Recombinant glutathione-S-transferase A3 protein regulates the angiogenesis-related genes of erythrocytes in thiram induced tibial lesions.

Tibial dyschondroplasia (TD) is a skeletal deformity disease in broilers that occurs when vascularization in the growth plate (GP) is below normal. Although, blood vessels have been reported to contribute significantly in bone formation. Therefore, in the current study, we have examined the mRNA expression of angiogenesis-related genes in erythrocytes of thiram induced TD chickens by qRT-PCR and performed histopathological analysis to determine regulatory effect of recombinant Glutathione-S-Transferase A3 (rGSTA3) protein in response to the destructive effect of thiram following the injection of rGSTA3 protein. Histopathology results suggested that, blood vessels of GPs were damaged in thiram induced TD chicken group (D), it also affected the area and density of blood vessels. In the 20 and 50 µg·kg-1 of rGSTA3 protein-administered groups, E and F vessels appeared to be normal and improved on day 6 and 15. Furthermore, qRT-PCR results showed that rGSTA3 protein significantly (P < .05) up-regulated the expression of the most important angiogenesis-related integrin family genes ITGA2, ITGA5, ITGB2, ITGB3, ITGAV. The expression level of other genes including TBXA2R, FYN, IQGAP2, IL1R1, GIT1, RAP1B, RPL17, RAC2, MAML3, PTPN11, VAV1, PTCH1, NCOR2, CLU and ITGB3 up-regulated on dosage of rGSTA3 protein. In conclusion, angiogenesis is destroyed in thiram induced TD broilers, and rGSTA3 protein injection improved the vascularization of GPs by upregulating the angiogenesis related genes most importantly integrin family genes ITGAV, ITGA2, ITGB2, ITGB3, ITGA5.

Synthesis of Tilmicosin Nanostructured Lipid Carriers for Improved Oral Delivery in Broilers: Physiochemical Characterization and Cellular Permeation.

This study aimed to develop nanostructured lipid carriers (NLCs) for improved oral absorption of tilmicosin (TMS) in broilers. Thus, palmitic acid, lauric acid, and stearic acid were selected as solid lipids to formulate TMS-pNLCs, TMS-lNLCs, and TMS-sNLCs, respectively. They showed similar physicochemical properties and meanwhile possessed excellent storage and gastrointestinal stability. The TMS interacted with the lipid matrix and was encapsulated efficiently in NLCs in an amorphous structure. NLCs could enhance oral absorption of TMS compared to 10% tilmicosin phosphate solution in broilers, among which the TMS-sNLCs were the most efficient drug delivery carriers, with a relative oral bioavailability of 203.55%. NLCs could inhibit the efflux of P-glycoprotein (P-pg) toward TMS, which may be involved with improved oral absorption. Taken together, these types of solid lipids influenced the enhanced level of NLCs toward oral bioavailability of TMS, and the sNLCs proved to be the most promising oral delivery carriers of TMS.

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality.

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1ß in mammalian cells. Our present study shows that IL-1ß is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1ß in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1ß did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1ß. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1ß into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1ß inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1ß on the development of embryo reduced in 20 ng/mL IL-1ß supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1ß can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.

First detection and genetic characterization of ungulate tetraparvovirus 2 and ungulate tetraparvovirus 4 in special livestock on the Qinghai-Tibet Plateau in China.

Tetraparvovirus, formerly known as Partetravirus, is a newly discovered genus in the family Parvoviridae that is considered phylogenetically distinct from other parvoviruses. However, nothing is known about the prevalence of Tetraparvovirus in special livestock living on the Qinghai-Tibet Plateau of China, such as Tibetan pigs and Tibetan sheep. A pair of special primers was designed based on the conserved regions in the genome of ungulate tetraparvovirus 2 (P-PARV4) and ungulate tetraparvovirus 4 (O-PARV4) and was used to detect P-PARV4 in domestic pigs and Tibetan pigs and O-PARV4 in ovines and Tibetan sheep. The results showed a 15.59 and 9.38% prevalence of P-PARV4 in domestic pigs (18.96% in Gansu Province and 11.76% in Qinghai Province) and Tibetan pigs (14.28% in Gansu Province and 4.44% in Qinghai Province), respectively, and a 7.31 and 5.20% prevalence of O-PARV4 in ovines (6.61% in Gansu Province and 8.00% in Qinghai Province) and Tibetan sheep (4.55% in Gansu Province and 5.50% in Qinghai Province), respectively. The prevalence of P-PARV4 was 14.76% (31/210) for ≤1-month-old pigs and 10.58% (20/189) for > 1-month-old pigs, and the positive rates of O-PARV4 were 7.65% (18/235) for ≤1-month-old sheep and 5.05% (11/218) for > 1-month-old sheep. The phylogenetic analysis of NS1, VP1, VP2 and the whole PARV4-related provirus genome demonstrated that both P-PARV4 and O-PARV4 sequences in this study were more closely related to the sequences of other strains discovered in the same genus of animals. The identity analyses for the full-length VP2 genomes of O-PARV4 revealed 98.84-100.00% sequence identity among the 7 strains and the previously reported strain, which was 98.60-99.28% for P-PARV4. In the present study, for the first time, we have provided comprehensive information regarding the widespread infection of P-PARV4 and O-PARV4 in special livestock on the Qinghai-Tibet Plateau in China. Our present findings highlight the importance of epidemiologic surveillance to limit the spread of Tetraparvovirus in livestock at high altitudes in China.

FGF10 enhances yak oocyte fertilization competence and subsequent blastocyst quality and regulates the levels of CD9, CD81, DNMT1, and DNMT3B.

The fusion of sperm and oocytes determines the fertilization competence and subsequent development of embryos, which, in turn, can be affected by various proteins and DNA methylation. However, several factors in this whole regulation process remain unknown, especially in yaks. Here, we report that fibroblast growth factor 10 (FGF10) is an important growth factor that can enhance the maturation rate of yak oocytes and the motility of frozen spermatozoa. Subsequent blastocyst quality was also improved by increasing the total cell number and level of pregnancy-associated protein in blastocysts. These effects were significantly high in the group that received the 5 ng/ml FGF10 treatment, during both in vitro maturation (IVM) and capacitation. Our data show that the effects of FGF10 were dose-dependent at vital steps of embryogenesis in vitro. Furthermore, quantitative polymerase chain reaction, western blot analysis, and immunofluorescence demonstrated that the levels of CD9, CD81, DNMT1, and DNMT3B in both mature cumulus-oocyte complexes and capacitated sperms were regulated by FGF10, which was also highly expressed in the group treated with 5 ng/ml FGF10 during both IVM and capacitation. From our present study, we concluded that FGF10 promotes yak oocyte fertilization competence and subsequent blastocyst quality, and could also regulate CD9, CD81, DNMT1, and DNMT3B to optimize sperm-oocyte interactions and DNA methylation during fertilization.

Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance.

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.

Comparative Genomic Analysis of the ICESa2603 Family ICEs and Spread of erm(B)- and tet(O)-Carrying Transferable 89K-Subtype ICEs in Swine and Bovine Isolates in China.

Integrative and conjugative elements (ICEs) of the ICESa2603 family have been isolated from several species of Streptococcus spp.; however, the comparative genomic and evolutionary analyses of these particular ICEs are currently only at their initial stages. By investigating 13 ICEs of the ICESa2603 family and two ICESa2603 family-like ICEs derived from diverse hosts and locations, we have determined that ICEs comprised a backbone of 30 identical syntenic core genes and accessory genes that were restricted to the intergenic sites or the 3'-end of the non-conserved domain of core genes to maintain its function. ICESa2603 family integrase IntICE Sa 2603 specifically recognized a 15-bp att sequence (TTATTTAAGAGTAAC) at the 3'-end of rplL, which was highly conserved in genus Streptococcus. Phylogenetic analyses suggest that extensive recombination/insertion and the occurrence of a hybrid/mosaic in the ICESa2603 family were responsible for the significant increase in ICE diversity, thereby broadening its host range. Approximately 42.5 and 38.1% of the tested Streptococcus suis and Streptococcus agalactiae clinical isolates respectively contained ICESa2603 family Type IV secretion system (T4SS) genes, and 80.5 and 62.5% of which also respectively carried int ICE Sa 2603, indicating that ICESa2603 family is widely distributed across these bacteria. Sequencing and conjugation transfer of a novel sequence type ST303 clinical S. suis isolate HB1011 demonstrated that the 89K-subtype ICESsuHB1011 retained its transferrable function, thereby conferring tetracycline and macrolide resistance.

Genetic diversity of Streptococcus suis isolated from three pig farms of China obtained by acquiring antibiotic resistance genes.

BACKGROUND: Acquiring antibiotic resistance genes may change an organism's genetic characteristics and the effect of antibiotics, resulting in a rapid transmission of microbial pathogens. The objectives of this experiment were to identify the features of Streptococcus suis (S. suis) isolated from three pig farms in China which are geographically isolated. RESULTS: Among the isolates, 56.52% were sequence type 7 (ST7), followed by ST1 (26.09%), indicating that ST7 prevails in China, as revealed by multi-locus sequence typing (MLST). Statistical analysis indicated an association between geography, sequence types and antibiotic resistance genotypes. 66.67% of the isolates in Sichuan province presented a (ermB(-) + mefA(-) + tetO(-) + tetM(-)) + ST7 type. The tetM(+) +ST7 type was the most prevalent in Jiangsu province, whereas the strains from Hebei province had a phenotype ermB(+) +tetO(+) +ST1 (63.64%). Pulsed-field gel electrophoresis (PGFE) pattern A2 with 100% similarity reflected the clonal dissemination between Sichuan and Jiangsu provinces. Strains carrying or not carrying antibiotic resistance genes presented different PFGE patterns in Hebei province. CONCLUSION: ST7 is widespread in many regions of China and a clonal dissemination occurred between Sichuan and Jiangsu provinces in diseased pigs. However, ST1 strains with macrolide and tetracycline resistance (ermB(+) +tetO(+) +ST1) isolated from a farm in Hebei province demonstrated that the genetic diversity was contributed by horizontal acquiring of ermB and tetO carrying elements.

Overexpression of an ABC transporter and mutations of GyrA, GyrB, and ParC in contributing to high-level ciprofloxacin resistance in Streptococcus suis type 2.

Streptococcus suis is a pathogen of zoonotic diseases. Moreover, the emergence of fluoro-quinolones (FQs) resistance in this pathogen has severe consequences for pigs and human health. In this study, the molecular mechanism of FQs resistance in S. suis type 2 (SS2) sensitive strains isolated from pigs was assessed after in vitro induction of resistance against the most frequently used FQs: ciprofloxacin, norfloxacin, and enrofloxacin. Proteome analysis, sequencing and real-time RT-PCR results strongly established an overexpression of an ABC transporter protein (other than SatAB) and topoisomerase mutations in GyrA (Ser81Arg), GyrB (Glu354Lys), and ParC (Ser79Phe) in contributing to high level ciprofloxacin resistance in SS2. Due to the overexpression of the ABC transporter, intracellular ciprofloxacin concentrations were significantly lower in the resistant strains than those of sensitive strains after 20, 35, and 60 min exposures to ciprofloxacin (p < 0.05). It was concluded that improper use of FQs is one of the main causes of the emergence of this zoonotic pathogen as a multiresistant organism against commonly used antibiotics. The existence of an efflux-like protein is an incentive to find new drug targets to avoid the spread of FQs-resistant S. suis isolates in pigs and the human population.