Mesomycoplasma ovipneumoniae from goats with respiratory infection: pathogenic characteristics, population structure, and genomic features.

BACKGROUND: Mycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism. RESULTS: Toxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes (ycjT and group_1595) and glucosidase-related genes (atpD_2), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities. CONCLUSIONS: The population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

Proteomic analysis of exosome-like vesicles from Fasciola gigantica adult worm provides support for new vaccine targets against fascioliasis.

BACKGROUND: Extracellular vesicles (EVs) released by helminths play an important role in parasite-host communication. However, little is known about the characteristics and contents of the EVs of Fasciola gigantica, a parasitic flatworm that causes tropical fascioliasis. A better understanding of EVs released by F. gigantica will help elucidate the mechanism of F. gigantica-host interaction and facilitate the search for new vaccine candidates for the control and treatment of fascioliasis. METHODS: Two different populations of EVs (15k EVs and 100k EVs) were purified from adult F. gigantica culture media by ultracentrifugation. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and by the Zetasizer Nano ZSP high performance particle characterization system. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100k EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS). Mice were then vaccinated with excretory/secretory products (ESPs; depleted of EVs), 15k EVs, 100k EVs and recombinant F. gigantica heat shock protein 70 (rFg-HSP70) combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used as measures of vaccine protection. RESULTS: TEM analysis and nanoparticle tracking analysis indicated the successful isolation of two subpopulations of EVs (15k EVs and 100k EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100k EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT (endosomal sorting complex required for transport) pathway proteins and exosome markers, heat shock proteins and 14-3-3 proteins were identified in the 100k EVs. These results indicate that the isolated 100k EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion and virulence. Mice immunized with F. gigantica ESPs, 15k EVs, 100k EVs and rFg-HSP70 exhibited a reduction in fluke burden of 67.90%, 60.38%, 37.73% and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100k EVs, 15k EVs, ESP and rFg-HSP70 induced significant production of specific immunoglobulins in sera, namely IgG, IgG1 and IgG2a. CONCLUSION: The results of this study suggest that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination and the diagnosis of fascioliasis.

Immunological pathogenesis of Bovine E. coli infection in a model of C. elegans.

BACKGROUND: Cattle industry is critical for China's livestock industry, whereas E. coli infection and relevant diseases could lead huge economic loss. Traditional mammalian models would be costly, time consuming and complicated to study pathological changes of bovine E. coli. There is an urgent need for a simple but efficient animal model to quantitatively evaluate the pathological changes of bovine-derived E. coli in vivo. Caenorhabditis elegans (C. elegans) has a broad host range of diverse E. coli strains with advantages, including a short life cycle, a simple structure, a transparent body which is easily visualized, a well-studied genetic map, an intrinsic immune system which is conservable with more complicated mammalians. RESULTS: Here, we considered that O126 was the dominant serotype, and a total of 19 virulence factors were identified from 41 common E. coli virulence factors. Different E. coli strains with diverse pathogenicity strengths were tested in C. elegans in E. coli with higher pathogenicity (EC3/10), Nsy-1, Sek-1 and Pmk-1 of the p38 MAPK signaling pathway cascade and the expression of the antimicrobial peptides Abf-3 and Clec-60 were significantly up-regulated comparing with other groups. E. coli with lower pathogenicity (EC5/13) only activated the expression of Nsy-1 and Sek-1 genes in the p38 MAPK signaling pathway, Additionally, both groups of E. coli strains caused significant upregulation of the antimicrobial peptide Spp-1. CONCLUSION: Thirteen E. coli strains showed diverse pathogenicity in nematodes and the detection rate of virulence factors did not corresponding to the virulence in nematodes, indicating complex pathogenicity mechanisms. We approved that C. elegans is a fast and convenient detection model for pathogenic bacteria virulence examinations.

Isolation and Phylogenetic Analysis of a Hunnivirus Strain in Water Buffaloes From China.

In recent years, hunniviruses have been reported in a variety of animal species from many countries. Here, hunnivirus was detected in fecal samples from water buffaloes and named as BufHuV-GX-2106. The samples were inoculated into cultures of MDBK cells supplemented with TPCK trypsin and the BufHuV-GX-2106 strain was stably passaged and replicated. Electron microscopic analysis showed the BufHuV-GX-2106 virus particles were spherical and 20~30 nm in diameter. The complete genome of a plaque purified sample of BufHuV-GX-2106 was determined and analyzed. Genomic analysis revealed that the whole sequence of BufHuV-GX-2106 was ~7,601 nucleotides (nt) in length and consisted of a large open reading frame of 6,759nt, a 5'UTR, a 3'UTR and a poly(A) tail. The complete genome sequence of BufHuV-GX-2106 shares 68-85% nucleotide identities with other known hunnivirus strains, indicating high genetic heterogeneity among these viruses. Phylogenetic analysis showed that BufHuV-GX-2106 belonged to the Hunnivirus A species and was more closely related to ovine hunnivirus than other known viruses of this type. This study describes the first isolation and complete genome sequence of a hunnivirus strain from water buffaloes. In addition, this study will help to understand the mechanisms involved in the pathogenesis of Hunnivirus A among different animal species.

Recombinant Expression And Indirect ELISA For COWP And HSP70 Proteins From Cryptosporidium andersoni.

Cryptosporidium spp. infect cattle at a high rates, and reduce milk production. Cryptosporidiosis has caused economic losses for the dairy industry. Studies in Western countries have shown that Cryptosporidium can also infect humans. Therefore, the development of methods for the early detection of Cryptosporidium is an important public health objective. Total RNA isolated from C. andersoni was used as template for generating cDNA encoding the COWP and HSP70 proteins. The recombinant plasmid, pET-32a(+)-COWP-HSP70, was constructed by double digestion and subcloning. The expression of the three recombinant proteins was induced in Escherichia coli BL21 using isopropyl-ß-D-thiogalactopyranoside. The antigenicity of the recombinant proteins was examined using western blotting and indirect ELISA. The identities of the COWP fusion protein (CFP), HSP70 fusion protein (HFP), and COWP-HSP70 fusion protein (CHFP) were confirmed by BLAST searches of known sequences in GenBank respectively. The ELISA and western blot analyses indicated that all three of the proteins were highly immunogenic and antigenic. An indirect ELISA method was developed using the three recombinant proteins as coating antigens for the analysis of 40 clinical samples. The results showed that CHFP was the best candidate antigen for clinical testing, with a detection rate of 100%, compared with general parasitological screening. Above of all, the recombinant CHFP protein represents the best candidate antigen among three ones for detecting anti-Cryptosporidium antibodies in clinical samples. The development of the indirect ELISA lays the foundation for further research in immunodiagnosis and disease prevention of cryptosporidiosis.

Genetic Analysis of Avian Coronavirus Infectious Bronchitis Virus in Yellow Chickens in Southern China over the Past Decade: Revealing the Changes of Genetic Diversity, Dominant Genotypes, and Selection Pressure.

The high mutation rates of infectious bronchitis virus (IBV) pose economic threats to the poultry industry. In order to track the genetic evolutionary of IBV isolates circulating in yellow chickens, we continued to conduct the genetic analyses of the structural genes S1, E, M, and N from 64 IBV isolates in southern China during 2009-2017. The results showed that the dominant genotypes based on the four genes had changed when compared with those during 1985-2008. Based on the S1 gene phylogenetic tree, LX4-type (GI-19) was the most dominant genotype, which was different from that during 1985-2008. The second most dominant genotype was LDT3-A-type, but this genotype disappeared after 2012. New-type 1 (GVI-1) isolates showed increasing tendency and there were four aa (QKEP) located in the hypervariable region (HVR) III and one aa (S) insertion in all the New-type 1 isolates. Both the analyses of amino acid entropy and molecular evolutionary rate revealed that the variations from large to small were S1, E, M, and N. Purifying selection was detected in the S1, E, M, and N gene proteins, which was different from the positive selection during 1985-2008. Six isolates were confirmed to be recombinants, possibly generated from a vaccine virus of the 4/91-type or LDT3-A-type and a circulating virus. The estimated times for the most recent common ancestors based on the S1, E, M, and N genes were the years of 1744, 1893, 1940, and 1945, respectively. Bayesian skyline analysis revealed a sharp decrease in genetic diversity of all the four structural genes after 2010 and since late 2015, the viral population rapidly rose. In conclusion, the IBVs circulating in southern China over the past decade have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines to counteract the emerging LX4-type and New-type IBVs.

Detection and genomic characterization of Bovine papillomavirus isolated from Chinese native cattle.

Bovine papillomaviruses (BPV) are small circular DNA viruses which can be widely spread in herd, inducing cattle tumours, therefore, leading economic losses in dairy and beef production industries. BPV-leads symptoms include cutaneous papillomas, fibropapillomas, urinary bladder and oesophageal carcinoma. As one of the most important producers of beef in the world, China has not provided systematic research to prevent the harm of BPV, particularly in papillomavirus molecular characterization which presents among Chinese native cattle which was known to have higher disease resistance. In this study, skin papilloma was observed and samples were collected following by histopathological analysis. We analysed all neoplasms samples and reviewed their degrees in acanthosis and/or hyperkeratosis. Full-length genomic sequencing was applied for all four isolated strains (JX180408, LA150909, HX160815, and BS160810) to exploring the molecular reason why BPV currently prevalent in Chinese native cattle. As a result, we identified that these four isolates were classified as BPV-1 and clustered into the Deltapapillomavirus genera. Our study also identified that BPV 1 isolates from Chinese indigenous cattle breeds belong to subtypes A which has a closer genetic background compare with their common ancestor and suggest it can be a more ancestral species. European isolates more recently diverged group (group B) contained almost exclusively European samples. In this study, we analysed the similarity of ORF between Chinese isolated BPV 1 and BPV 1 reference strains and listed results. This study provides the complete genomic characterization of BPVs circulating in Chinese native cattle breeds for the first time, which provide a detailed description of how diverse strains may cause skin tumour among Chinese local breed cattle therefore critical for further epidemiological study of relevant diseases.

[Sequencing and Serologic Identification of S1 Genes of Infectious Bronchitis Viruses Isolated during 2012-2013 in Guangxi Province, China].

We wished to ascertain the prevalence as well as the genetic and antigenic variation of infectious bronchitis viruses (IBVs) circulating in the Guangxi Province of China in recent years. The S1 gene of 15 IBV field isolates during 2012-2013 underwent analyses in terms of the similarity of amino-acid sequences, creation of phylogenetic trees, recombination, and serologic identification. Similarities in amino-acid sequences among the 15 isolates of the S1 gene were 54.3%-99.6%, and 43.3%-99.3% among 15 isolates and reference strains. Compared with the vaccine strain H120, except for GX-YL130025, the other 14 isolates showed a lower similarity of amino-acid sequences of the S1 gene (65.1-81.4%). Phylogenetic analyses of the S1 gene suggested that 15 IBV isolates were classified into eight genotypes, with the predominant genotype being new-type II. Recombination analyses demonstrated that the S1 gene of the GX-NN130048 isolate originated from recombination events between vaccine strain 4/91 and a LX4-like isolate. Serotyping results suggested that seven serotypes prevailed during 2012-2013 in Guangxi Province, and that only one isolate was consistent with the vaccine strain H120 in serotype (which has been used widely in recent years). The serotype of recombinant isolate GX-NN130048 was different from those of its parent strains. These results suggested that not only the genotype, but also the serotype of IBV field isolates in Guangxi Province had distinct variations, and that increasing numbers of genotypes and serotypes are in circulation. We showed that recombination events can lead to the emergence of new serotypes. Our study provides new evidence for understanding of the molecular mechanisms of IBV variations, and the development of new vaccines against IBVs.

Heterogeneous nucleation of ß-type precipitates on nanoscale Zr-rich particles in a Mg-6Zn-0.5Cu-0.6Zr alloy.

Zirconium (Zr) is an important alloying element to Mg-Zn-based alloy system. In this paper, we report the formation of the ß-type precipitates on the nanoscale Zr-rich particles in a Mg-6Zn-0.5Cu-0.6Zr alloy during ageing at 180°C. Scanning transmission electron microscopy examinations revealed that the nanoscale Zr-rich [0001]α rods/laths are dominant in the Zr-rich core regions of the as-quenched sample after a solution treatment at 430°C. More significantly, these Zr-rich particles served as favourable sites for heterogeneous nucleation of the Zn-rich ß-type phase during subsequent isothermal ageing at 180°C. This research provides a potential route to engineer precipitate microstructure for better strengthening effect in the Zr-containing Mg alloys.

The formation of symmetric SiC bi-nanowires with a Y-shaped junction.

Transmission electron microscopy was used to investigate the formation mechanism of SiC bi-nanowires (BNW) with a Y-shaped junction. Different from the previously reported growth mechanisms, our study suggests that when two individual nanowires that are growing through the vapor-liquid-solid mechanism meet with a proper angle, they will merge and form a straight and symmetric BNW with its two side-branches maintaining their original crystallographic orientations and sharing the same crystallographic growth direction, which can be (110), (112) or (113), depending on the meeting angle between the two initial nanowires. According to our observations, a growth model of SiC BNWs with a Y-shaped junction was proposed. The radii and the microstructure of the BNWs are controlled to a certain extent by the meeting angle and the radii of the two SiC single NWs.