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Background: Dried blood spot (DBS) technology is valuable in providing simple means of storing blood samples from wildlife with small blood volumes. Methods designed for heavy metal analysis on DBS become more useful if extended to elements of nutritional significance., Purpose: (1) Development of procedures for measuring Mn, Fe, Co, Cu, Zn, Se and Mo in DBS; (2) use the designed methods in health assessments of Galápagos land iguanas (Conolophus species)., Procedures: Elements were measured by inductively coupled plasma/mass spectrometry (ICP-MS) following acid digestion of whole blood or DBS from the same animal for direct comparison. Study animals comprised free-ranging iguanas from separate islands in the Galápagos archipelago., Main Findings: DBS spikes (Mn, Fe, Co, Cu, Zn, Se and Mo) demonstrated accuracy to ∼100 ppb; reporting limits were set there except for Fe and Zn which were set at 1000 ppb. Plasma samples - generally preferable for nutritional element diagnostics - were submitted from Galápagos land iguanas along with DBS as part of a large-scale health assessment. In plasma versus DBS concentration comparisons, Fe, Cu, Se and Mn correlated well with R^2 values of 0.799, 0.818, 0.896 and 0.899, respectively, and slopes ranging 0.88 - 1.3. Co and Zn showed greater scatter. Mo had insufficient points above its reporting limit and offered advantages for toxicity assessments. Bland-Altman diagrams showed flat scatter between 2x standard deviation boundaries with no undue trends except for Mn which had few points above its reporting limit. Bias, defined as the average difference [DBS - plasma] divided by the average value, was relatively low throughout, with values of - 19.3 % (Fe), - 48.7 % (Co), - 19.6 % (Cu), - 6.9 % (Zn), - 21.4 % (Se) and + 40.7 % (Mn). Normal distribution assessment of iguana Cu, Zn, Se and Fe plasma values showed unanticipated divergences between two species., Conclusions: The DBS approach for nutritional element analysis offers a suitable methodology for determining crucial elements Mn, Fe, Co, Cu, Zn, Se, and Mo in veterinary samples. Analyses of samples from Conolophus species revealed interesting divergences particularly for Cu, Zn, Se and Fe, elements generally associated with defense against oxidative stress., Competing Interests: Declaration of Competing Interest The authors confirm that there are no existing conflicts of interest related to the presented work., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in nondomestic felids have been documented in North America, South America, Africa, Europe, and Asia. Between March 2020 and February 2021, at nine institutions across three continents, infection was confirmed in 16 tigers ( Panthera tigris ), 14 lions ( Panthera leo ), three snow leopards ( Panthera uncia ), one cougar ( Puma concolor ), and one Amur leopard cat ( Prionailurus bengalensis e uptilurus ) ranging from 2 to 21 yr old (average, 10 yr). Infection was suspected in an additional 12 tigers, 4 lions, and 9 cougars. Clinical signs (in order of most to least common) included coughing, ocular and/or nasal discharge, wheezing, sneezing, decreased appetite, lethargy, diarrhea, and vomiting. Most felids recovered uneventfully, but one geriatric tiger with comorbidities developed severe dyspnea and neurologic signs necessitating euthanasia. Clinical signs lasted 1-19 d (average, 8 d); one tiger was asymptomatic. Infection was confirmed by various methods, including antigen tests and/or polymerase chain reaction (PCR) of nasal or oral swabs, tracheal wash, and feces, or virus isolation from feces or tracheal wash. Infection status and resolution were determined by testing nasal swabs from awake animals, fecal PCR, and observation of clinical signs. Shedding of fecal viral RNA was significantly longer than duration of clinical signs. Postinfection seropositivity was confirmed by four institutions including 11 felids (5 lions, 6 tigers). In most instances, asymptomatic or presymptomatic keepers were the presumed or confirmed source of infection, although in some instances the infection source remains uncertain. Almost all infections occurred despite using cloth facemasks and disposable gloves when in proximity to the felids and during food preparation. Although transmission may have occurred during momentary lapses in personal protective equipment compliance, it seems probable that cloth masks are insufficient at preventing transmission of SARS-CoV-2 from humans to nondomestic felids. Surgical or higher grade masks may be warranted when working with nondomestic felids.

The endemic Grand Cayman or blue iguana ( Cyclura lewisi ) is endangered. Beginning in 2015 significant morbidity and mortality occurred in captive and wild blue iguanas within Grand Cayman's Queen Elizabeth II Botanic Park (QEIIBP). Investigation identified a novel Helicobacter sp., provisionally named Helicobacter sp. Grand Cayman Blue Iguana 1 (GCBI1), as the cause. Invasive green iguanas ( Iguana iguana ) are believed to play a role in GCBI1 transmission to the blue iguana; however, the origin and transmission pathways have not been determined. To assess the likelihood of blue iguanas asymptomatically harboring GCBI1, in May 2022 population-level screening of captive blue iguanas at QEIIBP was conducted on half ( n = 102) of the captive blue iguana population ( n = 201) including half of each age class. Helicobacter sp. GCBI1 is closely related to a chelonian Helicobacter sp. and 10 sympatric wild north Antillean sliders ( Trachemys decussata angusta ) were sampled in October 2019. Combined choana/cloacal swabs were screened by a GCBI1-specific quantitative polymerase chain reaction (qPCR) assay. All samples were negative, suggesting that GCBI1 is not present asymptomatically in the captive blue iguana population or in north Antillean sliders. These results provide support for the hypothesis that GCBI1 is periodically introduced to captive and wild blue iguanas from another species or source.

Blood samples were obtained from the dorsal cervical sinus of free-ranging hawksbill sea turtles ( Eretmochelys imbricata ) collected at Glover's Reef Marine Reserve, 42 km east of the coast of Belize, for hematology and plasma biochemistry analysis. Unknown sex, subadult turtles ( N = 32) were sampled in 2013 ( n = 22) and 2017 ( n = 10). To provide a more robust data set, parameters that did not have statistically significant differences were pooled and treated as a single population. Eleven hematologic parameters were evaluated; of these, five were pooled. Twenty-three plasma biochemical parameters were evaluated; of these, 15 were pooled. The PCV observed in this study (mean 33.44%) was double that observed in two studies of juvenile hawksbills in Dubai (means 17% and 16%), whereas the total WBC count was half that observed in immature and adult hawksbills sampled in the Galápagos (mean 2.91 × 10 3 versus 5.3 × 103/µl). Total protein and albumin were lower than regionally similar, adult female hawksbills in Brazil (means 3.36 versus 5.45 g/dl and 0.93 versus 2.11 g/dl, respectively). Globulins were higher (mean 2.43 versus 1.06 and 0.5 g/dl), driving the albumin:globulin ratio lower than that observed in two studies of juvenile hawksbills in Dubai (0.4:1 versus 1.1:1 and 1:1, respectively). These findings represent a geographically distinct population from previous reports, highlight the variability in blood parameters from disparate populations, and reaffirm the vital importance of considering a multitude of variables when interpreting reptilian blood values. The similarities in the majority of values observed in 2013 and 2017 provide confidence in the stability of these parameters in this population.

A multiantigen print immunoassay (MAPIA) and rapid test (RT) developed and validated for detection of mycobacterial antibodies in elephants ( Elephas maximus and Loxodonta africana ) was assessed in Malayan tapir ( Tapirus indicus ). Retrospective analysis of banked serum from one Mycobacterium bovis infected and seven presumably uninfected tapir was performed by MAPIA and RT. A sample collected 2 mon prior to the death of a culture-confirmed M. bovis -infected tapir served as a positive control. Seroreactivity of this sample was demonstrated via both MAPIA and RT testing. Seven uninfected animals, including four without postmortem evidence of mycobacterial disease and three that remain healthy, were negative controls; none demonstrated seroreactivity to key antigens with either test. These results suggest that MAPIA and RT have potential utility for rapid detection of M. bovis infection in Malayan tapir.

Cefovecin is a third-generation cephalosporin antibiotic with an efficacy of 2 wk following a single injection in domestic dogs and cats. A high degree of plasma protein binding to cefovecin has been proposed as one of the mechanisms responsible for the long elimination half-life, but protein binding has not been evaluated extensively in nondomestic species. In this study, a review of the current literature was conducted, and pharmacokinetic data were compiled for species in which cefovecin has been evaluated thus far. Additionally, in vitro cefovecin protein binding was evaluated in plasma from 22 nondomestic species representing a broad range of taxa. Animals of the order Carnivora demonstrated protein-binding levels of >98%, which is supportive of the long elimination half-life seen in related species. Protein binding was highly variable in Artiodactyl and Perissodactyl species, with dolphins ( Tursiops truncatus ) and barasingha ( Rucervus duvaucelii ) displaying high protein binding (99.12% to >99%), but not gazelles ( Eudorcas thomsonii ) or equids (91.76-92.70%). Cefovecin was not highly bound in any reptiles or birds, corresponding to short half-lives reported for these taxa. These results suggest that a high percentage of plasma protein binding in vitro may predict in which species cefovecin may exhibit a long half-life and duration of action in vivo. These findings may aid in selecting species for cefovecin pharmacokinetic research and for empirical treatment of infections caused by susceptible bacteria.

The Burmese roofed turtle ( Batagur trivittata ), a critically endangered freshwater turtle, is endemic to Myanmar. Once thought to be extinct, remnant wild populations were discovered in 2001 and limited captive individuals identified in pagoda ponds or confiscated from fishers in Myanmar. These and their offspring are maintained in five facilities in Myanmar and form the basis of a conservation program (habitat protection, captive breeding, nest protection, egg collection, head-starting, and release). Prerelease health screenings were performed in 2014 and 2018 at Yadanabon Zoological Gardens, a head-starting facility in Limpha Village, and Lawkanandar Wildlife Park. One hundred forty-three turtles were assessed (37 male, 50 female, 56 juveniles [too young to determine sex]; two females were assessed in both years), age range of 1 to 12 y (one unknown age adult founder), and body mass range of 0.111 to 32.72 kg. Health evaluations both years included physical examination and combined choanal/cloacal swab samples for polymerase chain reaction testing of the potential chelonian pathogens intranuclear coccidia, Mycoplasma , Herpesvirus, Ranavirus, and Adenovirus (not all tests performed each year). In 2018, cloacal swabs from 30 and 20 turtles at the Yadanabon Zoological Gardens and Lawkanandar Wildlife Park, respectively, were cultured for Salmonella . All turtles were assessed as healthy based on normal physical examination findings, and all had negative test results. Prerelease health screening, such as performed in this study, is an important component of release, reintroduction, and translocation projects to prevent introduction of novel pathogens into naïve wild populations.

Ceftiofur crystalline free acid (CCFA) is a third-generation, oil-based, cephalosporin antimicrobial marketed as a once weekly treatment in cattle and swine, and as a two-time dose with 10-day duration in horses. Because handling and restraint times can be reduced, long-acting antibiotic preparations are particularly useful for treatment of nondomestic species. This study evaluated the pharmacokinetics of CCFA in ringneck doves ( Streptopelia risoria ). A single intramuscular (IM) injection of CCFA at 50 mg/kg was administered to each of 30 doves, and blood was collected from subsamples of 6 birds at predetermined sampling times (i.e., with a postinjection range of 0.5 to 192 hr). All ringneck doves were scheduled for euthanasia because of reasons unrelated to the study; this was performed at the conclusion of the study; and complete postmortem and histopathologic examinations were performed. Plasma concentrations of CCFA remained above the minimum inhibitory concentration (1.0 µg/ml; observed for most avian pathogenic bacteria) for 108 hr. No abnormalities were identified on individual birds before and after clinical pathology results (i.e., hematocrits and plasma biochemistry profiles), and only minimal gross and histopathologic changes such as mild tissue inflammation at the injection site were observed. Based on these results, one IM injection of CCFA at 50 mg/kg seems to be a potential option for treatment of ringneck doves.

The Blue Iguana Recovery Programme maintains a captive breeding and head-starting program for endangered Grand Cayman blue iguanas (Cyclura lewisi) on Grand Cayman, Cayman Islands. In May 2015, program staff encountered two lethargic wild Grand Cayman blue iguanas within the Queen Elizabeth II Botanic Park (QEIIBP). Spiral-shaped bacteria were identified on peripheral blood smears from both animals, which molecular diagnostics identified as a novel Helicobacter species (provisionary name Helicobacter sp. GCBI1). Between March 2015 and February 2017, 11 Grand Cayman blue iguanas were identified with the infection. Two of these were found dead and nine were treated; five of the nine treated animals survived the initial infection. Phylogenetic analysis of the 16S rRNA gene suggests Helicobacter sp. GCBI1 is most closely related to Helicobacter spp. in chelonians. We developed a Taqman qPCR assay specific for Helicobacter sp. GCBI1 to screen tissue and/or blood samples from clinical cases, fecal and cloacal samples from clinically healthy Grand Cayman blue iguanas, including previously infected and recovered iguanas, and iguanas housed adjacent to clinical cases. Fecal and/or cloacal swab samples were all negative, suggesting that Grand Cayman blue iguanas do not asymptomatically carry this organism nor shed this pathogen per cloaca post infection. Retrospective analysis of a 2014 mortality event affecting green iguanas (Iguana iguana) from a separate Grand Cayman location identified Helicobacter sp. GCBI1 in two of three cases. The source of infection and mode of transmission are yet to be confirmed. Analysis of rainfall data reveal that all infections occurred during a multi-year dry period, and most occurred shortly after the first rains at the end of seasonal drought. Additionally, further screening has identified Helicobacter sp. GCBI1 from choanal swabs of clinically normal green iguanas in the QEIIBP, suggesting they could be asymptomatic carriers and a potential source of the pathogen., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: One author (ISP) was commercially employed through the duration of the study and manuscript preparation (Island Veterinary Services and Newmarket Vets4Pets); funding from these businesses was limited to salary support. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger ( Panthera tigris jacksoni ) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers ( Panthera tigris altaica ) in the same building and three lions ( Panthera leo krugeri ) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids.

The blue iguana ( Cyclura lewisi ) is an endangered rock iguana species native to Grand Cayman, in the Cayman Islands. Health assessments were conducted on captive and free-roaming iguanas in 2001 and 2003-2014 and were performed in the summer wet season (June-July) of 2003-2004 and 2010-2014 and in the winter dry season (November-December) of 2001 and 2005-2009. Morphometric data were recorded from iguanas when blood samples were collected: 903 samples were collected and data from 890 samples from 775 iguanas were included. Samples were analyzed for hematology, plasma biochemistry, protein electrophoresis, mineral panels, 25-hydroxyvitamin D, and testosterone. Reference intervals were created for captive subadults, captive adults, and free-roaming adults when data were sufficient. Significant differences among these groups were described, as were differences on the basis of sex, season, and origin (captive vs free-roaming). In captive iguanas, most analytes were significantly different between subadults and adults, mature heterophils and copper were significantly higher in the dry season, zinc levels were significantly higher in the wet season, and cholesterol and triglycerides were significantly higher in adult females than adult males. Testosterone in adult males was significantly higher in the dry season. These results will aid in future health assessments and disease investigations in wild and captive populations of blue iguanas and are of comparative value for other Cyclura species that are free-roaming, captive, and, especially, in similar conservation release programs.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species. IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health., (Copyright © 2020 McAloose et al.)

This report describes the identification and characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a Malayan tiger in a U.S. zoo.

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife., Competing Interests: The authors have declared that no competing interests exist.

Feces ( n = 226; 2004-2015) from healthy captive and wild blue iguanas ( Cyclura lewisi ) from Grand Cayman, Cayman Islands, were examined for endoparasites. Parasites identified included Nyctotherus sp. and Entamoeba sp. cysts and trophozoites, trichomonad trophozoites, and oxyurid and trichostrongylid eggs. Endoparasites from postmortem examinations ( n = 13) included adult and larval nematodes: Ozolaimus megatyphlon , Ozolaimus monhystera , Alaeuris travassosi , Atractis mega , and an unidentified species of Oswaldocruzia . Entamoeba spp. were more likely in captive juveniles of both sexes than captive or wild adults of either sex; Entamoeba spp. were more likely in captive adult females than captive adult males; trichomonad trophozoites were more likely in adult captive and wild iguanas of both sexes than in captive juveniles of either sex; and Nyctotherus spp. were more likely in juvenile captive males than captive adult males or females and more likely in adult wild males than captive juvenile males. Trichostrongylid eggs were more likely in adult wild females than adult captive females and more likely in captive and wild adults of both sexes than in captive juveniles of both sexes. Oxyurid eggs were more likely in adult captive and wild iguanas of both sexes than captive juveniles of either sex. Blue iguanas have a variety of endoparasites regardless of age, sex, or captive vs wild status, with no type found exclusively in either captive or wild populations. Ectoparasites from wild adults included adult ticks ( Amblyomma torrei ) and a single adult mite ( Hirstiella trombidiformis ). All are new host records for this species and Grand Cayman. Knowledge of parasite status of captive and wild populations is important to evaluate the relative risk of introduction of captive animals into wild populations.

The land iguanas, Conolophus pallidus and Conolophus subcristatu are large and charismatic lizards endemic to the Galápagos archipelago, but little information exists on their normal health parameters. The former is restricted to Santa Fe island, while C. subcristatus inhabits the islands of the central and western region of the archipelago. Both species are classified as vulnerable by the IUCN Red List of Threatened Species. As part of a population health assessment authorized by the Galápagos National Park, wild adult iguanas from three islands (North Seymour, South Plazas, and Santa Fe) were captured in July 2018. Data from a single C. subcristatus X Amblyrhynchus cristatus hybrid captured on South Plazas is also included. We analyzed blood samples drawn from 52 healthy wild adult land iguanas captured on three islands. An iSTAT portable blood analyzer was used to obtain values for pH, lactate, pO2, pCO2, HCO3-, sO2%, hematocrit, packed cell volume (PCV), hemoglobin Na, K, iCa, and glucose. Standard laboratory hematology techniques were employed for PCV determination; resulting values were also compared to the hematocrit values generated by the iSTAT. Body temperature, heart rate, respiratory rate, and body measurements were also recorded and compared to previously published data for the marine iguana (Amblyrhynchus cristatus), which shares a common ancestor with the land iguana. The data reported here provide preliminary baseline values that may be useful in comparisons between captive and wild populations, between wild populations, and in detecting changes in health status among Galápagos land iguanas affected by anthropogenic threats, climate change, or natural disturbances., Competing Interests: The authors have declared that no competing interests exist.

The Bronx River in Bronx, New York, US spans an area of significant human development and has been subject to historic and ongoing industrial contamination. We evaluated the health of freeranging native common snapping turtles ( Chelydra serpentina) and nonnative invasive red-eared sliders ( Trachemys scripta) in a segment of the Bronx River between May and July 2012. In 18 snapping turtles and nine sliders, complete physical examinations were performed, ectoparasites collected, and blood was analyzed for contaminants (mercury, thallium, cadmium, arsenic, lead, selenium, oxychlordane, alpha-chlordane, dieldrin, DDD, DDE, polychlorinated biphenyls). Complete blood counts and the presence of hemoparasites were determined in 16 snapping turtles and nine sliders. Swabs of the choana and cloaca were screened for ranavirus, adenovirus, herpesvirus, and Mycoplasma spp. by PCR in 39 snapping turtles and 28 sliders. Both turtle species exhibited bioaccumulation of various environmental contaminants, particularly organochlorines and polychlorinated biphenyls. Molecular screening revealed a unique herpesvirus in each species. A Mycoplasma sp. previously isolated from emydid turtles was detected in red-eared sliders while a unique Mycoplasma sp. was identified in common snapping turtles. Ranaviruses and adenoviruses were not detected. Our study established a baseline health assessment to which future data can be compared. Moreover, it served to expand the knowledge and patterns of health markers, environmental contaminants, and microorganisms of freeranging chelonians.

Since cardiovascular, respiratory, and metabolic systems of reptiles are affected by temperature, accurate measurements are of great importance in both captive husbandry and research. Ectothermic animals generally have core body temperatures close to ambient temperature but can differ from the immediate environment if they are using sunlight to thermoregulate. Many zoological facilities and exotic pet caregivers have begun using infrared temperature guns to assess ambient temperatures of reptile enclosures but there are currently few studies assessing the efficacy of these devices for measuring the body temperatures of reptiles. Conolophus subcristatus , Conolophus pallidus (Galápagos land iguanas), and Amblyrhynchus cristatus X C. subcristatus hybrid are robust land iguanas endemic to the Galápagos archipelago. By comparing the infrared body temperature measurements of land iguanas against virtual simultaneous collection of cloacal temperatures obtained using a thermocouple thermometer, we sought to assess the efficacy of this non-invasive method. We found that internal body temperature can be predicted with a high level of accuracy from three external body temperature sites, providing a good non-invasive method that avoids the capture of animals., Competing Interests: The authors declare that they have no competing interests.

From 2005 to 2017, a total of 334 fresh fecal samples was obtained from 236 captive and free-ranging Grand Cayman iguanas ( Cyclura lewisi) in a captive breeding and release program. One-hundred-sixteen samples were obtained from individual captive iguanas living in enclosures with natural substrate, 161 samples from captive iguanas living in elevated wire-bottom enclosures, and 57 samples from free-ranging wild iguanas. These samples were cultured to isolate subspecies of Salmonella enterica. as part of prerelease health evaluations, and to determine baseline health assessments of captive and wild populations of these iguanas. There was a 5.45% (95% confidence interval [CI]: 1.14-15.12) prevalence of S. enterica in iguanas housed on natural substrate ( n = 51), 3.85% (CI: 1.42-8.19) in iguanas housed in elevated wire-bottom enclosures ( n = 157), and 6.06% (CI: 0.74-20.23) in wild free-ranging iguanas ( n = 38). These results demonstrate no significant difference ( P = 0.73) in S. enterica prevalence among these housing conditions. The incidence of S. enterica from 2005 to 2017 in the population sampled was 4.19% (CI 3.10-5.29). Ten different serotypes of S. enterica were isolated from 14 iguanas. Salmonella enterica ser. Saintpaul was the most frequent isolate. Annual S. enterica prevalence was calculated for iguanas housed in different enclosure types, for free-roaming wild iguanas, and for all cultured iguanas. The highest yearly prevalence was 23.08% (CI: 5.04-53.81) in iguanas tested in 2007 ( n = 21). No Salmonella enterica were cultured in 2005, 2008, 2009, 2010, 2011, 2012, and 2015. These results suggest that the shedding of S. enterica was not significantly different between housing types or between captive versus wild iguanas and therefore that release of captive iguanas did not significantly affect the health of the wild population or their exposure to S. enterica.

Langur alphaherpesvirus (HVL), a provisionally named alphaherpesvirus in the Simplexvirus genus, was first identified in 1991 at the Bronx Zoo in wild-origin silvered langurs ( Trachypithecus cristatus) and their descendants. HVL is closely related to B virus ( Macacine alphaherpesvirus 1) based on serologic and genetic data, but its natural history and zoonotic potential remain unknown. A cohort study was undertaken to describe the epidemiology, clinical impact, and potential management implications of this virus in a naturally infected, zoo-based population of silvered langurs. Opportunistic surveillance sampling from 1991 through 2015 resulted in 235 serum samples and 225 mucosal swabs from 75 individuals. A total of 43 individuals (57.3%) were seropositive for HVL within this period. Seroprevalence increased significantly with age, and indirect evidence suggested a peak in transmission at the onset of sexual maturity. These findings were similar to the behavior of other simplexviruses in their adapted hosts. Yearly cumulative incidence declined significantly through the study period, with zero or one new case detected each year from 2007 through 2015. The density of this population decreased within the study period for management reasons unrelated to HVL infection, and a change in age distribution or less-frequent contacts may have contributed to low transmission. In addition, clinical signs of simplexvirus infection were rare, and virus isolation was negative on all mucosal swabs, suggesting that viral shedding was infrequent. Yearly period seroprevalence remained relatively constant with a median of 45.8%, likely because of the extended survival of infected individuals within the population. Maintenance of a naturally occurring, novel virus with unknown zoonotic potential in a zoo population for over 25 yr highlights the importance of biosecurity and biosafety for management of silvered langurs and all primate species.

Southern River terrapins ( Batagur affinis) are among the most critically endangered turtles in the world. To augment the Cambodia population, a head-start program was established for the endemic subspecies Batagur affinis edwardmolli in 2006, and in 2015, prerelease health assessments were performed on 70 subadults (hatch years, 2006-2011). Combined choanal/cloacal swab samples ( n = 70) were collected and screened by polymerase chain reaction (PCR) for Mycoplasma, herpesvirus, and ranavirus. Cloacal samples ( n = 50) were also collected and cultured for Salmonella sp. Of 70 tested samples, six (8.6%) were positive for Mycoplasma, and all other PCR and culture test results were negative. Phylogenetic analysis of the 16S ribosomal RNA gene placed the Mycoplasma sp. from B. affinis edwardmolli in the chelonian Mycoplasma cluster that groups within the Mycoplasma pulmonis clade. This mollicute was not associated with clinical disease (defined as observable clinical abnormalities, such as depression, lethargy, respiratory signs, and anorexia) and is likely part of the endemic microbial flora of these terrapins.

We conducted health assessments on 113 free-ranging raccoons ( Procyon lotor ) in Central Park, New York City, US, in February 2010, September 2010, and November 2011 in conjunction with a trap-vaccinate-release program to control a raccoon rabies epizootic. Five individuals were sampled at two time points for 118 raccoon examinations in total. We tested 13 of 13 and 8 of 13 euthanized raccoons for rabies and canine distemper virus (CDV), respectively, by antigen testing on brain tissue; all were negative for both viruses. Endoparasitism was the most common necropsy finding, with definitive identification of Baylisascaris procyonis in six of eight (75%) necropsied raccoons. Multiple intestinal parasites were detected in feces of living raccoons, including ascarid-type ova in 25 of 80 (31%) raccoons, with B. procyonis confirmed in one sample. Median blood lead level was 7.3 μg/dL (n=104). Rabies virus neutralizing antibody titer was ≥0.5 IU/mL in 9 of 88 (10%) raccoons naive to rabies vaccination and in 13 of 20 (65%) previously vaccinated raccoons. The majority of raccoons we tested were seropositive for canine parvovirus-2 (54/59, 92%) and Toxoplasma gondii (39/60, 65%). Fewer were seropositive for Rickettsia rickettsii (3/30, 10%). None were seropositive for CDV (n=108), canine adenovirus-1 (n=60), or Borrelia burgdorferi (n=30). Ectoparasites found during 16 of 118 (13.6%) physical examinations included Ixodes texanus ticks (15/118, 12.7%) and Trichodectes octomaculatus lice (1/118, 0.8%). We detected Campylobacter jejuni in 5 of 79 (6%) fecal samples. We detected 11 Salmonella enterica serotypes in 70 of 111 (63.1%) enteric cultures, the most common of which were Salmonella Newport (20/70, 29%) and Salmonella Oranienburg (20/70, 29%). These results indicate that raccoons in Central Park likely are involved in the environmental occurrence and potential disease transmission of a variety of infectious and noninfectious diseases of concern for human, wildlife, and domestic animal health.

The use of frozen fecal samples in enrichment media to detect Salmonella spp. strains was evaluated in Indian star tortoises (Geochelone elegans) to determine the utility of this test method for field collection. Fresh fecal samples were collected from 10 captive adult Indian star tortoises. Each sample was split, with one portion in enrichment media at room temperature for 1 or 2 days before submission to a reference laboratory for standard enteric culture. The other was placed in the same enrichment media and frozen at -20°C for 2 wk. Afterwards, it was transferred to a -80°C freezer for 1 mo before submission to the same reference laboratory. These freezer temperatures and holding times were selected to replicate typical field collection procedures. Salmonella enterica was isolated from all 10 fresh samples but from only six frozen samples. Statistically, results showed no significant difference between the two methods; however, this may be due to the limited sample size, and if so, utilization of frozen fecal samples to determine Salmonella spp. prevalence may underestimate the actual prevalence.

Uropygial, or preen, glands are found in a variety of avian species including penguins. These glands have a multitude of functions and can develop a variety of conditions including impaction, rupture, adenitis, squamous metaplasia, and neoplasia of various types, with squamous cell carcinoma the most commonly reported. A case series of uropygial gland squamous cell carcinoma in five penguins at the Wildlife Conservation Society's Central Park Zoo is described. Most birds were aged (>10 yr) with a history of chronic, recurrent uropygial gland problems including impaction, rupture, abscess formation, or a combination of conditions. Before and after neoplasia diagnosis, these cases were managed conservatively, and palliative care was provided. Because many of these cases were preceded by chronic inflammation, it is possible this inflammation predisposed the uropygial gland to neoplastic transformation, and more aggressive treatment early in the disease process may therefore be warranted.

All anuran specimens in the Wildlife Conservation Society's collections testing positive for Batrachochytrium dendrobatidis (Bd) were treated with itraconazole and then studied after treatment to assess the long-term effects of itraconazole and the drug's effectiveness in eliminating Bd carriers. Twenty-four individuals and eight colonies of 11 different species (75 total specimens) tested positive for Bd via polymerase chain reaction (PCR) on multicollection survey. All positive individuals and colonies were treated with a 0.01% itraconazole bath solution and retested for Bd via one of two PCR methodologies within 14 days of treatment completion, and all were negative for Bd. A total of 64 animals received secondary follow-up PCR testing at the time of death, 6-8 mo, or 12-15 mo post-treatment. Fourteen animals (14/64, 21.9%) were PCR positive for Bd on second follow-up. The highest percentage positive at second recheck were green-and-black poison dart frogs (Dendrobates auratus; 5/5 specimens, 100%), followed by red-eyed tree frogs (Agalychnis callidryas; 4/11, 36.4%), grey tree frogs (Hyla versicolor; 1/3, 33.3%), and green tree frogs (Hyla cinera; 3/11, 27.3%). Re-testing by PCR performed on 26/28 individuals that died during the study indicated 11/26 (42.3%) were positive (all via DNA extracted from formalin-fixed paraffin-embedded skin sections). However, there was no histologic evidence of chytridiomycosis in any of 27/28 individuals. The small number of deceased animals and effects of postmortem autolysis limited the ability to determine statistical trends in the pathology data, but none of the necropsied specimens showed evidence of itraconazole toxicity. Problems with itraconazole may be species dependent, and this report expands the list of species that can tolerate treatment. Although itraconazole is effective for clearance of most individuals infected with Bd, results of the study suggest that repeat itraconazole treatment and follow-up diagnostics may be required to ensure that subclinical infections are eliminated in amphibian collections.

Nonspecific responses to intradermal tuberculin skin testing (TST) have been frequently documented in silvered langurs (Trachypithecus cristatus ultima) at the Wildlife Conservation Society's Bronx Zoo. A commercial rapid lateral flow immunoassay (RT) for the identification of Mycobacterium tuberculosis and M. bovis antibodies was employed to assess this test's correlation with TST results and tuberculosis status. Twenty-two silvered langurs were tested with both TST and RT. TST results were characterized into three groups as nonspecific tuberculin responses (n=9), responses to avian tuberculin only (n=6), or negative (n=7). Rapid lateral flow test results were negative in 20 of 22 individuals with equivocal results in two avian tuberculin responders. There was no difference in RT results between TST groups (P = 0.178). Rapid lateral flow test results were not in agreement with mammalian old tuberculin TST results (kappa = 0.08) but showed raw agreement with tuberculosis status (proportion of overall agreement = 0.909). Results suggest the RT assay may useful in the confirmation of nonspecific TST responders in silvered langurs.

Seven captive adult male guanacos (Lama guanicoe) weighing 112.0 +/- 10.9 kg (mean +/- standard deviation) were anesthetized with a combination of medetomidine (90.0 +/- 8.8 microg/kg), ketamine (2.7 +/- 0.3 mg/kg), and butorphanol (0.3 +/- 0.03 mg/kg) administered intramuscularly to evaluate its anesthetic and cardiopulmonary effects. Inductions were smooth and rapid, with a mean time to initial effect of 3 +/- 1.5 min and a mean time to recumbency of 5.1 +/- 3.1 min. Anesthesia was predictable, smooth, and characterized by excellent muscle relaxation. Spontaneous ventilation was maintained throughout anesthesia in all animals. Marked bradycardia ranging from 24 to 52 beats/min was noted across all time points for all individuals. Median heart rates decreased during the procedures, but median heart rate, temperature, respiratory rate, and end-tidal carbon dioxide values over the 20 min monitoring period were not significantly different. Mean arterial partial pressure of oxygen (PaO2) on initial sampling was 65.9 +/- 14.8 mm Hg, with six of seven animals exhibiting hypoxemia (PaO2 <80 mm Hg). After oxygen supplementation for 20 min, mean PaO2 values showed statistically significant increases to a mean value of 127.7 +/- 32.4 mm Hg (P = 0.0014). Mean arterial partial pressure of carbon dioxide (PaCO2) showed a significant increase over the monitoring period (P = 0.0004), and mild hypoventilation (PaCO2 >45 mm Hg) was noted in four animals. Mean total duration of procedure time was 23.3 +/- 3.3 min. All guanacos received 0.45 +/- 0.04 mg/kg atipamezole and 2.7 +/- 0.25 mg/kg naltrexone administered intramuscularly for anesthetic antagonism. Recoveries were smooth and uncomplicated. Mean time to sternal recumbency after antagonist administration was 7.7 +/- 4.5 min, and time to successful standing was 12.9 +/- 5.0 min, with all animals standing on first attempt.

Hepatic hemosiderosis is one of the most common postmortem findings in captive callitrichid species. Noninvasive evaluation of hematologic iron analytes has been used to diagnose hepatic iron storage disease in humans, lemurs, and bats. This study evaluated the relationship between hematologic iron analyte values (iron, ferritin, total iron binding capacity, and percent transferrin saturation) and hepatic hemosiderosis in callitrichids at the Wildlife Conservation Society's Central Park and Bronx Zoos. Results revealed that both ferritin and percent transferrin saturation levels had strong positive correlations with hepatic iron concentration (P<0.001, r=0.77, n=20; P<0.001, r=0.85, n=10, respectively). Serum iron levels positively correlated with hepatic iron concentration (P=0.06, r=0.56, n=11), but this finding was not significant. Serum total iron binding capacity did not significantly correlate with hepatic iron concentration (P=0.47, r=0.25, n=10). Both ferritin and hepatic iron concentration positively correlated with severity of hepatic iron deposition on histology (P<0.05, r=0.49, n=21; P<0.001, r=0.67, n=21, respectively). This study suggests that ferritin, serum iron concentration, and percent transferrin saturation are convenient, noninvasive, antemortem methods for assessing severity of hemosiderosis in callitrichids.

Thiafentanil oxalate, previously known as A-3080, is a synthetic opioid used for chemical immobilization of a variety of nondomestic hoofstock species. This study compared the combination of thiafentanil oxalate, medetomidine, and ketamine (TMK; 0.09 +/- 0.02 mg/kg, 0.01 +/- 0.003 mg/kg, and 1.36 +/- 0.33 mg/kg, respectively) with the combination of medetomidine and ketamine (MK; 0.09 +/- 0.02 mg/kg and 3.48 +/- 0.55 mg/kg, respectively) for anesthetization of 17 captive male axis deer (Axis axis) for vasectomy. Nine deer received TMK and eight deer received MK via projectile syringe during the months of January and February, 2005. Mean induction and arousal times, vital signs, and arterial blood gas values were monitored and compared. All animals received supplemental oxygen during the surgical procedure. Animals receiving TMK were reversed with naltrexone (100 mg/mg thiafentanil) and atipamazole (5 mg/mg medetomidine). Animals receiving MK were reversed with atipamazole (5 mg/mg medetomidine). Two MK animals and three TMK animals required supplementation with ketamine i.v. immediately upon handling. Six of the nine animals immobilized with TMK required intubation for positive-pressure ventilation. Two of these six animals also required isoflurane to maintain anesthesia. Mean induction time was 3.5 +/- 2.0 min in the TMK group, and 9.8 +/- 6.7 min in the MK group. Despite shorter mean induction times, animals anesthetized with TMK experienced unpredictable inductions, apnea, muscle rigidity, limb movement, and significant respiratory and metabolic lactic acidosis. MK resulted in smoother inductions, better respiratory function, and less adverse metabolic disturbances, and thus was considered superior to TMK for anesthesia in captive axis deer at the dosages tested.

Published serum cholesterol values in captive western lowland gorillas (Gorilla gorilla gorilla) are much higher than human ranges, with a national mean of 7.36 mmol/L (284 mg/dl, n = 863). Complete blood lipid profiles were examined in 15 captive gorillas. High-density lipoprotein (HDL) was found to decrease more rapidly with age than total cholesterol, resulting in an increasing ratio of cholesterol HDL with age. The ratio of apolipoprotein B to apolipoprotein Al also increased with age. Establishment of a database of blood lipid values for captive gorillas with correlative analysis of animals with known atherosclerosis status may help to identify sensitive predictors of coronary heart disease risk.

Two adult Wied's marmosets (Callithrix kuhlii) presented with jaundice, anemia, and weight loss. Death of one individual was attributed to renal tubular necrosis; liver and kidney were positive for Leptospira antigen by immunohistochemical (IHC) staining. The second animal was negative for antigen by IHC staining, but serologically positive for Leptospira borgpetersenii serovar ballum with an eightfold titer increase in paired samples, and was euthanized because of unresponsiveness to treatment. Environmental contamination by mice was suspected as the Leptospira source.

A 7-mo-old male alpaca (Lama pacos) presented with an abnormal lowered posture of the head and neck and reluctance to walk. Cervical radiographs demonstrated atlantooccipital luxation. Successful manual closed reduction was achieved while the animal was anesthetized, resulting in complete return to normal gait and posture.

Two adult North American river otters (Lontra canadensis) and an adult red panda (Ailurus fulgens fulgens) at three separate institutions died within 22 hr after receiving single 2.5- to 2.7-mg/kg doses of melarsomine dihydrochloride administered in the epaxial musculature as a treatment for filarid nematodes. One otter had a suspected Dirofilaria immitis infection, the other had a confirmed D. lutrae infection, and the red panda had a confirmed Dirofilaria sp. infection, presumably with D. immitis. Postmortem examinations revealed similar gross lesions, although they were less severe in the red panda. The trachea and primary bronchi contained abundant foamy fluid, the lungs were mottled with areas of consolidation, and the pulmonary parenchyma exuded abundant fluid at the cut section. Histologic evaluation revealed acute pulmonary edema, which resulted in respiratory failure and death. There may have been direct pulmonary cellular toxicity of melarsomine dihydrochloride or a severe systemic anaphylactic reaction to antigens released after parasite death. An idiosyncratic drug reaction or a low therapeutic index of melarsomine probably caused the death of the three individuals. Melarsomine dihydrochloride use should be avoided in North American river otters and red pandas.

An outbreak of West Nile virus (WNV) in and around New York City during the late summer of 1999 was the cause of extensive mortality among free-ranging birds. Within the Bronx Zoo/Wildlife Conservation Park, viral activity was also observed and produced some morbidity and mortality among specimens in the zoo's bird collection and probably caused morbidity in at least one specimen from the zoo's mammal collection. To determine the extent of the outbreak and attempt to ascertain the temporal appearance of virus within the park, a serologic survey of birds and mammals was performed. The survey showed that 34% of tested birds (125 of 368; 124 species) were positive for antibody to WNV. The virus caused a disease to infection ratio of 22% (27 of 125) among birds with a 70% (19 of 27) case fatality rate. In contrast, only 8% of the mammals (9 of 117; 35 species) possessed antibody to WNV and there was no virus-associated mortality. Testing of banked and fresh sera obtained from both birds and mammals revealed that there was no evidence of WNV circulation before the 1999 outbreak and that birds introduced into the park were not the source of the New York outbreak. West Nile virus RNA was detected in tissues from one bird that died in February 2000, long after the end of the mosquito transmission season. The potential importance of zoologic parks as possible sentinels for emerging diseases is discussed.

Serum or heparinized plasma samples were obtained between 1994 and 1996 from 20 male and 20 female adult free-ranging Pacific walrus (Odobenus rosmarus divergens) from St. Lawrence Island and Round Island, Alaska. Samples were screened for antibodies to some potentially pathogenic bacteria and viruses. No sample had detectable antibody to Brucella spp. Three of 40 (8%) had low antibody titers to Leptospira interrogans serovars. Phocine distemper virus antibodies were not detected. Serologic responses to one or more caliciviruses (San Miguel sea lion virus 12 or vesicular exanthema of swine serotypes E54, F55, G55, 1934B) were detected in 18% (seven of 40) walrus. Antibodies to one or more subtypes of influenza A virus (H10, N2, N3, N5, N6, N7) were detected in 21% (eight of 38). Periodic screening of free-ranging populations for exposure to infectious diseases has become an important component of bio-monitoring programs to facilitate understanding and detecting trends in marine mammal populations.

Reptiles can harbor pathogenic microorganisms asymptomatically and serve as potential reservoirs of infection for humans, domestic animals, and other reptiles. Infectious diseases are also problematic for free-ranging reptile populations and are an important consideration in reptile reintroduction and translocation projects. There have been limited serologic studies of free-ranging reptiles for evidence of exposure to potential pathogens. In the present study, serum or plasma samples from five male and five female free-ranging Venezuelan anacondas (Eunectes murinus) were screened for antibodies to eastern, western, and Venezuelan equine encephalitis viruses, vesicular stomatitis virus, ophidian paramyxovirus, 19 Leptospira interrogans serovars, and Cryptosporidium serpentes. Antibodies to these agents were not detected, or antibody titers were low and possibly nonspecific. These results for the limited number of anacondas surveyed suggest that they do not serve as significant reservoirs for these infectious agents at this location.

The GnRH analogue deslorelin, in long-acting implants, was used in an attempt to temporarily control reproduction or aggression in wild carnivores in southern Africa and the USA. In the southern African study, 6 mg deslorelin was administered to cheetahs (eight females, four males), one female leopard and wild dogs (six females, one male) housed in groups, and 12 mg deslorelin was administered to two lionesses. None of the animals became pregnant after deslorelin administration apart from one wild dog that was mated at the initial treatment-induced oestrus. Two wild dogs and one lioness came into oestrus 12 and 18 months after deslorelin administration, respectively, thus demonstrating that the anti-fertility effects of deslorelin are reversible. Two lionesses and four cheetahs underwent oestrus without allowing mating 2-14 days after treatment. Simultaneous administration of progestins to three bitches and one lioness did not suppress oestrus. Male cheetahs had no spermatozoa on day 82 after treatment and did not impregnate two untreated females. Of three untreated female wild dogs housed with treated males, only the first female to enter oestrus (21 days after deslorelin administration) became pregnant. One month after treatment, plasma testosterone concentrations of male dogs were at basal values. In the USA study, three male sea otters that had been treated with 6 mg deslorelin ceased antagonistic behaviour and blood testosterone concentrations and size of the testes were still sharply reduced 24 months after treatment. Male red (n = 7) and grey (n = 5) wolves received 6 mg deslorelin in December 1998 but no effects on seasonal spermatogenesis and behaviour were observed. In a black-footed cat, sperm production, libido and aggressiveness decreased in response to treatment with 3 mg deslorelin and penile spines were not observed within 3 months after treatment, but were observed again 4-6 months later. Treatment of female red (n = 5) and grey (n = 5) wolves with deslorelin in December 1999 triggered preseason oestrus and mating, which were followed by one abortion and one successful pregnancy. Contraception was achieved in female Fennec foxes (n = 7) and two lionesses, which was observed in the foxes by an absence of increases in faecal progesterone concentrations. In two male bush dogs, administration of 3 mg deslorelin once or twice was insufficient to suppress reproductive function or behaviour.

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.

West Nile fever caused fatal disease in humans, horses, and birds in the northeastern United States during 1999. We studied birds from two wildlife facilities in New York City, New York, that died or were euthanatized and were suspected to have West Nile virus infections. Using standard histologic and ultrastructural methods, virus isolation, immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction, we identified West Nile virus as the cause of clinical disease, severe pathologic changes, and death in 27 birds representing eight orders and 14 species. Virus was detected in 23/26 brains (88%), 24/ 25 hearts (96%), 15/18 spleens (83%), 14/20 livers (70%), 20/20 kidneys (100%), 10/13 adrenals (77%), 13/ 14 intestines (93%), 10/12 pancreata (83%), 5/12 lungs (42%), and 4/8 ovaries (50%) by one or more methods. Cellular targets included neurons and glial cells in the brain, spinal cord, and peripheral ganglia; myocardial fibers; macrophages and blood monocytes; renal tubular epithelium; adrenal cortical cells; pancreatic acinar cells and islet cells; intestinal crypt epithelium; oocytes; and fibroblasts and smooth muscle cells. Purkinje cells were especially targeted, except in crows and magpies. Gross hemorrhage of the brain, splenomegaly, meningoencephalitis, and myocarditis were the most prominent lesions. Immunohistochemistry was an efficient and reliable method for identifying infected cases, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses. In contrast, the in situ hybridization probe pWNV-E (WN-USAMRIID99) reacted only with West Nile virus. These methods should aid diagnosticians faced with the emergence of West Nile virus in the United States.

Twelve babirusa (Babyrousa babyrussa) (four females/eight males) were immobilized 30 times during a 4-yr interval. Significantly higher premedication and immobilizing doses were needed for females than for males (P < 0.05). An i.m. preanesthetic xylazine dose of 1.88 +/- 0.37 mg/kg (range = 1.20-2.12 mg/kg) was used for females and 1.22 +/- 0.16 mg/kg (range = 0.82-1.43 mg/kg) for males. After xylazine, the animals were induced with i.m. tiletamine/zolazepam; females received 2.20 +/- 0.47 mg/kg (range = 1.78-3.33 mg/kg) and males received 1.71 +/- 0.34 mg/kg (range = 1.08-2.05 mg/kg). Anesthesia was reversed with yohimbine (0.14 +/- 0.03 mg/kg; range = 0.07-0.20 mg/kg) and flumazenil (1 mg flumazenil/20 mg zolazepam) either i.m. or i.v. This anesthetic combination produced smooth induction, good relaxation, and sufficient immobilization to perform routine diagnostic and therapeutic procedures (venipuncture, hoof and tusk trims, transportation, radiographs, ultrasound examination, weight determinations, and skin biopsies). Supplemental ketamine HCl or isoflurane was administered to two animals to effectively deepen or prolong the anesthetic plane, with no resultant adverse effects.

From January through November 1994, 32% (7/22) of koi carp (Cyprinus carpio) maintained in indoor aquariums developed proliferative cutaneous lesions that consisted of single to multiple 2-10-mm whitish to pink fleshy masses usually associated with fin rays. Although scaleless koi were more commonly affected (3/6) than were normally scaled koi (4/16), the difference in incidence rates was not significant (chi2 text, P > 0.05). Lesions typically resolved spontaneously in 1-3 wk, occasionally persisted for >3 mo, and recurred in several fish after 2-5 mo. Fish were otherwise asymptomatic. Wet mount preparations from lesions were densely cellular and consisted of hyperplastic epidermal cells of normal morphology without parasites or inflammatory cells. Histologically, biopsies were consistent with papillomas and were characterized by a marked benign epidermal hyperplasia without inclusion bodies or inflammatory infiltrate. Transmission electron microscopic examination revealed intranuclear and intracytoplasmic herpesvirus virions. Virus isolation attempts were unsuccessful.

During the fall of 1995 and the spring of 1996, 77 statistically comparable tick collections, comprising 792 specimens, were made from adults of the Russian spur-thighed tortoise, Testudo graeca nikolskii, at 4 sites along Russia's Black Sea coast. These are the first tick collections reported from T. g. nikolskii since its recognition as a taxonomic entity. All ticks were determined to be Hyalomma (Hyalomma) aegyptium, a common tortoise parasite in southern Russia that in 1930 was erroneously designated the type of subgenus Hyalommasta. Male ticks were recovered from more tortoises (67) than were females (57) or immatures (14), and nymphs were seen only in the fall. Significantly more ticks parasitized male tortoises than females, perhaps because males of T. g. nikolskii have larger home ranges. However, no functional relationship was found between tortoise ventral surface area and degree of tick infestation. Like other tortoise ticks, H. aegyptium is expected to decline in concert with its increasingly threatened hosts.

Mallards and black ducks (n = 409) killed by hunters during the 1980 and 1981 hunting seasons in Pennsylvania (Susquehanna River and Crawford County) were examined to evaluate the effectiveness of regulations that converted the studied areas from lead to steel shotgun pellets in 1977-1978. Gizzards were examined for ingested lead and steel shot, and liver specimens were analyzed for lead. Since there is no evidence to suggest that ducks preferentially ingest steel or lead shot, it was concluded that ducks with steel shot would have contained lead shot. Therefore, we concluded that the conversion to steel shotgun pellets accounted for the decreased prevalence of ingested lead shot from 11.2% to 5.6%. Toxic concentrations of lead (greater than or equal to 6 ppm, wet weight) in the liver were found in 6 of 23 ducks that contained lead shot, whereas only 2 of 386 ducks without lead pellets had toxic concentrations. It was concluded that the conversion to steel shotgun pellets in the studied areas probably has decreased the exposure of ducks to lead shot, thereby decreasing the potential for lead poisoning.

The necropsy files of the National Zoological Park and Baltimore Zoological Society were reviewed for cases of distal extremity necrosis (DEN) in birds. Nineteen cases of DEN occurred following either trauma or frostbite. Six birds developed an apparently primary type of DEN in which no predisposing factors were obvious clinically. The toes and feet were most commonly involved, and in several cases the beak was also affected. Some pathologic evidence is provided that certain cardiovascular lesions may predispose birds to DEN by compromising circulation of the extremities.