Emergence of genetic diversity and multi-drug resistant Clostridium perfringens from wild birds.

BACKGROUND: Clostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited. RESULT: In this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species. CONCLUSION: C. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.

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.

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 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.

Mining metagenomic data to gain a new insight into the gut microbial biosynthetic potential in placental mammals.

Mammals host a remarkable diversity and abundance of gut microbes. Biosynthetic gene clusters (BGCs) in microbial genomes encode biologically active chemical products and play an important role in microbe-host interactions. Traditionally, the exploration of gut microbial metabolic functions has relied on the pure culture method. However, given the limited amounts of microbes being cultivated, insights into the metabolism of gut microbes in mammals continued to be very limited. In this study, we adopted a computational pipeline for mining the metagenomic data (named taxonomy-guided identification of biosynthetic gene clusters, TaxiBGC) to identify experimentally verified BGCs in 373 metagenomes across 53 mammalian species in an unbiased manner. We demonstrated that polyketides (PKs) and nonribosomal peptides (NRPs) are representative of mammals, and the products derived from them were associated with cell-cell communication and resistance to inflammation. Large carnivores had the highest number of BGCs, followed by large herbivores and small mammals. We also observed that the large mammals had more common BGCs that aid in the biosynthesis of a variety of natural products. However, small mammals not only had fewer BGCs but were also unique to each species. Our results provide novel insights into the mining of metagenomic data sets to identify active BGCs and their products across mammals.IMPORTANCEThe gut microbes host numerous biosynthetic gene clusters (BGCs) that biosynthesize natural products and impact the host's physiology. Historically, our understanding of BGCs in mammalian gut microbes was largely based on studies on cultured isolates; however, only a small fraction of mammal-associated microbes have been investigated. The biochemical diversity of the mammalian gut microbiota is poorly understood. Metagenomic sequencing contains data from a vast number of organisms and provides information on the total gene content of communities. Unfortunately, the existing BGC prediction tools are designed for individual microbial genomes. Recently, a BGC prediction tool called the taxonomy-guided identification of biosynthetic gene clusters (TaxiBGC) that directly mine the metagenome was developed. To gain new insights into the microbial metabolism, we used TaxiBGC to predict BGCs from 373 metagenomes across 53 mammalian species representing seven orders. Our findings elucidate the functional activities of complex microbial communities in the gut.

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.

Multi-omic Analyses Shed Light on The Genetic Control of High-altitude Adaptation in Sheep.

Sheep were domesticated in the Fertile Crescent and then spread globally, where they have been encountering various environmental conditions. The Tibetan sheep has adapted to high altitudes on the Qinghai-Tibet Plateau over the past 3000 years. To explore genomic variants associated with high-altitude adaptation in Tibetan sheep, we analyzed Illumina short-reads of 994 whole genomes representing ∼ 60 sheep breeds/populations at varied altitudes, PacBio High fidelity (HiFi) reads of 13 breeds, and 96 transcriptomes from 12 sheep organs. Association testing between the inhabited altitudes and 34,298,967 variants was conducted to investigate the genetic architecture of altitude adaptation. Highly accurate HiFi reads were used to complement the current ovine reference assembly at the most significantly associated ß-globin locus and to validate the presence of two haplotypes A and B among 13 sheep breeds. The haplotype A carried two homologous gene clusters: (1) HBE1, HBE2, HBB-like, and HBBC, and (2) HBE1-like, HBE2-like, HBB-like, and HBB; while the haplotype B lacked the first cluster. The high-altitude sheep showed highly frequent or nearly fixed haplotype A, while the low-altitude sheep dominated by haplotype B. We further demonstrated that sheep with haplotype A had an increased hemoglobin-O2 affinity compared with those carrying haplotype B. Another highly associated genomic region contained the EGLN1 gene which showed varied expression between high-altitude and low-altitude sheep. Our results provide evidence that the rapid adaptive evolution of advantageous alleles play an important role in facilitating the environmental adaptation of Tibetan sheep.

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.