Dried blood spot analysis for elements of nutritional concern as demonstrated in studies of Galápagos land iguanas (Conolophus species).

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.

HELICOBACTER SCREENING OF GRAND CAYMAN BLUE IGUANA (CYCLURA LEWISI) AND NORTH ANTILLEAN SLIDER (TRACHEMYS DECUSSATA ANGUSTA) ON GRAND CAYMAN, CAYMAN ISLANDS.

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.

SARS-COV-2 INFECTION AND LONGITUDINAL FECAL SCREENING IN MALAYAN TIGERS (PANTHERA TIGRIS JACKSONI), AMUR TIGERS (PANTHERA TIGRIS ALTAICA ), AND AFRICAN LIONS (PANTHERA LEO KRUGERI) AT THE BRONX ZOO, NEW YORK, USA.

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.

BURMESE ROOFED TURTLE (BATAGUR TRIVITTATA) DISEASE SCREENING IN MYANMAR.

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.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

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.

Health assessment of Conolophus subcristatus, Conolophus pallidus, and C. subcristatus X Amblyrhynchus cristatus hybrid (Galápagos land iguanas).

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.

HEALTH ASSESSMENT OF FREE-RANGING CHELONIANS IN AN URBAN SECTION OF THE BRONX RIVER, NEW YORK, USA.

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.