University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln USDA National Wildlife Research Center - Staff U.S. Department of Agriculture: Animal and Plant Publications Health Inspection Service 2018 CLINICOPATHOLOGIC FEATURES OF INFECTION WITH NOVEL BRUCELLA ORGANISMS IN CAPTIVE WAXY TREE FROGS (PHYLLOMEDUSA SAUVAGII) AND COLORADO RIVER TOADS (INCILIUS ALVARIUS) Kelly E. Helmick American College of Zoological Medicine Michael M. Garner American College of Zoological Medicine Jack Rhyan Daniel Bradway Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc Part of the Life Sciences Commons Helmick, Kelly E.; Garner, Michael M.; Rhyan, Jack; and Bradway, Daniel, "CLINICOPATHOLOGIC FEATURES OF INFECTION WITH NOVEL BRUCELLA ORGANISMS IN CAPTIVE WAXY TREE FROGS (PHYLLOMEDUSA SAUVAGII) AND COLORADO RIVER TOADS (INCILIUS ALVARIUS)" (2018). USDA National Wildlife Research Center - Staff Publications. 2067. https://digitalcommons.unl.edu/icwdm_usdanwrc/2067 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA National Wildlife Research Center - Staff ubP lications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Journal of Zoo and Wildlife Medicine 49(1): 153–161, 2018 Copyright 2018 by American Association of Zoo Veterinarians CLINICOPATHOLOGIC FEATURES OF INFECTION WITH NOVEL BRUCELLA ORGANISMS IN CAPTIVE WAXY TREE FROGS (PHYLLOMEDUSA SAUVAGII) AND COLORADO RIVER TOADS (INCILIUS ALVARIUS) Kelly E. Helmick, D.V.M., Dipl. A.C.Z.M., Michael M. Garner, D.V.M., Dipl. A.C.V.P., Jack Rhyan, D.V.M., M.S., and Daniel Bradway, B.S. Abstract: Two novel and distinct Brucella strains were recovered from 5 of 10 adult, sex undetermined, captive waxy tree frogs (Phyllomedusa sauvagii) and two of five adult, sex undetermined, captive Colorado river toads (Incilius alvarius) held in a zoologic collection with clinical and pathologic findings of bacterial disease. These amphibians originated from three separate private breeding facilities over several years and exhibited disease 9–49 mo following release from quarantine. Common presenting signs were vague but included focal abscessation, weight loss, change in coloration, anorexia, and decreased perching. Two waxy tree frogs and one Colorado river toad recovered with supportive care and antimicrobial treatment based on susceptibility testing. Micro- granulomatosis, subcutaneous and renal abscessation, femoral osteomyelitis, and multicentric infection were the most common histologic findings. The organisms were identified antemortem in samples from subcutaneous abscesses, cloaca, and skin and from a variety of organ systems postmortem, and demonstrated a consistent susceptibility pattern. Initial isolates were misidentified as Ochrobactrum anthropi. Polymerase chain reaction and sequencing of the 16S rRNA gene identified the two organisms as novel Brucella strains similar to Brucella inopinata–like sp. and other novel organisms within the emerging ‘‘BO clade.’’ Brucella strain oaks (isolated from waxy tree frogs) and Brucella strain leathers (isolated from Colorado river toads) differed from each other by 16 of 571 base pairs in a region of chromosome 2, and did not closely match any previous GenBank entries. This report describes the clinicopathologic features of infection by these bacteria in two amphibian species and expands the range of novel Brucella organisms from amphibian reservoirs. Key words: Amphibians, Brucella, Colorado river toad, Incilius alvarius, Phyllomedusa sauvagii, waxy tree frog. INTRODUCTION reservoir while raising questions regarding poten- tial zoonotic risks and population health concerns Brucellosis is a major disease concern in that could affect the captive breeding and man- humans and domesticated animals worldwide. agement of taxa of which some species are Animal reservoirs are the source of human undergoing a global decline. infection, which is most often associated with All Brucella species have a specific insertion Brucella melitensis, Brucella abortus, and Brucella sequence, IS711, and core species share identical suis.3 With the isolation of marine mammal 16S rRNA sequences.13,16,22,25 All core species are Brucella species from clinically affected hu- from mammals and include B. melitensis, B. suis, B. mans,12,19 new wildlife reservoirs are emerging as abortus, Brucella canis, Brucella neotamae, Brucella a potential source for human brucellosis. Brucella- ovis, Brucella pinnepedialis, Brucella ceti, and Bru- like organisms have been isolated from a variety cella papionis.AtypicalBrucella exhibit slight of amphibians,5,7,13,18,20,25 expanding the wildlife differences in 16S rRNA sequence, biochemical features, growth rate, and motility. Atypical From the Woodland Park Zoo, 601 N. 59th St., Seattle, Brucella spp. include Brucella inopinata and B. Washington 98103, USA (Helmick), Northwest ZooPath, inopinata–like (human),4,16,22 Brucella microti (voles, West Main St., Monroe, Washington 98272, USA (Gar- Microtus arvalis),11,14,15 Brucella vulpes (red fox, ner), US Department of Agriculture, National Wildlife Vulpes vulpes),17 and numerous isolates collected Research Center, Fort Collins, Colorado 80521, USA from a diversity of amphibians, including African (Rhyan), and Washington Animal Disease Diagnostic Lab bullfrog (Pyxicephalus edulis),5 big-eyed tree frog and Paul G. Allen School for Global Animal Health, (Leptopelis vermiculatus),7 cane toad (Rhinella ma- Pullman, Washington 99164-7034, USA (Bradway). Pre- 18 sent address (Helmick): Smithsonian Conservation Biology rina, formerly Chaunus [Bufo] marinus), Argentine 20 Institute, 1500 Remount Rd., Front Royal, Virginia 22630, horned (Pac-Man) frog (Ceratophrys ornata), and USA. Correspondence should be directed to Dr. Helmick White’s tree frog (Litoria caerulea),24 as well as red- ([email protected]). eyed tree frog (Agalychnis callidryas), Amazonian 153 154 JOURNAL OF ZOO AND WILDLIFE MEDICINE milk frog (Trachycephalus resinifictrix), tomato frog mg/ml, Intervet Inc., Millsboro, Delaware 19966, (Dyscophus antongilii), and Cranwell’s horned frog USA) 100 mg/kg po q 14 days for five doses. All (Ceratophrys cranwelli).13 These atypical Brucella frogs from group 2 survived the 49-day quarantine organisms, distinct from currently described Bru- period and tested negative for chytrid fungus from cella sp. and genetic relatives, pose an undeter- skin swab samples. mined risk for human, domesticated animal, and Two groups (group 3, group 4) of Colorado wildlife populations. river toads were obtained from the same private Native to the semiarid Chaco Plain of Argenti- facility located in Washington, USA, approxi- na, Bolivia, Brazil, and Paraguay, waxy tree frogs mately 1 yr apart. Group 3 consisted of two adult (Phyllomedusa sauvagii) are an arboreal amphibian toads of undetermined age and sex, listed as species threatened by the international pet trade ‘‘undetermined place of hatch.’’ Group 4 consist- and habitat alteration through agriculture and ed of three adult toads, captive bred, of undeter- wood harvesting.1 The Colorado river toad (In- minedageandsex.Healthhistoryand cilius alvarius) is a common terrestrial amphibian preshipment testing were not available for either native to the arid lowlands, grasslands, and group. All toads survived the quarantine period mountain canyons of southern California, Arizo- (81 days for group 3 and 31 days for group 4) and na, and New Mexico in the United States, tested negative for chytrid fungus on skin swab extending south into Mexico.9 This report de- samples. During quarantine all toads were treated scribes the clinicopathologic features of two novel for intestinal nematodes with fenbendazole (Pan- Brucella strains from two species of captive acur, 100 mg/ml) 50 mg/kg po sid for 3–10 days. amphibians, Brucella strain oaks, isolated from Group 3 toads were successfully treated for South American waxy tree frogs (P. sauvagii), and presumptive ivermectin toxicosis following iver- Brucella strain leathers, isolated from Colorado mectin (Vetrimec 1%, Norbrook Laboratories river toads (I. alvarius), from a zoologic collection Limited, Newry, Co. Down, Northern Ireland) in the United States. administered 0.2 mg/kg po once for intestinal nematodiasis. Symptoms of bloating, lethargy, CASE REPORT and increased sternal recumbency with a Two groups (group 1, group 2) of waxy tree ‘‘spread-out’’ appearance developed in both toads frogs were obtained from private facilities ap- approximately 72 hr following ivermectin treat- proximately 1 yr apart. Group 1 consisted of six ment, and both toads responded to supportive adult frogs of undetermined age and sex from a care. Group 4 toads were not treated with private breeder located in California, USA, cap- ivermectin during quarantine. Group 3 toads tive bred from wild-caught frogs originating from underwent quarantine during the same time Paraguay. Group 2 consisted of four adult frogs, period as group 1 frogs but were housed in a captive bred, of undetermined age and sex from a separate enclosed room with separate air flow. private facility located in Nevada, USA, but may Case 1 is a waxy tree frog that originated from have originated from the same breeder from group 1. Approximately 14 mo following release which group 1 frogs were obtained. Health history from quarantine, the frog presented