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To improve the diagnosis of Staphylococcus aureus mastitis and its potential antibiotic resistance in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for simultaneous species identification and detection of penicillin, erythromycin, and tetracycline resistance genes. The assay was first verified using 85 clinical isolates of S. aureus, and its effectiveness was evaluated by testing 99 mastitic milk samples. The multiplex PCR assay had high detection sensitivity in samples from both groups (100% agreement with single-gene PCR), with detection limits of 100–103 colony-forming units (cfu)/ml. The detection limits of the current assay for nuc, blaZ, erm B, erm C, tet K, and tet M genes were 101, 101, 102, 100, 102, and 103 cfu/ml, respectively. There was a good correlation between genotypic analysis by PCR and phenotypic determination using S. aureus species identification and susceptibility testing methods. High diagnostic sensitivities and specificities were shown for the target resistance genes ( blaZ gene, 97.2% and 92.9%; ERM resistance genes, 100% and 95.6%; TET resistance genes, 100% and 96%). Results suggested that the current PCR assay could be used as an alternative diagnostic method in the routine diagnosis for rapid, sensitive, and specific detection of S. aureus and its associated antibiotic resistance genes in mastitic milk samples.

It has been reported that sodium fluoride suppressed proliferation and induced apoptosis in osteoblasts. However, the details about the mechanism at work in bone metabolism are limited. In this study, we further investigated the mechanisms of NaF on proliferation and apoptosis in the primary cultured mouse osteoblasts, which were exposed to different concentration of NaF (10(-6)-5 × 10(-4) M). We examined the effect of NaF on proliferation, cell cycle, apoptosis, oxidative stress, and the protein level of insulin-like growth factor-I (IGF-I) in osteoblasts. All the tested NaF inhibited proliferation and arrested cell cycle at S phase in osteoblasts, and further demonstrated to induce apoptosis in osteoblasts. On the other hand, we found that NaF increased oxidative stress and decreased protein expression of IGF-I. Our study herein suggested that NaF caused proliferation suppression, and apoptosis may contribute to decrease IGF-I expression and increased oxidative stress damage by NaF in the primary mouse osteoblasts.

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.

An evaluation of a rapid enzyme-linked immunosorbent assay (ELISA) and colloidal gold based one-step strip assay for cephalexin (CEX) residue detection in milk and beef is described. A monoclonal antibody (mAb) against CEX was produced using cephalexin-bovine serum albumin (CEX-BSA) conjugate as the immunogen, which exhibited no cross-reactivity with applied chemicals in the studied concentration range. The detection limit of rapid ELISA was calculated as 0.39 microg/kg in PBS and 19.5 microg/kg in beef and milk, which was quite lower than the European Union Maximum Residue Limit (MRL) of 100 microg/kg in milk and 200 microg/kg in muscle. Spiked samples were detected with a mean recovery of 82.8-124% and coefficient of variation of 4.88-25%, which indicated a good agreement with the spiked concentration. Accuracy and reproducibility were determined using spiked samples with four different final concentrations of 1, 2, 5, and 10 microg/kg of CEX (n = 7). Mean intra-assay variation of 6.67% and inter-assay variation of 10.66% were obtained. In contrast, the strip test for CEX had a visual detection limit of 0.5 microg/kg, which could be evaluated within 3-10 min. However, positive samples should be further quantified by more sensitive and accurate competitive indirect ELISA method. In conclusion, the described strip test is rapid, simple, and cost-effective as well as sensitive and specific enough for reliable and accurate on-site screening.

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bOrnithobacterium rhinotracheale is a Gram-negative bacterium associated with respiratory diseases in many avian species, with worldwide distribution, and it causes significant economic loss to the poultry industry. In this study, the isolation and characterization of O. rhinotracheale small-colony variants (SCVs) are described for the first time. O. rhinotracheale isolates (n ‫؍‬ 27) were recovered from tracheal samples (n ‫؍‬ 321) collected from different avian species with clinical signs of respiratory disease. Of the 27 O. rhinotracheale isolates, 21 (77.8%) showed SCVs in their primary cultures. Five O. rhinotracheale SCV isolates showed high levels of stability and were chosen for further characterization with their wildtype (WT) isolates. Stable O. rhinotracheale SCVs were oxidase negative, while their WT isolates were positive. Growth curves for stable O. rhinotracheale SCVs indicated lower growth rates and longer lag phases than for their WT isolates. Furthermore, it was possible to increase the efficacy of the broth medium in supporting the growth of O. rhinotracheale WT isolates by supplementing it with 5% fetal bovine serum (FBS) and 2% IsoVitaleX Enrichment. Antibiotic susceptibility tests showed that O. rhinotracheale SCVs had higher MIC values than their WT isolates. This study suggests that successful antibiotic treatment of respiratory diseases associated with O. rhinotracheale must take into consideration the resistance patterns of O. rhinotracheale SCVs. Intracellular persistence in murine RAW 264.7 macrophages revealed that O. rhinotracheale SCV28 had higher survival rates than its WT isolate. Finally, small-colony variants may be important contributors to the pathogenesis of O. rhinotracheale.

Staphylococcus aureus is a major pathogen associated to bovine mastitis and has the ability to form a slow-growing population termed the small colony variants (SCVs). From 20 samples of 5 chronic S. aureus cases, 1 SCV isolate (SCV102) was recovered simultaneously with 1 of 8 S. aureus isolates. SCV102 showed auxotrophy for thymidine and had a slow growth rate. Intracellular persistence in human mammary epithelial cells (HBL100cell line) monolayer revealed that SCV102 isolate had minimal cytopathological effects compared with its parent strains. SCV102 isolate and its parent strain S. aureus 101 indicate similar resistant pattern to four antibiotics. On the contrary, the minimal inhibitory concentrations values for chloramphenicol and sulfadimethoxine were much higher in SCV102 than that of S. aureus 101. To the best of our knowledge this is the first time the isolation of S. aureus SCV102 from a persistent bovine mastitis has been reported in Beijing (China). This study suggests that SCV102 isolate may be an important contributor to persistent bovine mastitis.