Quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages in non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus

In 13 of 43 non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus, two truncated beta-hemolysin (hlb) genes were demonstrated by PCR and sequencing, and one truncated hlb gene was located beside the integrase (int) gene of phage origin. The staphylokinase (sak) gene was detected in all 13 isolates in which the truncated hlb genes were detected by PCR. Enterotoxin A (sea) and enterotoxin P (sep) genes were also detected in 5 and 2 of the 13 isolates, respectively. Moreover, the scn and chp genes encoding staphylococcal complement inhibitor (SCIN) and chemotaxis inhibitory protein of S. aureus (CHIPS) were detected in 13 and 4 of the 13 isolates, respectively. The bacteriophage induced by mitomycin C treatment was able to lysogenize one beta-hemolysin-producing isolate of S. aureus, and the sak and scn genes were detected from the lysogenized isolate. These results suggest quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages among non-beta-hemolysin-producing bovine isolates of S. aureus.     

Assessment of the ideal ratios of digestible essential amino acids for pacu,Piaractus mesopotamicus,juveniles by the amino acid deletion method

The present study aimed to determine the ideal ratios of digestible essential amino acids (EAAs) for pacu (Piaractus mesopotamicus) juveniles by the amino acid (AA) deletion method. A completely randomized design which consisted of 11 treatments and three replicates each was used. The treatments included a control diet (CD) containing 55% of nonpurified natural ingredients and 45% of purified synthetic amino acids and ingredients, and other ten isonitrogenous and isoenergetic EAA limiting diets (LDs), each being deficient in 44.4 ± 0.02% of the respective EAA. Pacu juveniles with initial average body weight of 6.22 ± 0.09 g were distributed among 33 fiber glass tanks. Fish were fed with semipurified and extruded diets for 113 days two times a day until apparent satiation. The ideal ratio of each dietary EAA was calculated on the basis of the relationship between body N retention and amount of EAA deleted from the respective EAA LD. Based on the AA deletion method, the ideal ratios of digestible EAAs for pacu juveniles, relative to lysine requirement of 100% were estimated as: methionine 14.6%, threonine 35.0%, tryptophan 6.6%, arginine 62.8%, histidine 13.6%, isoleucine 26.3%, leucine 43.7%, phenylalanine 27.2%, and valine 35.8%.     

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary‐infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3–21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin‐positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.     

Factorial models to estimate isoleucine requirements for broilers

The objective of this work was to determine the efficiency of utilization (EU) and produce factorial models for optimal isoleucine (Ile) intake. Six dose–response trials were carried out, three for males and three for females, with 640 Ross 308 in each studied phase. The initial (1–14 days), grower (15–28 days) and finisher (29–42 days) phases were evaluated to cover the growing phase of the broiler chicken. In total, eight treatments were randomly distributed to four replicates of 20 birds each. The treatments consisted of seven crescent levels of Ile and one counter proof to ensure that Ile was the first limiting amino acid in the diet. Dilution technique was applied to produce the levels of Ile and keep the amino acid ratio with lysine. The EU was determined to account for whole body or partitioned for feather‐free body (Bff) and feather. Two distinct factorial models were adjusted, M1 and M2. The M2 model was evaluated for one or two EU, being denominated as M2 and M3. When the efficiency was partitioned, the values of 53% and 69% for feather and Bff were determined. The optimal Ile intake estimated for each model were of 275, 908, 1,412 mg of Ile/bird/day (M1); 258, 829, 1,321 mg of Ile/bird/day (M2); and 284, 835, 1,288 mg of Ile/bird/day (M3) for initial, grower and finisher phases respectively. The EU partitioned for feather‐free body and feather reduced the biased of the model M3. Overall, higher values of Ile intake are estimated when model M1 is used, which may be the difference in account for body weight gain (M1) or only protein gain (M2 and M3) to estimate the amount of amino acid required for broiler.     

Antinephritis and radical scavenging activity of prenylflavonoids

Antinephritis activity of 5 prenylflavonoids similar to glabridin (1-5), isolated from Morus alba, Artocarpus communis, Glycyrrhiza uralensis and G. inflata, was evaluated in mice with glomerular disease (Masugi-nephritis). Oral administrations of artonin E (2) or licochalcone A (4) for 10 days (30 mg kg(-1) day(-1)) reduced the amount of urinary protein excretion compared to nephritic mice. ESR spectroscopy demonstrated that morusin (1) and licorisoflavan A (5) increased the radical intensity of sodium ascorbate by about two times. Morusin, licoricidin (3), licochalcone A and licorisoflavan A showed weak scavenging activity against superoxide anion radical.     

Manipulation of fish germ cell: visualization, cryopreservation and transplantation

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.     

Analysis of antibody responses by commercial western blot assay in horses with alveolar echinococcosis

Commercial western blot (WB) assay was used to detect serum antibodies specific to Echinococcus multilocularis in 23 horses in which infection was confirmed by postmortem inspection at a slaughterhouse. Livers contained from 1 to >20 nodular lesions; foci diameter ranged from 1 to 25 mm. Antibody tests of serum from all 23 animals were negative for antigen bands at 7, 16, 18, and 26-28 kDa, which show specificity in the serum of human patients. However, sera from two infected horses with the largest nodules (diameter, 25 mm) showed positive response to one of the 22-kDa and 30-kDa antigen bands. It may be possible to diagnose E. multilocularis infection in horses based on the detection of these bands on commercial WB assay.     

Effect of amino acid substitutions in the epitope regions of pyolysin from Arcanobacterium pyogenes

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY, cholesterol-dependent cytolysin) family of bacterial toxins. Recently, we demonstrated that the epitopes of monoclonal antibodies (mAbs) S, H, C, and G lie in the regions of amino acids regions 55-73, 123-166, 482-506, and 482-506 of PLO, respectively, by the reaction of mAbs with truncated PLOs. In this study, we substituted the amino acids in these epitope regions of PLO by site-directed mutagenesis and examined the effect of these amino acid substitutions. Mutants I70S/R71A/L73S, Y131S/P132S, and L163S/P164S for mAbs H or S completely lost the hemolytic activity of the proteins, but these mutants still bound to erythrocyte membranes. Mutants L495S/W497S and W500S/W501S for mAbs C and G also completely lost their hemolytic activity, but still bound to erythrocyte membranes. In the undecapeptide region of PLO, the cysteine residue required for thiol activation is replaced with alanine. Therefore, we substituted Ala-492 of the undecapeptide region for Cys. The hemolytic activity of this mutant A492C decreased by adding hydrogen peroxide or storing at 4 degrees C, and the decreased hemolytic activity was restored by adding L-cysteine.     

Structural gene and strain specificity of a novel cysteine protease produced by Staphylococcus aureus isolated from a diseased chicken

Recently whole genome sequencing of Staphylococcus aureus has revealed the genes encoding cysteine proteases such as staphopain and SspB. In this study, we cloned and sequenced the structural gene (ScpA) encoding a cysteine (thiol) protease of S. aureus strain CH-91 from a chicken with dermatitis using polymerase chain reaction (PCR) and inverse PCR methods. The sequence information revealed a coding sequence (CDS) of 1200 nucleotides encoding the ScpA preproenzyme of 399 amino acids with a molecular mass of 45,071 Da. The deduced amino acid sequence of the ScpA differed at many positions from those of staphopain and SspB with identities of 64 and 42%, respectively. In the Southern blot analysis with a total DNA of S. aureus strain CH-91, the ScpA probe hybridized with a single 7.7 kb XbaI fragment or 2.8 and 0.8 kb EcoRI fragments, whereas the staphopain and SspB probes did not hybridize with these DNA fragments. These results suggest that this ScpA gene is a single-copy gene and is a novel gene, which is not found in the published whole genome sequences of S. aureus. In immunoblot, PCR, and Southern blot assays, the ScpA or its gene was detected in high protease-producing strains from chickens, but was not recognized in bovine and porcine strains or low protease-producing avian strains. These results indicate that the ScpA of CH-91 type may be specific to the high protease-producing strains of S. aureus from chickens, namely, there is a strain specificity of the ScpA.     

Response of juvenile pacu (Piaractus mesopotamicus Holmberg, 1887) to balanced digestible protein

This 45‐day work aimed to determine the response of juvenile pacu (Piaractus mesopotamicus) to balanced digestible protein (BDP) and to use these responses to determine whether the optimum economic levels of BDP would differ depending on the form in which the fish is sold. Six isoenergetic diets containing 163, 201, 238, 272, 315 and 348 g/kg BDP (dry‐matter basis) were prepared through the serial dilutions of a high‐protein diet with the low‐protein diet. Fish (initial average body weight, 10.82 ± 0.14 g) were fed with respective experimental diets three times a day until apparent satiation. The optimum biological level of BDP was calculated as 326 g/kg (dry‐matter basis) by the quadratic regression model for maximum body weight gain of juvenile pacu. To maximize economic returns (US$/kg) for different end products, the optimum economic levels of BDP were calculated as 311, 317 and 319 g/kg (dry‐matter basis) by an economic model for whole‐body, eviscerated and sliced juvenile pacu respectively. This finding revealed that obtaining maximum biological performance of fish in the case of high feed costs or low prices of the end product in consumer market would substantially reduce the economic returns.