First screening for tick-borne pathogens in Chinese Milu deer (Elaphurus davidianus).

Ticks are primary vectors for many tick-borne pathogens (TBPs) and pose a serious threat to veterinary and public health. Information on the presence of TBPs in Chinese Milu deer (Elaphurus davidianus) is limited. In this study, a total of 102 Chinese Milu deer blood samples were examined for Anaplasma spp., Theileria spp., Babesia spp., Rickettsia spp., and Borrelia spp., and three TBPs were identified: Anaplasma phagocytophilum (48; 47.1 %), Candidatus Anaplasma boleense (47; 46.1%), and Theileria capreoli (8; 7.8 %). Genetic and phylogenetic analysis of the 16S rRNA and 18S rRNA confirmed their identity with corresponding TBPs. To our knowledge, this is the first report on Candidatus A. boleense and T. capreoli detection in Chinese Milu deer. A high prevalence of A. phagocytophilum with veterinary and medical significance was identified in endangered Chinese Milu deer, which could act as potential zoonotic reservoirs. The identification of the TBPs in Chinese Milu deer provides useful information for the prevention and control of tick-borne diseases.

Effects of Age and Season on Blood Parameters of Captive Giant Pandas: A Pilot Study.

The giant panda, Ailuropoda melanoleuca, serves as a flagship species for biodiversity conservation, embodying the intersection of ecological, evolutionary, and anthropogenic forces shaping the natural world. Hematological parameters serve as crucial indicators for assessing the physiological status of animals. However, our understanding of blood parameters and hemorheology in captive giant pandas under non-anesthetic conditions is limited. In this study, from September 2018 to August 2020, we collected blood samples from captive giant pandas under non-anesthetic conditions. Twelve captive giant pandas, ranging in age from 2 to 28 years, were divided into three groups based on their age, and the variations in basic blood parameters and hemorheological parameters across four seasons were analyzed. This provided baseline data for future blood sample comparisons in non-anesthetized captive giant pandas. Additionally, we observed seasonal changes in hematological morphology, hemorheology, and serum enzymes. Moreover, seasonality had a regulatory effect on hemorheological parameters and negatively impacted blood viscosity. Age influenced changes in serum enzymes, serum protein content, and serum metabolites, indicating differences in overall metabolic processes among giant pandas of different age groups. Whether factors such as season and climate contribute to environmental stress in captive giant pandas requires further investigation. The findings of this study may help to protect the stability of the giant panda population better and provide a reference for the medical care of captive giant pandas.

Comparative proteomics in captive giant pandas to identify proteins involved in age-related cataract formation.

Approximately 20% of aged captive giant pandas (Ailuropoda melanoleuca) have cataracts that impair their quality of life. To identify potential biomarkers of cataract formation, we carried out a quantitative proteomics analysis of 10 giant pandas to find proteins differing in abundance between healthy and cataract-bearing animals. We identified almost 150 proteins exceeding our threshold for differential abundance, most of which were associated with GO categories related to extracellular localization. The most significant differential abundance was associated with components of the proteasome and other proteins with a role in proteolysis or its regulation, most of which were depleted in pandas with cataracts. Other modulated proteins included components of the extracellular matrix or cytoskeleton, as well as associated signaling proteins and regulators, but we did not find any differentially expressed transcription factors. These results indicate that the formation of cataracts involves a complex post-transcriptional network of signaling inside and outside lens cells to drive stress responses as a means to address the accumulation of protein aggregates triggered by oxidative damage. The modulated proteins also indicate that it should be possible to predict the onset of cataracts in captive pandas by taking blood samples and testing them for the presence or absence of specific protein markers.

Mechanism of inulin in colic and gut microbiota of captive Asian elephant.

BACKGROUND: Gut microbiota have a complex role on the survivability, digestive physiology, production, and growth performance in animals. Recent studies have emphasized the effects of prebiotics therapy on the gut disease, but the relationship between elephant gut-related diseases and prebiotics remains elusive. Here, a case study was undertaken to evaluate the mechanism of inulin treatment in colic in Asian elephant (Elephas maximus Linnaeus). METHODS: Fecal samples were collected from a sick elephant and four healthy elephants. Analysis of microbial profile was carried out by 16S rRNA sequencing, and the short chain fatty acids were tested by gas chromatography. The physiological function of "inulin-microbiota" of elephant was verified in mice by fecal microbial transplantation (FMT). The expression of related proteins was determined by Western blotting and qPCR. RESULTS: (1) Eating inulin can cure gut colic of the sick elephant and changed gut microbiota. (2) It was found that "inulin microbiota" from the post-treatment elephants can promote the proliferation of intestinal cells, increase the utilization of short chain fatty acids (SCFAs), maintain intestinal barrier, and reduce the inflammation in mice. (3) The mechanism was inulin-gut microbiota-SCFAs-immune barrier. CONCLUSIONS: Inulin contributed to rehabilitate the gut microbiota and gut immune barrier of the elephant with colic. This provides reasonable verification for using prebiotics to treat the colic in captive elephants. Prebiotics will foresure play an increasingly important role in disease prevention and treatment of captive animals in the future. Video Abstract.

Development of short-target primers for species identification in biological studies of Carnivora.

Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling-based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high-performance short-target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short-target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short-target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.

Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats.

Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high > 3000 m above sea level and low < 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (PGWAS = 3.62 × 10-25) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection.

A rare case of giant panda cancer: Pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer of the digestive system common among humans. However, it is rarely reported in wild animals. In 2018, a giant panda died in the Beijing Zoo. During subsequent histological observation of the pancreas, it was discovered that the glandular epithelial cells had lost the pancreatic acinar structure, tubular areas with obvious structure in the pancreas, and the ductal epithelium was substituted by high columnar mucus cells. Masson staining showed that there were several fibrous tissue proliferative reactions around the ductal adenocarcinoma and immunohistochemical staining revealed that CK7 and CK19 were positively expressed in the pancreatic tissue. Therefore, the pathological diagnosis indicated that the panda had PDAC. In this paper, the panda's living conditions and pathological diagnosis results are examined, with the aim of providing a reference point for the future diagnosis of wild animal tumors.

A survey of Cryptosporidium prevalence among birds in two zoos in China.

BACKGROUND: Cryptosporidiosis is an important zoonotic protozoan disease worldwide, but few studies on this disease have been performed in wild birds; thus, our knowledge of this disease is insufficient, even in zoo birds. Animals in zoos are possible zoonotic disease reservoirs, potentially resulting in zoonotic agent spillover to humans; accordingly, our understanding of such phenomena should be improved. METHODS: A total of 263 fresh fecal samples from 43 avian species were randomly collected from the Beijing Zoo and Harbin North Forest Zoo and screened for the prevalence of Cryptosporidium by 18S rRNA gene sequencing. Cryptosporidium species were distinguished based on the combined results of phylogenetic tree and genetic distance analyses conducted with the inclusion of seven avian Cryptosporidium species and 13 avian Cryptosporidium genotypes. The genetic diversity of Cryptosporidium parvum among different hosts, including humans, cattle, dogs, and birds, and the genetic diversity of avian C. parvum among avian hosts in China, Iraq and Brazil were determined based on C. parvum 18S rRNA haplotypes. RESULTS: The results of PCR targeting the 18S rRNA gene revealed that 1.9% (5/263) of the samples were Cryptosporidium-positive. Four of the five Cryptosporidium-positive samples originated from white cranes (Grus leucogeranus), and one originated from a flamingo (Phoenicopteridae). Avian C. parvum isolates, including the isolates examined in the present study, showed gene flow with other isolates from different types of hosts, including humans, cattle and dogs, indicating that zoo birds potentially pose zoonotic and pathogenic risks to humans and animals. Additionally, gene flow between avian C. parvum isolates from China and Brazil was detected. CONCLUSIONS: To the best of our knowledge, our results demonstrate C. parvum infection in a flamingo (Phoenicopteridae) and white cranes (Grus leucogeranus) for the first time. The results of our study provide an important reference for understanding the host range, biological characteristics, and molecular epidemiology of C. parvum.

Long-Read Genome Sequencing Provides Molecular Insights into Scavenging and Societal Complexity in Spotted Hyena Crocuta crocuta.

The spotted hyena (Crocuta crocuta) is a large and unique terrestrial carnivore. It is a particularly fascinating species due to its distinct phenotypic traits, especially its complex social structure and scavenging lifestyle, with associated high dietary exposure to microbial pathogens. However, the underlying molecular mechanisms related to these phenotypes remain elusive. Here, we sequenced and assembled a high-quality long-read genome of the spotted hyena, with a contig N50 length of ∼13.75 Mb. Based on comparative genomics, immunoglobulin family members (e.g., IGKV4-1) showed significant adaptive duplications in the spotted hyena and striped hyena. Furthermore, immune-related genes (e.g., CD8A, LAG3, and TLR3) experienced species-specific positive selection in the spotted hyena lineage. These results suggest that immune tolerance between the spotted hyena and closely related striped hyena has undergone adaptive divergence to cope with prolonged dietary exposure to microbial pathogens from scavenging. Furthermore, we provided the potential genetic insights underlying social complexity, hinting at social behavior and cognition. Specifically, the RECNE-associated genes (e.g., UGP2 and ACTR2) in the spotted hyena genome are involved in regulation of social communication. Taken together, our genomic analyses provide molecular insights into the scavenging lifestyle and societal complexity of spotted hyenas.

RNA-Seq analysis in giant pandas reveals the differential expression of multiple genes involved in cataract formation.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is an endangered mammalian species native to China. Fewer than 2500 giant pandas are known to exist, many of which are bred in captivity as a means to preserve and repopulate the species. Like other captive mammals, giant pandas acquire age-related cataracts, reducing their quality of life. Recent comparative genome-wide methylation analysis revealed 110 differentially methylated genes associated with cataract formation including six also associated with the formation of age-related cataracts in humans. RESULTS: To investigate the pathological pathway in greater detail, here we used RNA-Seq analysis to investigate the differential expression profiles of genes in three giant pandas with cataracts and three healthy controls. We identified more than 700 differentially expressed genes, 29 of which were selected for further analysis based on their low q-value. We found that many of the genes encoded regulatory and signaling proteins associated with the control of cell growth, migration, differentiation and apoptosis, supporting previous research indicating a key role for apoptosis in cataract formation. CONCLUSION: The identification of genes involved in the formation of age-related cataracts could facilitate the development of predictive markers, preventative measures and even new therapies to improve the life of captive animals.

A novel missense mutation in the HSF4 gene of giant pandas with senile congenital cataracts.

Cataracts are a common cause of visual impairment and blindness in mammals. They are usually associated with aging, but approximately one third of cases have a significant genetic component. Cataracts are increasingly prevalent among aging populations of captive giant pandas (Ailuropoda melanoleuca) and it is therefore important to identify genetic determinants that influence the likelihood of cataract development in order to distinguish between congenital and age-related disease. Here we screened for cataract-related genetic effects using a functional candidate gene approach combined with bioinformatics to identify the underlying genetic defect in a giant panda with congenital cataracts. We identified a missense mutation in exon 10 of the HSF4 gene encoding heat shock transcription factor 4. The mutation causes the amino acid substitution R377W in a highly conserved segment of the protein between the isoform-specific and downstream hydrophobic regions. Predictive modeling revealed that the substitution is likely to increase the hydrophobicity of the protein and disrupt interactions with spatially adjacent amino acid side chains. The mutation was not found in 13 unaffected unrelated animals but was found in an unrelated animal also diagnosed with senile congenital cataract. The novel missense mutation in the HSF4 gene therefore provides a potential new genetic determinant that could help to predict the risk of cataracts in giant pandas.

A novel missense mutation in the gene encoding major intrinsic protein (MIP) in a Giant panda with unilateral cataract formation.

BACKGROUND: Cataracts are defects of the lens that cause progressive visual impairment and ultimately blindness in many vertebrate species. Most cataracts are age-related, but up to one third have an underlying genetic cause. Cataracts are common in captive zoo animals, but it is often unclear whether these are congenital or acquired (age-related) lesions. RESULTS: Here we used a functional candidate gene screening approach to identify mutations associated with cataracts in a captive giant panda (Ailuropoda melanoleuca). We screened 11 genes often associated with human cataracts and identified a novel missense mutation (c.686G > A) in the MIP gene encoding major intrinsic protein. This is expressed in the lens and normally accumulates in the plasma membrane of lens fiber cells, where it plays an important role in fluid transport and cell adhesion. The mutation causes the replacement of serine with asparagine (p.S229N) in the C-terminal tail of the protein, and modeling predicts that the mutation induces conformational changes that may interfere with lens permeability and cell-cell interactions. CONCLUSION: The c.686G > A mutation was found in a captive giant panda with a unilateral cataract but not in 18 controls from diverse regions in China, suggesting it is most likely a genuine disease-associated mutation rather than a single-nucleotide polymorphism. The mutation could therefore serve as a new genetic marker to predict the risk of congenital cataracts in captive giant pandas.

Malaria parasites and related haemosporidians cause mortality in cranes: a study on the parasites diversity, prevalence and distribution in Beijing Zoo.

BACKGROUND: Malaria parasites and related haemosporidian parasites are widespread and may cause severe diseases in birds. These pathogens should be considered in projects aiming breeding of birds for purposes of sustained ex situ conservation. Cranes are the 'flagship species' for health assessment of wetland ecosystems, and the majority of species are endangered. Malaria parasites and other haemosporidians have been reported in cranes, but the host-parasite relationships remain insufficiently understood. Morbidity of cranes due to malaria has been reported in Beijing Zoo. This study report prevalence, diversity and distribution of malaria parasites and related haemosporidians in cranes in Beijing Zoo and suggest simple measures to protect vulnerable individuals. METHODS: In all, 123 cranes (62 adults and 61 juveniles) belonging to 10 species were examined using PCR-based testing and microscopic examination of blood samples collected in 2007-2014. All birds were maintained in open-air aviaries, except for 19 chicks that were raised in a greenhouse with the aim to protect them from bites of blood-sucking insects. Bayesian phylogenetic analysis was used to identify the closely related avian haemosporidian parasites. RESULTS: Species of Plasmodium (5 lineages), Haemoproteus (1) and Leucocytozoon (2) were reported. Malaria parasites predominated (83% of all reported infections). The overall prevalence of haemosporidians in juveniles was approximately seven-fold higher than in adults, indicating high susceptibility of chicks and local transmission. Juvenile and adult birds hosted different lineages of Plasmodium, indicating that chicks got infection from non-parent birds. Plasmodium relictum (pSGS1) was the most prevalent malaria parasite. Mortality was not reported in adults, but 53% of infected chicks died, with reports of co-infection with Plasmodium and Leucocytozoon species. All chicks maintained in the greenhouse were non-infected and survived. Species of Leucocytozoon were undetectable by commonly used PCR protocol, but readily visible in blood films. CONCLUSION: Crane chicks often die due to malaria and Leucocytozoon infections, which they likely gain from wild free-living birds in Beijing Zoo. Molecular diagnostics of crane Leucocytozoon parasites needs improvement. Because the reported infections are mainly chick diseases, the authors recommend maintaining of juvenile birds in vector-free facilities until the age of approximately 6 months before they are placed in open-air aviaries.

Immobilization of wild giant panda (Ailuropoda melanoleuca) with dexmedetomidine-tiletamine-zolazepam.

OBJECTIVE: To assess the effects and utility of dexmedetomidine combined with tiletamine and zolazepam (dexMTZ) to immobilize the wild giant panda. STUDY DESIGN: Prospective clinical study. ANIMALS: Seven giant pandas (Ailuropoda melanoleuca), five males and two females, aged 7-20 years and weighing 69.2-132.9 kg. METHODS: Once an animal was located, prior data on the individual was reviewed and the panda's previously estimated body weight was used to calculate the volumes of drugs to administer: dexmedetomidine (dexM; 8 µg kg(-1) ; 0.5 mg mL(-1) ) and tiletamine-zolazepam (TZ; 2 mg kg(-1) ; 50 mg mL(-1) ). The mixture was injected intramuscularly (IM) using the Dan-Inject pistol system. When the panda was immobilized, it was weighed, a physical examination was performed and a blood sample collected. Every 5 minutes, the heart rate (HR), respiratory rate (fR ), rectal temperature (T), noninvasive systolic arterial pressure (SAP), capillary refill time (CRT), mucous membrane color and pulse quality were recorded. After all procedures had been completed, atipamezole (40 µg kg(-1) ) was injected IM. RESULTS: A single injection of dexMTZ resulted in the immobilization of all seven giant pandas. The median (range) of anesthetic agents administered was dexM 8.4 µg kg(-1) (7.3-10.5 µg kg(-1) ) and TZ 2.0 mg kg(-1) (1.8-2.5 mg kg(-1) ). The palpebral reflex was lost 8 (7-12) minutes after the injection. Most of the physiological variables remained in the acceptable range. All procedures were completed in approximately 1 hour. Six out of the seven (85.7%) giant pandas recovered smoothly; one panda had a rough recovery. CONCLUSIONS AND CLINICAL RELEVANCE: DexMTZ produced a satisfactory immobilization and a smooth recovery for wild giant pandas while allowing approximately 55 minutes for planned noninvasive procedures.

DENTAL ABNORMALITIES OF EIGHT WILD QINLING GIANT PANDAS (AILUROPODA MELANOLEUCA QINLINGENSIS), SHAANXI PROVINCE, CHINA.

Eight adult (six male and two female) wild Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) from China National Foping Nature Reserve were tracked, and their dental data collected and recorded from October 2010 to April 2014. Each panda had dental abnormalities of varying severity. Dental wear and fracture were the most common conditions. Absent teeth were common, with premolars missing most often. Mild caries were present in five molar teeth between two animals. Different degrees of dental plaque and calculus occurred in all animals but without severe periodontal disease. Two animals with severe dental abnormalities died due to intestinal problems. Large segments of bamboo were found in their intestinal tracts, and intestinal perforation and ulcers were evident, indicating dental abnormalities can be an important factor in the health of wild giant pandas and may lead to death. Further research with larger sample sizes of wild and captive giant pandas will be required to substantiate the relationship between dental abnormalities and mortality in giant pandas.