A RETROSPECTIVE REVIEW OF THE CLINICAL UTILITY OF FECAL BACTERIAL ENTERIC PATHOGEN CULTURES IN MAMMALS WITHIN A ZOOLOGICAL COLLECTION AND THE USE OF FECAL CYTOLOGY FOR OPTIMIZATION OF DIAGNOSTIC TESTING.

To determine the diagnostic value of fecal bacterial enteric pathogen cultures (FBEPC) as part of routine preventive medicine protocols in terrestrial mammals housed in a zoological collection, this study investigated the clinical utility of FBEPC results in context of subsequent clinical actions and how its use was rationalized after adjunct use of fecal cytology as a first-line diagnostic tool. Retrospective results (n = 692) that included a routine FBEPC panel of a commercial diagnostic laboratory, including Aeromonas, Salmonella, Campylobacter, Plesiomonas, Shigella, Yersinia, and Edwardsiella, of 417 mammals were organized into preventive (P; n = 485), diagnostic (D; n = 177), or recheck (R; n = 30) samples; for P and D samples, findings were assigned a "clinical significance factor" of 1 to 5 according to culture results and subsequent clinical actions. A score of 3 or higher indicated positive growth of potentially pathogenic bacterial organisms, of which there were 50 FBEPC (P n = 27, D n = 20, R n = 3). The difference in mean clinical significance factor for P versus D samples was significant. Aeromonas spp. were most frequently isolated (n = 32), followed by Salmonella spp. (n = 8) and Plesiomonas shigelloides (n = 8), then Campylobacter spp. (n = 5). There was no growth of Yersinia enterocolitica, Shigella spp., or Edwardsiella spp. In the absence of clinical evidence of gastrointestinal disease, treatment was initiated in only two cases with isolated Campylobacter spp. Implementation of fecal cytology as an initial step in fecal evaluation resulted in a prompt, substantial reduction in number of ordered FBEPC (mean n = 12/month before and n = 5/month after implementation). The findings in this study suggest that FBEPC for these bacterial species has limited value as a screening tool in preventive medicine protocols for the mammalian orders best represented in this study. The use of fecal cytology led to a more targeted and cost-effective use of FBEPC. Fecal cytology as an initial step in preventative and diagnostic testing protocols is recommended.

COMPARISON BETWEEN STANDARD ZOO QUARANTINE PRACTICES AND RISK-BASED MANAGEMENT OF ANIMAL TRANSFERS: A RETROSPECTIVE ANALYSIS OF AVIAN ACQUISITION MORBIDITY AND MORTALITY (2013-2018).

Beginning 01 January 2016, for all animals transferred into the collection, Disney's Animal Kingdom used a risk-based process that relies on pathogen hazard identification and then assessment and mitigation of the risk for each identified pathogen hazard. Data on morbidity, mortality, and disease introduction was compared for birds transferred in pre- and post-risk-based processes used during the period 2013-2018. When assessing morbidity within 3 mo of acquisition, logistic regression revealed that birds entering "straight to collection" had lower morbidity rates than those entering with either "standard quarantine" or "risk-based standard quarantine" methods (P = 0.032). Mortality within 3 mo of acquisition was not significantly different between straight-to-collection and standard quarantine or risk-based standard quarantine methods (P = 0.40). In addition, no transmissible pathogens of concern were introduced with acquired birds using either method. Implementation of a risk-based approach to animal transfers between zoos and aquariums may be an alternative method that does not pose a greater risk to animal morbidity or mortality while still protecting the collection from disease introduction.

Koala retrovirus diversity, transmissibility, and disease associations.

BACKGROUND: Koalas are infected with the koala retrovirus (KoRV) that exists as exogenous or endogenous viruses. KoRV is genetically diverse with co-infection with up to ten envelope subtypes (A-J) possible; KoRV-A is the prototype endogenous form. KoRV-B, first found in a small number of koalas with an increased leukemia prevalence at one US zoo, has been associated with other cancers and increased chlamydial disease. To better understand the molecular epidemiology of KoRV variants and the effect of increased viral loads (VLs) on transmissibility and pathogenicity we developed subtype-specific quantitative PCR (qPCR) assays and tested blood and tissue samples from koalas at US zoos (n = 78), two Australian zoos (n = 27) and wild-caught (n = 21) in Australia. We analyzed PCR results with available clinical, demographic, and pedigree data. RESULTS: All koalas were KoRV-A-infected. A small number of koalas (10.3%) at one US zoo were also infected with non-A subtypes, while a higher non-A subtype prevalence (59.3%) was found in koalas at Australian zoos. Wild koalas from one location were only infected with KoRV-A. We observed a significant association of infection and plasma VLs of non-A subtypes in koalas that died of leukemia/lymphoma and other neoplasias and report cancer diagnoses in KoRV-A-positive animals. Infection and VLs of non-A subtypes was not associated with age or sex. Transmission of non-A subtypes occurred from dam-to-offspring and likely following adult-to-adult contact, but associations with contact type were not evaluated. Brief antiretroviral treatment of one leukemic koala infected with high plasma levels of KoRV-A, -B, and -F did not affect VL or disease progression. CONCLUSIONS: Our results show a significant association of non-A KoRV infection and plasma VLs with leukemia and other cancers. Although we confirm dam-to-offspring transmission of these variants, we also show other routes are possible. Our validated qPCR assays will be useful to further understand KoRV epidemiology and its zoonotic transmission potential for humans exposed to koalas because KoRV can infect human cells.

STRECK CELL PRESERVATIVE STABILIZES KOALA ( PHASCOLARCTOS CINEREUS) WHOLE BLOOD FOR COMPLETE BLOOD COUNTS.

Wildlife health assessments at remote sites may lead to delayed testing of whole blood for complete blood counts (CBC) resulting in artifacts affecting clinical interpretation. Streck Cell Preservative (SCP) is a proprietary liquid stabilization reagent designed to preserve human leukocytes and may be applicable to wildlife health assessments when immediate processing of blood is not possible. The purpose of this study was to determine if SCP adequately preserved EDTA-anticoagulated whole blood from koalas ( Phascolarctos cinereus) for up to 14 days. Blood from 12 captive adult koalas was collected and combined with SCP in a 1 : 1 ratio and refrigerated. Paired samples of SCP treated and untreated blood had CBCs performed at five time-points over 14 days. Streck Cell Preservative extended koala EDTA-anticoagulated whole blood viability to 14 days by decreasing cellular lysis. Species- and method-specific reference intervals for SCP should be generated to avoid clinical misinterpretation, especially when evaluating anemia.

RISK ANALYSIS: CHANGING THE QUARANTINE PARADIGM?

As modern zoos evolved from menageries, quarantine practices were developed to protect animal collections from the introduction of diseases that might arrive with new animals from wild populations and, more recently, from other managed populations. As the understanding of, and ability to diagnose, diseases grew tremendously in recent decades, quarantine protocols became more stringent and standardized as the tolerance for disease introduction decreased. These practices resulted in healthier animal collections, and the introduction of transmissible pathogens of concern became rarer. Sustainable population management in zoos has further lessened the reliance on taking animals from the wild, and the knowledge of disease risk in such situations has deepened. Today animals being transferred between modern zoological institutions have well-documented medical histories complemented with robust collection health histories. This improved understanding has reduced concerns about disease transmission in many cases. Consequently, more formal use of risk analysis to drive transfer-related examination, diagnostic testing, and quarantine decision making was adopted by a number of zoos and aquariums that have modified their quarantine practices and use risk analysis to reduce quarantine requirements, in some cases moving animals directly into collections after transportation. This commentary discusses the rationale for and changes to quarantine processes for animal acquisitions in zoos and aquariums, using risk analysis to direct decision making.

Resolution of a Localized Granuloma Caused by Mycobacterium avium-intracellulare Complex on the Cere of a Bruce's Green Pigeon (Treron waalia).

A 3-year-old female Bruce's green pigeon (Treron waalia) was presented with granulomatous inflammation of the cere and underlying tissues with osteomyelitis and bone proliferation of the dorsal premaxilla. Biopsy and culture revealed the presence of Mycobacterium avium-intracellulare complex, and multi-antimicrobial treatment was initiated with clarithromycin, ethambutol, rifabutin, and enrofloxacin. The cere lesion improved and no evidence of systemic granulomas was observed over 4 months of treatment, although leukocytosis and monocytosis persisted. Five months after discontinuation of antibiotic therapy, the white blood cell count had normalized, but distal beak irregularities and partial recurrence of the mass were present. The bird died 15 months after discontinuation of antibiotic therapy and necropsy revealed no evidence of active mycobacteriosis of the beak or cere. This report documents an unusual clinical presentation of mycobacteriosis, in addition to its successful resolution.

KOALA RETROVIRUS: A REVIEW.

Koala retrovirus (KoRV) is a gammaretrovirus that has been identified in both captive and free-ranging koalas ( Phascolarctos cinereus ) with variable geographic distribution in Australia. KoRV is capable of both exogenous and endogenous transmission, which provides an interesting research platform for scientists to study active retrovirus endogenization into a host genome and offers veterinary scientists an opportunity to examine the clinical consequences of KoRV infection in koalas. Causation between KoRV and frequently recognized clinical conditions associated with immune suppression and neoplasia in koalas has not been definitively established, however research continues to evaluate a potential association. Three KoRV variants, KoRV-A, KoRV-B, and KoRV-J, have been the most thoroughly described and preliminary evidence suggests KoRV variability may be fundamental in host pathogenicity. In addition to reviewing what is currently known about KoRV, this article discusses treatment, management, and future research directions.

LESSONS FROM A RETROSPECTIVE ANALYSIS OF A 5-YR PERIOD OF PRESHIPMENT TESTING AT SAN DIEGO ZOO: A RISK-BASED APPROACH TO PRESHIPMENT TESTING MAY BENEFIT ANIMAL WELFARE.

The preshipment examination, with associated transmissible disease testing, has become standard practice in the movement of animals between zoos. An alternative disease risk-based approach, based on a comprehensive surveillance program including necropsy and preventive medicine examination testing and data, has been in practice since 2006 between the San Diego Zoo and San Diego Zoo Safari Park. A retrospective analysis, evaluating comprehensive necropsy data and preshipment testing over a 5-yr study period, was performed to determine the viability of this model for use with sending animals to other institutions. Animals (607 birds, 704 reptiles and amphibians, and 341 mammals) were shipped to 116 Association of Zoos and Aquariums (AZA)-accredited and 29 non-AZA-accredited institutions. The evaluation showed no evidence of the specific transmissible diseases tested for during the preshipment exam being present within the San Diego Zoo collection. We suggest that a risk-based animal and institution-specific approach to transmissible disease preshipment testing is more cost effective and is in the better interest of animal welfare than the current industry standard of dogmatic preshipment testing.

LESSONS FROM A RETROSPECTIVE ANALYSIS OF A 5-YR PERIOD OF QUARANTINE AT SAN DIEGO ZOO: A RISK-BASED APPROACH TO QUARANTINE ISOLATION AND TESTING MAY BENEFIT ANIMAL WELFARE.

Quarantine is designed primarily to prevent the introduction of transmissible diseases to zoological collections. Improvements in preventive medicine, disease eradication, and comprehensive pathology programs call into question current industry quarantine standards. Disease risk analysis was used at the San Diego Zoo (SDZ) and the SDZ Safari Park to eliminate quarantine isolation and transmissible disease testing for animals transferred between the two institutions. To determine if a risk-based approach might be valid between other institutions and SDZ, we reviewed quarantine data for animals arriving at SDZ from 81 Association of Zoos and Aquariums (AZA)-accredited and 124 other sources (e.g., non-AZA-accredited institutions, private breeders, private dealers, governmental bodies) over a 5-yr period (2009-2013). No mammal or herptile failed quarantine due to transmissible diseases of concern. Approximately 2.5% of incoming birds failed quarantine due to transmissible disease; however, all 14 failed individuals were obtained from three nonaccredited sources (private breeders, confiscation). The results of our study suggest that a risk-based approach could be used to minimize or eliminate quarantine for the transfer of animals from institutions with comprehensive disease surveillance programs and/or preshipment testing practices. Quarantine isolation with testing remains an essential defense against introducing transmissible diseases of concern when there is a lack of health knowledge about the animals being received.

Esophageal dissection and hematoma associated with obstruction in an Indian elephant (Elephas maximus indicus).

A 42-year-old female Indian elephant (Elephas maximus indicus) developed a sudden onset of excessive salivation and dysphagia. Esophageal obstruction was suspected; possibly related to palm frond ingestion. Esophageal endoscopy revealed a mat of plant material in the distal esophagus. An initial attempt at relieving the obstruction was unsuccessful, but subsequent use of custom-made instruments along with insufflation and hydropulsion enabled partial removal of the material. Postimmobilization care included aggressive intravenous and rectal fluids, anti-inflammatory and antibiotic administration, and fasting. Despite treatment, the dysphagia persisted and the elephant was euthanized due to lack of improvement and grave prognosis. Postmortem examination revealed remaining plant material in the esophagus, complicated by an esophageal dissection, mural hematoma, and secondary bacterial infection. Iatrogenic trauma may have contributed to the extent of esophageal injury. Although treatment was ultimately unsuccessful, the supportive care employed could potentially aid recovery in cases of less severe esophageal trauma.

Dental onlay bridge-like prosthesis in three koalas (Phascolarctos cinereus): the use of Premise trimodal composite to prevent interproximal Eucalyptus spp. retention and infection.

Three koalas (Phascolarctos cinereus) presented with moderate to severe interproximal space accumulation with Eucalyptus spp. at the San Diego Zoo. Premise trimodal composite was used to create dental onlay bridge-like prostheses to eliminate open, enlarged interproximal spaces. The prostheses prevented further leaf material accumulation within the interproximal spaces and consequently reduced periodontal disease. Aesthetically, the prosthesis replicates normal tooth coloration and appearance and wears at a similar rate to surrounding teeth. Prosthetic repair or replacement may be required, so periodic examination every 3-6 mo is recommended.

Gastrointestinal torsions and intussusception in northern koalas (Phascolarctos cinereus) at San Diego Zoo (1976-2012).

The recent classification as threatened status of the northern koala (Phascolarctos cinereus) by the Australian Government highlights the importance of the conservation and health management of this iconic Australian marsupial. This case series describes gastrointestinal torsion and intussusception in six northern koalas (three males, three females, 2-11 yr old) at the San Diego Zoo from 1976 to 2012. Two koalas died shortly after presentation. Diagnoses of ileocecal intussusception, resulting from enteritis in one case and cecal torsion in the other, were made at postmortem examination. One koala died 4 days after an exploratory laparotomy, with negative findings, and an acute double colonic intussusception was diagnosed at postmortem examination. Two small intestinal mesenteric torsion and one proximal colon mesenteric torsion cases were successfully corrected surgically. In the case of colonic mesenteric torsion, the koala had recurrent clinical signs 2 wk later, and a second surgery requiring resection and anastomosis of ischemic jejunum was performed, with the koala dying shortly afterward. One koala with small intestinal torsion had a recurrence of torsion 22 mo later and subsequently died. The koala with the second case of small intestinal torsion remains alive 14 mo postsurgical correction. All six koalas presented with signs of colic that included anorexia, lethargy, depression, acute abdominal distension, abdominal stretching, decreased fecal output, open-mouth gasping, or a combination of symptoms. Abdominal radiographs may show stacked gastrointestinal linear gas patterns and contrast stasis. Prevalence of torsion and intussusception is low at this institution (2%), although recurrence in individuals is common (50%) and overall survival is poor (83%), which emphasizes the importance of timely recognition, surgical correction, and postoperative management. While inciting etiologies were unable to be determined in these cases, monitoring generalized gastrointestinal health and differing Eucalyptus sp. effects on individual koala's gastrointestinal function, parasite control, and stress minimization through standardized husbandry practices are likely important.

Computed tomography and magnetic resonance for the advanced imaging of the normal nasal cavity and paranasal sinuses of the koala (Phascolarctos cinereus).

The objective of this study is to describe computed tomography (CT) and magnetic resonance (MR) for the cross-sectional imaging of the normal anatomy of the nasal cavity and paranasal sinuses of the koala (Phascolarctos cinereus), to provide reference figures for gross anatomy with corresponding CT and MR images and to compare the features of the nasal cavity and paranasal sinuses of the normal koala with that reported in other domestic species. Advanced imaging can be used to aid in diagnosis, to plan surgical intervention, and to monitor therapeutic responses to diseases of the nasal passages in koalas. One clinically normal koala was anesthetized twice for the separate acquisition of dorsal CT scan images and transverse, dorsal, and sagittal MR images of its nasal cavity and paranasal sinuses. Sagittal and transverse CT planes were reformatted. Three fresh koala skulls were also transected in one of each transverse, sagittal, and dorsal planes and photographed. The CT and MR images obtained were matched with corresponding gross anatomic images and the normal bone, tissues and airway passages were identified. All anatomic structures were readily identifiable on CT, magnetic resonance imaging (MRI), and gross images. CT and MRI are both valuable diagnostic tools for imaging the nasal cavities and paranasal sinuses of koalas. Images obtained from this project can be used as baseline references for future comparison with diseased koalas to help with diagnosis, surgical intervention, and response to therapy.

Metabolic bone disease in juvenile koalas (Phascolartcos cinereus).

Due to climate restrictions in parts of North America and Europe, koalas (Phascolarctos cinereus) are housed indoors. Koala young (joeys) raised indoors are susceptible to the development of metabolic bone disease (MBD) due to a lack of exposure to natural ultraviolet light to themselves and their female parents (dams). In this retrospective study, radiographs from 27 koala joeys born at four zoos in North America and two zoos in Europe were evaluated for signs of MBD. Eight of the joeys were radiographically diagnosed with MBD and four additional joeys were considered suspect MBD cases; in two joeys absence or presence of MBD could not be determined. All joeys had mild to severe hip and shoulder dysplasia. There were significant associations between a lack of exposure to UV light and MBD development and between MBD and the degree of severity of hip and shoulder dysplasia. It is recommended to house breeding female koalas and their joeys outdoors whenever possible.

Serum vitamin D levels in free-ranging koalas (Phascolarctos cinereus).

Due to climatic conditions in Northern America and Europe, koalas (Phascolarctos cinereus) are often housed indoors. Koala joeys raised in these environments are susceptible to the development of metabolic bone disease due to a lack of exposure to solar ultraviolet radiation to themselves and their dam. As an initial step toward describing vitamin D sufficiency and adequately measuring responses to supplementation, vitamin D values were calculated by using serum collected from 20 free-ranging koalas on St. Bees Island, Queensland, Australia. Vitamin D values ranged from 8.1 to 30.4 pg/ml (18.4 +/- 5.5 pg/ml) for 1, 25-hydroxyvitamin D, and from 1 to 14 nM/L (7.4 +/- 3.0 nM/L) for 25-hydroxyvitamin D. These koala serum vitamin D values are unusually low when compared with eutherian mammals. Although this study was limited in numbers and in the geographically range of the koalas sampled, it does suggest that the koala's requirement for vitamin D is low. Therefore, supplementation to prevent disease may be relatively easy to achieve because low doses will likely meet requirements. Caution should be taken to avoid intoxication if supplementing vitamin D in koalas.

An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo.

Leukemia and lymphoma account for more than 60% of deaths in captive koalas (Phascolarctos cinereus) in northeastern Australia. Although the endogenizing gammaretrovirus koala endogenous retrovirus (KoRV) was isolated from these koalas, KoRV has not been definitively associated with leukemogenesis. We performed KoRV screening in koalas from the San Diego Zoo, maintained for more than 45 y with very limited outbreeding, and the Los Angeles Zoo, maintained by continuously assimilating captive-born Australian koalas. San Diego Zoo koalas are currently free of malignant neoplasias and were infected with only endogenous KoRV, which we now term subtype "KoRV-A," whereas Los Angeles Zoo koalas with lymphomas/leukemias are infected in addition to KoRV-A by a unique KoRV we term subtype "KoRV-B." KoRV-B is most divergent in the envelope protein and uses a host receptor distinct from KoRV-A. KoRV-B also has duplicated enhancer regions in the LTR associated with increased pathology in gammaretroviruses. Whereas KoRV-A uses the sodium-dependent phosphate transporter 1 (PiT1) as a receptor, KoRV-B employs a different receptor, the thiamine transporter 1 (THTR1), to infect cells. KoRV-B is transmitted from dam to offspring through de novo infection, rather than via genetic inheritance like KoRV-A. Detection of KoRV-B in native Australian koalas should provide a history, and a mode for remediation, of leukemia/lymphoma currently endemic in this population.

Fat-soluble vitamin and mineral comparisons between zoo-based and free-ranging koalas (Phascolarctos cinereus).

As part of a health investigation on koalas at San Diego Zoo, serum samples were analyzed from 18 free-ranging and 22 zoo-based koalas, Phascolarctos cinereus. Serum concentrations of calcium, chloride, cobalt, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium, zinc, and vitamins A, E, and 25(OH)D3 were quantified. Calcium, chloride, molybdenum, selenium, and vitamin E concentrations were significantly higher in zoo-based koalas than in free-ranging koalas, whereas magnesium, manganese, phosphorus, and zinc concentrations were significantly higher in the free-ranging koalas. No significant differences were found between genders. The results from this study will help to establish a starting point for determining target circulating nutrient concentrations in koalas.

A retrospective and prospective study of megaesophagus in the parma wallaby (Macropus parma) at the San Diego Zoo, California, USA.

At the San Diego Zoo (California, USA), 22 cases of megaesophagus were diagnosed in the parma wallaby (Macropus parma), yielding a prevalence of 21.1%. Parma wallabies often have no clinical signs until severe and chronic dilation of the esophagus is present. Clinical signs of advanced disease include weight loss, swelling of the cervical region, regurgitation without reswallowing of ingesta, short flight distance, depression, collapse, dyspnea, and sudden death. Retrospective and prospective studies at the San Diego Zoo and a multi-institutional survey in the United States were used to try to determine the cause of megaesophagus. The retrospective study did not identify an etiology. The prospective study revealed megaesophagus and severely delayed esophageal transit time in eight of eight animals. Myasthenia gravis, lead toxicosis, toxoplasmosis, and thyroid disease were eliminated as possible causes. Of 286 living and dead parma wallabies surveyed at other institutions, three cases of esophageal diverticulum and one case of megaesophagus were reported. The cause of megaesophagus in parma wallabies was not determined.

A retrospective and prospective study of megaesophagus in the parma wallaby (Macropus parma) at the San Diego Zoo, California, USA.

At the San Diego Zoo (California, USA), 22 cases of megaesophagus were diagnosed in the parma wallaby (Macropus parma); a prevalence of 21.1%. Parma wallabies often have no clinical signs until severe and chronic dilation of the esophagus is present. Clinical signs of advanced disease include weight loss, swelling of the cervical region, regurgitation without reswallowing of ingesta, short flight distance, depression, collapse, dyspnea, and sudden death. Retrospective and prospective studies at the San Diego Zoo and a multi-institutional survey in the United States were used to try to determine the cause of megaesophagus. The retrospective study did not identify an etiology. The prospective study revealed megaesophagus and severely delayed esophageal transit time in eight of eight animals. Myasthenia gravis, lead toxicosis, toxoplasmosis, and thyroid disease were eliminated as possible causes. Of 286 living and dead parma wallabies surveyed at other institutions, three cases of esophageal diverticulum and one case of megaesophagus were reported. The cause of megaesophagus in parma wallabies was not determined.