Addition of olive (olea europaea) leaf extract as a source of natural antioxidant in mutton meatball stored at refrigeration temperature.

This study was aimed to evaluate the effects of using different levels of olive (Olea europaea) leaf extract on fresh and preserved mutton meatballs. Meatballs were divided into four different groups and treated as T0 (0), T1 (0.1), T2 (0.2) and T3 (0.3%), respectively based on olive leaf extract supplementation. Days of intervals of experiment were 0, 5, 10 days. Samples were preserved at 4˚C for up to 10 days. Different types of analysis such as, sensory (color, flavor, juiciness and overall acceptability), proximate composition (CP %), physicochemical (pH), biochemical (POV, FFA and TBARS) and microbiological (TVC, TCC and TYMC) were determined. Color, flavor and acceptability reduced significantly (p < 0.05) with the increase of the storage periods. Values of the studied quality parameters in all the treatment groups differed significantly (p < 0.05). Based on our findings 0.3% olive leaf extract is found suitable to add in mutton meatballs as a source of natural antioxidant.

[Analysis of influencing factors of shunt dysfunction after transjugular intrahepatic portosystemic shunt in liver cirrhosis accompanied with portal vein thrombosis].

Objective: To investigate the efficacy of shunt after transjugular intrahepatic portosystemic shunt (TIPS) in liver cirrhosis accompanied with portal vein thrombosis (PVT). Methods: Forty-four cases with liver cirrhosis accompanied with PVT who underwent TIPS treatment from January 2015 to May 2018 were retrospectively analyzed. Clinical baseline data of the patients were collected. Portal vein pressure gradient (PVPG) before and after the surgery was recorded. Shunt patency was observed at 3, 6, 12, 18 and 24 months after the surgery. The influencing factors were determined by univariate and multivariate analysis. Results: Transjugular intrahepatic portosystemic shunt was successfully established in all 44 cases. The postoperative PVPG was lower than preoperative (P < 0.01). The shunt patency rate after TIPS in PVT was 18.2% (n = 8). The cumulative shunt patency rates at 3, 6, 12, 18, and 24 months after surgery were 95.5%, 90.7%, 90.7%, 86.8% and 74.4%, respectively. Univariate analysis showed that diabetes history, platelet level and prothrombin time-international normalized ratio were associated with postoperative shunt dysfunction. Multivariate analysis showed that diabetes history (P = 0.007, OR = 28.606) was an independent risk factor for postoperative shunt dysfunction. Conclusion: TIPS is a safe and feasible procedure, which can effectively reduce the portal pressure in liver cirrhosis accompanied with PVT. Diabetic patients have a higher risk of postoperative shunt dysfunction. Therefore, clinical intervention should be strengthened for high-risk patients.

Prevalence and antimicrobial-resistance phenotypes and genotypes of Escherichia coli isolated from raw milk samples from mastitis cases in four regions of China.

OBJECTIVES: The objective was to find the differences in the prevalence and resistance of Escherischia coli isolated from raw milk samples from mastitis cases in four regions of China. METHODS: A total of 750 bovine raw milk samples from mastitis cases were collected from four regions of China over two seasons. Antimicrobial resistance against 29 antimicrobial agents was determined, and 27 drug-resistant genes were tested. RESULTS: Eighty-three strains (11.1%) of E. coli were isolated and identified. No significant differences in the number of E. coli isolates were observed between the two sampling seasons in the same regions (P>0.05). However, a significant difference in E. coli prevalence was found among the four different regions (P<0.01). The isolates were most frequently resistant to penicillin (100%), acetylspiramycin (100%), lincomycin (98.8%), oxacillin (98.8%) and sulphamethoxazole (53%). All the E. coli strains were multiresistant to at least three antimicrobial classes, and the most frequent multidrug-resistance patterns for the isolates were resistant to three (36.1%) or four (39.8%) classes of drugs simultaneously. The blaTEM gene (n=69; 83.1%) was the most frequently detected resistance gene. The most frequent gene combinations were a four-gene pattern of blaCTX-M-sulII-blaTEM-sulI (n=13; 15.7%) and a three-gene pattern of blaCTX-M-aph (3)-II-blaTEM (n=11; 13.3%). CONCLUSIONS: This study indicated that there is a high incidence of E. coli with a great variation in resistance patterns and resistance genes; this is a matter of great concern for public and animal health in China.

Antimicrobial resistance and virulence genes of Streptococcus isolated from dairy cows with mastitis in China.

Streptococcus is a major mastitis-causing pathogen in dairy cows. To investigate the prevalence, antimicrobial resistance and virulence gene of Streptococcus in mastitic milk, a total of 735 mastitic raw milk samples from dairy cows in 11 provinces of China were collected and tested. Antimicrobial resistance of Streptococcus isolates was determined by disc diffusion against 8 classes 29 antimicrobial agents, and Streptococcus resistant genes and virulence genes were determined by PCR and agarose gel electrophoresis. A total of 64 (8.71%) isolates of Streptococcus were isolated and identified using biochemical profiling, including 22 isolates of Streptococcus agalactiae, 13 isolates of Streptococcus dysgalactiae, and 29 isolates of Streptococcus uberis. Out of 64 resistant Streptococcus isolates, all isolates (100%) were resistant to 3 or more antimicrobials. The most frequency (n = 18, 28.12%) of the isolates were multi-resistant to 5-7 antimicrobials and the highest multi-resistant number was 29 (n = 1, 1.56%). Streptococcus isolates had the highest resistance rate to tetracycline (98.44%) and oxacillin (98.44%), followed by penicillin G (96.88%) and doxycycline (96.88%), and the lowest resistance was observed with respect to ciprofloxacin (1.56%). A total of 16 antimicrobials resistance genes with 25 combination patterns were detected in the isolates. The gene combination of Sul1/Sul2/Sul3 + gyrA/parC + cat1/cat2 was the most common pattern (12.5%). The correlation between resistant phenotypes and resistance genes in Streptococcs was 35.87%. A total of 7 virulence genes were detected and 59 (92.19%) isolates harbored at least one gene. Twenty-four classes of gene patterns were found in the isolates and the patterns of bca (9.38%) and cfb (9.38%) were the most prevalent form. In conclusion, the issue of drug resistance of Streptococcus is still a great concern in cattle health in China.

Polymorphic microsatellite loci developed from the Hong Kong oyster (Crassostrea hongkongensis).

Forty polymorphic microsatellite loci were developed from Crassostrea hongkongensis using an enriched partial genomic library with magnetic beads. The polymorphism of these loci was assessed in 30 individuals from a wild population. The allele number of the polymorphic markers ranged from 2 to 13, with an average of 5.8 per locus. The polymorphism information content ranged from 0.032 to 0.891 and 37 loci presented a medium or high level of polymorphism. The observed and expected heterozygosity values ranged from 0.033 to 1.000 and 0.033 to 0.931, respectively. Of the 40 loci, 28 were found to conform to Hardy-Weinberg equilibrium (HWE), whereas the remaining 12 showed a significant departure from HWE. The availability of these markers will aid future genetic studies in C. hongkongensis.

Simultaneous determination of 38 veterinary antibiotic residues in raw milk by UPLC-MS/MS.

A selective and rapid method has been developed to determine, simultaneously, 38 veterinary antibiotic residues in raw milk by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One milliliter of raw milk was diluted with 0.5 mL water and 3 mL acetonitrile, then purified using an Oasis HLB cartridge. The eluates were evaporated by nitrogen drying and then reconstituted to 4 mL with water/acetonitrile (8:1) before being injected into the UPLC-MS/MS system. The results indicated recoveries of 68-118% for 14 ß-lactams, 79-118% for eight quinolones, 71-106% for eight sulfonamides, 76-116% for four tetracyclines, 78-106% for three macrolides, and 88-103% for one lincosamides, with coefficients of variation less than 15% for intraday and interday precisions. The limit of quantification for all antibiotics was 0.03-10 µg kg(-1). This methodology was then applied to field-collected real raw milk samples and trace levels of four antibiotics were detected.

[The characteristics of Bacillus thuringiensis strain YBT833 and its transformants that containing different ICP genes].

Four different transformants were selected by transferring cry1C into Bacillus thuringiensis strain YBT833. Southern blot and Plasmid profiles results all proved that cry1C was transferred into strain YBT833. However, it was found by PCR analysis that transformant YBT833-1 kept all indigenous ICP(insecticidal crystal protein) genes of strain YBT833 while transformant YBT833-2 lost cry1Ab, and transformant YBT833-3 lost all ICP genes. SDS-PAGE showed that transformants of YBT833-1, YBT833-2 and YBT833 all had 130 kDa and 65 kDa peptide bands, but transformant YBT833-3 did not have 65 kDa peptide band. Bioassay results showed that the toxicities of all transformants to Spodoptera exigua, Heliothis armigera and Plutella xylostella were lower than that of strain YBT833, except the toxicity of transformant YBT833-2 to P. xylostella which was obviously higher than that of YBT833.

[Transfer of cry1C gene into Bacillus thuringiensis by electroporation to construct strain with broader insecticidal activity].

Three Transformants were selected by transferring cry1C into Bacillus thuringiensis strain YBT1535. Plasmid profiles, PCR and Southern blot result all proved that cry1C had been transferred into strain YBT1535. Bioassay results showed that the transformants of strain YBT1535 displayed significantly higher toxicity against Spodoptera exigua than strain YBT1535, but the toxicities against Heliothis armigera and Plutella xylostella did not rise except transformant YBT1535-2.

[Cloning of plasmid pBMB2062 in Bacillus thuringiensis strain YBT-1520 and construction of plasmid vector with genetic stability].

A small plasmid pBMB2062 in Bacillus thuringiensis strain YBT-1520 was cloned and sequenced. Its 2,062 bp sequence contains two potential open reading frames (orfs). The orf1 and orf2 encode a tentative replication initial protein consisting of 289 amino acid residues and a tentative replication protein consisting of 80 amino acid residues, respectively. Two homological plasmids were found by Blast searching. There are 23 nucleotides difference occurring among three of the plasmids. The difference occurred in the orf1 causes different encoding capability. Comparing with the orf1 in pBMB2062, the orf1 in the homological plasmids are truncated, one at the N-terminal and another at the C-terminal. cDNA synthesis and PCR detection showed that the mRNA corresponding to orf1 in pBMB2062 really occurs. Shuttle vectors were constructed based on pBMB2062 and showed the ability to express insecticidal crystal gene. Under nonselective condition, recombinant plasmids based on pBMB2062 were genetically stable.

[The influence of the 20 kDa protein from Bacillus thuringiensis subsp. israelensis on the cytolytic activity of CytA].

In order to understand the influence of the 20 kDa protein on the cytolytic activity of CytA protein, a set of PCR primers were designed to amplify 20 kDa protein and CytA protein genes. The genes were ligated to E. coli expression vector pUHE24 and transformed into E. coli XL1 and DH5 alpha. The clones, LZ20, LZcytA and LZ20A containing 20 kDa protein gene, cytA protein gene and 20 kDa-cytA genes were obtained respectively. The influence of the clones on the growth of E. coli cells was determined under induction of IPTG. The results showed that the growth of LZ20 cells were not affected, LZcytA cells were killed, and the growth of LZ20A cells were not affected. It was suggested that expression product of 20 kDa protein gene protected the host cells from the cytolytic effect of CytA protein. This was supported by using different host strains.

Formycin triphosphate as a probe for the ATP binding site involved in the activation of guanylate cyclase.

Formycin A triphosphate (FTP), a fluorescent analog of ATP, slightly increased basal guanylate cyclase activity, but significantly potentiated guanylate cyclase activity stimulated by atrial natriuretic factor (ANF) in rat lung membranes. FTP potentiated ANF-stimulated guanylate cyclase activity with an EC50 at about 90 microM and inhibited ATP-stimulated guanylate cyclase activity with an IC50 at about 100 microM. These results indicate that FTP binds more tightly than ATP for the same binding site. Therefore, FTP would be an excellent tool for studying the ATP binding site.