Coincidence of low genetic diversity and increasing population size in wild gaur populations in the Khao Phaeng Ma Non-Hunting Area, Thailand: A challenge for conservation management under human-wildlife conflict.

The gaur (Bos gaurus) is found throughout mainland South and Southeast Asia but is listed as an endangered species in Thailand with a decreasing population size and a reduction in suitable habitat. While gaur have shown a population recovery from 35 to 300 individuals within 30 years in the Khao Phaeng Ma (KPM) Non-Hunting Area, this has caused conflict with villagers along the border of the protected area. At the same time, the ecotourism potential of watching gaurs has boosted the local economy. In this study, 13 mitochondrial displacement-loop sequence samples taken from gaur with GPS collars were analyzed. Three haplotypes identified in the population were defined by only two parsimony informative sites (from 9 mutational steps of nucleotide difference). One haplotype was shared among eleven individuals located in different subpopulations/herds, suggesting very low genetic diversity with few maternal lineages in the founder population. Based on the current small number of sequences, neutrality and demographic expansion test results also showed that the population was likely to contract in the near future. These findings provide insight into the genetic diversity and demography of the wild gaur population in the KPM protected area that can inform long-term sustainable management action plans.

Immobilization of captive plains zebras (Equus quagga) with a combination of etorphine hydrochloride, acepromazine, and xylazine hydrochloride.

The plains zebra (Equus quagga) is a zebra species commonly kept in zoos around the world. However, they are not tame like their domestic relatives and are difficult to immobilize. We immobilized 30 captive plains zebra with a combination of etorphine hydrochloride (2-4 mg), acepromazine (8 mg), and xylazine hydrochloride (30 or 50 mg) to perform physical examination and blood sample collection for disease diagnostics. Physiological parameters including heart rate, respiratory rate, body temperature, and hemoglobin oxygen saturation were recorded. All zebras exhibited satisfactory anesthesia and fully recovered without re-narcotization. The results suggest that etorphine hydrochloride-acepromazine-xylazine hydrochloride combination for plains zebra immobilization is a safe and sufficient regimen for short procedures such as wellness examinations and sample collection.

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.

Subspecies in the Sarus Crane Antigone antigone revisited; with particular reference to the Australian population.

Subspecies are often less well-defined than species but have become one of the basic units for legal protection. Evidence for the erection or synonymy of subspecies therefore needs to be founded on the best science available. Here we show that there is clear genetic disjunction in the Sarus Crane Antigone antigone, where previously the variation had appeared to be clinal. Based on a total sample of 76 individuals, analysis of 10 microsatellite loci from 67 samples and 49 sequences from the mitochondrial control region, this research establishes that the Australian Sarus Crane A. a. gillae differs significantly from both A. a. antigone (South Asia) and A. a. sharpii (Myanmar and Indochina). A single sample from the extinct Philippine subspecies A. a luzonica clustered with A. a. gillae, hinting at the potential for a more recent separation between them than from A. a. antigone and A. a. sharpii, even though A. a. sharpii is closer geographically. The results demonstrate that failure to detect subspecies through initial genetic profiling does not mean discontinuities are absent and has significance for other cases where subspecies are dismissed based on partial genetic evidence. It could also be potentially important for sourcing birds for reintroduction to the Philippines.

Operationalizing One Health Employing Social-Ecological Systems Theory: Lessons From the Greater Mekong Sub-region.

The idea of the interdependency of the health of humans, animals, and ecosystems emerged from the interplay of theory and concepts from medicine, public health and ecology among leading thinkers in these fields during the last century. The rationale for One Health and its focus on the "human, animal, and environmental interface" stems from this legacy and points to transdisciplinary, ecological and complex systems approaches as central to One Health practice. Demonstration of One Health's efficacy, its wider adoption and continual improvement require explicit operational criteria and evaluation metrics on this basis. Social-Ecological Systems Theory with its unique conception of resilience (SESR) currently offers the most well-developed framework for understanding these approaches and development of performance standards. This paper describes operational criteria for One Health developed accordingly, including a protocol currently being tested for vector borne disease interventions. Wider adoption of One Health is most likely to occur as One Health practitioners gain an increasing familiarity with ecological and complex systems concepts in practice employing a transdisciplinary process. Two areas in which this inevitably will be required for significant further progress, and where the beginnings of a foundation for building upon exist, include: (1) Emerging and re-emerging zoonotic diseases, and (2) successful implementation of the United Nations (UN) Sustainable Development Goals (SDGs). The former includes the challenge of stemming the threat of new microbial pathogens, anti-microbial resistant variants of existing pathogens, as well as resurgence of malaria and other recalcitrant diseases. The applicability of SESR in this regard is illustrated with two case examples from the Greater Mekong Subregion, Avian Influenza (H5N1) and Liver Fluke (Opisthorchis viverrini). Each is shown to represent a science and policy challenge suggestive of an avoidable social-ecological system pathology that similarly has challenged sustainable development. Thus, SESR framing arguably is highly applicable to the SDGs, which, to a large extent, require consideration of human-animal-environmental health linkages. Further elaboration of these One Health operational criteria and metrics could contribute to the achievement of many of the SDGs.

The complete mitochondrial genome of Fea's muntjac (Muntiacus feae Thomas and Doria, 1889) with phylogenetic analysis†.

The complete mitochondrial genome (mitogenome) of the rare Fea's muntjac (Muntiacus feae) was sequenced (GenBank accession nos. MG857662-MG857664). The mitogenome was found to be 16,355 bp in length with base compositions of 33.16% A, 24.59% C, 13.46% G, and 28.78% T and a GC content of 38.06%. The genome is comprised of 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop). Phylogenetic analysis revealed that Fea's muntjac is more closely related to Black muntjac (M. cronifrons) than to Red muntjac (M. muntjak). These data will be useful for further studies on the genetic diversity and molecular phylogenetic relationship of the genus Muntiacus.

SUCCESSFUL LAPAROSCOPIC OVIDUCTAL ARTIFICIAL INSEMINATION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA) IN THAILAND.

Captive breeding of clouded leopards (Neofelis nebulosa) is challenging because of mating incompatibility, high incidence of teratospermia in males, and inconsistent ovulation patterns in females. Assisted reproductive techniques, therefore, are necessary to overcome these issues and maintain the genetic diversity in the captive population. The objective was to use laparoscopic oviductal artificial insemination (AI) to breed genetically valuable females (n = 4; aged 4.5-5 yr) that were unsuccessfully paired. Fecal hormone metabolites (estrogen and progesterone) were extracted and measured by enzyme immunoassay for monitoring of ovarian activity 45 days before and 65 days after laparoscopic AI. For timed insemination, females were injected with 200 IU equine chorionic gonadotropin and 1,000 IU porcine luteinizing hormone (pLH) at the 82-hr interval. Ovarian assessment was performed by laparoscopy 44 hr after pLH administration. One nulliparous female out of four presented two ovulation sites on each ovary. The single female that had ovulated was inseminated with chilled semen collected from two males (8 × 106 and 2.7 × 106 motile spermatozoa, respectively, in each oviduct). A significant increase in fecal progesterone concentrations was observed after AI with a concentration peak (500 µg/g dry feces) detected on day 24 after pLH injection, which was then sustained for more than 45 days after the pLH injection. The delivery of two cubs occurred on day 92 after pLH. Microsatellite marker analysis determined that both cubs were sired by the same male. This is the first report of a successful oviductal AI in the clouded leopard.

The impact of ovarian stimulation protocol on oocyte quality, subsequent in vitro embryo development, and pregnancy after transfer to recipients in Eld's deer (Rucervus eldii thamin).

Propagating genetically valuable individuals through oocyte collection, in vitro fertilization (IVF) and embryo transfer is critical to maintain sustainable populations of the endangered Eld's deer. The objectives of this study were to assess the impact of exogenous FSH injections on (1) the number and in vitro competence of oocytes collected and (2) the developmental potential of resulting IVF embryos after transfer into recipients during the breeding season (February-April). In a pilot experiment, estrus synchronization was conducted in three surplus females (using intravaginal progesterone-releasing devices, CIDRG for 14 days and injections of buserelin (a GnRH agonist). Five days after CIDR removal, ovaries were excised, minced and a total of 133 oocytes were recovered. Following in vitro maturation (IVM) and IVF, 63% of the oocytes formed embryos but only 5% reached the blastocyst stage. In a subsequent study, follicle numbers and diameters were compared between synchronized does stimulated with 0 or 80 mg FSH (-FSH and +FSH; n = 8 does in each group) and oocytes collected either by laparoscopic ovum pick-up or ovariectomy. FSH stimulation increased the main follicular diameter from 2-3 mm to 4-5 mm (P < 0.05) but not the oocyte number (∼20/donor) or the percentage of good quality oocytes (57%) regardless of the treatment. FSH stimulation did not either affect the percentage of cleaved embryos after IVF (25-35%; P > 0.05). Lastly, embryos at the 2-to 8-cell stage (from either + FSH or -FSH groups) were transferred into the oviducts of 11 synchronized recipients. With the +FSH embryos, three pregnancies failed between 90 and 120 days of gestation and two fawns that were born preterm (Days 215 and 224 of gestation) died at birth. In the -FSH group one healthy female fawn was born on Day 234 of gestation. This is the first report of a successful in vitro embryo production and subsequent birth of a live Eld's deer fawn. Further investigations are required to improve IVM/IVF success and the developmental potential of the embryos.

Genetic diversity of the captive Asian tapir population in Thailand, based on mitochondrial control region sequence data and the comparison of its nucleotide structure with Brazilian tapir.

The Asian tapir (Tapirus indicus) has been classified as Endangered on the IUCN Red List of Threatened Species (2008). Genetic diversity data provide important information for the management of captive breeding and conservation of this species. We analyzed mitochondrial control region (CR) sequences from 37 captive Asian tapirs in Thailand. Multiple alignments of the full-length CR sequences sized 1268 bp comprised three domains as described in other mammal species. Analysis of 16 parsimony-informative variable sites revealed 11 haplotypes. Furthermore, the phylogenetic analysis using median-joining network clearly showed three clades correlated with our earlier cytochrome b gene study in this endangered species. The repetitive motif is located between first and second conserved sequence blocks, similar to the Brazilian tapir. The highest polymorphic site was located in the extended termination associated sequences domain. The results could be applied for future genetic management based in captivity and wild that shows stable populations.

The complete mitochondrial genome of the Asian tapirs (Tapirus indicus): the only extant Tapiridae species in the old world.

Asian tapir (Tapirus indicus) is categorized as Endangered on the 2008 IUCN red list. The first full-length mitochondrial DNA (mtDNA) sequence of Asian tapir is 16,717 bp in length. Base composition shows 34.6% A, 27.2% T, 25.8% C and 12.3% G. Highest polymorphic site is on the control region as typical for many species.

Locational diversity of alpha satellite DNA and intergeneric hybridization aspects in the Nomascus and Hylobates genera of small apes.

Recently, we discovered that alpha satellite DNA has unique and genus-specific localizations on the chromosomes of small apes. This study describes the details of alpha satellite localization in the genera Nomascus and Hylobates and explores their usefulness in distinguishing parental genome sets in hybrids between these genera. Fluorescence in situ hybridization was used to establish diagnostic criteria of alpha satellite DNA markers in discriminating small ape genomes. In particular we established the genus specificity of alpha satellite distribution in three species of light-cheeked gibbons (Nomascus leucogenys, N. siki, and N. gabriellae) in comparison to that of Hylobates lar. Then we determined the localization of alpha satellite DNA in a hybrid individual which resulted from a cross between these two genera. In Nomascus the alpha satellite DNA blocks were located at the centromere, telomere, and four interstitial regions. In Hylobates detectable amounts of alpha satellite DNA were seen only at centromeric regions. The differences in alpha satellite DNA locations between Nomascus and Hylobates allowed us to easily distinguish the parental chromosomal sets in the genome of intergeneric hybrid individuals found in Thai and Japanese zoos. Our study illustrates how molecular cytogenetic markers can serve as diagnostic tools to identify the origin of individuals. These molecular tools can aid zoos, captive breeding programs and conservation efforts in managing small apes species. Discovering more information on alpha satellite distribution is also an opportunity to examine phylogenetic and evolutionary questions that are still controversial in small apes.

Assessment of ovarian activity in captive goral (Naemorhedus griseus) using noninvasive fecal steroid monitoring.

To date, there is no information on gonadal steroidogenic activity of female goral (Naemorhedus griseus), a threatened species of Thailand. Captive goral populations have been established to produce animals for ex situ conservation and reintroduction, but as yet none are self-sustaining. The objectives of the present study were to (1) determine the influence of season on ovarian steriodogenic function; and (2) examine the relationship between gonadal hormone excretion and sexual behaviors throughout the year. Fecal samples were collected 5 to 7 days/wk for 15 months from 8 adult females housed at Omkoi Wildlife Breeding Center in Thailand and analyzed for ovarian steroid metabolites using validated enzyme immunoassays. Observations of sexual behaviors and mating were conducted each morning for 30 min/session. Based on fecal estrogen and progestagen metabolite concentrations, the overall estrous cycle length was about 21 days, with a 2- to 3-day follicular phase and an 18- to 20-day luteal phase. Sexual behaviors, most notably tail-up, increased for 2 to 3 days during the time estrogens were elevated during mating. Fecal progestagens were elevated during luteal phases and increased further during gestation, which lasted approximately 7 months. The lactation period was 5 months, and females were anestrus for 2 to 5 of those months, with the exception of one that cycled continuously throughout. Two females conceived around 2 months postpartum and so were pregnant during lactation. Birth records over the past 21 years indicated young are born throughout the year. This combined with the hormonal data suggests that female gorals are not strongly seasonal, at least in captivity, although there was considerable variation among females in estrogen and progestagen patterns. In conclusion, fecal steroid metabolite monitoring is an effective means of assessing ovarian function in this species and will be a useful tool for breeding management and planned development of assisted reproductive techniques such as artificial insemination and embryo transfer.

Parasite and viral species richness of Southeast Asian bats: Fragmentation of area distribution matters.

Interest in bat-borne diseases and parasites has grown in the past decade over concerns for human health. However, the drivers of parasite diversity among bat host species are understudied as are the links between parasite richness and emerging risks. Thus, we aimed at exploring factors that explain macro and microparasite species richness in bats from Southeast Asia, a hotspot of emerging infectious diseases. First, we identified bat species that need increased sampling effort for pathogen discovery. Our approach highlights pathogen investigation disparities among species within the same genus, such as Rhinolophus and Pteropus. Secondly, comparative analysis using independent contrasts method allowed the identification of likely factors explaining parasite and viral diversity of bats. Our results showed a key role of bat distribution shape, an index of the fragmentation of bat distribution, on parasite diversity, linked to a decrease for both viral and endoparasite species richness. We discuss how our study may contribute to a better understanding of the link between parasite species richness and emergence.

Gender, season and management affect fecal glucocorticoid metabolite concentrations in captive goral (Naemorhedus griseus) in Thailand.

Chinese goral (Naemorhedus griseus) are a threatened species in Thailand and the focus of captive breeding for possible reintroduction. However, little is known of their biology or what factors in the captive environment affect welfare. Our objective was to determine the impact of gender, season, and management on goral adrenal activity. We hypothesized that differences in fecal glucocorticoid concentrations would be related to animal density. Fecal samples were collected 3 days/week for 1 year from 63 individuals (n = 32 males, 31 females) at two facilities that house the majority of goral in Thailand: Omkoi Wildlife Sanctuary (Omkoi), an off-exhibit breeding center that houses goral in individual pens (16 pens; n = 8 males, 8 females) and in small family groups (8 pens; n = 8 males, 8 females); and the Chiang Mai Night Safari (NS), a zoo that maintains 31 goral (n = 17 males, 14 females) in one large pen. Glucocorticoid metabolite concentrations were higher in male than female goral at Omkoi throughout the year, and there was a seasonal effect on adrenal activity (p<0.05). Goral at Omkoi and NS were used to test the effect of animal density on fecal glucocorticoid excretion of goral housed in similar-sized enclosures. Overall, the highest levels were found at NS (n = 31 adults/pen; 27 m2 per animal) compared to Omkoi (n = 2 adults/pen; 400 m2 per animal) (p<0.05). Overall findings support our hypothesis that animal density and aspects of the captive environment impact adrenal steroid activity in captive goral. In addition, gender and season also had significant effects on glucocorticoid metabolite production. Potential stressors pertaining to the welfare of this species were identified, which will guide future efforts to improve management and create self-sustaining and healthy populations of this threatened species.

Seasonality of fecal androgen and glucocorticoid metabolite excretion in male goral (Naemorhedus griseus) in Thailand.

There is no information on the endocrinology of Chinese goral (Naemorhedus griseus), a high priority species for captive breeding and reintroduction in Thailand. This study characterized fecal androgen and glucocorticoid metabolites in male goral at Omkoi Wildlife Sanctuary to investigate seasonal relationships. Fecal samples were collected 3 days/week for 1 year from eight adult males. Mean androgen metabolite concentrations were greater (P<0.05) during the rainy season (289.82±9.18ng/g) and winter (224.09±11.97ng/g) compared to the summer (195.48±8.23ng/g), and were related to breeding activity. A similar pattern was observed for glucocorticoid concentrations (22.10±0.72ng/g compared to 21.98±0.98ng/g compared to 15.30±0.48ng/g), respectively, and this resulted in a positive correlation between the two hormones (P<0.05). There also were positive correlations between fecal androgen metabolite concentrations and temperature (P<0.05) and day length (P<0.05). In summary, this is the first study to assess endocrine function in male goral, and results showed seasonal variation in testicular and adrenal steroidogenic function, with greater activity in the rainy season and winter. Given that resources for captive male goral are consistent throughout the year, reproduction may be regulated primarily by photoperiod in this species.

Environmental management procedures following fatal melioidosis in a captive chimpanzee (Pan troglodytes).

A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.

A case report concerning male gametes rescued from a Siamese Eld's deer (Rucervus eldii siamensis): post-thawed testicular and epididymal sperm quality and heterologous zona pellucida binding ability.

In the present study, the quality of frozen-thawed epididymal and testicular sperm recovered from a Siamese Eld's deer was examined. The epididymal sperm quality was assessed in fresh, cold-stored at 4°C and frozen-thawed samples. Zona binding ability of the frozen-thawed epididymal samples with Burmese Eld's deer oocytes was also evaluated. Testicular sperm extracted from tissues frozen at -80 or -196°C for one month were examined for membrane and DNA integrity. Epididymal sperm retained their quality for up to 24 hr of cold storage at 4°C. The percentages of sperm motility, intact membrane, intact acrosome and intact DNA were 30, 46.5, 27 and 89.5% in the frozen and thawed epididymal sperm, and the average ability to bind with oocytes was 92.5 ± 64 sperm/oocytes. Around 70% of the sperm extracted from testicular tissues cryopreserved at -196 and -80°C for one month showed an intact membrane. In conclusion, epididymal and testicular sperm survived for more than 13 hr post-mortem. Furthermore, cold storage at 4°C and cryopreservation at -196 and -80°C maintain the quality of epididymal and testicular sperm. This study represents a model for male gamete rescue in endangered Eld's deer.

Gene conversion and purifying selection shape nucleotide variation in gibbon L/M opsin genes.

BACKGROUND: Routine trichromatic color vision is a characteristic feature of catarrhines (humans, apes and Old World monkeys). This is enabled by L and M opsin genes arrayed on the X chromosome and an autosomal S opsin gene. In non-human catarrhines, genetic variation affecting the color vision phenotype is reported to be absent or rare in both L and M opsin genes, despite the suggestion that gene conversion has homogenized the two genes. However, nucleotide variation of both introns and exons among catarrhines has only been examined in detail for the L opsin gene of humans and chimpanzees. In the present study, we examined the nucleotide variation of gibbon (Catarrhini, Hylobatidae) L and M opsin genes. Specifically, we focused on the 3.6~3.9-kb region that encompasses the centrally located exon 3 through exon 5, which encode the amino acid sites functional for the spectral tuning of the genes. RESULTS: Among 152 individuals representing three genera (Hylobates, Nomascus and Symphalangus), all had both L and M opsin genes and no L/M hybrid genes. Among 94 individuals subjected to the detailed DNA sequencing, the nucleotide divergence between L and M opsin genes in the exons was significantly higher than the divergence in introns in each species. The ratio of the inter-LM divergence to the intra-L/M polymorphism was significantly lower in the introns than that in synonymous sites. When we reconstructed the phylogenetic tree using the exon sequences, the L/M gene duplication was placed in the common ancestor of catarrhines, whereas when intron sequences were used, the gene duplications appeared multiple times in different species. Using the GENECONV program, we also detected that tracts of gene conversions between L and M opsin genes occurred mostly within the intron regions. CONCLUSIONS: These results indicate the historical accumulation of gene conversions between L and M opsin genes in the introns in gibbons. Our study provides further support for the homogenizing role of gene conversion between the L and M opsin genes and for the purifying selection against such homogenization in the central exons to maintain the spectral difference between L and M opsins in non-human catarrhines.

Systemic neosporosis in a white rhinoceros.

Neosporosis was diagnosed in a 16-year-old female white rhinoceros (Ceratotherium simum) that died suddenly without clinical signs. Histopathology revealed disseminated protozoan tachyzoites in liver, adrenal cortex, kidney, and intestine, with morphology compatible with either Toxoplasma or Neospora. The organism was identified as Neospora caninum with the use of primary rabbit anti-N. caninum antibody immunohistochemistry and polymerase chain reaction. The exact source of infection remains unknown, but it is suspected that N. caninum oocysts were ingested from the soil.