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.

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.

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.

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.

Brain macrophages harbor latent, infectious simian immunodeficiency virus.

: The current review examines the role of brain macrophages, that is perivascular macrophages and microglia, as a potential viral reservoir in antiretroviral therapy (ART) treated, simian immunodeficiency virus (SIV)-infected macaques. The role, if any, of latent viral reservoirs of HIV and SIV in the central nervous system during ART suppression is an unresolved issue. HIV and SIV infect both CD4 lymphocytes and myeloid cells in blood and tissues during acute and chronic infection. HIV spread to the brain occurs during acute infection by the infiltration of activated CD4 lymphocytes and monocytes from blood and is established in both embryonically derived resident microglia and monocyte-derived perivascular macrophages. ART controls viral replication in peripheral blood and cerebrospinal fluid in HIV-infected individuals but does not directly eliminate infected cells in blood, tissues or brain. Latently infected resting CD4 lymphocytes in blood and lymphoid tissues are a well recognized viral reservoir that can rebound once ART is withdrawn. In contrast, central nervous system resident microglia and perivascular macrophages in brain have not been examined as potential reservoirs for HIV during suppressive ART. Macrophages in tissues are long-lived cells that are HIV and SIV infected in tissues such as gut, lung, spleen, lymph node and brain and contribute to ongoing inflammation in tissues. However, their potential role in viral persistence and latency or their potential to rebound in the absence ART has not been examined. It has been shown that measurement of HIV latency by HIV DNA PCR in CD4 lymphocytes overestimates the size of the latent reservoirs of HIV that contribute to rebound that is cells containing the genomes of replicative viruses. Thus, the quantitative viral outgrowth assay has been used as a reliable measure of the number of latent cells that harbor infectious viral DNA and, may constitute a functional latent reservoir. Using quantitative viral outgrowth assays specifically designed to quantitate latently infected CD4 lymphocytes and myeloid cells in an SIV macaque model, we demonstrated that macrophages in brain harbor SIV genomes that reactivate and produce infectious virus in this assay, demonstrating that these cells have the potential to be a reservoir.

Myeloid and CD4 T Cells Comprise the Latent Reservoir in Antiretroviral Therapy-Suppressed SIVmac251-Infected Macaques.

Human immunodeficiency virus (HIV) eradication or long-term suppression in the absence of antiretroviral therapy (ART) requires an understanding of all viral reservoirs that could contribute to viral rebound after ART interruption. CD4 T cells (CD4s) are recognized as the predominant reservoir in HIV type 1 (HIV-1)-infected individuals. However, macrophages are also infected by HIV-1 and simian immunodeficiency virus (SIV) during acute infection and may persist throughout ART, contributing to the size of the latent reservoir. We sought to determine whether tissue macrophages contribute to the SIVmac251 reservoir in suppressed macaques. Using cell-specific quantitative viral outgrowth assays (CD4-QVOA and MΦ-QVOA), we measured functional latent reservoirs in CD4s and macrophages in ART-suppressed SIVmac251-infected macaques. Spleen, lung, and brain in all suppressed animals contained latently infected macrophages, undetectable or low-level SIV RNA, and detectable SIV DNA. Silent viral genomes with potential for reactivation and viral spread were also identified in blood monocytes, although these cells might not be considered reservoirs due to their short life span. Additionally, virus produced in the MΦ-QVOA was capable of infecting healthy activated CD4s. Our results strongly suggest that functional latent reservoirs in CD4s and macrophages can contribute to viral rebound and reestablishment of productive infection after ART interruption. These findings should be considered in the design and implementation of future HIV cure strategies.IMPORTANCE This study provides further evidence that the latent reservoir is comprised of both CD4+ T cells and myeloid cells. The data presented here suggest that CD4+ T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies.

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.

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.

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.

Persistent Peripheral Nervous System Damage in Simian Immunodeficiency Virus-Infected Macaques Receiving Antiretroviral Therapy.

Human immunodeficiency virus (HIV)-induced peripheral neuropathy is the most common neurologic complication associated with HIV infection. In addition to virus-mediated injury of the peripheral nervous system (PNS), treatment of HIV infection with combination antiretroviral therapy (cART) may induce toxic neuropathy as a side effect. Antiretroviral toxic neuropathy is clinically indistinguishable from the sensory neuropathy induced by HIV; in some patients, these 2 processes are likely superimposed. To study these intercurrent PNS disease processes, we first established a simian immunodeficiency virus (SIV)/pigtailed macaque model in which more than 90% of animals developed PNS changes closely resembling those seen in HIV-infected individuals with distal sensory neuropathy. To determine whether cART alters the progression of SIV-induced PNS damage, dorsal root ganglia and epidermal nerve fibers were evaluated in SIV-infected macaques after long-term suppressive cART. Although cART effectively suppressed SIV replication and reduced macrophage activation in the dorsal root ganglia, PGP 9.5 immunostaining and measurements of epidermal nerve fibers in the plantar surface of the feet of treated SIV-infected macaques clearly showed that cART did not normalize epidermal nerve fiber density. These findings illustrate that significant PNS damage persists in SIV-infected macaques on suppressive cART.

Neuroinflammation and virus replication in the spinal cord of simian immunodeficiency virus-infected macaques.

Studies of neurologic diseases induced by simian immunodeficiency virus (SIV) in Asian macaques have contributed greatly to the current understanding of human immunodeficiency virus pathogenesis in the brain and peripheral nervous system. Detailed investigations into SIV-induced alterations in the spinal cord, a critical sensorimotor relay point between the brain and the peripheral nervous system, have yet to be reported. In this study, lumbar spinal cords from SIV-infected pigtailed macaques were examined to quantify SIV replication and associated neuroinflammation. In untreated SIV-infected animals, there was a strong correlation between amount of SIV RNA in the spinal cord and expression of the macrophage marker CD68 and the key proinflammatory mediators tumor necrosis factor and CCL2. We also found a significant correlation between SIV-induced alterations in the spinal cord and the degree of distal epidermal nerve fiber loss among untreated animals. Spinal cord changes (including elevated glial fibrillary acidic protein immunostaining and enhanced CCL2 gene expression) also were present in SIV-infected antiretroviral drug-treated animals despite SIV suppression. A fuller understanding of the complex virus and host factor dynamics in the spinal cord during human immunodeficiency virus infection will be critical in the development of new treatments for human immunodeficiency virus-associated sensory neuropathies and studies aimed at eradicating the virus from the central nervous system.

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.