NIR-II fluorescent Ag2Se polystyrene beads in a lateral flow immunoassay to detect biomarkers for breast cancer.

Fluorescent lateral flow immunoassay (LFA), one tool in point of care testing (POCT) systems for breast cancer, has attracted attention because it is quick, simple, and convenient. However, samples and the constituent material exhibit autofluorescence in the visible region, which is a very large obstacle in the development of fluorescent LFAs. The autofluorescence of biological samples is scarcely found in the second near-infrared (NIR-II) range and samples scatter and absorb less NIR-II light than visible light. Here, we report an NIR-II QD-LFA platform using the NIR-II fluorescent Ag2Se quantum dots (QDs) with 1020 nm emission encapsulated into polystyrene beads as fluorescent probes. The NIR-II LFA platform was established to detect breast cancer tumour markers (CEA and CA153) within 15 min with a low limit of detection (CEA: 0.768 ng mL-1, CA153: 1.192 U mL-1), high recoveries (93.7% ~ 108.8%), and relative standard deviations (RSDs) of less than 10%. This study demonstrated the potential of NIR-II Ag2Se polystyrene beads as a fluorescent probe in LFA for rapid and accurate identification of biomarkers. They are suited for use in professional situations.

Antimicrobial susceptibility and multilocus sequence typing of Clostridium perfringens isolated from yaks in Qinghai-Tibet plateau, China.

Clostridium perfringens (C. perfringens) is an opportunistic pathogen that cause necrotic enteritis, food poisoning and even death in animals. In this study, we explored the prevalence, antibiotic resistance and genetic diversity of Clostridium perfringens isolated from yak in the Qinghai-Tibet plateau, China. A total of 744 yak fecal samples were collected and assessed for toxin genes, antimicrobial susceptibility and multilocus sequence typing (MLST). Results indicated that 144 out of 744 (19.35%) yak fecal samples were tested to be positive for C. perfringens, 75% (n = 108, 108/144) were C. perfringens type A, 17.36% (n = 25, 25/144) were C. perfringens type C, 2.78% (n = 4, 4/144) were C. perfringens type D, and 4.86% (n = 7, 7/144) were C. perfringens type F. In addition, 2.78% (n = 4, 4/144) of the isolates were positive for cpb2 toxin gene. Antimicrobial susceptibility testing revealed that 98.61% (142/144) of the isolates showed multiple-antibiotic resistance. According to MLST and phylogenetic tree, 144 yak-derived C. perfringens isolates had an average of 12.95 alleles and could be divided into 89 sequence types (STs) and clustered in 11 clonal complexes (CCs). The most of isolates belong to type A with a considerable genetic diversity, having Simpson index up to 0.9754. MLST and phylogenetic analysis showed that the isolates under the same clade came from multiple regions. Cross-transmission among isolates and interconnectedness were observed in the genetic evolution. According to the study, the most of the isolates exhibited broad-spectrum antibacterial resistance, diverse alleles, and multiple lethal toxin genes of C. perfringens.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

Gut Virome of the World's Highest-Elevation Lizard Species (Phrynocephalus erythrurus and Phrynocephalus theobaldi) Reveals Versatile Commensal Viruses.

The gut virome is a reservoir of diverse symbiotic and pathogenic viruses coevolving with their hosts, and yet limited research has explored the gut viromes of highland-dwelling rare species. Using viral metagenomic analysis, the viral communities of the Phrynocephalus lizards living in the Qinghai-Tibet Plateau were investigated. Phage-encoded functional genes and antibiotic resistance genes (ARGs) were analyzed. The viral communities of different lizard species were all predominated by bacteriophages, especially the Caudovirales order. The virome of Phrynocephalus erythrurus living around the Namtso Lake possessed a unique structure, with the greatest abundance of the Parvoviridae family and the highest number of exclusive viral species. Several vertebrate-infecting viruses were discovered, including caliciviruses, astroviruses, and parvoviruses. Phylogenetic analyses demonstrated that the virus hallmark genes of bacteriophages possessed high genetic diversity. After functional annotation, the majority of phage-associated functional genes were classified in the energy metabolism category. In addition, plenty of ARGs belonging to the multidrug category were discovered, and five ARGs were exclusive to the virome from Phrynocephalus theobaldi. This study provided the first insight into the structure and function of the virome in highland lizards, contributing to the protection of threatened lizard species. Also, our research is of exemplary significance for the gut virome research of lizard species and other cold-blooded and highland-dwelling animals, prompting a better understanding of the interspecific differences and transmission of commensal viruses. IMPORTANCE The Phrynocephalus lizards inhabiting the Qinghai-Tibet Plateau (QTP) are considered to be the highest-altitude lizard species in the world, and they have been added to the IUCN list of threatened species. Living in the QTP with hypoxic, arid, natural conditions, the lizards presented a unique pattern of gut virome, which could provide both positive and negative effects, such as the enrichment of functional genes and the dissemination of antibiotic resistance genes (ARGs). This work provides the foundation for further research on the gut virome in these endangered lizard species and other cold-blooded and highland-dwelling animals, contributing to the maintenance of ecological balance on the plateau.

Next-Generation Sequencing Reveals Four Novel Viruses Associated with Calf Diarrhea.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Metagenomic insights into the diversity of carbohydrate-degrading enzymes in the yak fecal microbial community.

BACKGROUND: Yaks are able to utilize the gastrointestinal microbiota to digest plant materials. Although the cellulolytic bacteria in the yak rumen have been reported, there is still limited information on the diversity of the major microorganisms and putative carbohydrate-metabolizing enzymes for the degradation of complex lignocellulosic biomass in its gut ecosystem. RESULTS: Here, this study aimed to decode biomass-degrading genes and genomes in the yak fecal microbiota using deep metagenome sequencing. A comprehensive catalog comprising 4.5 million microbial genes from the yak feces were established based on metagenomic assemblies from 92 Gb sequencing data. We identified a full spectrum of genes encoding carbohydrate-active enzymes, three-quarters of which were assigned to highly diversified enzyme families involved in the breakdown of complex dietary carbohydrates, including 120 families of glycoside hydrolases, 25 families of polysaccharide lyases, and 15 families of carbohydrate esterases. Inference of taxonomic assignments to the carbohydrate-degrading genes revealed the major microbial contributors were Bacteroidaceae, Ruminococcaceae, Rikenellaceae, Clostridiaceae, and Prevotellaceae. Furthermore, 68 prokaryotic genomes were reconstructed and the genes encoding glycoside hydrolases involved in plant-derived polysaccharide degradation were identified in these uncultured genomes, many of which were novel species with lignocellulolytic capability. CONCLUSIONS: Our findings shed light on a great diversity of carbohydrate-degrading enzymes in the yak gut microbial community and uncultured species, which provides a useful genetic resource for future studies on the discovery of novel enzymes for industrial applications.

Characterization of Metagenome-Assembled Genomes and Carbohydrate-Degrading Genes in the Gut Microbiota of Tibetan Pig.

Tibetan pig is an important domestic mammal, providing products of high nutritional value for millions of people living in the Qinghai-Tibet Plateau. The genomes of mammalian gut microbiota encode a large number of carbohydrate-active enzymes, which are essential for the digestion of complex polysaccharides through fermentation. However, the current understanding of microbial degradation of dietary carbohydrates in the Tibetan pig gut is limited. In this study, we produced approximately 145 gigabases of metagenomic sequence data for the fecal samples from 11 Tibetan pigs. De novo assembly and binning recovered 322 metagenome-assembled genomes taxonomically assigned to 11 bacterial phyla and two archaeal phyla. Of these genomes, 191 represented the uncultivated microbes derived from novel prokaryotic taxa. Twenty-three genomes were identified as metagenomic biomarkers that were significantly abundant in the gut ecosystem of Tibetan pigs compared to the other low-altitude relatives. Further, over 13,000 carbohydrate-degrading genes were identified, and these genes were more abundant in some of the genomes within the five principal phyla: Firmicutes, Bacteroidetes, Spirochaetota, Verrucomicrobiota, and Fibrobacterota. Particularly, three genomes representing the uncultivated Verrucomicrobiota encode the most abundant degradative enzymes in the fecal microbiota of Tibetan pigs. These findings should substantially increase the phylogenetic diversity of specific taxonomic clades in the microbial tree of life and provide an expanded repertoire of biomass-degrading genes for future application to microbial production of industrial enzymes.

Seroprevalence of Toxoplasma gondii infection in Tibetan sheep in Tibet, China.

In the present investigation, the seroprevalence of Toxoplasma gondii antibodies in 455 Tibetan sheep in Tibet, China, was examined using an indirect hemagglutination test. Of these, 26 (5.7%) Tibetan sheep were seropositive at the cut-off of 1:64 serum dilution. The seroprevalence ranged from 2.2% to 8.9% among Tibetan sheep of <1-yr-old, 1-3-yr-old, and >3-yr-old, but the differences among the age groups were not significant (P > 0.05). The prevalence in male Tibetan sheep (2.8%) was lower than that in female Tibetan sheep (6.6%), but the difference was not statistically significant (P > 0.05). The results of this survey indicated the presence of T. gondii infection in Tibetan sheep, which may cause economic losses to the local livestock industry and which poses a potential threat to human health in this area.

Experimental infection of Balb/c nude mice with Hepatitis E virus.

BACKGROUND: Several animal species can reportedly act as reservoirs for Hepatitis E virus (HEV), a zoonotic pathogen. HEV and antibody to the virus have been detected in a variety of animals including rodents. Pig and rat models for HEV have been established for HEV, but a nude mouse has not yet been developed. METHODS: Balb/c nude mice were inoculated with swine HEV, both orally and via intravenous injection to insure infection. Negative control and experimental contact-exposed groups of mice were also included in the study. The liver, spleen, kidney, jejunum, ileum, cecum and colon of each mouse from all three groups were collected for reverse transcription nested polymerase chain reaction (RT-nPCR) detection, indirect immunofluorescence observation and histopathologic examination. The sera from nude mice were tested for anti-HEV IgG by enzyme linked immunosorbent assay (ELISA). Activities of liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), as well as total bilirubin (TBIL) were also measured in the sera of the nude mice. RESULTS: HEV antigens and HEV RNA were detected in liver, spleen, kidney, jejunum, ileum and colon both by indirect immunofluorescence and by RT-nPCR in all of the inoculated and in one of the contact-exposed nude mice. Histopathological changes were observed in the liver and spleen of these mice. Infected mice showed increased levels of AST, ALP, and anti-HEV IgG in sera. The livers of contact-exposed mice showed obvious histopathological damage. CONCLUSION: Nude mice could be readily infected by HEV isolated from pigs. The nude mouse may therefore be a useful animal model for studying the pathogenesis of HEV.