Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections

Background

Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC.A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.

Results

The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.

Conclusions

The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.     

Myoelectric activity in the intestine of cows with strangulating obstruction of the distal small intestine

Myoelectric activity in 2 cows instrumented with permanent electrodes in the ileum, cecum, proximal loop of the ascending colon (PLAC), and spiral colon was analyzed after an obstruction developed in the distal small intestine. Results were compared with patterns from a group of 7 normal cows. Myoelectric activity in the ileum immediately orad to the occlusion was characterized by abolition of the migrating myoelectric complex (MMC) and a constant pattern of strong spike bursts of long duration. Cyclic activity was present in all parts of the large intestine, and propagation of phase III activity was evident from proximal to distal. A slight degree of disorganization in phase III propagation was restricted to the spiral colon. Activity cycles tended to be shorter in the cecum and PLAC of both cows with colic than in normal cows, and the intensity of spiking activity was generally lower. Changes in duration of the MMC in the spiral colon (bovine colonic MMC, bcMMC) were inconsistent, but the intensity of spiking activity tended to be lower in phases I and II of both cows compared to controls. The organization of phase III in several spindles typical of the bovine spiral colon was not disrupted, but phase IV of the bcMMC occurred only infrequently. Organized cyclic activity occurred in the large intestine of both cows despite complete disruption of the small intestinal MMC, indicating the presence of mechanisms able to initiate and regulate coordinated myoelectric patterns in the large intestine independent of the small intestine.     

Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.)

Wheat endosperm storage proteins, namely gliadins and glutenins, are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 6 glutenin loci and at 3 -gliadin loci were identified from a set of 200 hexaploid wheat cultivars grown primarily in France using SDS PAGE. At Glu-A1, Glu-B1 and Glu-D1, encoding high molecular weight glutenin subunits (HMW-GS), 3, 8 and 5 alleles were observed respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 5 and 11 alleles were identified at loci Glu-A3 and Glu-B3 respectively. Four alleles were observed at Glu-D3 loci. Omega-gliadin diversity was also very high, as 7, 13 and 9 alleles were found at Gli-A1, Gli-B1 and Gli-D1, respectively. A total of 147 (or 149) patterns resulted from the genetic combination of the alleles encoding at the six glutenin loci (or Glu-1 and Gli-1 loci). Although Glu-1 and Glu-3 loci were located on different chromosome arms and were theoretically independent, some associations were revealed due to pedigree relatedness between some French wheat cultivars. The usefulness of allelic identification of LMW-GS together with HMW-GS and gliadins for future genetic and technological wheat improvement is discussed.     

Effects of casein and fat content on water self-diffusion coefficients in casein systems: a pulsed field gradient nuclear magnetic resonance study

The water self-diffusion coefficients in casein matrixes were measured using a pulsed field gradient spin-echo nuclear magnetic resonance technique (PFG-SE NMR). The dependence of the water self-diffusion coefficient on the casein concentration and the aqueous phase composition is reported in both a rehydrated native phosphocaseinate dispersion and a concentrated casein retentate. A model has been proposed to explain the different behavior of the water self-diffusion coefficient in the two casein systems. This model demonstrates that the water self-diffusion cannot be simply explained by the water content only. So, taking into account the specific effect of each constituent of the aqueous dispersing phase, the water self-diffusion reduction induced by the casein micelle can be modeled. The effect of fat on the water self-diffusion coefficients was investigated. Anhydrous milk fat-reconstituted retentate samples were used in order to estimate the obstruction effect of fat globules in the modeling process. The dependence of the self-diffusion coefficient of water on the fat and casein content is reported. A general model included the effect of the aqueous phase composition, and the obstruction effects of casein micelles and fat globules were proposed. This model was validated for water self-diffusion coefficients in industrial fatty retentates.     

Gradients of abundance and biomass across reserve boundaries in six Mediterranean marine protected areas: Evidence of fish spillover?

Marine protected areas (MPAs) are considered as an effective tool in marine coastal management, and considered able to enhance local fisheries through adult fish spillover. Indirect evidence of fish spillover could be obtained by horizontal gradients in fish abundance. To address this question, the existence of gradients of fish abundance and biomass across marine reserve boundaries was assessed in six Mediterranean MPAs using underwater visual censuses performed at various distances from the core of the MPA, in integral reserve (IR), to buffer zone (BZ) and fished areas. A reserve effect was evidenced with higher values of fish species richness (×1.1), abundance (×1.3), and biomass (×4.7) recorded inside MPAs compared to adjacent fished areas. Linear correlations revealed significant negative gradients in mean fish biomass in all the reserves studied after the effect of habitat had been removed, whereas negative gradients in abundance were less conspicuous. Generalized additive models suggested two main patterns of biomass gradients, with a sharp decrease at the IR-BZ boundary or at the BZ-fished area boundary. It was estimated that fish spillover beneficial to local fisheries occurred mostly at a small spatial scale (100s of metres). The existence of regular patterns of negative fish biomass gradients from within MPAs to fished areas was consistent with the hypothesis of adult fish biomass spillover processes from marine reserves and could be considered as a general pattern in this Mediterranean region.     

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r² = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.