Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts.

IMPORTANCE: Environmental factors like climate change and captive breeding can impact the gut microbiota and host health. Therefore, conservation efforts for threatened species may benefit from understanding how these factors influence animal microbiomes. Parabasalid protists are members of the mammalian microbiota that can modulate the immune system and impact susceptibility to infections. However, little is known about parabasalids in reptiles. Here, we profile reptile-associated parabasalids in wild and captive reptiles and find that captivity has minimal impact on parabasalid prevalence or diversity. However, because reptiles are cold-blooded (ectothermic), their microbiotas experience wider temperature fluctuation than microbes in warm-blooded animals. To investigate whether extreme weather patterns affect parabasalid-host interactions, we analyzed the gene expression in reptile-associated parabasalids and found that temperature differences significantly alter genes associated with host health. These results expand our understanding of parabasalids in this vulnerable vertebrate group and highlight important factors to be taken into consideration for conservation efforts.

MILBEMYCIN OXIME (INTERCEPTOR) TREATMENT OF PYCNOGONID SEA SPIDER INFESTATION IN THREE SPECIES OF CORALS.

A coral reef system at the Steinhart Aquarium in San Francisco, CA, USA experienced a population explosion of pycnogonid sea spiders (Arthropoda: Class Pycnogonida) with subsequent deleterious health effects on the corals in the system. Sixteen coral colonies across three species (Stylophora pistillata, Pocillopora damicornis, and Acropora tenuis) were chosen from this system for milbemycin oxime immersion therapy trials, with the goal of decreasing or eradicating the sea spider population with minimal detrimental effect to the corals. Corals underwent two milbemycin immersion treatments, administered 1 wk apart, at the previously published aquatic invertebrate dose of 0.016 parts per million (ppm; mg/L), but therapy did not reduce the number of sea spiders. Doubling the dose to 0.032 ppm milbemycin and repeating this immersion therapy 1/wk for three treatments successfully reduced the sea spiders. Histopathology was used to assess the health of the corals and tolerance to therapy, and posttreatment biopsies confirmed that there were no adverse effects to any of the three species of coral. Repeated 1/ wk treatments of milbemycin oxime immersion therapy at 0.032 ppm appears to be both safe and effective for reducing the numbers of pycnogonid sea spiders in the stony corals S. pistillata, P. damicornis, and A. tenuis.

Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts.

Parabasalid protists recently emerged as keystone members of the mammalian microbiota with important effects on their host's health. However, the prevalence and diversity of parabasalids in wild reptiles and the consequences of captivity and other environmental factors on these symbiotic protists are unknown. Reptiles are ectothermic, and their microbiomes are subject to temperature fluctuations, such as those driven by climate change. Thus, conservation efforts for threatened reptile species may benefit from understanding how shifts in temperature and captive breeding influence the microbiota, including parabasalids, to impact host fitness and disease susceptibility. Here, we surveyed intestinal parabasalids in a cohort of wild reptiles across three continents and compared these to captive animals. Reptiles harbor surprisingly few species of parabasalids compared to mammals, but these protists exhibited a flexible host-range, suggesting specific adaptations to reptilian social structures and microbiota transmission. Furthermore, reptile-associated parabasalids are adapted to wide temperature ranges, although colder temperatures significantly altered the protist transcriptomes, with increased expression of genes associated with detrimental interactions with the host. Our findings establish that parabasalids are widely distributed in the microbiota of wild and captive reptiles and highlight how these protists respond to temperature swings encountered in their ectothermic hosts.

APPARENT PREVALENCE, DIVERSITY, AND ASSOCIATED LESIONS OF PERIORBITAL NEMATODES IN A POPULATION OF BARRED OWLS (STRIX VARIA) FROM NORTHERN CALIFORNIA, USA.

Over the last four decades, Barred Owls (Strix varia) have expanded their range to include much of western North America, including California. This expansion is suspected to have contributed to declining populations of a closely related species, the federally threatened Northern Spotted Owl (Strix occidentalis caurina). As a result, understanding potential health threats to Barred Owls has implications for Spotted Owl health and recovery. From 2016 to 2020, 69 Barred Owls were collected to determine the apparent prevalence of periorbital nematode infection, to identify the parasite species present, and to investigate the potential pathologic effects on their hosts. The nematodes were morphologically identified as Oxyspirura and Aprocta spp. On the basis of phylogenetic analyses, they were clearly divergent from published sequences of other species within these genera. Overall, 34 (49%) Barred Owls were infected with periorbital nematodes, with Oxyspirura sp. infections being much more common (94%) than Aprocta sp. (18%). Histopathology revealed varying severity of conjunctivitis in infected owls. Despite the frequency of infection and subsequent inflammation, parasite burden was not associated with reduced body weight in these owls. As a result, the potential health effect of these nematodes is unclear. Further taxonomic characterization is needed to determine potential novelty of these nematodes.

Nephrolithiasis in a giant Pacific octopus Enteroctopus dofleini.

Managed care of cephalopods can be complicated by numerous factors including infectious and non-infectious disease, environmental stressors, and anatomic and physiological changes associated with senescence. The current report describes a unique case of nephrolithiasis in a senescent, >2 yr old female Pacific octopus Enteroctopus dofleini housed in a public aquarium. Clinical signs included generalized external pallor, inappetence progressing to anorexia, lethargy, and a slow healing mantle abrasion over a period of a year. Due to the animal's declining condition, humane euthanasia was elected. At necropsy, multiple, small, approximately 1-5 mm diameter crystalline deposits were reported throughout all sections of the renal appendages. Histopathology identified a large crystal expanding and rupturing a focal tubule, causing necrosis, ulceration, and hemocytic infiltration. Crystalline stone analysis revealed that the nephrolith was composed of 100% ammonium acid urate. Additional changes to the digestive gland included marked atrophy with fibrosis correlated to the animal's history of hyporexia/anorexia secondary to senescence. To our knowledge, this is the first report of nephrolithiasis in E. dofleini.

Ocular anatomy of the black pacu (Colossoma macropomum): gross, histologic, and diagnostic imaging.

OBJECTIVE: To describe the ocular anatomy of the black pacu (Colossoma macropomum), a freshwater teleost fish of the Amazon River basin, including an unusual choroid laden with adipose tissue. PROCEDURES: Three adult black pacu were anesthetized and examined clinically and with ocular ultrasonography, then euthanized. Three fish were euthanized and their heads imaged immediately postmortem using computed tomography. One fish was euthanized and its exenterated eyes imaged by high-resolution magnetic resonance imaging. The exenterated eyes of all seven fish were fixed in formalin; eyes from three fish were examined grossly and histologically. Additionally, archived histologic sections from two smaller black pacu specimens were examined. RESULTS: Findings were consistent among the ocular imaging modalities used. Intrinsic to the sclera were circumferential ossicles and scleral cartilage. The lens was spherical and protruded through the ovoid pupil with an aphakic space inferiorly when the accommodative mechanism was relaxed under anesthesia. Both a small falciform process and epiretinal vasculature were present in the posterior segment. The retina was cone-rich, and processes of the retinal pigment epithelium enveloped the photoreceptor outer segments. Remarkably, the choroid occupied one-third of the anteroposterior length of the globe; histology confirmed that the bulk of the choroid was composed of adipose tissue. CONCLUSIONS: The eye of the pacu overall is typical of teleosts but has the notable and consistent finding of a substantive store of choroidal fat of unknown function.

Widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus infections.

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.

Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease.

UNLABELLED: Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens. IMPORTANCE: Inclusion body disease (IBD) is a common infectious disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of virtually every emerging infectious human disease. We searched for candidate causative agents in snakes diagnosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also to filoviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolution.

An outbreak of Chlamydophila psittaci in an outdoor colony of Magellanic penguins (Spheniscus magellanicus).

An outbreak of Chlamydophila psittaci occurred in an outdoor colony of 63 Magellanic penguins (Spheniscus magellanicus) at the San Francisco Zoo. Affected penguins presented with inappetence, lethargy, and light green urates. Hematologic and serum biochemical findings were consistent with chronic inflammation. Penguins did not respond to initial supportive and antimicrobial therapy, and 3 died. Necropsy results of the 3 birds revealed hepatomegaly and splenomegaly, and histologic lesions included necrotizing hepatitis, splenitis, and vasculitis. Chlamydophila psittaci infection was confirmed by results of Gimenez staining, immunohistochemistry, and tissue polymerase chain reaction assay. As additional birds continued to present with similar clinical signs, the entire colony of penguins was prophylactically treated with a 30-day minimum course of doxycycline, administered orally or intramuscularly or as a combination of both. Despite treatment, 9 additional penguins died during a 3-month period. Pathologic results from these birds revealed renal and visceral gout (n = 4), cardiac insufficiency (n = 2), sepsis from a suspected esophageal perforation (n = 2), and no gross lesions (n = 1). During the outbreak, 4 birds presented with seizures, 5 developed dermatitis, and nearly 90% of birds in the colony showed severe keratoconjunctivitis, believed to be related to drug therapy with doxycycline. We report the clinical and pathologic features of Chlamydophila psittaci infection in an outdoor colony of penguins and the associated challenges of treatment.

Survey of minerals and fat-soluble vitamins in captive black and white ruffed lemurs (Varecia variegata).

Serum and whole blood samples from 64 clinically normal captive black and white ruffed lemurs (Varecia variegata), aged 6 mo to 32 yr, were analyzed to survey mineral and fat-soluble vitamin concentrations. All animals were fed a commercial primate food and a wide range of fruits and vegetables. Specific commercial diet information was available for 52 animals that were fed one of 10 different diets. Data analysis showed no differences in the analytes attributable to sex or access to natural ultraviolet light. Serum phosphorus (range: 1.4-3.1 mmol/L) was significantly higher and retinol (range: 0.38-1.23 micromol/L) was significantly lower in young animals (< or =4 yr). Iron (range: 17.2-77.0 micromol/L) and copper (range: 10.7-53.3 micromol/L) were much higher than concentrations reported in other free-ranging lemur species, and in some animals were at levels considered potentially toxic in domestic animals. Magnesium (range: 0.66-2.04 mmol/L), sodium (range: 111-201 mmol/L), and potassium (range: 2.0-6.8 mmol/L) ranged both lower and higher than concentrations considered adequate for a mammal, but were similar to concentrations reported in wild red ruffed lemurs (Varecia rubra), a closely related species. Selenium (range: 3.5-7.7 micromol/L) was within the range expected for a mammal, but higher than concentrations reported in wild V rubra. Zinc (range: 9.2-62.7 micromol/L) was similar to concentrations reported in V. rubra. Calcidiol (range: <12.5-144.8 nmol/L) and retinol (range: 0.38-2.95 micromol/L) were both lower and higher than concentrations reported in V. rubra. Lower serum calcidiol concentration correlated with lower commercial dietary vitamin D3. Alpha-tocopherol (range: 1.2-17.6 micromol/L) and y-tocopherol (range: 0.3-3.9 micromol/L) were within a range expected in a captive frugivorous primate but higher than concentrations found in wild V. rubra.

Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment.

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.

Survey and clinical application of serum iron, total iron binding capacity, transferrin saturation, and serum ferritin in captive black and white ruffed lemurs (Varecia variegata variegata).

Serum samples from 63 clinically normal captive black and white ruffed lemurs (Varecia variegata variegata) were analyzed to survey serum iron, total iron binding capacity, transferrin saturation, and serum ferritin levels. Data analysis showed no differences in these analytes attributable to sex, but significantly higher levels of serum iron, transferrin saturation, and serum ferritin in older animals. The survey data were examined in light of two black and white ruffed lemurs that were treated for iron overload with serial phlebotomies. Prior to therapy, both phlebotomized lemurs had excess hepatic iron deposition, but had serum iron, transferrin saturation, and serum ferritin below the upper limits observed in the survey animals, suggesting that some clinically normal animals included in the survey may have accumulated excess systemic iron. Serial phlebotomy therapy reduced serum iron, transferrin saturation, and serum ferritin in both animals. Three years after the conclusion of therapy in the one remaining case, serum iron and transferrin saturation have risen substantially, whereas serum ferritin has risen slightly. Serum iron, transferrin saturation, and serum ferritin may be useful predictors of systemic iron stores in this species, though the correlation between these parameters and systemic iron stores needs to be determined.