Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Exploration of Parascaris species in three different Equus populations in China.

BACKGROUND: The roundworms, Parascaris spp., are important nematode parasites of foals and were historically model organisms in the field of cell biology, leading to many important discoveries. According to karyotype, ascarids in Equus are commonly divided into Parascaris univalens (2n = 2) and Parascaris equorum (2n = 4). METHODS: Here, we performed morphological identification, karyotyping and sequencing of roundworms from three different hosts (horses, zebras and donkeys). Phylogenetic analysis was performed to study the divergence of these ascarids based on cytochrome c oxidase subunit I (COI) and internal transcribed spacer (ITS) sequences. RESULTS: Karyotyping, performed on eggs recovered from worms of three different Equus hosts in China, showed two different karyotypes (2n = 2 in P. univalens collected from horses and zebras; 2n = 6 in Parascaris sp. collected from donkeys). There are some differences in the terminal part of the spicula between P. univalens (concave) and Parascaris sp. (rounded). Additionally, it was found that the egg's chitinous layer was significantly thicker in Parascaris sp. (> 5 µm) than P. univalens (< 5 µm) (F(2537) = 1967, P < 0.01). Phylogenetic trees showed that the sequences of Parascaris from Equus hosts were divided into two distinct lineages based on sequences of the COI and ITS. CONCLUSIONS: Comparing the differences in roundworms collected from three different Equus hosts, this study describes a Parascaris species (Parascaris sp.) with six chromosomes in donkeys. It is worth noting that the thickness of the chitinous layer in the Parascaris egg may serve as a diagnostic indicator to distinguish the two roundworms (P. univalens and Parascaris sp.). The Parascaris sp. with six chromosomes in donkeys in the present study may be a species of P. trivalens described in 1934, but the possibility that it is a new Parascaris species cannot be ruled out. Both karyotyping and molecular analysis are necessary to solve the taxonomic problems in Parascaris species.

Revived Amplicon Sequence Variants Monitoring in Closed Systems Identifies More Dormant Microorganisms.

The large number of dormant microorganisms present in the environment is an important component of microbial diversity, and neglecting dormant microorganisms would be disruptive to all research under the science of microbial diversity. However, current methods can only predict the dormancy potential of microorganisms in a sample and are not yet able to monitor dormant microorganisms directly and efficiently. Based on this, this study proposes a new method for the identification of dormant microorganisms based on high-throughput sequencing technology: Revived Amplicon sequence variants (ASV) Monitoring (RAM). Pao cai (Chinese fermented vegetables) soup was used to construct a closed experimental system, and sequenced samples were collected at 26 timepoints over a 60-day period. RAM was used to identify dormant microorganisms in the samples. The results were then compared with the results of the currently used gene function prediction (GFP), and it was found that RAM was able to identify more dormant microorganisms. In 60 days, GFP monitored 5045 ASVs and 270 genera, while RAM monitored 27,415 ASVs and 616 genera, and the RAM results were fully inclusive of the GFP results. Meanwhile, the consistency of GFP and RAM was also found in the results. The dormant microorganisms monitored by both showed a four-stage distribution pattern over a 60-day period, with significant differences in the community structure between the stages. Therefore, RAM monitoring of dormant microorganisms is effective and feasible. It is worth noting that the results of GFP and RAM can complement and refer to each other. In the future, the results obtained from RAM can be used as a database to extend and improve the monitoring of dormant microorganisms by GFP, and the two can be combined with each other to build a dormant microorganism detection system.

Seroprevalence and risk factor analysis of Toxoplasma gondii infection in Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China.

Toxoplasmosis, caused by Toxoplasma gondii, is a worldwide zoonosis. The aim of the present study was to detect the seroprevalence of T. gondii infection and associated risk factors among Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China. Blood samples from 112 Siberian tigers and 22 giant pandas were tested for immunoglobulin G (IgG) against T. gondii by enzyme-linked immunosorbent assay (ELISA). The seroprevalence of T. gondii infection was 7.14% among Siberian tigers and 9.09% among giant pandas. No risk factors were found to be significantly associated with seroprevalence (P > 0.05). This is the first study to evaluate T. gondii infection in Siberian tigers on a large scale in China, and it also updates the information regarding the positivity rate of T. gondii infection among giant pandas in China.

The genetic characteristics of Sarcoptes scabiei from Chinese serow (Capricornis milneedwardsii) and goral (Naemorhedus goral arnouxianus) compared with other mites from different hosts and geographic locations using ITS2 and cox1 sequences.

Scabies is a common parasitic disease in many mammalian species, caused by the infestation of Sarcoptes scabiei. There is no consistent conclusion on whether Sarcoptes mites from different hosts or geographic locations have apparent genetic divergence. In this study, we collected and morphologically identified S. scabiei from Chinese serow and goral, and we described the genetic diversity of S. scabiei and other mites based on phylogenetic analyses of the ITS2 and cox1 sequence fragments, including data available in GenBank. The mites isolated from Chinese serow and goral were S. scabiei, and they were morphologically similar. The phylogenetic trees and haplotype networks showed that S. scabiei from other locations worldwide did not cluster according to host divergence or geographical distribution. Additionally, the Fst values were - 0.224 to 0.136 and - 0.045 to 1 between S. scabiei from different hosts, including humans and domestic and wild animals, based on partial ITS and cox1 sequences. Worldwide S. scabiei samples formed three clusters (with H2, H5, and H12 at their centers) in the ITS and one cluster (with C9 at the center) in the cox1 haplotype phylogenetic network. The S. scabiei collected from Chinese serow and goral were morphologically similar and had the same genotype. A study on the genetic characteristics of S. scabiei from Chinese serow and goral together with other mites from different hosts and geographic locations around the world showed no obvious divergence. These findings indicated that scabies likely is a zoonotic disease and that the global prevalence of scabies is probably related to the worldwide trade of domestic animals.

The chromosome-scale genome of the raccoon dog: Insights into its evolutionary characteristics.

The raccoon dog (Nyctereutes procyonoides) is an invasive canid species native to East Asia with several distinct characteristics. Here, we report a chromosome-scale genome of the raccoon dog with high contiguity, completeness, and accuracy. The intact taste receptor genes, expanded gene families, and positively selected genes related to digestion, absorption, foraging, and detoxification likely support the omnivory of raccoon dogs. Several positively selected genes and raccoon dog-specific mutations in TDRD6 and ZP3 genes may explain their high reproductivity. Enriched GO terms in energy metabolism and positively selected immune genes were speculated to be closely related to the diverse immune system of raccoon dogs. In addition, we found that several expanded gene families and positively selected genes related to lipid metabolism and insulin resistance may contribute to winter sleep of the raccoon dog. This high-quality genome provides a valuable resource for understanding the evolutionary characteristics of this species.

Gene rearrangements in the mitochondrial genome of ten ascaris species and phylogenetic implications for Ascaridoidea and Heterakoidea families.

The Ascaridoidea family and Heterakoidea family are the most common and typical representative of large parasites. Although our understanding of these parasites' diversity has expanded by analyses of some mitochondrial genes, there is limited information on these species' evolutionary rates. Here we determined ten complete mitogenome sequences of five subfamilies of Ascaridoidea and one subfamily of Heterakoidea. The phylogenetic tree divided the Ascaridoidea into six monophyletic major clades, and the divergence time of Heterakoidea family and Ascaridoidea family can be placed during the early Carboniferous Period (300-360 Mya). The reconstruction of the ancestral state showed that the gene orders of all species in Ascaridoidea were conserved, and the Heterakoidea had obvious genome rearrangement. The conserved blocks between them were divided into five and the main types are tandem-duplication/random loss (TDRL). These results will help to better understand the gene rearrangements and evolutionary position of ascaris species.

Equus roundworms (Parascaris univalens) are undergoing rapid divergence while genes involved in metabolic as well as anthelminic resistance are under positive selection.

BACKGROUND: The evolution of parasites is often directly affected by the host's environment. Studies on the evolution of the same parasites in different hosts are of great interest and are highly relevant to our understanding of divergence. METHODS: Here we performed whole-genome sequencing of Parascaris univalens from different Equus hosts (horses, zebras and donkeys). Phylogenetic and selection analyses were performed to study the divergence and adaptability of P. univalens. RESULTS: At the genetic level, multiple lines of evidence indicate that P. univalens is mainly separated into two clades (horse-derived and zebra & donkey-derived). This divergence began 300-1000 years ago, and we found that most of the key enzymes related to glycolysis were under strong positive selection in zebra & donkey-derived roundworms, whereas the lipid-related metabolic system was under positive selection in horse-derived roundworms, indicating that the adaptive evolution of metabolism has occurred over the past few centuries. In addition, we found that some drug-related genes showed a significantly higher degree of selection in diverse populations. CONCLUSIONS: This work reports the adaptive evolution and divergence trend of P. univalens in different hosts for the first time. Its results indicate that the divergence of P. univalens is a continuous, dynamic process. Furthermore, the continuous monitoring of the effects of differences in nutritional and drug histories on the rapid evolution of roundworms is conducive to further understanding host-parasite interactions.

First isolation of Trichuris from Chinese serow, Capricornis milneedwardsii (Cetartiodactyla: Caprinae).

Whipworms are soil-transmitted helminths that can infect a variety of animals. A Chinese serow possibly infected by whipworms was found during a wildlife disease surveillance project in Baima Snow Mountain National Nature Reserve, Yunnan, China, in 2021. As convergent evolution is common in coinhabiting parasites, a mitochondrial gene sequence (cox1) and ribosomal gene sequence (ITS1) were used to identify species similar to Trichuris from Chinese serow. The phylogenetic trees and genetic distances of ten Trichuris samples from a Chinese serow together with other Trichuris spp. that have been previously reported were analysed based on the cox1 and ITS1 sequences. The combined results of the phylogenetic tree and genetic distances based on cox1 gene showed that the whipworms in Chinese serow are T. skrjabini. However, the whipworms in the present study were divided into two apparent clades in the phylogenic trees constructed by the cox1 sequences (Clades A and B) and the ITS1 sequences (Clades C and D). In addition, the Fst and Nm values were 0.82 and 0.23 between Clade A and Clade B for the cox1 gene, and 0.30 and 0.45 between Clade C and Clade D for the ITS1 sequences; both indicators showed low gene flow among the clades. Therefore, the genetic population structure of T. skrjabini was illustrated.

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.

Chromosome-scale assembly and whole-genome sequencing of 266 giant panda roundworms provide insights into their evolution, adaptation and potential drug targets.

Helminth diseases have long been a threat to the health of humans and animals. Roundworms are important organisms for studying parasitic mechanisms, disease transmission and prevention. The study of parasites in the giant panda is of importance for understanding how roundworms adapt to the host. Here, we report a high-quality chromosome-scale genome of Baylisascaris schroederi with a genome size of 253.60 Mb and 19,262 predicted protein-coding genes. We found that gene families related to epidermal chitin synthesis and environmental information processes in the roundworm genome have expanded significantly. Furthermore, we demonstrated unique genes involved in essential amino acid metabolism in the B. schroederi genome, inferred to be essential for the adaptation to the giant panda-specific diet. In addition, under different deworming pressures, we found that four resistance-related genes (glc-1, nrf-6, bre-4 and ced-7) were under strong positive selection in a captive population. Finally, 23 known drug targets and 47 potential drug target proteins were identified. The genome provides a unique reference for inferring the early evolution of roundworms and their adaptation to the host. Population genetic analysis and drug sensitivity prediction provide insights revealing the impact of deworming history on population genetic structure of importance for disease prevention.

Investigation of the Efficacy of Pyrantel Pamoate, Mebendazole, Albendazole, and Ivermectin against Baylisascaris schroederi in Captive Giant Pandas.

Baylisascaris schroederi is one of the main health risks threatening both wild and captive giant pandas. The administration of anthelmintics is a common method to effectively control B. schroederi infection, but there is a notable risk of anthelmintic resistance (AR) after long-term, constant use of anthelmintics. Four anthelmintics-pyrantel pamoate (PYR), mebendazole (MBZ), albendazole (ABZ), and ivermectin (IVM)-were each administered separately at intervals of 2 months to 22 enrolled giant pandas. The fecal egg count reduction (FECR) proportions were calculated by both the Markov chain Monte Carlo (MCMC) Bayesian mathematical model and the arithmetic mean. AR was assessed based on the criteria recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). The estimated prevalence of B. schroederi infection was 34.1%. After treatment with PYR, MBZ, ABZ, and IVM, it was determined that MBZ, ABZ, and IVM were efficacious against B. schroederi, while nematodes were suspected to be resistant to PYR according to the fecal egg count reduction (FECR) proportions.