Sublethal effects of parasitism on ruminants can have cascading consequences for ecosystems.

Parasitic infections are common, but how they shape ecosystem-level processes is understudied. Using a mathematical model and meta-analysis, we explored the potential for helminth parasites to trigger trophic cascades through lethal and sublethal effects imposed on herbivorous ruminant hosts after infection. First, using the model, we linked negative effects of parasitic infection on host survival, fecundity, and feeding rate to host and producer biomass. Our model, parameterized with data from a well-documented producer­caribou­helminth system, reveals that even moderate impacts of parasites on host survival, fecundity, or feeding rate can have cascading effects on ruminant host and producer biomass. Second, using meta-analysis, we investigated the links between helminth infections and traits of free-living ruminant hosts in nature. We found that helminth infections tend to exert negative but sublethal effects on ruminant hosts. Specifically, infection significantly reduces host feeding rates, body mass, and body condition but has weak and highly variable effects on survival and fecundity. Together, these findings suggest that while helminth parasites can trigger trophic cascades through multiple mechanisms, overlooked sublethal effects on nonreproductive traits likely dominate their impacts on ecosystems. In particular, by reducing ruminant herbivory, pervasive helminth infections may contribute to a greener world.

HEMATOLOGY AND BLOOD CHEMISTRY VALUES IN CUBAN CROCODILES (CROCODYLUS RHOMBIFER) HOUSED AT THE ZAPATA SWAMP CROCODILE FARM, CUBA.

We report hematology and biochemistry reference intervals (RI) for the critically endangered Cuban crocodile (Crocodylus rhombifer). In November 2019, we sampled 43 adult crocodiles (6 male, 37 female) under human care at the Zapata Swamp Crocodile Farm in Matanzas, Cuba. These crocodiles are part of a breeding program for the species registered by the Convention on International Trade in Endangered Species (CITES). Visual health evaluations were performed immediately after manual restraint, and blood was collected from the postoccipital sinus. We performed packed cell volume (PCV), total solids (TS), complete blood counts (CBC), and biochemistry profiles for each crocodile on the day of sampling. Mean PCV (n = 42) was 21.1 ± 5.0% and TS (n = 42) 7.3 ± 1.2 mg/dl, respectively. Absolute white blood cell (WBC) (n = 40) was 9.6 ± 5.7 ｘ 109/L. Similar to other crocodilian species, the dominant leukocyte was lymphocytes (70.7 ± 10.4%), followed by heterophils (18.7 ± 9.7%). Two of the crocodiles had a high heterophil:lymphocyte ratio (0.87 and 0.74), although on visual exam they were both considered healthy. The range of creatine kinase was 41-1,482 U/L, and the higher values may be a reflection of muscle exertion at time of handling. Limitations to the study included skewed sex ratios and high lipemia and hemolysis in most samples collected. These are the first reference intervals reported for this species, including the first descriptions of WBC morphology. These data are valuable for the management of animals at the Zapata Swamp Crocodile Farm, for comparison with free-living Cuban crocodiles in Cuba, and for comparison with those managed under human care outside of Cuba.

Staff perceptions of COVID-19 impacts on wildlife conservation at a zoological institution.

Coronavirus Disease 2019 (COVID-19) has impacted virtually all aspects of a global society. By using a transdisciplinary team and methodology, our study highlights the importance of utilizing a One Health approach to address global health and conservation challenges in tandem. We examined how conservation conducted by an accredited zoological institution was altered by the pandemic. In July 2020, we surveyed a select subset of Saint Louis Zoo employees to understand how these staff members perceived their conservation work to be affected during this time. Additionally, in November and December 2020, seven survey respondents were interviewed virtually for qualitative data. Our hypothesis was that lack of funding, reduced ability to travel, and shifting priorities among conservation professionals, as well as their respective institutions, would be significant barriers to conservation efforts. Our survey results revealed that the top three perceived challenges to conservation projects at the Saint Louis Zoo due to the COVID-19 pandemic were lack of funding (83.9%), reductions in Zoo visitors (56.3%), and inability/lack of ability to access field sites and laboratories (55.2%). Respondents also indicated that the top three most important Zoo conservation activities before the COVID-19 pandemic were (1) local/national/global wildlife management and recovery programs, (tie 2) caring for animals in the Zoo's collection, and (tie 2) genetic breeding programs. At least half of respondents indicated that the pandemic had significantly or slightly decreased the ability of staff to perform all three activities. Results supported our hypothesis that reduced funding, limited travel, and shifting priorities were challenges to zoological conservation at this institution. Although travel restrictions will likely persist, continual funding will be critical for maintaining conservation. Our results also indicate that reductions in Zoo visitors were another perceived barrier to conservation and that staff needed to find novel ways to connect with the public. Results from this study may help zoos and aquariums better understand the unique pandemic-associated challenges that threaten conservation at their institutions and may be helpful in designing programs and projects in a post-pandemic world.

Conservation medicine and One Health in zoos: Scope, obstacles, and unrecognized potential.

Zoo veterinarians and allied professionals have been contributing to conservation medicine (CM) and One Health (OH) activities for more than two decades. Although the Association of Zoos and Aquariums (AZA) considers conservation a key part of its mission, little published material exists about the extent of AZA work in CM/OH or the challenges and opportunities associated with these endeavors. To better understand the current scope of CM/OH in zoos, we surveyed 53 AZA-accredited institutions from April through October of 2016. We obtained information on CM/OH infrastructure, support for expansion in this area, and strategies to overcome perceived obstacles hindering CM/OH from becoming a core AZA activity. Survey results revealed that while most zoos favor greater investment in CM/OH programs, awareness, and understanding of CM/OH across the broader zoo community and public is lacking. The majority of respondents stated that overcoming this challenge is paramount to attaining support for CM/OH initiatives. In spite of these obstacles, survey respondents highlighted many positive developments in CM/OH. We found that 84% of zoos surveyed are actively engaged in CM/OH activities, and 12% house formal CM/OH programs. Another 8% of respondents said their institutions were developing CM/OH programs. Perhaps most noteworthy, we found that zoo size did not have a significant bearing on the financial amount allocated toward an institution's CM/OH activities. This suggests that all zoos, regardless of size, can make meaningful contributions to the growing movement of CM/OH and help redefine the role of zoos within this movement.

FIELD ANESTHESIA AND GONADAL MORPHOLOGY OF IMMATURE WESTERN SANTA CRUZ TORTOISES (CHELONOIDIS PORTERI).

Evaluation of sex ratios is a critical component of chelonian captive breeding programs and may become increasingly useful to assess the demographics of free-living populations. In many reptile species, the sex of immature animals cannot be determined based on external features. Endoscopic sex identification is an accurate and safe method to identify the sex of immature individuals of some chelonian species. A number of studies describe this technique in controlled, hospital settings and report significant interspecies variations in gonad morphology; however, there are few reports describing this technique in field conditions. In the current study, the gonadal morphology of 40 immature Western Santa Cruz tortoises (Chelonoidis porteri) on Santa Cruz Island in Galapagos, Ecuador, was assessed. A previously described endoscopic protocol was used to perform sex identification under field conditions. Tortoises were anesthetized using an intramuscular injection of ketamine (10 mg/kg) and medetomidine (0.1 mg/kg), which provided an adequate plane of anesthesia. The medetomidine was reversed with atipamezole (0.5 mg/kg). Field conditions presented challenges such as limited control over lighting, suboptimal patient positioning, and restricted power supply for endoscopy equipment. The immature testicle in Western Santa Cruz tortoises was oval, reddish pink, and tightly adhered to the coelomic membrane ventral to the kidney. The surface of the gonads resembled other species with the notable exception that the ovaries lacked a significant number of primordial follicles. These gonadal characteristics were consistent, with only one individual identified as undetermined sex of the 40 samples. This field-based endoscopic gonadal evaluation was a safe and sensitive technique for determining the sex of free-living immature Western Santa Cruz Galapagos tortoises.

Assessment of hematologic and corticosterone response in free-living eastern box turtles (Terrapene carolina carolina) at capture and after handling.

Hematology is a common tool for wildlife health assessments. Manual leukocyte counts are required in reptiles, however, disagreement between quantification methods has been observed in some chelonians. This study determined agreement between two methods of leukocyte quantification, eosinophilic leukopet, and blood film white blood cell (WBC) estimates, in free-living eastern box turtles (Terrapene carolina carolina). Simultaneously, we assessed the impact of handling duration on both leukocyte quantity and corticosterone levels. We collected blood at capture (<2 min from disturbance) and again before release 30-150 min later from 92 box turtles at six sites in Illinois and Tennessee. Constant and proportional error was present in the leukopet results for WBC, lymphocytes, and basophils compared to the estimate method. Both methods were in agreement for heterophils, monocytes, and eosinophils. Agreement between the methods was significantly more likely at WBC counts below 23,241/µl. All hematologic parameters were significantly higher at the final blood draw compared to the initial blood draw using both WBC determination methods, except relative eosinophil and basophil counts. Corticosterone levels varied with time, with maximum concentrations reached at 54 min postcapture, followed by a rapid return to baseline levels. Corticosterone level was not significantly associated with any hematologic parameter or sex. This study provides a framework for understanding the effects of animal handling methodology and diagnostic modality when evaluating hematologic health in box turtles.

Circulating nutrient concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Punta San Juan, Peru.

Free-ranging Humboldt penguin (HP, Spheniscus humboldti) populations are under pressure from resource competition with industrial fisheries, habitat loss, and El Niño Southern Oscillation events. Foraging patterns for this top marine predator change during periods of aberrant oceanographic conditions and scarce fish stock numbers. These radical dietary fluctuations can lead to poor fertility, early embryonic death, poor hatchability, suppressed immune function, high chick mortality, and illness. To understand the variability of nutrient status in reproductive seasons, we measured select circulating nutrient concentrations (fat-soluble vitamins A, D, E, and carotenoids, fatty acids, amino acids, minerals, and electrolytes) of 105 HP at Punta San Juan, Peru during the first reproductive seasons of 2007 and 2008. We determined significant differences in nutrient status between sexes, years of sampling, and reproductive stages. Males (4.5 ± 0.38 kg) weighed more than females (4.0 ± 0.29 kg) and exhibited higher concentrations of vitamin A (0.71 ± 0.11 vs. 0.61 ± 0.12 µg/ml) and docosahexaenoic acid (6.70 ± 1.61 vs. 5.65 ± 1.59%). Males also displayed lower concentrations of ß-carotene (0.01 ± 0.01 vs. 0.012 ± 0.001 µg/ml) and phosphorus (3.43 ± 0.83 vs. 4.40 ± 1.66 mg/dl). Comparison between the 2 years showed most circulating amino acid concentrations were higher in 2007. Significant differences in circulating amino acids and vitamins were also noted between different reproductive stages. These results demonstrate concentrations of nutrients can vary due to the physiological state of the animal, as well as the overall dynamics of their marine ecosystem habitat.

SURVEY OF ANTITUBERCULOSIS DRUG ADMINISTRATION AND ADVERSE EFFECTS IN ELEPHANTS IN NORTH AMERICA.

Tuberculosis, caused by Mycobacterium tuberculosis, is a disease causing morbidity and mortality in captive elephants (Elephas maximus and Loxodonta africana) as well as free-ranging individuals. Elephants in North America diagnosed with tuberculosis are often treated with antituberculosis drugs, unlike livestock species, which has necessitated the development of treatment guidelines adapted from recommendations for humans. There are few published reports describing empirical treatment, which may be complicated by poor patient compliance, interruptions in drug administration, and adverse effects. A survey of elephants in North America was conducted to compile information on treatment protocols, including drugs, dosages, routes of administration, serum drug concentrations, and adverse effects of antituberculosis treatment. Responses were received regarding 182 elephants, 12 of which were treated prophylactically or therapeutically with antituberculosis drugs. Treatment protocols varied among elephants, and included various combinations of isoniazid, rifampin, pyrazinamide, ethambutol, enrofloxacin, levofloxacin, and ethionamide. Serum drug concentrations also varied considerably among and within individuals. Facility staff reported 5 elephants (out of 7 treated elephants with responses) that exhibited clinical signs that may have been associated with antituberculosis drugs or treatment procedures. Anorexia, decreased water intake, constipation, depression, ataxia, limb paresis, and tremors were among the signs observed. Most adverse effects were reported to be moderate or severe, resulting in interruption of the treatment. The results from this survey provide veterinarians and elephant managers with valuable historical data to make informed clinical management decisions regarding antituberculosis therapy in elephants.

Transcriptomic alterations in the brain of painted turtles (Chrysemys picta) developmentally exposed to bisphenol A or ethinyl estradiol.

Developmental exposure of turtles and other reptiles to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), can stimulate partial to full gonadal sex-reversal in males. We have also recently shown that in ovo exposure to either EDC can induce similar sex-dependent behavioral changes typified by improved spatial learning and memory or possibly feminized brain responses. Observed behavioral changes are presumed to be due to BPA- and EE-induced brain transcriptomic alterations during development. To test this hypothesis, we treated painted turtles (Chrysemys picta) at developmental stage 17, incubated at 26°C (male-inducing temperature), with 1) BPA (1 ng/µl), 2) EE (4 ng/µl), or 3) vehicle ethanol (control group). Ten months after hatching and completion of the behavioral tests, juvenile turtles were euthanized, brains were collected and frozen in liquid nitrogen, and RNA was isolated for RNA-Seq analysis. Turtles exposed to BPA clustered separately from EE-exposed and control individuals. More transcripts and gene pathways were altered in BPA vs. EE individuals. The one transcript upregulated in both BPA- and EE-exposed individuals was the mitochondrial-associated gene, ND5, which is involved in oxidative phosphorylation. Early exposure of turtles to BPA increases transcripts linked with ribosomal and mitochondrial functions, especially bioenergetics, which has been previously linked with improved cognitive performance. In summary, even though both BPA and EE resulted in similar behavioral alterations, they diverge in the pattern of neural transcript alterations with early BPA significantly upregulating several genes involved in oxidative phosphorylation, mitochondrial activity, and ribosomal function, which could enhance cognitive performance.

Serologic Evidence for Influenza C and D Virus among Ruminants and Camelids, Africa, 1991-2015.

Influenza D virus has been identified in America, Europe, and Asia. We detected influenza D virus antibodies in cattle and small ruminants from North (Morocco) and West (Togo and Benin) Africa. Dromedary camels in Kenya harbored influenza C or D virus antibodies, indicating a potential new host for these viruses.

FIRST RECORD OF HEMATOLOGIC VALUES IN FREE-LIVING AND CAPTIVE MANED SLOTHS (BRADYPUS TORQUATUS; XENARTHA, BRADYPODIDAE).

Bradypus torquatus is a rare and endemic sloth species from the Atlantic Forest, Brazil. Due to a lack of medical information including hematologic reference parameters for the species, hematologic baseline values were determined using samples from 14 clinically healthy B. torquatus , under captive (n = 7) and free-living (n = 7) conditions in Bahia State, Brazil. Additionally, the morphology of the blood cells is presented, with a demonstration that the Barr body chromosome may assist with sex determination of the species. The Barr body chromosome was present in all seven females and absent in all males. Many erythrocytes were approximately the size of small lymphocytes, with red blood cells exhibiting anisocystosis, normochromia, and apparent macrocytosis, compared with domestic animals. This study provides the first published hematologic values and cell morphology for B. torquatus . However, further studies are suggested using an automated hematology analyzer with larger sample sizes so that reference intervals may be established and hematologic values better understood for sex, habitat type, and age cohorts.

Species-specific transmission of novel picornaviruses in lemurs.

UNLABELLED: The roles of host genetics versus exposure and contact frequency in driving cross-species transmission remain the subject of debate. Here, we used a multitaxon lemur collection at the Saint Louis Zoo in the United States as a model to gain insight into viral transmission in a setting of high interspecies contact. Lemurs are a diverse and understudied group of primates that are highly endangered. The speciation of lemurs, which are endemic to the island of Madagascar, occurred in geographic isolation apart from that of continental African primates. Although evidence of endogenized viruses in lemur genomes exists, no exogenous viruses of lemurs have been described to date. Here we identified two novel picornaviruses in fecal specimens of ring-tailed lemurs (Lemur catta) and black-and-white ruffed lemurs (Varecia variegata). We found that the viruses were transmitted in a species-specific manner (lesavirus 1 was detected only in ring-tailed lemurs, while lesavirus 2 was detected only in black-and-white ruffed lemurs). Longitudinal sampling over a 1-year interval demonstrated ongoing infection in the collection. This was supported by evidence of viral clearance in some animals and new infections in previously uninfected animals, including a set of newly born triplets that acquired the infection. While the two virus strains were found to be cocirculating in a mixed-species exhibit of ring-tailed lemurs, black-and-white ruffed lemurs, and black lemurs, there was no evidence of cross-species transmission. This suggests that despite high-intensity contact, host species barriers can prevent cross-species transmissions of these viruses. IMPORTANCE: Up to 75% of emerging infectious diseases in humans today are the result of zoonotic transmission. However, a challenge in understanding transmission dynamics has been the limited models of cross-species transmission. Zoos provide a unique opportunity to explore parameters defining viral transmission. We demonstrated that ongoing virus transmission in a mixed lemur species exhibit was species specific. This suggests that despite high contact intensity, host species barriers contribute to protection from cross-species transmission of these viruses. While the combinations of species might differ, most zoological parks worldwide commonly feature mixed-species exhibits. Collectively, this report demonstrates a widely applicable approach toward understanding infectious disease transmission.

Effects of developmental exposure to bisphenol A and ethinyl estradiol on spatial navigational learning and memory in painted turtles (Chrysemys picta).

Developmental exposure of turtles and other reptiles to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE2, estrogen present in birth control pills), can induce partial to full gonadal sex-reversal in males. No prior studies have considered whether in ovo exposure to EDCs disrupts normal brain sexual differentiation. Yet, rodent model studies indicate early exposure to these chemicals disturbs sexually selected behavioral traits, including spatial navigational learning and memory. Thus, we sought to determine whether developmental exposure of painted turtles (Chrysemys picta) to BPA and EE2 results in sex-dependent behavioral changes. At developmental stage 17, turtles incubated at 26°C (male-inducing temperature) were treated with 1) BPA High (100µg /mL), 2) BPA Low (0.01µg/mL), 3) EE2 (0.2µg/mL), or 4) vehicle or no vehicle control groups. Five months after hatching, turtles were tested with a spatial navigational test that included four food containers, only one of which was baited with food. Each turtle was randomly assigned one container that did not change over the trial period. Each individual was tested for 14 consecutive days. Results show developmental exposure to BPA High and EE2 improved spatial navigational learning and memory, as evidenced by increased number of times spent in the correct target zone and greater likelihood of solving the maze compared to control turtles. This study is the first to show that in addition to overriding temperature sex determination (TSD) of the male gonad, these EDCs may induce sex-dependent behavioral changes in turtles.

Changes in human health parameters associated with a touch tank experience at a zoological institution.

Association of Zoos and Aquariums (AZA) institutions provide a variety of benefits to visitors. However, one area that has received little study is the direct human health benefits from zoo and aquarium visits. With the increase in stress related non-infectious diseases in industrialized countries, understanding the extent of these benefits is important. We studied the effects on visitor stress of an experience at a touch tank exhibit featuring stingrays, sharks, and horseshoe crabs. Stress was measured by physiological and psychological parameters. Heart rate was recorded before, during, and after interacting with the animals, and mood was assessed before and after the experience using a psychological instrument. Multilevel models of heart rate show a quadratic trend, with heart rate elevated (b = -3.01, t = 26.4, P < 0.001) and less variable (b = 3.60, t = 15.9, P < 0.001) while touching the animals compared to before or after. Wilcoxon signed-rank tests on mood data suggest that most visitors felt happier (V = 174.5, P < 0.001), more energized (V = 743.5, P < 0.001), and less tense (V = 5618, P < 0.001) after the experience. This suggests that interacting with animals led to a physiological response during interactions reminiscent of a theme park experience along with a decrease in mental stress. The effects of confounding variables such as crowd size are also discussed. Further studies should be conducted to help deepen our understanding of the health benefits of experiences at AZA institutions.

COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

GASTROINTESTINAL PARASITES OF CAPTIVE AND FREE-LIVING LEMURS AND DOMESTIC CARNIVORES IN EASTERN MADAGASCAR.

Fecal samples from captive and free-living lemurs at Ivoloina Zoological Park (IZP) and domestic carnivores from six villages surrounding IZP were evaluated between July and August 2012. Free-living lemurs from Betampona Natural Reserve (BNR), a relatively pristine rainforest fragment 40 km away, were also evaluated in November 2013. All 33 dogs sampled (100%) and 16 of 22 cats sampled (72.7%) were parasitized, predominantly with nematodes (strongyles, ascarids, and spirurids) as well as cestodes and protozoans. Similar types of parasites were identified in the lemur populations. Identification of spirurid nematodes and protozoans in the lemur fecal samples were of concern due to previously documented morbidity and mortality in lemurs from these parasitic agents. Twelve of 13 free-living (93%) and 31 of 49 captive (63%) lemurs sampled at IZP had a higher parasite prevalence than lemurs at BNR, with 13 of 24 (54%) being parasitized. The lemurs in BNR are likely at risk of increased exposure to these parasites and, therefore, increased morbidity and mortality, as humans and their domestic animals are encroaching on this natural area.

Effects of the environmental estrogenic contaminants bisphenol A and 17α-ethinyl estradiol on sexual development and adult behaviors in aquatic wildlife species.

Endocrine disrupting chemicals (EDCs), including the mass-produced component of plastics, bisphenol A (BPA) are widely prevalent in aquatic and terrestrial habitats. Many aquatic species, such as fish, amphibians, aquatic reptiles and mammals, are exposed daily to high concentrations of BPA and ethinyl estradiol (EE2), estrogen in birth control pills. In this review, we will predominantly focus on BPA and EE2, well-described estrogenic EDCs. First, the evidence that BPA and EE2 are detectable in almost all bodies of water will be discussed. We will consider how BPA affects sexual and neural development in these species, as these effects have been the best characterized across taxa. For instance, such chemicals have been in many cases reported to cause sex-reversal of males to females. Even if these chemicals do not overtly alter the gonadal sex, there are indications that several EDCs might demasculinize male-specific behaviors that are essential for attracting a mate. In so doing, these chemicals may reduce the likelihood that these males reproduce. If exposed males do reproduce, the concern is that they will then be passing on compromised genetic fitness to their offspring and transmitting potential transgenerational effects through their sperm epigenome. We will thus consider how diverse epigenetic changes might be a unifying mechanism of how BPA and EE2 disrupt several processes across species. Such changes might also serve as universal species diagnostic biomarkers of BPA and other EDCs exposure. Lastly, the evidence that estrogenic EDCs-induced effects in aquatic species might translate to humans will be considered.

Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles (Chrysemys picta).

Environmental chemicals can disrupt endocrine signaling and adversely impact sexual differentiation in wildlife. Bisphenol A (BPA) is an estrogenic chemical commonly found in a variety of habitats. In this study, we used painted turtles (Chrysemys picta), which have temperature-dependent sex determination (TSD), as an animal model for ontogenetic endocrine disruption by BPA. We hypothesized that BPA would override TSD and disrupt sexual development. We incubated farm-raised turtle eggs at the male-producing temperature (26°C), randomly assigned individuals to treatment groups: control, vehicle control, 17ß-estradiol (E2, 20ng/g-egg) or 0.01, 1.0, 100µgBPA/g-egg and harvested tissues at hatch. Typical female gonads were present in 89% of the E2-treated "males", but in none of the control males (n=35). Gonads of BPA-exposed turtles had varying amounts of ovarian-like cortical (OLC) tissue and disorganized testicular tubules in the medulla. Although the percentage of males with OLCs increased with BPA dose (BPA-low=30%, BPA-medium=33%, BPA-high=39%), this difference was not significant (p=0.85). In all three BPA treatments, SOX9 patterns revealed disorganized medullary testicular tubules and ß-catenin expression in a thickened cortex. Liver vitellogenin, a female-specific liver protein commonly used as an exposure biomarker, was not induced by any of the treatments. Notably, these results suggest that developmental exposure to BPA disrupts sexual differentiation in painted turtles. Further examination is necessary to determine the underlying mechanisms of sex reversal in reptiles and how these translate to EDC exposure in wild populations.

Health assessment of free-living eastern box turtles (Terrapene carolina carolina) in and around the Maryland Zoo in Baltimore 1996-2011.

Health data for free-living eastern box turtles (Terrapene carolina carolina) at the Maryland Zoo in Baltimore were analyzed. One hundred and eighteen turtles were captured on or near zoo grounds over the course of 15 yr (1996-2011), with recapture of many individuals leading to 208 total evaluations. Of the 118 individuals, 61 were male, 50 were female, and 7 were of undetermined sex. Of the 208 captures, 188 were healthy, and 20 were sick or injured. Complete health evaluations were performed on 30 turtles with physical examination records, complete blood counts (CBCs), and plasma biochemistry profiles. Eight animals were sampled more than once, yielding 40 total samples for complete health evaluations of these 30 individuals. The 40 samples were divided into healthy (N=29) and sick (N=11) groups based on clinical findings on physical examination. Samples from healthy animals were further divided into male (N=17) and female (N= 12) groups. CBC and biochemistry profile parameters were compared between sick and healthy groups and between healthy males and females. Sick turtles had lower albumin, globulin, total protein (TP), calcium, phosphorous, sodium, and potassium than healthy animals. Sick turtles also had higher heterophil to lymphocyte ratios. Healthy female turtles had higher leukocyte count, eosinophil count, total solids, TP, globulin, cholesterol, calcium, and phosphorous than healthy males. Banked plasma from all 40 samples was tested for antibodies to Mycoplasma agassizii and Mycoplasma testudineum via enzyme-linked immunosorbent assay. One sample from a clinically healthy female was antibody positive for M. agassizii; none were positive for M. testudineum. This study provides descriptive health data for eastern box turtles and CBC and biochemistry profile information for T. carolina carolina at and near the Maryland Zoo in Baltimore. It also reports low serologic evidence of exposure to mycoplasmosis.

Serosurveillance and the first detection of Bourbon virus RNA in a wildlife host.

Bourbon virus (BRBV) is an emerging pathogen that can cause severe and fatal disease in humans. BRBV is vectored by Amblyomma americanum (lone star ticks), which are widely distributed across the central, southern, and eastern United States. Wildlife species are potentially important for the maintenance and transmission of BRBV, but little is known about which species are involved, and what other factors play a role in the exposure to BRBV. To assess the exposure risk to BRBV among wildlife in the St. Louis area, we collected sera from 98 individuals, representing 6 different mammalian species from two locations in St. Louis County: Tyson Research Center (TRC) and WildCare Park (WCP) from fall 2021 to spring 2023. The sera were used in a BRBV neutralization assay to detect neutralizing antibodies and RT-qPCR for viral RNA analysis. We also sampled and compared the abundance of A. americanum ticks at the two locations and modeled which factors influenced BRBV seropositivity across species. In TRC, we observed a high rate of seropositivity in raccoons (Procyon lotor, 23/25), and white-tailed deer (Odocoileus virginianus, 18/27), but a low rate in opossums (Didelphis virginiana, 1/18). Neutralizing antibodies were also detected in sampled TRC bobcats (Lynx rufus, 4/4), coyotes (Canis latrans, 3/3), and a red fox (Vulpes vulpes, 1/1). The virological analysis identified BRBV RNA in one of the coyote serum samples. In contrast to TRC, all sera screened from WCP were negative for BRBV-specific neutralizing antibodies, and significantly fewer ticks were collected at WCP (31) compared to TRC (2,316). Collectively, these findings suggest that BRBV circulates in multiple wildlife species in the St. Louis area and that tick density and host community composition may be important factors in BRBV ecology.