Nodular and neoplastic skin lesions in an aquarium-managed group of Sabana Surinam toads Pipa parva.

Although skin disease is a common cause of morbidity and mortality in amphibians, published reports of integumentary conditions affecting skin-brooding anurans are extremely limited. This case series describes the clinical, macroscopic, and histopathologic features of nodular skin lesions in an aquarium-managed population of Sabana Surinam toads Pipa parva, a fully aquatic, skin-brooding species native to South America. The skin lesions represented an ongoing clinical concern in this group, affecting approximately 10-20% of animals throughout the study period, and were observed exclusively in females, suggesting an association with the females' specialized cutaneous reproductive anatomy. Multiple animals died or were euthanized due to skin lesions, which were histologically complex and encompassed a range of hyperplastic, cystic, and neoplastic changes (with internal metastases in one animal). Cultures and special stains showed evidence of mixed polymicrobial infection, including occasional fungal hyphae and acid-fast organisms, but were predominated by Gram-negative bacteria. Lack of a significant response to various environmental modulations and therapeutic interventions indicates that the pathogenesis of the skin lesions is multifactorial. Additional research into the reproductive physiology and ideal environmental conditions (both social and physical) for this species will likely help identify new strategies for prevention and treatment of skin disease.

A RETROSPECTIVE SURVEY OF NEOPLASIA IN MANAGED GIRAFFES (GIRAFFA CAMELOPARDALIS).

Giraffes (Giraffa camelopardalis) are commonly managed in zoos and conservation programs worldwide, but the current understanding of the occurrence and progression of neoplastic disease in this species is limited by the scarcity of published reports. This study collated documented cases of neoplasia on the basis of gross and histologic evaluation of ante- and postmortem samples. In total, 30 giraffes from 22 institutions across the United States were included. Subspecies was not reported in all cases, but those identified included Masai (Giraffa camelopardalis tippelskirchi), Rothschild (Giraffa camelopardalis rothschildi), and reticulated subspecies (Giraffe camelopardalis reticulata). Thirteen animals died natural deaths, 15 were euthanized, and 2 were alive at the time of this article. A total of 38 tumors were reported and classified as 18 different diagnoses, including leiomyoma (7), adenoma (4), luteoma (4), lymphoma (4), pheochromocytoma (3), squamous cell carcinoma (3), adenocarcinoma (2), ameloblastic fibroma (1), carcinomatosis of undetermined cell lineage (1), cavernous hemangioma (1), cystic granulosa cell tumor (1), dysgerminoma (1), fibrosarcoma (1), leukemia (1), lipoma (1), pituitary nerve sheath tumor (1), rhabdomyosarcoma (1), and teratoma (1). Multiple concurrent neoplastic lesions were documented in six cases. Mesenchymal tumors (18) were the majority of neoplasms. The most prevalent location, regardless of tumor type, was the female reproductive tract (14). Twenty-four neoplastic lesions were incidental findings at necropsy, whereas eight neoplasms were considered to be the primary cause of death. The findings reported here identify multiple neoplastic lesions in giraffes and could provide insight to the future management of this species.

SPINAL NEMATODIASIS IN A LINED FLAT-TAIL GECKO (UROPLATUS LINEATUS).

A 5-y-old female lined flat-tail gecko (Uroplatus lineatus) presented for acute onset of lethargy and paraplegia and was subsequently euthanized. Histologic examination of the spinal cord revealed a verminous myelitis comprising moderate, multifocal, necrotizing myelitis with intramedullary adult and larval nematodes. Molecular data and morphology indicate a cosmocercid nematode, most likely of the genus Raillietnema, a diverse taxon reported to parasitize reptiles, amphibians, and teleost fish. To the authors' knowledge this is the first report of spinal nematodiasis in a reptile species, and the first report of spinal parasitism causing hind-limb paraplegia in a reptile.

Infectious Virus Persists in CD4+ T Cells and Macrophages in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques.

Understanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T cells in compartments other than blood and lymph nodes is unclear. Macrophages (Mϕ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T cell and Mϕ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected Mϕs. Surprisingly, the numbers of CD4+ T cells, monocytes, and Mϕs carrying infectious genomes in blood and spleen were at comparable frequencies (∼1 infected cell per million). We also demonstrate that ex vivo viruses produced in the Mϕ QVOA are capable of infecting activated CD4+ T cells. These results strongly suggest that latently infected tissue Mϕs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+ T cell and Mϕ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Mϕs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Mϕs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies.IMPORTANCE This study suggests that CD4+ T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.

Intraepidermal Nerve Fiber Analysis in Human Patients and Animal Models of Peripheral Neuropathy: A Comparative Review.

Analysis of intraepidermal nerve fibers (IENFs) in skin biopsy samples has become a standard clinical tool for diagnosing peripheral neuropathies in human patients. Compared to sural nerve biopsy, skin biopsy is safer, less invasive, and can be performed repeatedly to facilitate longitudinal assessment. Intraepidermal nerve fiber analysis is also more sensitive than conventional nerve histology or electrophysiological tests for detecting damage to small-diameter sensory nerve fibers. The techniques used for IENF analysis in humans have been adapted for large and small animal models and successfully used in studies of diabetic neuropathy, chemotherapy-induced peripheral neuropathy, HIV-associated sensory neuropathy, among others. Although IENF analysis has yet to become a routine end point in nonclinical safety testing, it has the potential to serve as a highly relevant indicator of sensory nerve fiber status in neurotoxicity studies, as well as development of neuroprotective and neuroregenerative therapies. Recently, there is also interest in the evaluation of IENF via skin biopsy as a biomarker of small fiber neuropathy in the regulatory setting. This article provides an overview of the anatomic and pathophysiologic principles behind IENF analysis, its use as a diagnostic tool in humans, and applications in animal models with focus on comparative methodology and considerations for study design.

Lymphocyte-Dominant Encephalitis and Meningitis in Simian Immunodeficiency Virus-Infected Macaques Receiving Antiretroviral Therapy.

A retrospective neuropathologic review of 30 SIV-infected pigtailed macaques receiving combination antiretroviral therapy (cART) was conducted. Seventeen animals with lymphocyte-dominant inflammation in the brain and/or meninges that clearly was morphologically distinct from prototypic SIV encephalitis and human immunodeficiency virus encephalitis were identified. Central nervous system (CNS) infiltrates in cART-treated macaques primarily comprised CD20+ B cells and CD3+ T cells with fewer CD68+ macrophages. Inflammation was associated with low levels of SIV RNA in the brain as shown by in situ hybridization, and generally was observed in animals with episodes of cerebrospinal fluid (CSF) viral rebound or sustained plasma and CSF viremia during treatment. Although the lymphocytic CNS inflammation in these macaques shared morphologic characteristics with uncommon immune-mediated neurologic disorders reported in treated HIV patients, including CNS immune reconstitution inflammatory syndrome and neurosymptomatic CSF escape, the high prevalence of CNS lesions in macaques suggests that persistent adaptive immune responses in the CNS also may develop in neuroasymptomatic or mildly impaired HIV patients yet remain unrecognized given the lack of access to CNS tissue for histopathologic evaluation. Continued investigation into the mechanisms and outcomes of CNS inflammation in cART-treated, SIV-infected macaques will advance our understanding of the consequences of residual CNS HIV replication in patients on cART, including the possible contribution of adaptive immune responses to HIV-associated neurocognitive disorders.

Histologic Findings in Captive American Horseshoe Crabs (Limulus polyphemus).

Histopathology of 61 captive American horseshoe crabs (HSCs; Limulus polyphemus) is reviewed. HSC organs evaluated histologically included body wall (chitin, epidermis, dermis, and skeletal muscle), hepatopancreas, gut, gonads, book gills, eyes, heart, brain, and coxal gland. In descending order, lesions were most frequently identified in compound eye, body wall, book gills, hepatopancreas, chitinous gut, nonchitinous gut, heart, and brain; lesions were not observed in coxal gland or gonads. Hemocytes (also called amoebocytes) surrounded infectious agents and occluded ulcers. Large hemocyte aggregates had a central eosinophilic coagulum (ie, hemocyte coagulum). Cutaneous ulceration (34/60 cases), branchitis (29/48 cases), and ophthalmitis (17/20 cases) were common lesions and consistently associated with fungi, which were invasive into subjacent tissues, and/or bacteria, which were usually superficial. Fungal culture was performed in 3 cases and isolated Fusarium spp., although fungal morphology varied and multiple fungal species may have been present. Presumptive green algae were associated with ulceration in 1 case with minimal to no inflammation. Presumptive cyanobacteria were identified within a biofilm overlying the gills in 4 of 48 cases and were not invasive. Multifocal, random hepatopancreatitis was identified in 16 of 57 cases, 10 of which were associated with bacteria. Metacercarial cysts were identified in 25 of 61 cases and associated with minimal to no inflammation. Depleted eosinophilic globules in hepatopancreatic interstitial cells were interpreted as decreased nutritional status in 12 of 57 cases.

FATAL RANAVIRUS INFECTION IN A GROUP OF ZOO-HOUSED MELLER'S CHAMELEONS (TRIOCEROS MELLERI).

A group of five juvenile Meller's chameleons (Trioceros melleri) experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle (Terrapene carolina carolina) group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.

Characterization of an Unusual Mesenchymal Tumor in the Proventriculus of a Free-ranging Red-tailed Hawk (Buteo jamaicensis).

An adult, female red-tailed hawk (Buteo jamaicensis) was presented for examination after being found unable to fly on a private citizen's property. Further diagnostic testing revealed a vascular, soft tissue mass arising from the serosal surface of the proximal proventriculus. Postmortem histological and immunohistochemical examination of the mass revealed an unusual mesenchymal tumor with features consistent with a leiomyoma or low-grade gastrointestinal stromal tumor.

An SIV/macaque model targeted to study HIV-associated neurocognitive disorders.

Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.

CAUSES OF MORTALITY IN CAPTIVE PANAMANIAN GOLDEN FROGS ( ATELOPUS ZETEKI) AT THE MARYLAND ZOO IN BALTIMORE, 2001-2013.

The Maryland Zoo in Baltimore is home to the largest captive assurance population of the critically endangered Panamanian golden frog ( Atelopus zeteki). With the ongoing extinction that is occurring worldwide in amphibians, the need for amphibian captive assurance populations is growing, and few mortality reviews on amphibian species exist. Necropsy and histopathologic examination of animals that die in captivity can help identify population-level disease problems, direct research needs in amphibian medicine and husbandry, and improve the success of captive breeding programs. This study reviews postmortem findings from 406 frogs, greater than 1 yr of age, which died in this population from 2001 to 2013. Frogs were categorized by age and sex, and the cause of mortality was determined. Dermatitis associated with filamentous-type fungal organisms was the most common cause of mortality in both age and sex categories and accounted for one-third of frog deaths in this study (36.0%; n = 146 out of 406 frogs). Other major causes of mortality included renal disease, gastrointestinal disease, septicemia, and a previously undescribed myopathy condition associated with a tetany syndrome. Increased mortality of frogs occurred during the breeding season, highlighting the need for further research into methods to minimize mortality during this time.

CLINICAL EFFECT OF HEMOPARASITE INFECTIONS IN SNOWY OWLS ( BUBO SCANDIACUS).

Vector-borne hemoparasites are commonly found in avian species. Plasmodium spp., the causative agent of avian malaria, are intraerythrocytic parasites that can cause signs ranging from subclinical infection to severe acute disease. In raptor species, most hemoparasites are associated with subclinical infection and are generally not treated when seen on blood evaluation. This case series reviews five cases of hemoparasite infection in snowy owls ( Bubo scandiacus). These animals were infected with a variety of hemoparasites, including Plasmodium, Haemoproteus, and Leukocytozoon spp. Death of one of these birds due to hemoparasite burden led to a change in the monitoring for and treatment of subclinical hemoparasitic infections in this species. Three subsequently infected snowy owls have been treated with primaquine and chloroquine. The birds that were treated survived infection, and parasite burdens in peripheral blood diminished. Postulated reasons for increased morbidity and mortality associated with hemoparasitic infections in captive snowy owls, as opposed to other raptor species, include stress, concurrent disease, novel pathogen exposure, and elevated environmental temperatures.

PIGMENTED VILLONODULAR SYNOVITIS IN A RETICULATED GIRAFFE (GIRAFFA CAMELOPARDALIS).

: A 17-yr-old, female, captive-born reticulated giraffe ( Giraffa camelopardalis ) presented with acute-onset lameness of the right metacarpophalangeal (fetlock) joint. Despite multiple courses of treatment, the lameness and swelling progressively worsened over a 3.5-yr period, and the giraffe was euthanized. At necropsy, gross and microscopic changes in the right, front fetlock and associated flexor tendon sheath included villous synovial hyperplasia and the formation of discrete pigmented nodules within synovial membranes. Histologically, the nodules were composed of abundant, fibrous connective tissue with heavy macrophage infiltration, hemosiderin deposition, and distinctive, multinucleated cells that resembled osteoclasts. These findings were consistent with pigmented villonodular synovitis (PVNS), a rare condition affecting both humans and animals. Although the pathophysiology of PVNS is poorly understood, lesions exhibit features of both neoplastic and reactive inflammatory processes. This case report represents, to the authors' knowledge, the first description of PVNS in a nondomestic ungulate.

Tracking Epidermal Nerve Fiber Changes in Asian Macaques: Tools and Techniques for Quantitative Assessment.

Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus-associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.

Loss of corneal sensory nerve fibers in SIV-infected macaques: an alternate approach to investigate HIV-induced PNS damage.

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker ßIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings.

A Switch from Glial to Neuronal Gene Expression Alterations in the Spinal Cord of SIV-infected Macaques on Antiretroviral Therapy.

Despite antiretroviral therapy (ART), HIV-associated peripheral neuropathy remains one of the most prevalent neurologic manifestations of HIV infection. The spinal cord is an essential component of sensory pathways, but spinal cord sampling and evaluation in people with HIV has been very limited, especially in those on ART. The SIV/macaque model allows for assessment of the spinal cord at key time points throughout infection with and without ART. In this study, RNA was isolated from the spinal cord of uninfected, SIV+, and SIV + ART animals to track alterations in gene expression using global RNA-seq. Next, the SeqSeek platform was used to map changes in gene expression to specific cell types. Pathway analysis of differentially expressed genes demonstrated that highly upregulated genes in SIV-infected spinal cord aligned with interferon and viral response pathways. Additionally, this upregulated gene set significantly overlapped with those expressed in myeloid-derived cells including microglia. Downregulated genes were involved in cholesterol and collagen biosynthesis, and TGF-b regulation of extracellular matrix. In contrast, enriched pathways identified in SIV + ART animals included neurotransmitter receptors and post synaptic signaling regulators, and transmission across chemical synapses. SeqSeek analysis showed that upregulated genes were primarily expressed by neurons rather than glia. These findings indicate that pathways activated in the spinal cord of SIV + ART macaques are predominantly involved in neuronal signaling rather than proinflammatory pathways. This study provides the basis for further evaluation of mechanisms of SIV infection + ART within the spinal cord with a focus on therapeutic interventions to maintain synaptodendritic homeostasis.

Proliferative thyroid lesions in three diplodactylid geckos: Nephrurus amyae, Nephrurus levis, and Oedura marmorata.

Over a 5-mo period, three diplodactylid geckos housed at the National Aquarium were diagnosed with proliferative thyroid lesions: a rough knob-tail gecko (Nephrurus amyae), a smooth knob-tail gecko (Nephrurus levis), and a marbled velvet gecko (Oedura marmorata). Clinical signs included an intraoral mass or ventral throat swelling (or both), oral bleeding, and weight loss. Both of the knob-tail geckos died. The histologic diagnosis for the rough knob-tail gecko was thyroid carcinoma with metastases to the liver and lungs, and thyroid carcinoma with no metastases was reported in the smooth knob-tail gecko. A thyroidectomy was performed on the marbled velvet gecko with a histologic diagnosis of adenomatous hyperplasia. Postoperative weight loss and bradycardia resolved following oral supplementation with levothyroxine. The animal is in normal health 10 mo post-surgery. Five other diplodactylid geckos in the collection remain unaffected, giving a 38% prevalence of proliferative thyroid lesions (3/8). The etiology remains undetermined. This is the first report of a cluster of proliferative thyroid lesions in geckos.

Research-Relevant Background Lesions and Conditions in Common Avian and Aquatic Species.

Non-mammalian vertebrates including birds, fish, and amphibians have a long history of contributing to ground-breaking scientific discoveries. Because these species offer several experimental advantages over higher vertebrates and share extensive anatomic and genetic homology with their mammalian counterparts, they remain popular animal models in a variety of fields such as developmental biology, physiology, toxicology, drug discovery, immunology, toxicology, and infectious disease. As with all animal models, familiarity with the anatomy, physiology, and spontaneous diseases of these species is necessary for ensuring animal welfare, as well as accurate interpretation and reporting of study findings. Working with avian and aquatic species can be especially challenging in this respect due to their rich diversity and array of unique adaptations. Here, we provide an overview of the research-relevant anatomic features, non-infectious conditions, and infectious diseases that impact research colonies of birds and aquatic animals, including fish and Xenopus species.

Amphibian Renal Disease.

Amphibians are a remarkably diverse group of vertebrates with lifestyles ranging from fully aquatic to entirely terrestrial. Although some aspects of renal anatomy and physiology are similar among all amphibians, species differences in nitrogenous waste production and broad normal variation in plasma osmolality and composition make definitive antemortem diagnosis of renal disease challenging. Treatment is often empirical and aimed at addressing possible underlying infection, reducing abnormal fluid accumulation, and optimizing husbandry practices to support metabolic and fluid homeostasis. This article reviews amphibian renal anatomy and physiology, provides recommendations for diagnostic and therapeutic options, and discusses etiologies of renal disease.