually affected more than tadpole, which only have keratinized mouthparts. * CS – Sudden death with no obvious signs to progressive lethargy, excessive shedding, ventral erythrma and petechiation, deformities of keratin beaks in tadpoles, cutaneous lesions on tips of toes. * Shedding numerous small discolored skin fragments rather than large translucent to white sheets that are normally shed. * Gross lesions – Skin in postmetamorphic amphibs and keratinized mouthparts in larval anurans. * Hyperplasia, excessive shedding, necrosis of digits/feet, dehydration, lethargy, abnormal behavior. Depigmentation of tadpole mouthparts. * Bs (first described 2013) * Restricted to salamanders and newts. * Near-extinction of the fire salamander, Salamandra salamandra in the Netherlands. * Found in Europe, Asia. NOT North America. * CS – Skin erosions all over the body, ataxia, anorexia, death within 7 days of infection. * Gross lesions – Multifocal superficial erosions and extensive epidermal ulcerations, excessive shedding of skin, anorexia, apathy, ataxia, death.

Answer 27

* Dx confirmation * Cytology – Detection of zoosporangia provides rapid diagnosis. * Biopsies of drink patch or toe tips often productive. * PCR or qPCR can be done on skin swabs from mouthparts of live anuran larvae and from the ventral pelvic patch, hind legs, and feet of live postmetamorphic, fresh dead, frozen, or ethanol-preserved postmetamorphic amphibs. * PCR even more sensitive than histo or wet mount exams. * Newly developed PCR for Bs. * Histo * Bd = Multifocal hyperplastic and hyperkeratotic dermatitis, sloughing of keratin, numerous fungal zoosporangia. * Epidermal hyperplasia and orthokeratotic hyperkeratosis with intracellular chytrid thalli within the straum corneum. * Bs = Erosive skin lesions associated with presence of numerous intracellular colonial thalli that spreads over the epidermis with marked necrosis of the adjacent keratinocytes. * Hyperplasia and hyperkeratosis ABSENT. * Multifocal necrosis of keratinocytes associated with numerous chytrid-type fungal thalli. * Prominent necrosis with development of erosions and ulcers as well as frequent thalli with multiple internal septa should raise suspicion for Bsal. * Zoosporangia can be found intracellular within keratinocytes. * EM, IHC.

Answer 28

* Tx/prevention * Itraconazole baths * 0.01% in 0.6% saline 5 minutes daily for 11 consecutive days. * Safe and effective tx in most situations, different concentrations and durations reported. * Voriconazole baths/sprays * 125 mg/L for 7 days. * Used in a few species, effectively treated a salamander. * Chloramphenicol bath * Effectively treated green tree frogs. * Terbinafine dissolved in ethanol and diluted for bath * Popular treatment, commercially available as athlete’s foot spray. * Increased temps * Effective for some spp, gradual increase in environmental temp over a period of two days, then maintained at that temp, up to 10 consecutive days reported. * Elevating temp to 25C for 10 days has been effective for Bs. * Disinfectants that effective kill Bd: * Virkon – Ozone (potassium peroxymonosulfate), sodium dodecylbenzenesulfonate, sulfamic acid, and inorganic buffers at 2 g/L. * Dilute bleach (4%). * Quarantine recommendations: at LEAST 2 months (60 days). * PCR on arrival and 7 weeks later.

Answer 29

* Rhabdias spp (fam Rhabdiasidae) – Amphibian lungworm (40+ spp). * Free-ranging and captive anurans and caudates. * Direct LC. * Hermaphroditic adults in lung produce eggs or larvae that are swallowed and pass through the GIT, shed in feces. * In environment, larvae develop into male and female adults, mate to produce infective larvae that penetrate the skin of the host and migrate internally to the lung. * Gross – Mottled white-black worms within the airway lumen of lungs. * Histo – Scant platymyarian musculature, vacuolated lateral chords, uterus with developing larvae, intestinal tract containing abundant dark pigmented material. * Embryonated eggs or larvae can be seen in fecals or tracheal wash specimens. Fecal parasite or oropharyngeal mucus exams. * Ddx – digenetic trematodes genus Haematoloechus, monogenean trematodes genus Pseudodiplorchis. * Infected amphibians should be isolated and strict hygiene practiced, preventing spread to other amphibians or superinfection of the original animal. * Baths with ivermectin or levamisole weekly for 12+ weeks may be needed to reduce or eliminate Rhabdias spp.

Answer 30

* Strongyloides spp – Proliferative enteritis in free-ranging and captive anurans. * Direct LC. * Larvated eggs or adults observed in fresh fecals. * May cause severe intestinal lesions, PLE and generalized malnutrition. * Oral fenbendazole and levamisole applied o the pelvic patch has been used to decrease both Strongyloides and Rhabdias.

Answer 31

* Capillarid nematode – Pseudocapillaroides xenopi. * Infects epidermis of free-ranging and captive African clawed frogs. * Excessive skin shedding and roughening, discoloration of the skin surface and multifocal cutaneous erosions or ulcers. * Heavy infections cause a wasting syndrome. * Bioperculate eggs. * Larvated eggs within the uterus of the female is a feature unique to this species. * Dx – Eggs or adults on histo or wet mount of shed skin fragments or skin scrape, PCR. * Cutaneous hemorrhage and exfoliation, nematodes and ova can be found in mucus and skin scrapings. * Tx with thiabendazole PO and levamisole SQ or ivermectin and levamisole baths effective.

Answer 32

* Digenetic trematodes * Clinostomum marginatum – Head of red spotted newts, interferes with feeding. * Encystment of metacercariae of Ribeiroia ondatrae within limb buds of anurans assoc with limb malformations. * Digeneans in genus Haemotoloechus common in lungs of anurans. * Gorgoderid digenetic trematodes of genera Gorgoderina and Phyllodistomum inhabit Wolffian ducts and UB of anurans and caudates, subclinical infections. * Praziquantel has been used in many amphibian spp without ill effects, may eliminate adult and larval trematodes and cestodes.

Answer 33

* Myxozoans * Genus Cystodiscus develop spore-forming plasmodia in gallbladder of anurans, caudates, caecilians. Incidental. * Most renal myxozoa are also incidental but fatalities have been reported. * Frog kidney enlargement disease caused by Hoferellus anurae occurred in wild-caught African hyperoliid frogs.

Answer 34

* Protozoa * Opalinids, flagellates, ciliates normal commensals in GIT of amphibians. * Trichodina incidental on wet mounts, skin, gills. * Tetrahymena invasive to skin and muscle, disseminate to visceral organs of tadpoles. * Frequent water changes and improved sanitation and filtration may help eliminate ciliated protozoa in aquatic amphibians. * Trypanosoma ranarum flagellates incidental on blood smears. * Quinidine therapy may be used with signs of anemia. * Sudden death may occur, splenomegaly common post mortem, trypanosomes can be seen on impression smears of spleen. * Cryptosporidiosis – apicomplexan. * Cryptosporidium fragile colonizes superficial gastric epithelium of black-spined toads. * Wt loss, mild gastric epithelial hyperplasia without assoc inflammation. * Oral tetracyclines and paromomycin sulfate may be warranted with high densities of ciliated protozoa in feces or gastric washes. * Variety of apicomplexans – Hematozoon spp, Lankesterella spp. * Intestinal coccidiosis – Eimeria, Isospora, Goussia spp. Not significant.

Answer 35

* Entamoeba ranarum – GIT commensal, dz rare. * Transmission via ingestion of cysts. * Direct LC, rapid spread within collection. * Cysts durable in environment, are ingested, then trophozoite stage excysts to mature in the colon. * Directly attack colonic mucosa, liver and kidneys can be infected. * CS – Anorexia, dehydration, wasting. * Stools change in consistency – liquid, tinged with blood. * Dx – Accurate ID of cyst or trophozoite from fecal sample or colonic wash. * Direct for active trophozoites, cysts found on direct smears or with floats. * Detection of cyst may be enhanced with Lugol’s iodine. * 4-16 nuclei. * Tx – Metronidazole 50 mg/kg PO q14 days or daily for 3-5 days with fluid therapy. Decrease dose if animal starts showing neuro dz.

Answer 36

* Clinical techniques: * Fluid samples with low TP and low cell counts lacking cytologic evidence of infection more likely to be associated with organ dysfunction or physiologic and environmental factors. * Frog blood volume considered 10%; 10% withdrawn safety from healthy frogs and 5% from ill frogs. 0.5 mL can be taken from a 10 gram frog. * Venipuncture (Anuran): * Lingual venous plexus. * Avoid breaking mandible. * CTA used to draw the tongue forward. * Vessels on underside of tongue and buccal floor. * Puncture with needle and use a HCT tube to draw blood up. * Samples can be contaminated with saliva and mucus. * Ventral (midline) abdominal vein * Insert needle cranio-dorsal, midway between sternum and pelvis. * Femoral vein, cardiocentesis (visualize, penetrate apex of ventricle). * Venipuncture (Caudate) – Ventral tail vein, ventral abdominal vein (if sedated, otherwise concern with autotomy), cardiocentesis. * Venipuncture (Caecilians) – Cardiocentesis. * Lithium heparin preferred anticoagulant. * Does not affect the values of plasma calcium, dosium, or ammonia. * EDTA lyses RBCS. * Ammonium heparin gives falsely elevated NH3 levels. * Sodium heparin should be avoided for evaluation of electrolytes. * Blood smears should be made immediately. * Preheparinize syringes may lead to lower and inconsistent values. * CBC performed same as avian and reptile spp. * Amphibians have neutrophils (peroxidase-positive). * Natt & Herrick’s solution or “eosinophil indirect method” i.e. Avian Leukopette to count granulocytes (only recommended in spp it has been verified for). * N:L ratio positively correlates with corticosterone. * Because of presence of injured epithelial cells after handling, amphibian monocyte counts may show a relative and absolute increase on subsequent leukocyte profiles. * Slender salamanders (genus Batrachoceps) – Majority of erythrocytes are anucleate in normal individuals. * Rickettsiae, parasites, and viral inclusions may be detected on smears. * Trypanosomes, microfilariae are extraerythrocytic. * Hemogregarines are intraerythrocytic. * Low levels probably not clinically significant. * Normal blood values may be influenced by environmental conditions, life cycle stage, nutrition, other variables. * Lymph can be used for biochemistry analysis.

Answer 37

* Acute mortalities * Most commonly due to infectious dz, sudden environmental change, or toxins. * Chytrid, ranavirus, lungworm, bacterial septicemia. * Tank flooding or drying, temp changes. * Chloramine, chlorine, ammonia, bleach, insecticides. * Bd often causes death without clinical signs. * Ranaviruses implicated in mass mortality events – hemorrhages, edema. * Septicemia usually gram-negatives, gram-positives also. * Atypical myco and chlamydophila result in chronic granulomatous dz.

Answer 38

* Coelomic distension * Fluid or soft tissue (ova, fat), or rarely gas. * Hydrocoelom/lymphedema * Coelomic cavity effusion – hydrocoelom. * Generalized lymphatic system swelling – lymphedema. * Rounded appearance over pelvis, loss of linea alba, exophthalmos, limb swelling. * Major causes include cutaneous lesions, infectious dz, renal or hepatic disease, environmental problems. * Chytrid, ranavirus, bacterial septicemia. * Cytology or histo and PCR can differentiate. * Cytology, culture and susceptibility of fluid. * Renal disease common. * Infectious dz capable of direct renal damage include local or systemic bacterial, viral, fungal agents i.e. mucormyosis, chromomycosis, zygomycosis, myxozoan and microsporidial parasites. * Noninfectious causes of renal dz – Dehydration, neoplasia, nutrition (oxalates, high protein, hypovit A), and toxins (aminoglycosides, heavy metals, polyvinyl chloride glue). * Hepatic disease via hepatocyte necrosis, impaired intrahepatic circulation, low plasma protein levels. * Inappropriate husbandry – exposure to cool temps has been associated with transient hydrocoelom, resolves without intervention. * Obesity – Coelomic cavity often firm on palpation. * Repro activity – egg masses, may be retained until suitable environmental cues are received. * May have focal edema around the upper thighs. * Impaction, FBs – Gas accumulation can be severe, bacterial overgrowth can lead to sepsis. * Ocular dz may predispose consumption of foreign material. * Organomegaly – Neoplasia and granulomatous disease. * Lymphoma, GI carcinomas, nephroblastomas, sertolic cell tumors, pancreatic carcinomas. * Focal granulomatous lesions. * Atypical myco, chlamydiosis, fungal infections most commonly reported cause of granulomas.

Answer 39

* GI signs. * Tissue prolapse. * Intestinal, urinary, or repro. * Cloacal tissue generally small, smooth, pink. * Colon smooth to ridged with tubular appearance. * Central lumen or feces may be visible on impression smear. * UB rounded with thin wall and fluid contents. * Oviduct includes narrow, coiled tubules, may have longitudinal lines and eggs. * Cloacal and colon prolapses most common followed by UB. * Common causes of prolapse – Parasitism, intussusception, GI FB, neoplasia, septicemia, toxemia, dehydration, extraluminal GI blockage i.e. renal neoplasia, toxins, hypoglycemia, hypocalcemia, malnutrition. * Tissue impression smears and FNA of tissue may help ID. * Iatrogenic gastric prolapse has been reported in leopard frogs ax with clove oil, resolves during recovery. * Diarrhea – Differentiate from polyuria. * Inappropriate temp, humidity, diets high in carbs. * Amoebiasis, coccidiosis, some metazoans i.e. Strongyloides, Acanthocephalus ranae. * Ciliates typically nonpathogenic. * Mucor amphibiorum can be primary cause of gastroenteritis. * Ranaviruses can cause intestinal distension and hemorrhage, diarrhea.

Answer 40

EVALUATION OF BONE MINERALIZATION BY COMPUTED TOMOGRAPHY IN WILD AND CAPTIVE EUROPEAN COMMON SPADEFOOTS (PELOBATES FUSCUS), IN RELATION TO EXPOSURE TO ULTRAVIOLET B RADIATION AND DIETARY SUPPLEMENTS van Zijll Langhout M, Struijk RP, Könning T, van Zuilen D, Horvath K, van Bolhuis H, Maarschalkerweerd R, Verstappen F. Journal of Zoo and Wildlife Medicine. 2017 Sep;48(3):748-56. Highest Hounsfield units (HU) in: Adults with dietary supplementation (no UVB) than those with only UVB Juveniles with only UVB (no dietary supplementation) compared to only dietary supplementation (no UVB) Adults (nocturnal) can get sufficient vitamin D from diet, while juveniles rely on UVB radiation Conclusion: CT is a practical tool for assessing bone mineralization in European common toads, and European common toad adults are not dependent on UVB for vitamin D3 and juveniles are more dependent on access to UVB radiation.

Answer 41

Cardiovascular effects of alfaxalone and propofol in the bullfrog, Lithobates catesbeianus. Williams CJ, Alstrup AK, Bertelsen MF, Jensen HM, Leite CA, Wang T. Journal of Zoo and Wildlife Medicine. 2018 Mar;49(1):92-8. Alfaxalone at 10 mg/kg caused: Sedation and loss of righting reflex Decreased respiratory rate Increased heart rate (\> 60 min when IV) Increased MAP (decreased after 10 min IV) Did not provide analgesia (no loss of noxious stimulus response) Propofol IV caused increased MAP only for first 5 minutes No change to heart rate Alfaxalone 10mg/kg IM pilot - loss of RR within 5min, loss of muscle tone 10min, recovery at 60min, no loss of nociception (toe pinch), RR declined , HR stable Conclusions: Alfaxalone at 10 mg/kg IV or IM in bullfrogs caused an increase in heart rate and a transient increase in MAP, while propofol only transiently increased MAP.

Answer 42

Comparison of subcutaneous administration of alfaxalone–midazolam–dexmedetomidine with ketamine–midazolam–dexmedetomidine for chemical restraint in juvenile blue poison dart frogs (Dendrobates tinctorius azureus) Yaw TJ, Mans C, Martinelli L, Sladky KK. . Journal of Zoo and Wildlife Medicine. 2020 Jan;50(4):868-73. Ketamine-midazolam-dexmedetomidine caused: Sawhorse stance/more limb rigidity (more pronounced after antagonists delivered) Decreased heart rate (min 20 -50) Increased respiratory rate Alfaxalone-midazolam-dexmedetomidine caused decreased respiratory rate and heart rate (min 15-50) Both protocols caused loss of righting reflex, similar recovery times, and some gastric prolapse Gastric prolapse more prevalent in AMD group Conclusions: Ket-midaz-dexmed decreased HR, increased RR, and caused limb rigidity. Alfax-midaz-dexmed decreased HR and RR; some gastric prolapse (AMD \> KMD)

Answer 43

Anesthetic Efficacy of MS-222 in White's Tree Frogs (Litoria caerulea) Krisp AR, Hausmann JC, Sladky KK, Mans C. Journal of Herpetological Medicine and Surgery. 2020 Jan;30(1):38-41. MS-222 at 0.5 g/L produced light sedation and loss of palpebral reflex in most frogs Corneal and righting reflexes were present in all frogs MS-222 at 2.0 g/L produced surgical anesthesia within 20 min with all reflexes lost except corneal reflex Time dependent decreased in heart rate Dose and time dependent respiratory depression. Concentration for MS-222 to produce general anesthesia much lower in leopard frogs (0.4 g/L) Conclusions: In White’s tree frogs, MS-222 at 0.5 g/L produces sedation but 2 g/L produces general anesthesia with time dependent decrease in heart rate and dose and time dependent respiratory depression.

Answer 44

Inhalatory isoflurane anesthesia in mountain chicken frogs (Leptodactylus fallax). Barbon AR, Routh A, Lopez J. Journal of Zoo and Wildlife Medicine. 2019 Jun;50(2):453-6. Bag with frog filled with 5% iso and O2. Intubated with 2.5-3.5mm cuffed tubes that were shortened and maintained on gas anesthesia for surgery. ETT was inflated PRIOR to placement in the trachea so that it sealed off the larynx. Ventilated manually Heart rate decreased over time Mtn Ckn frogs do not have a trachea or bronchi. Larynx is directly connected to lung Conclusions: You can provide inhalant anesthesia via a modified endotracheal tube in a mountain chicken frog.

Answer 45

Lethal and sublethal amphibian host responses to batrachochytrium dendrobatidis exposure are determined by the additive influence of host resource availability. Rumschlag SL, Boone MD. The Journal of Wildlife Diseases. 2020 Apr;56(2):338-49. Mortalities from Bd were typically within 24 hours, sooner if low food BD exposure and low food availability combined led to: Lowest survival rates in American toads Lowest growth rates in northern leopard frogs BD exposure did not influence survival of northern leopard frogs and cricket frogs Cricket frogs were unaffected by BD exposure Conclusions: Bd and low food availability influence survival of the American toad and growth of the northern leopard frog. Bd did not influence either in the cricket frog.

Answer 46

BATRACHOCHYTRIUM DENDROBATIDIS IN A CAPTIVE COLLECTION OF GREEN SALAMANDERS (ANEIDES AENEUS), LONG-TAILED SALAMANDERS (EURYCEA LONGICAUDA), AND TWO-LINED SALAMANDERS (EURYCEA BISLINEATA) Bauer KL, Steeil JC, Walsh TF, Evans MJ, Klocke B, Gratwicke B, Siegal-Willott JL, Neiffer DL. Journal of Zoo and Wildlife Medicine. 2018 Jun;49(2):454-9. First salamander found dead acutely without clinical signs, three others died during treatment Diagnosed via cytology and histopathology (dermatitis and hyperkeratosis with visible chytrid) Treated other Eurycea spp. salamanders with 0.01% itraconazole baths and had no mortalities Chytrid swabbing protocol = ventral surface 10x + each foot 5 times Conclusions: Difference in mortality and fungal load suggests Aneides sp. may be more susceptible to Bd than Eurycea sp.

Answer 47

SEASONALITY IN BATRACHOCHYTRIUM DENDROBATIDIS DETECTION IN AMPHIBIANS IN CENTRAL OKLAHOMA, USA Watters JL, McMillin SL, Marhanka EC, Davis DR, Farkas JK, Kerby JL, Siler CD. Journal of Zoo and Wildlife Medicine. 2019 Jun;50(2):492-7. Key Points: 42% were positive, none had clinical signs Most common positives: Blanchard’s cricket frog, American bullfrog, southern leopard frog Highest loads found on Anaxyrus woodhousii, Gastrophryne olivacea, Pseudacris fouquettei Prevalence highest in FALL \> spring \> summer Conclusions: Highest prevalence of Bd was in the FALL then spring. Positive PCR were negative for lesions. More rainfall = higher prevalence. Blanchard’s cricket frog was the most common positive (of the species that had \>10 animals caught)

Answer 48

Higher Infection Prevalence in Amphibians Inhabiting Human-made Compared to Natural Wetlands Spencer R. Siddons, Marin C. Bray, and Catherine L. Searle Journal of Wildlife Diseases, 56(4), 2020, pp. 823–836 Anthropogenic practices can decrease Bd growth and transmission of Bd: Increased salinity pH \<4 or \>8 Pesticides Increased or decreased water temp (optimal range for Bd is 17-25\*C) Habitat loss in tropics (due to increased temps from less canopy cover?) Key Points: Bd prevalence higher at human-made sites than natural No difference in frog dentistry community composition Conclusions: Human-made wetlands may play a role in sustaining frog communities, but may also pose a higher risk for Bd infection than natural wetlands.

Answer 49

Estimating occurrence, prevalence, and detection of amphibian pathogens: Insights from occupancy models. Mosher BA, Brand AB, Wiewel AN, Miller DA, Gray MJ, Miller DL, Grant EH. Journal of wildlife diseases. 2019 Jul;55(3):563-75. Background: Ranavirus (an iridovirus) = generalist pathogen linked to die-offs of amphibians in US Larval amphibians especially susceptible Affinity for vascular endothelial cells Key Points: pH does not affect ranavirus prevalence Ranavirus detection probability was higher (6.7x higher odds of detection) in tail samples than livers Ranavirus prevalence high for wood frogs than for spotted salamanders in Maryland Conclusions: Can monitor non-lethally for ranavirus with PCR on tail clips.

Answer 50

Clinicopathologic features of infection with novel Brucella organisms in captive waxy tree frogs (Phyllomedusa sauvagii) and Colorado River Toads (Incilius Alvarius) Helmick KE, Garner MM, Rhyan J, Bradway D. Journal of Zoo and Wildlife Medicine. 2018 Mar;49(1):153-61. Key Points Novel Brucella infection in amphibians, caused femoral osteomyelitis, SQ and renal abscesses, sepsis. No single organ system involved in all cases, no repro pathology SQ abscessation more specific sign, otherwise vague (color changes, lethargy, anorexia, wt. loss) Treatment with systemic enro, surgical removal of localized lesions, chloramphenicol baths effective in some cases Need PCR to differentiate from Ochrobactrum anthropi Potential zoonotic risk, host species, geographic range, etc. not known for Brucella isolates from amphibians Take home message: Brucella species have been identified in amphibians, cause osteomyelitis, SQ and renal abscesses, sepsis.

Answer 51

Mycobacteriosis in a Zoo Population of Chinese Gliding Frogs (Rhacophorus dennysi) Due to Mycobacterium marinum Milnes EL, Delnatte P, Lentini A, May K, Ma J, Jamieson FB, Slavic D, Smith DA. Journal of Herpetological Medicine and Surgery. 2020 Jan;30(1):14-20. Cases: Symptomatic and asymptomatic Chinese gliding frogs with internal granulomas diagnosed with M. marinum Clinical signs: gallbladder empyema (n=2), found dead (n=2), dermal lesions (ulcerative or proliferative) Pneumonia (n=4) and internal granulomas (all) GenoType Mycobacterium CM assay incorrectly assigned isolate as MtbC due to cross reaction Sequencing correctly identified it as M. marinum Most common Mycobacterium spp in amphibians: M. marinum, M. fortuitum, M. ulcerans, M. xenopi No reports of Mycobacterium tuberculosis complex Conclusions: M. marinum caused death, dermal lesions, gallbladder empyema, or no clinical signs in Chinese gliding tree frogs.

Answer 52

DETECTION AND REPORTING OF RANAVIRUS IN AMPHIBIANS: EVALUATION OF THE ROLES OF THE WORLD ORGANISATION FOR ANIMAL HEALTH AND THE PUBLISHED LITERATURE Black Y, Meredith A, Price SJ. Journal of wildlife diseases. 2017 Jul;53(3):509-20. Ranavirus and chytridiomycosis are reportable to the OIE Ranavirus = iridovirus with patchy global distribution and broad host ranges (amphibians, reptiles, fish) OIE recommends sequencing all PCR positive results Does not recommend qPCR Conclusions: Ranavirus is an OIE-reportable disease and PCR positives should be sampled.

Answer 53

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA. Hardman RH, Sutton WB, Irwin KJ, McGinnity D, Reinsch SD, Freake M, Colclough P, Miller BT, Da Silva Neto JG, Souza M, Fitzpatrick B. Journal of Wildlife Diseases. 2020 Oct;56(4):803-14. Background: Batrachochytrium dendrobatidis is associated with amphibian mass mortality and extinctions Batrachochytrium salamandrivorans is highly lethal to salamanders Hellbender = fully aquatic salamander with declining populations Reported Bd mortalities in captive individuals (50% mortality in a head start program) Ranavirus causes vascular and epithelial damage Chinese giant salamander with ranavirus often have swollen digits and necrosis Key Points: Bd and ranavirus were found in all regions (Bd \> ranavirus) No Bsal No correlation with lesions and Bd zoospore load Some recaptured individuals had cleared Bd Toe lesions were seen in many hellbenders Some associated with increased ranavirus prevalence Ranavirus correlated with lower BCS Highest ranavirus load in middle TN Conclusions: Bd and ranavirus are found in hellbender habitats. Wild hellbenders commonly have toe lesions.

Answer 54

CAUSES OF MORTALITY IN CAPTIVE PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI) AT THE MARYLAND ZOO IN BALTIMORE, 2001–2013 Eustace R, Wack A, Mangus L, Bronson E. Journal of Zoo and Wildlife Medicine. 2018 Jun;49(2):324-34. Greatest number of deaths in younger frogs (201) Most frogs found dead (rather than euthanized) Most deaths during breeding season (Nov-May) Energetically demanding; frogs focus solely on breeding and often do not feed during this period Dermatitis was the most common cause of death: hyperkeratosis \> ulcers \> acanthosis \> necrosis Frequently associated with invasion by filamentous fungi (83.9%) like Zgomycetes and wartermolds Kidney disease caused 8% deaths and present in 41% frogs Clinical signs: hydrocooelom, lymphedema One clutch has polycystic kidney disease Lymphedema associated with renal disease, dermatitis, multifactorial, GI disease GI disease deaths were caused by mycotic enteritis, rarely parasites Tetany syndrome caused deaths in females Hind limbs are rigid and fix in extension or held dorsally over the height of the back Lacks a righting reflex Conclusions: Panamanian golden frogs died most commonly from dermatitis. Tetany syndrome is seen only in females. Deaths are most common in the breeding season.

Answer 55

A RETROSPECTIVE REVIEW OF POST-METAMORPHIC MOUNTAIN CHICKEN FROG (LEPTODACTYLUS FALLAX) NECROPSY FINDINGS FROM EUROPEAN ZOOLOGICAL COLLECTIONS, 1998 TO 2018 Ashpole IP, Steinmetz HW, Cunningham AA, Barbon A, Stidworthy MF, Garcia G, Sangster CR, López Journal of Zoo and Wildlife Medicine. 2021;52(1):133-44. Common findings in adult mountain chicken frogs in European zoos: Low body condition score (most common in adult and captive-bred frogs0 Most common organ system = gastrointestinal tract Most common process = intestinal inflammatory disease Most common neoplasia = intestinal/colonic adenocarcinoma (27.2%!) Associated with adhesions or fistula between colon and bladder More common in older frogs Enteric nematodiasis Juvenile mountain chicken frogs had inflammatory disease or musculoskeletal trauma Wild-born lived longer than captive-bred Lucke’s renal carcinoma in leopard frogs associated with ranid herpesvirus 1 Conclusions: Mountain chicken frogs are susceptible to gastrointestinal inflammatory disease and intestinal/colonic adenocarcinoma.

Answer 56

Analysis of Published Amphibian Neoplasia Case Reports. Hopewell E, Harrison SH, Posey R, Duke EG, Troan B, Harrison T. Journal of Herpetological Medicine and Surgery. 2020 Sep;30(3):148-55 Herpesvirus-induced, Luckes´ renal adenocarcinoma in wild northern leopard frogs Most common tumors: Chromatophoroma \> lymphoma/leukemia \> papilloma 50:50 benign:malignant, but chromatophoromas were malignant Most common species: Xenopus and axolotls Most common site: skin Most common treatment: surgical excision Improved prognosis Conclusions: Most common tumor in frogs is a benign chromatophoroma.

Answer 57

POSTMORTEM FINDINGS IN EIGHT SPECIES OF CAPTIVE CAECILIAN (AMPHIBIA: GYMNOPHIONA) OVER A TEN-YEAR PERIOD Flach EJ, Feltrer Y, Gower DJ, Jayson S, Michaels CJ, Pocknell A, Rivers S, Perkins M, Rendle ME, Stidworthy MF, Tapley B. Journal of Zoo and Wildlife Medicine. 2020 Jan;50(4):879-90.

Answer 58

Immersion in tricaine methanesulfonate (MS-222) is not sufficient for euthanasia of smokey jungle frogs (Leptodactylus pentadactylus). Balko JA, Posner LP, Chinnadurai SK. Journal of Zoo and Wildlife Medicine. 2019 Apr;50(1):89-95. Key Points: Initial excitement phase Death in 17/18 frogs in the study but is NOT sufficient for euthanasia Took too long Multiple episodes of regaining consciousness Take home: MS-222 at the doses used did result in death of most of the frogs but took too long to be humane. Return of spontaneous movement occurred following submersion.

Answer 59

MEASURING INTRAOCULAR PRESSURE IN WHITE'S TREE FROGS (LITORIA CAERULEA) BY REBOUND TONOMETRY: COMPARING DEVICE, TIME OF DAY, AND MANUAL VERSUS CHEMICAL RESTRAINT METHODS Hausmann JC, Krisp A, Sladky K, Miller PE, Mans C. J Zoo Wildl Med. 2017 Jun;48(2):413-419. In amphibians, aqueous humor flows out of the iridocorneal angle into two ciliary venous sinuses instead of an episcleral venous plexus. TonoLab = a rebound tonometer designed for the smaller cornea of rats TonoLab higher than TonoVet in morning and midday Diurnal variation in IOP has been reported (lower in the AM in bearded dragons) Lower IOPs in manually restrained animals compared to unrestrained (with TonoVet) Type II error or part of a “death feigning” response Anesthesia with MS-222 was not a significant change Anesthesia tends to decrease IOP in mammals due to reduced extraocular/adnexal muscle tone Conclusions: In White’s tree frogs, diurnal variation in IOP, manual restraint decreases IOP, and anesthesia with MS-222 does not significantly affect IOP.

Answer 60

Common abnormalities = corneal lipid keratopathy, corneal fibrosis, cataracts, lenticular sclerosis Lipid keratopathy common in mossy tree frogs, Mexican giant tree frogs, cuban tree frogs May be related to diet , vitellogenesis, environmental Cataracts were most common abnormality Lower IOP in eyes with cataracts in giant waxy monkey tree frogs Lenticular sclerosis was second most common finding; unrelated to IOP Canine “d” setting significantly higher than “p” setting for most animals - when measuring IOP in anurans Conclusions: Cataracts were most common abnormality and associated with lower IOP in giant waxy monkey tree frogs.

Answer 61

Plasma concentrations of ketoprofen and meloxicam after subcutaneous and topical administration in the smoky jungle frog (Leptodactylus pentadactylus). Balko JA, Watson MK, Papich MG, Posner LP, Chinnadurai SK. Journal of Herpetological Medicine and Surgery. 2018;28(3-4):89-92. Meloxicam only detected at the 3h time point with SQ injection and not detected at all after topical administration Ketoprofen detected at 3h, 8h, 24h Decline in ketoprofen over time was more variable in cutaneous route than SQ Conclusions: Topical and SQ ketoprofen could be systemically detected, but topical and SQ meloxicam were not.

Answer 62

THE PHARMACOKINETICS OF TOPICAL ITRACONAZOLE IN PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI) Rifkin A, Visser M, Barrett K, Boothe D, Bronson E. Journal of Zoo and Wildlife Medicine. 2017 Jun;48(2):344-51. Key Points: No adverse effects documented 0. 001% bath kept \>MIC for 1 hour: likely too low a dose and frequent treatments would be required. 0. 01% bath did not ever leave the skin enough to calculate AUC Repeated dosing lead to toxic accumulation Take Home Message: Systemic absorption and distribution of itraconazole (0.01% and 0.001%) in Panamanian golden frogs after topical bath. 0.01% bath found to exceed MIC for at least 36 hours, suggesting that it shouldn’t be dosed daily.

Answer 63

EFFICACY AND SAFETY OF CHLORAMPHENICOL TREATMENT FOR BATRACHOCHYTRIUM DENDROBATIDIS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) Anthony L. Ashley J. of Zoo and Wildlife Medicine, 52(1):373-378 (2021) Clinical signs of Bd in hellbenders: ventral erythema, excessive mucus, and rocking from side to side Chloramphenicol bath at 200 mg/L continuously x 2 weeks in clinically affected Bd hellbenders: Stopped deaths Positive at 1 month after treatment Negative at 2-3 years after treatment Treatments that did not prevent deaths in Bd-infected hellbenders: Chloramphenicol at 20 mg/L daily x 2 wks Itraconazole baths Conclusions: Chloramphenicol baths at 200 mg/L appeared to eliminate clinical signs and deaths caused by chytridiomycosis in captive hellbenders by decreasing the burden of Bd

Answer 64

Efficacy of subcutaneous implants to provide continuous plasma terbinafine in hellbenders (cryptobranchus alleganiensi) for future prophylactic use against chytridiomycosis. Hardman RH, Cox S, Reinsch SD, Schwartz HC, Skeba S, McGinnity D, Souza MJ, Miller DL. Journal of Zoo and Wildlife Medicine. 2021 Apr;52(1):300-5. Background: Batrachochytrium dendrobatidis (Bd) motile zoospores infect keratinized epithelium Disrupt skin osmoregulation Highly susceptible species: Panamanian golden frog Resistant species: many salamanders Bd-positive and healthy CA slender salamanders brought into captivity 100% die from Bd 100% mortality in head-started hellbenders despite high prevalence in wild Terbinafine: lipophilic antifungal that concentrates in skin even when given orally Terbinafine implants provided therapeutic levels in cottonmouths Key Points: Terbinafine implant in juvenile hellbenders → levels above Bd zoospores MIC for at least two time points Not above MIC for Bd zoosporangia Conclusions: Terbinafine implants may be effective as a prophylactic for hellbenders at risk of Bd.

Answer 65

**Vascular Tumors in Two Argentine Horned Frogs (Ceratophrys ornata)** Mitsuhiro Ikeda1, Hirotaka Kondo1, Yuki Iwata1, Iori Koizumi2, Naoto Tsunesumi3, Hisashi Shibuya1 Journal of Herpetological Medicine and Surgery, 32(2) : 119- 122 Key Points: - Most common tumors in amphibians are chromatophoroma, lymphoma/leukemia and papilloma - Case 1: splenic hemangiosarcoma - Case 2: lymphangioma - Described two vascular tumors of Argentine horned frogs and the usefulness of anti-CD31 antibody.

Answer 66

JHMS 2022 32(3):219-24 **Characterizing Tissue Enzyme Activities in the Marine Toad (Rhinella marina)** Cabot ML, Heugten KA, Smith D, Ridgley F, Minter LJ Key Points: - Elevated CK, LDH, & AST may suggest skeletal muscle damage - Elevated CK, LDH, ALP, & AST may suggest cardiac disease - None of the enzymes evaluated seem to be specific to the liver

Answer 67

**Hepatocellular toxicity of the metabolie emodin produced by the common buckthorn (Rhamnus cathartica) in green frog (lithobates clamitans) tadpoles**. Brenes R, Nguyen LM, Miller DL, Rohde ML. The Journal of Wildlife Diseases. 2022;58(2):341-347 Background - Common buckthorn – invasive plant spp, family Rhamnaceae, produces allelopathic substances and secondary compounds in leaves, bark, and fruits -- Secondary metabolite emodin – anthaquinone, deformities and mortality in inverts, fish, amphibian larvae; high concentrations – oxidative stress in liver, inflammation, congestion, necrosis, damage to lipids, DNA, proteins -- Clinical signs: dull skin color, mouth gaping/dyspnea, red skin, excess mucus, disorientation, seizures -- Metabolized in the liver - Tadpoles – liver function changes from hematopoietic in early developmental stages to metabolic; young tadpoles potentially more susceptible to toxicosis Key Points - Emodin concentration based on field sample ranges - 85% mortality of tadpoles at advanced developmental stage when exposed to 2.5 ppm emodin -- All displayed clinical signs: dyspnea, erythema, lethargy, spasms - Gross necropsy: enlarged nodular livers, accumulation of yellow/gold material (emodin powder) in gallbladder and biliary system - Histo: bridging portal fibrosis, marked bile duct proliferation Conclusions - Green frog tadpoles at advanced developmental stages had high levels of mortality and liver damage when exposed to emodin 2.5 ppm -- Liver damage: oxidative stress, hepatocyte death, fibrous replacement - The wide distribution of this invasive plant (in the Midwest) could be a threat to amphibian populations, especially early breeding-species.

Answer 68

**Fowler 10 Chapter 61 Update on Amphibian Chytrid Fungus** - Batrachochytrium dendrobatidis (Bd) (1998) -- Ubiquitous organisms -- Bd was the first chytrid to affect a vertebrate -- Morbidity and mortality are not simply based on the species of amphibian infected - Infects epidermal cells of amphibians, leading to impaired skin function - Second chytrid fungus → (2013) Batrachochytrium salmandrivorans (Bsal) -- Similar to Bd, main difference is that Bsal is more pathogenic to salamanders and Bd is more of a concern in anurans -- Bsal → skin ulcerations -- Bd → epidermal hyperplasia and hyperkeratosis - Most info here for Bd (specific Bsal info will be noted) - Tadpoles play a major role in maintenance in the environment → in their mouthparts and transitions to the hind limbs during metamorphosis - Nonamphibian hosts: dose-dependent mortality in zebrafish juvenies; gill recession in crayfish; attracted to and will adhere to/proliferate on keratinous toe scales of aquatic birds - Peptides and microbes in amphibian skin can be protective against Bd and Bsal to inhibit chytrid growth - Janthinobacterium lividum (bacterium isolated from skin of red-backed salamander) produces a metabolite (violacein) that has antifungal properties against Bd -- Can be protective when applied to other species - Some animals have physiological changes that help to fight infection -- Red-backed salamander increases food intake to keep weight up -- North American bullfrog tadpoles increase cardiac output by elevating stroke volume (rather than HR) to preserve energy to fight infection -- Tadpoles infected develop faster - Bd grows best b/w 17 and 25C - Bd susceptible to desiccation in drought environment - Wide range of chemicals w/ varying degrees of effect on Bd morbidity and mortality - Diagnosis relies heavily of PCR, secondarily histology -- Just because it is found, does not mean it is the cause of illness -- Need negative results several times over several weeks to confirm truly negative - Mainstay tx for years has been itraconazole 0.01% solution for 5 min x 11 days -- Sometimes kills pts over the infection -- Not as effective in tadpoles and can cause depigmentation - Good biosecurity is important in prevention and management -- Nitrile gloves have a cidal effect against Bd (latex and polyethylene do not) - Major threat to amphibian pops all over the world -- No single “magic bullet” for tx