Molecular epidemiology of environmental MRSA at an equine teaching hospital: introduction,circulation and maintenance

The role that environmental contamination might play as a reservoir and a possible source of Methicillin-resistant Staphylococcus aureus (MRSA) for patients and personnel at equine veterinary hospitals remains undefined, as the environment has only been monitored during outbreaks or for short periods. Therefore, the objectives of this study were to determine the monthly presence, distribution, and characteristics of environmental MRSA at an equine hospital, and to establish patterns of contamination over time using molecular epidemiological analyses. For this purpose, a yearlong active MRSA surveillance was performed targeting the environment and incoming patients. Antimicrobial susceptibility testing, SCCmec typing, PFGE typing, and dendrographic analysis were used to characterize and analyze these isolates. Overall, 8.6% of the surfaces and 5.8% of the horses sampled were positive for MRSA. The most common contaminated surfaces were: computers, feed-water buckets, and surgery tables-mats. Ninety percent of the isolates carried SCCmec type IV, and 62.0% were classified as USA500. Molecular analysis showed that new pulsotypes were constantly introduced into the hospital throughout the year. However, maintenance of strains in the environment was also observed when unique clones were detected for 2 consecutive months on the same surfaces. Additionally, pulsotypes were circulating throughout several areas and different contact surfaces of the hospital. Based on these results, it is evident that MRSA is constantly introduced and frequently found in the equine hospital environment, and that some contact surfaces could act as “hot-spots”. These contaminated surfaces should be actively targeted for strict cleaning and disinfection as well as regular monitoring.     

Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

A comparison between the spatial distribution pattern of flagellates and some physicochemical parameters in a waste stabilization pond

Protozoological and physicochemical analyses were performed in a waste stabilization pond at Sto. Tomas Atzingo, a small town near Mexico City, in order to discern the internal dynamics of the pond water through the study of the spatial distribution pattern of flagellates and the parallel water volume distribution pattern of some selected physicochemical determined parameters. The statistical method applied to the data obtained was by cluster analysis. The results showed a slight trend to heterogeneity in the system, when it was evaluated through the physicochemically determined parameters. On the other hand, the spatial distribution pattern of the flagellates pointed out a remarkable heterogeneity in the pond. In systems like the one studied where the main feature is an organic matter overload, such a difference in findings indicates a low correlation between the biological parameters, in this case the flagellates, and the physicochemical variables, that should be considered when evaluating the water quality of such systems, either through the physicochemical, the biological or both scopes.     

Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanase activity during the late trophophase. The low molecular mass (35 kDa) endoglucanase purified from cultured broths works optimally at 37 degrees C and pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures of 50 degrees C or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergents, by EDTA, and by the divalent cations Hg(2+) and Ag(2+). It is also inhibited to some extent by 10 mM Zn(2+), Fe(2+), and Mg(2+), but it is stimulated by Mn(2+). Enzyme activity is not affected by reducing agents. In the presence of low concentrations of water miscible organic solvents (20%) endoglucanase activity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activity is not affected by the water immiscible solvent ethyl acetate. Carboxymethylcellulose is the preferred substrate (K(m(app)) = 8.3 g/L; V(max(app)) = 1.1 microM/min). Hydrolysis of crystalline cellulosic substrates is very limited, but it is greatly enhanced by phosphoric acid swelling. The purified enzyme shows no activity toward disaccharides or aryl-glucosides. Its activity is inhibited by cellobiose.     

Effect of storage on wall-bound phenolics in green asparagus

The cell walls of green asparagus spears have been analyzed for their phenolic and carbohydrate composition as modified by postharvest storage. Esterified phenolic components were released by sequential alkaline hydrolysis and identified and quantified by diode array HPLC. Significant quantities of ferulic acid (FA) and its derivatives were found to increase at least 3-fold during storage, particularly in walls from the lower parts of the stem, where accompanying changes in sugar composition were also observed. In fresh asparagus, >60% of the total FA was in the form of diferulic acid, and this increased to approximately 70% after 3 days of storage. The main FA dehydrodimers were 8-8-, 8-O-4-, and 8-5-diferulates. These have been detected in other monocotyledonous and dicotyledonous plants, but as a smaller proportion of the total FA. The possible roles phenolic esters might have in relation to the mechanical, textural, and wound-response properties of asparagus spears are discussed.     

Hematology and serum chemistry values of the lesser rhea (Pterocnemia pennata) raised in Patagonian farms (Argentina).

Hematologic and serum chemistry values were determined for chicks, juveniles, and adult farmed lesser rheas (Prerocnemia pennata). Blood samples were taken during March-April 1998 from the brachial veins of lesser rhea, including 64 chicks, 24 juveniles, and 36 adults, raised in Patagonian farms (Argentina). Lesser rhea chicks had significantly lower erythrocyte counts, hematocrit, hemoglobin, red cell indices, and copper than did the juveniles and adult individuals. No significant differences were observed between females and males. Most values were similar to those reported for other ratites. The data obtained provide hematologic and serum chemistry values for lesser rhea from farms.     

Isolation of Babesia spp. from asymptomatic human beings

Babesiosis is a worldwide tick-borne protozoan disease of a variety of wild and domestic animals. For decades, various babesia strains were considered host-specific. However, babesiosis has been diagnosed as the cause of acute illness in 13 human beings in recent years, three of whom died. Studies of babesiosis in domestic animals indicate that for every clinically demonstrable case, there are hundreds of cases of latent infection. In an effort to determine if human babesia infections might be present in Mexico, we selected as our testing ground an endemic rural area along the Gulf Coast where continuous epizootics of equine, bovine, ovine and canine babesiosis were known to occur. Of the 101 individuals examined serologically by the indirect hemagglutination (IHA) test using Babesia canis as antigen, 38 reacted at titers from 1 : 10 to 1 : 80. Blood from these reactors was injected into splenectomized hamsters. Hamsters inoculated with blood from three of the individuals showed babesia in their peripheral blood. The growth of the organism was established by subpassages into additional hamsters. The persons from whom the organisms were isolated were asymptomatic for babesiosis. None of the 29 individuals residing in Mexico City, which represented the urban population in the survey, reacted in the IHA test. This is the first documented finding of the latent form of human babesiosis.     

Bacillary haemoglobinuria diagnosis by the peroxidase-antiperoxidase (PAP) technique.

A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B and C remained unstained by the PAP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.     

Effect of Flavonoids and Saponins Extracted from Black Bean (Phaseolus vulgaris L.) Seed Coats as Cholesterol Micelle Disruptors

Strategies for reducing plasma cholesterol have been focused on reducing the absorption or synthesis of cholesterol. The aim of this study was to correlate the content of flavonoids and saponins in black bean (Phaseolus vulgaris L.) seed coats extracts with a potential effect in lowering cholesterol absorption by the inhibition of cholesterol micellar solubility. Extracts with different flavonoids and saponins concentration were obtained from a Box-Behnken design used to optimize extraction temperature, stirring time and solvent composition. Major flavonoids and saponins were quantified by HPLC-PDA-ELSD and confirmed through mass spectrometry. Contrary to the flavonoid content, saponins were correlated to the inhibitory effect of cholesterol micelle solubility as an approach to a potential reduction of cholesterol absorption. Extracts with the highest saponin content strongly inhibited cholesterol micellization with values of 55.4?±?1.9 %, while stigmasterol used as control, only reached 12?±?2.3 % at the same concentration (5 mg/ml). The optimal extracting conditions for saponins were 25 °C, during 3 h in 85 % aqueous-methanol. Correlations of inhibitory effect of cholesterol micellar solubility with the content of each identified saponin suggested that the reduction of cholesterol micellization depends on the C-22 substitution of saponins.