Cloning and Expression of Pigeon-Derived Escherichia coli Type 1 Pilus Clusters and Analysis of Amino Acid Sequence Characteristics of Functional Proteins.

BACKGROUND: Type 1 pili, as an important virulence factor of E. coli, has certain homology between APEC and UPEC, but the homology degree is not clear enough. OBJECTIVES: This study aims to compare the homology between them. METHODS: The recombinant bacteria were constructed by homologous recombination. The pili were observed by TEM, and the hemagglutination characteristics were determined by MHSA. The complete gene sequence was determined by sequencing, and the amino acid sequences of the functional proteins of type 1 pili of APEC and UPEC were compared. RESULTS: TEM showed that they could express pili, which were slender, straight, and dense. Stable-pUC-fimBH has MHSA but stable-pUC-fimBG does not. The amino acid sequence similarity of FimB of NJ05 and UPEC was 98.8%, FimE was 99.4%, and the similarity between them was 51.5%. Compared with UPEC's type 1 pili FimC and FimD sequences, the similarity was 99.52% and 87.8%, respectively. The amino acid sequence of FimA of NJ05 was 89-96%, similar to UPEC, and the N-terminal and C-terminal amino acid sequences were exactly the same. The gene sequence and amino acid sequence similarity of FimH between them were both above 99%. The similarity of the pilus binding domain of FimH was 52.8%, but only 27.6% in the receptor binding domain. A few of the same amino acid residues were found in the corresponding regions of FimA, FimF, FimG, and FimH. CONCLUSIONS: The type 1 pili of APEC and UPEC come from the same origin, which is helpful to further reveal the pathogenic mechanism of E. coli infection in the poultry respiratory tract.

Screening of antigenic epitopes related to the adhesion of the avian Escherichia coli Type 1 Fimbrial Agglutinin Domain.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

DNAzyme-Amplified Electrochemical Biosensor Coupled with pH Meter for Ca2+ Determination at Variable pH Environments.

For more than 50% of multiparous cows, it is difficult to adapt to the sudden increase in calcium demand for milk production, which is highly likely to cause hypocalcemia. An electrochemical biosensor is a portable and efficient method to sense Ca2+ concentrations, but biomaterial is easily affected by the pH of the analyte solution. Here, an electrochemical biosensor was fabricated using a glassy carbon electrode (GCE) and single-walled carbon nanotube (SWNT), which amplified the impedance signal by changing the structure and length of the DNAzyme. Aiming at the interference of the pH, the electrochemical biosensor (GCE/SWNT/DNAzyme) was coupled with a pH meter to form an electrochemical device. It was used to collect data at different Ca2+ concentrations and pH values, and then was processed using different mathematical models, of which GPR showed higher detecting accuracy. After optimizing the detecting parameters, the electrochemical device could determine the Ca2+ concentration ranging from 5 µM to 25 mM, with a detection limit of 4.2 µM at pH values ranging from 4.0 to 7.5. Finally, the electrochemical device was used to determine the Ca2+ concentrations in different blood and milk samples, which can overcome the influence of the pH.

Molecular characterization of pigeon torque teno virus (PTTV) in Jiangsu province.

The torque teno virus (TTV) is a recently discovered DNA virus that has been detected in many different hosts, including humans, livestock and poultry. To date, there is no report of pigeon TTV (PTTV) from anywhere in the world. To investigate the distribution of PTTV in pigeons from the eastern Chinese province of Jiangsu and characterize their genomes, we employed PCR to detect PTTV in 144 samples collected from 6 pigeon plants in Jiangsu province, amplify complete genomes from representative samples and analyze genetic characteristics using bioinformatics. The results demonstrated that 71.5% (103/144) of samples were PTTV positive. The rate of sequence homology among the six PTTV complete genomes obtained from Jiangsu province ranged from 99.7% to 100%. Phylogenetic analysis suggested that PTTV genomes had a high degree of genetic similarity and were similar to chicken anemia virus that also had poultry as a host. Although with the same host, PTTV shared distant relationship with PiCV in both complete genome, Rep and Cap genes. The results of this study provided evidence that PTTV could be detected in Chinese pigeons at a high level, the evolutionary process of complete genome, Rep and Cap genes of Anelloviridae family had obvious divergence.

Epidemiology and genetic characteristics of pigeon circovirus (PiCV) in eastern China.

Pigeon circovirus (PiCV) is one of four viruses in the family Circoviridae that affect young pigeons around the world. We collected 158 serum or tissue samples from six poultry farms in eastern China to investigate the prevalence and genetic characteristics of PiCV in Chinese pigeons. We tested for PiCV using a PCR assay and found that PiCV was present in 80.7 % (88/109) of diseased pigeons and 63.3 % (31/49) of healthy pigeons; overall, 75.3 % (119/158) of samples were PiCV positive. One PiCV-positive sample from each poultry farm was randomly chosen for amplification of the complete PiCV genome by inverse primer PCR (IP-PCR). The six genomic PiCV strains were designated as AHBZ (KJ704801), HBLF-E2 (KJ704802), JSJN (KJ704803), NJPK-21 (KJ704804), SDDZ (KJ704805) and SHWH-AB4 (KJ704806). We compared these new PiCV genomes to six publicly available PiCV genomes and found that the Rep and Cap genes had sequence identity ranging from 93.8 % to 100 % and 79.1 % to 100 %, respectively. In a phylogenetic analysis, PiCV and eight other members of the genus Circovirus were sister to chicken anemia virus (CAV), the only member of genus Gyrovirus. The results of this study provide evidence that PiCV is present in Chinese pigeons at a high rate and that PiCV is a viral lineage that is distinct from CAV.

Detection and distribution of torque teno sus virus 1 in porcine reproductive and respiratory syndrome virus positive/negative pigs.

To investigate the detection rate and distribution of torque teno sus virus 1 (TTSuV1) in porcine reproductive and respiratory syndrome virus (PRRSV) positive/negative pigs, 2384 pathological tissues samples collected from 6 provinces of Eastern China from 2010 to 2013 were amplified using previously published PRRSV and TTSuV1 primers. The presence and viral load of TTSuV1 were investigated in a wide range of samples from 5 PRRSV positive/negative 4-week-old pigs by real-time TaqMan PCR. TTSuV1 was detected in 65.3% of 1115 PRRSV-positive samples, and 47.2% of 1269 negative samples. Viral DNA was most commonly detected in the immune organs, including spleen, lung, pancreas, and mesenteric and inguinal lymph nodes, followed by serum, liver, kidney, trachea, anal swabs, nasal swabs and sex glands of PRRSV-positive or negative pigs. TTSuV1 DNA loads in PRRSV-positive pigs increased from 2 to 5 times in almost all the corresponding parts compared with PRRSV-negative pigs. Statistical analysis showed that PRRSV may have a synergistic effect with TTSuV1, and promote the replication and proliferation of TTSuV1.

Synonymous codon usage in TTSuV2: analysis and comparison with TTSuV1.

Two species of the DNA virus Torque teno sus virus (TTSuV), TTSuV1 and TTSuV2, have become widely distributed in pig-farming countries in recent years. In this study, we performed a comprehensive analysis of synonymous codon usage bias in 41 available TTSuV2 coding sequences (CDS), and compared the codon usage patterns of TTSuV2 and TTSuV1. TTSuV codon usage patterns were found to be phylogenetically conserved. Values for the effective number of codons (ENC) indicated that the overall extent of codon usage bias in both TTSuV2 and TTSuV1 was not significant, the most frequently occurring codons had an A or C at the third codon position. Correspondence analysis (COA) was performed and TTSuV2 and TTSuV1 sequences were located in different quadrants of the first two major axes. A plot of the ENC revealed that compositional constraint was the major factor determining the codon usage bias for TTSuV2. In addition, hierarchical cluster analysis of 41 TTSuV2 isolates based on relative synonymous codon usage (RSCU) values suggested that there was no association between geographic distribution and codon bias of TTSuV2 sequences. Finally, the comparison of RSCU for TTSuV2, TTSuV1 and the corresponding host sequence indicated that the codon usage pattern of TTSuV2 was similar to that of TTSuV1. However the similarity was low for each virus and its host. These conclusions provide important insight into the synonymous codon usage pattern of TTSuV2, as well as better understangding of the molecular evolution of TTSuV2 genomes.

Complete genome sequence of a novel pigeon torque teno virus in china.

Torque teno virus is a nonenveloped single-stranded DNA virus infecting humans and nonprimate species. We report the complete genome sequence of a pigeon torque teno virus isolated from pigeons in Jiangsu Province, China, in 2012. This genome sequence will be useful for viral diagnostics and disease control.

Molecular characterization and epidemiological investigation of Pigeon circovirus isolated in eastern China.

To investigate the prevalence and genetic characterization of Pigeon circovirus (PiCV) circulating in Chinese flocks, the genomic DNA of 144 samples collected from pigeons in 6 different geographic regions of eastern China between 2009 and 2010 were amplified using previously published PiCV primers. The PiCV sequence was detected in 83 of 104 unhealthy pigeons (79.8%) and 25 of 40 healthy pigeons (62.5%). The overall positive rate was 75% for all samples. An inverse primer polymerase chain reaction (IP-PCR) assay was performed to amplify the full-length sequence from a random sample of each region, and 6 specific DNA fragments were gel-purified and sequenced. The 6 full-length sequences were designated as SHWH-AB4 (2,031 bp), NJPK-21 (2,035 bp), HBLF-E2 (2,031 bp), JSJN (2,039 bp), SDDZ (2,037 bp), and AHBZ (2,035 bp) after BLAST analyses. The phylogenic tree and amino acid comparison indicated that all the strains examined were derived from a common strain, but had undergone genetic mutations through time. Pairwise comparisons revealed 93.4%-100% amino acid identity for the putative replication-associated proteins and 67.5%-100% for the putative capsid proteins.