Hematological and biochemical parameters of giant pandas (Ailuropoda melanoleuca) in captive and semi-natural environments.

Physiological indexes like blood parameters have been widely used to monitor the health of free-roaming animals. Attempts to reintroduce one of China's most endangered species, the giant panda (Ailuropoda melanoleuca), have been hampered by a lack of data on its ecology and physiology. We examined three giant pandas' hematological and blood chemistry parameters in a soft release program and 30 captive giant pandas as controls and determined the reference intervals (RIs) for those blood parameters in the captive animals. Elevation, captivity status and the interaction of those factors were statistically significant for hematologic measures. Release pandas had significantly higher hemoglobin and hematocrit values after they moved to high elevation locations. We also found significant difference in the enzyme parameters between high and low elevation pandas such as higher aspartate aminotransferase, alanine aminotransferase, creatinine kinase, amylase and lower lactate dehydrogenase and alkaline phosphatase. Release pandas also had higher nutrition parameter values such as higher albumin, globulin and creatinine. The RI for blood parameters in our study provides a baseline to monitor the health of captive animals and forms the basis for assessing the health of free-roaming giant pandas in future reintroduction efforts.

Correction: Detection of novel coronaviruses in bats in Myanmar.

[This corrects the article DOI: 10.1371/journal.pone.0230802.].

Assessing Behavioral Risk Factors Driving Zoonotic Spillover Among High-risk Populations in Myanmar.

The increasing global emergence of zoonoses warrants improved awareness of activities that predispose vulnerable communities to greater risk of disease. Zoonotic disease outbreaks regularly occur within Myanmar and at its borders partly due to insufficient knowledge of behavioral risks, hindering participatory surveillance and reporting. This study employed a behavioral surveillance strategy among high-risk populations to understand the behavioral risks for zoonotic disease transmission in an effort to identify risk factors for pathogen spillover. To explore behavioral mechanisms of spillover in Myanmar, we aimed to: (1) evaluate the details around animal contact and types of interaction, (2) assess the association between self-reported unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) and animal contact activities and (3) identify the potential risk factors including behavioral practices of self-reported illness. Participants were enrolled at two community sites: Hpa-An and Hmawbi in Southern Myanmar. A behavioral questionnaire was administered to understand participants' animal exposures, behaviors and self-reported illnesses. From these responses, associations between (1) animal contact activities and self-reported unusual illnesses, and (2) potential risk factors and self-reported unusual illness were tested. Contact with poultry seemed to be very frequent (91.1%) and many participants reported raising, handling and having poultry in their houses as well as slaughtering or being scratched/bitten by them, followed by contact with rodents (57.8%) and swine (17.9%). Compared to participants who did not have any unusual symptoms, participants who had unusual symptoms in the past year were more likely to have sold dead animals (OR = 13.6, 95% CI 6.8-27.2), slaughtered (OR = 2.4, 95% CI 1.7-3.3), raised (OR = 3.4, 95% CI 2.3-5.0) or handled animals (OR = 2.1, 95% CI 1.2-3.6), and had eaten sick (OR = 4.4, 95% CI 3.0-6.4) and/or dead animals (OR = 6.0, 95% CI 4.1-8.8) in the same year. Odds of having reported unusual symptoms was higher among those involved in animal production business (OR = 3.4, 95% CI 1.9-6.2) and animal-involved livelihoods (OR = 3.3, 95% CI 1.5-7.2) compared to other livelihoods. The results suggest that there is a high level of interaction between humans, livestock and wild animals in communities we investigated in Myanmar. The study highlights the specific high-risk behaviors as they relate to animal contact and demographic risk factors for zoonotic spillover. Our findings contribute to human behavioral data needed to develop targeted interventions to prevent zoonotic disease transmission at human-animal interfaces.

Sylvatic Transmission of Chikungunya Virus among Nonhuman Primates in Myanmar.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Predicting the potential for zoonotic transmission and host associations for novel viruses.

Host-virus associations have co-evolved under ecological and evolutionary selection pressures that shape cross-species transmission and spillover to humans. Observed virus-host associations provide relevant context for newly discovered wildlife viruses to assess knowledge gaps in host-range and estimate pathways for potential human infection. Using models to predict virus-host networks, we predicted the likelihood of humans as hosts for 513 newly discovered viruses detected by large-scale wildlife surveillance at high-risk animal-human interfaces in Africa, Asia, and Latin America. Predictions indicated that novel coronaviruses are likely to infect a greater number of host species than viruses from other families. Our models further characterize novel viruses through prioritization scores and directly inform surveillance targets to identify host ranges for newly discovered viruses.

Salivary Gland Adenoma in a North Island Brown Kiwi (Apteryx mantelli).

A 7-month-old male North Island brown kiwi (Apteryx mantelli) was transferred from a European zoological park to a North American private aviary. The bird appeared clinically normal upon arrival, but within 30 days, it developed clinical disease signs of sneezing, stridor, and increased respiratory effort. Upon examination a 1-mm yellow plaque was identified on the glottis. After 14 days of empiric treatment with enrofloxacin, meloxicam, and itraconazole, on repeat examination, the glottal plaque had increased in size to a 7-mm-diameter friable mass attached to the right laryngeal cartilage and was partially obstructing the airway. Initially, 80% of the mass was manually dislodged, but within 4 days the tissue mass had again enlarged to approximately 3 mm × 4 mm × 4 mm, prompting surgical excision. On histopathologic examination, the mass was identified as a benign salivary gland adenoma without clear margins. Approximately 3 years postexcision, there is no evidence of tumor regrowth. The successful excision of a benign salivary tumor described in this report will serve as a valuable guide for future cases of similar neoplasms diagnosed in the oral cavity of kiwis and other avian species.

Living Safely With Bats: Lessons in Developing and Sharing a Global One Health Educational Resource.

As part of a public health behavior change and communication strategy related to the identification of a novel ebolavirus in bats in Sierra Leone in 2016, a consortium of experts launched an effort to create a widely accessible resource for community awareness and education on reducing disease risk. The resulting picture book, Living Safely With Bats, includes technical content developed by a consortium of experts in public health, animal health, conservation, bats, and disease ecology from 30 countries. The book has now been adapted, translated, and used in more than 20 countries in Africa and Asia. We review the processes used to integrate feedback from local stakeholders and multidisciplinary experts. We also provide recommendations for One Health and other practitioners who choose to pursue the development and evaluation of this or similar zoonotic disease risk mitigation tools.

Field metabolic rates of giant pandas reveal energetic adaptations.

Knowledge of energy expenditure informs conservation managers for long term plans for endangered species health and habitat suitability. We measured field metabolic rate (FMR) of free-roaming giant pandas in large enclosures in a nature reserve using the doubly labeled water method. Giant pandas in zoo like enclosures had a similar FMR (14,182 kJ/day) to giant pandas in larger field enclosures (13,280 kJ/day). In winter, giant pandas raised their metabolic rates when living at - 2.4 °C (36,108 kJ/day) indicating that they were below their thermal neutral zone. The lower critical temperature for thermoregulation was about 8.0 °C and the upper critical temperature was about 28 °C. Giant panda FMRs were somewhat lower than active metabolic rates of sloth bears, lower than FMRs of grizzly bears and polar bears and 69 and 81% of predicted values based on a regression of FMR versus body mass of mammals. That is probably due to their lower levels of activity since other bears actively forage for food over a larger home range and pandas often sit in a patch of bamboo and eat bamboo for hours at a time. The low metabolic rates of giant pandas in summer, their inability to acquire fat stores to hibernate in winter, and their ability to raise their metabolic rate to thermoregulate in winter are energetic adaptations related to eating a diet composed almost exclusively of bamboo. Differences in FMR of giant pandas between our study and previous studies (one similar and one lower) appear to be due to differences in activity of the giant pandas in those studies.

Movements of Indian Flying Fox in Myanmar as a Guide to Human-Bat Interface Sites.

Frugivorous bats play a vital role in tropical ecosystems as pollinators and seed dispersers but are also important vectors of zoonotic diseases. Myanmar sits at the intersection of numerous bioregions and contains habitats that are important for many endangered and endemic species. This rapidly developing country also forms a connection between hotspots of emerging human diseases. We deployed Global Positioning System collars to track the movements of 10 Indian flying fox (Pteropus giganteus) in the agricultural landscapes of central Myanmar. We used clustering analysis to identify foraging sites and high-utilization areas. As part of a larger viral surveillance study in bats of Myanmar, we also collected oral and rectal swab samples from 29 bats to test for key emerging viral diseases in this colony. There were no positive results detected for our chosen viruses. We analyzed their foraging movement behavior and evaluated selected foraging sites for their potential as human-wildlife interface sites.

EVALUATION OF PASSIVE TRANSFER IN NINE SPECIES OF CERVIDAE.

The reliability of packed cell volumes (PCV), total solids (TS), blood glucose (BG), γ-glutamyl transferase (GGT), and glutaraldehyde test in determining passive transfer of colostral immunoglobulins was investigated in nine species of cervids: axis deer (Axis axis), hog deer (Axis porcinus), sika deer (Cervus nippon), tufted deer (Elaphodus cephalophus), Père David's deer (Elaphurus davidianus), pudu (Pudu puda), sambar deer (Rusa unicolor), barasinga deer (Rucervus duvaucelli), and Eld's deer (Rucervus eldii). Individually the parameters evaluated were significant though imperfect predictors of passive transfer status in cervids. Interpreted collectively as a panel along with neonate condition, these tests were clinically helpful in diagnosing failure of passive transfer (FPT). Collectively interpreting test results as a panel along with clinical assessment of the animal is recommended. Some species-specific variations in TS, GGT, and glutaraldehyde test results were identified.

PHARMACOKINETICS AND CLINICAL SAFETY OF A SUSTAINED-RELEASE FORMULATION OF CEFTIOFUR CRYSTALLINE FREE ACID IN RINGNECK DOVES (STREPTOPELIA RISORIA) AFTER A SINGLE INTRAMUSCULAR INJECTION.

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.

SERUM BIOCHEMISTRY AND SELECT MINERAL PARAMETERS OF PRE-RELEASE SUNDA PANGOLINS (MANIS JAVANICA) FOLLOWING REHABILITATION IN VIETNAM.

Native to Southeast Asia, the Sunda pangolin (Manis javanica) is critically endangered largely because of poorly regulated wildlife trade, consumptive practices, and use in traditional Chinese medicine. Efforts to rescue and rehabilitate animals confiscated from the illegal trade are complicated by a general lack of knowledge surrounding the normal health and disease processes unique to the species. To provide clinical reference intervals for normal health states of Sunda pangolins, biochemical parameters were determined from rescued individuals in Vietnam that had undergone a 14-day observation period and met a set of criteria for release back into the wild. Blood samples were collected from 42 apparently healthy Sunda pangolins while anesthetized or awake. Packed cell volume (PCV) and total solids (TS) were determined manually, and serum biochemistry values were determined in-house with a benchtop analyzer. Additional biochemical and mineral parameters not included in the primary panel were determined from a subset of 10 pangolins through an external diagnostic laboratory. Overall reference intervals were calculated for PCV and TS (n = 29) and for standard serum biochemistry parameters (n = 42). Females and males demonstrated significant variation with respect to body mass, potassium (K+), and phosphorus, whereas age was a significant source of variation in alkaline phosphatase. Seasonal variation in glucose (GLU), creatinine (CRE), total proteins, sodium, calcium, and K+ was also observed. Comparisons between anesthetized and awake pangolins demonstrated significant variation in GLU, CRE, and K+. The parameters determined in this study can serve as a clinical reference for ex situ Sunda pangolin conservation efforts. In the context of wildlife rehabilitation, serial bloodwork allows for continued monitoring of patient health and should inform decision making regarding release readiness and timing.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Will the COVID-19 outbreak be a turning point for China's wildlife protection: New developments and challenges of wildlife conservation in China.

On 24 February 2020, at the brink of the global coronavirus disease 2019 (COVID-19) pandemic, the Chinese legislature adopted a decision to ban all terrestrial wildlife for food consumption. This decision set the tone for the revision of the Wildlife Protection Law (WPL), planned for release in late 2020. Both the terrestrial wildlife ban and revised WPL have the potential to greatly reduce consumption of wildlife and aid in prevention of future zoonotic disease outbreaks. The government still faces severe challenges in reducing wildlife use in traditional medicine, strengthening habitat and ecosystem conservation, committing to long-term WPL enforcement, and promoting community education and institutional reforms. However, recent progress in establishing a stricter ban on wildlife consumption, consolidating fragmented protected areas, and increased openness to public oversight are promising developments. While it is clear that global pandemics like COVID-19 can cause massive human suffering and disruption of economies; governments can no longer allow business as usual for the wildlife industry, regardless of the monetary or cultural values it brings. Here we discuss the latest development and limitations of the current wildlife legislation in China and the recommendations for improving Chinese wildlife conservation to better protect biodiversity and reduce risks of spreading zoonotic diseases to humans.

Use of a Portable Analyzer for Venous Blood Gas and Biochemistry Analysis in Free-Ranging Indian Flying Foxes (Pteropus giganteus) in Myanmar.

We determined venous blood gas, acid-base, and biochemical parameters for thirteen free-ranging Indian flying foxes (Pteropus giganteus) in Myanmar, using a handheld i-STAT analyzer with CG8+ and CHEM8 cartridges. For field-based projects, portable blood analyzers enable identification and management of electrolyte and acid-base imbalances and collection of physiologic data, but present logistical challenges.

CEFOVECIN PROTEIN BINDING AS A PREDICTOR FOR EXTENDED DURATION OF ACTION: A REVIEW OF CURRENT LITERATURE AND IN VITRO ANALYSIS IN MULTIPLE ZOOLOGICAL SPECIES.

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.

Gut microbiota composition and metabolomic profiles of wild and captive Chinese monals (Lophophorus lhuysii).

BACKGROUND: The Chinese monal (Lophophorus lhuysii) is an endangered bird species, with a wild population restricted to the mountains in southwest China, and only one known captive population in the world. We investigated the fecal microbiota and metabolome of wild and captive Chinese monals to explore differences and similarities in nutritional status and digestive characteristics. An integrated approach combining 16S ribosomal RNA (16S rRNA) gene sequencing and ultra-high performance liquid chromatography (UHPLC) based metabolomics were used to examine the fecal microbiota composition and the metabolomic profile of Chinese monals. RESULTS: The results showed that the alpha diversity of gut microbes in the wild group were significantly higher than that in the captive group and the core bacterial taxa in the two groups showed remarkable differences at phylum, class, order, and family levels. Metabolomic profiling also revealed differences, mainly related to galactose, starch and sucrose metabolism, fatty acid, bile acid biosynthesis and bile secretion. Furthermore, strong correlations between metabolite types and bacterial genus were detected. CONCLUSIONS: There were remarkable differences in the gut microbiota composition and metabolomic profile between wild and captive Chinese monals. This study has established a baseline for a normal gut microbiota and metabolomic profile for wild Chinese monals, thus allowing us to evaluate if differences seen in captive organisms have an impact on their overall health and reproduction.

Detection of novel coronaviruses in bats in Myanmar.

The recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. Of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, Coronavirus Disease 2019 (COVID-19), an epidemic of acute respiratory illness originating from Wuhan, China in December 2019. Viral detection, discovery, and surveillance activities were undertaken in Myanmar to identify viruses in animals at high risk contact interfaces with people. Free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. Sequences from positives were compared to known coronaviruses. Three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast Asian countries were detected for the first time in bats in Myanmar. Ongoing land use change remains a prominent driver of zoonotic disease emergence in Myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales.

First Report of a Novel Hepatozoon sp. in Giant Pandas (Ailuropoda melanoleuca).

The first report of giant pandas (Ailuropoda melanoleuca) infected with a novel Hepatozoon species is presented. An intraleukocytic parasite was detected via routine blood smear from a zoo-housed giant panda at the National Zoological Park. Ribosomal DNA sequences indicated a previously undescribed Hepatozoon species. Phylogenetic and distance analyses of the sequences placed it within its own branch, clustered with Old World species with carnivore (primarily ursid and mustelid) hosts. Retrospective and opportunistic testing of other individuals produced additional positive detections (17/23, 73.9%), demonstrating 100% prevalence (14/14) across five institutions. All animals were asymptomatic at time of sampling, and health implications for giant pandas remain unknown.