Outbreak of subclinical mastitis in a flock of dairy sheep associated with Burkholderia cepacia complex infection.

An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the Burkholderia cepacia complex (formerly Pseudomonas cepacia). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure B. cepacia cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for B. cepacia complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to B. cepacia genomovar III; the remaining isolate was identified as Burkholderia vietnamiensis (formerly B. cepacia genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify "B. cepacia" in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by B. cepacia complex and the first description of B. cepacia complex infection in sheep.

Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine.

The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar V(max) values, suggesting that the formation of the phosphatidyl-enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca(2+), and the optimum concentration was approximately 40 mM CaCl(2). K(Ca)(app) was calculated to be 8.6+/-0.74 mM for the hydrolytic and 10+/-0.97 mM for the transphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca(2+) concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388+/-37 mM. Km(app) for the lysophosphatidylcholine depended on Ca(2+) concentration and fell between 1 and 3 mM at CaCl(2) concentrations from 4 to 40 mM. SDS, TX-100, and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentrations at or above that of lysophosphatidylcholine.

Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435).

Immobilized lipase from Candida antarctica lipase B (Novozym 435) was effective in the synthesis of lysophosphatidylcholine (LPC). The transesterification of L-alpha-glycerophosphorylcholine (GPC) and vinyl laurate was carried out in a solvent free system or in the presence of 50% (v/v) t95%) were easily achieved. The lipase was selective for the sn10 times). High purity products could be produced by a decrease of the reaction temperature to induce precipitation of the product. The temperature needed depended on the fatty acid chain length. Thus, only lysophosphatidylcholine was produced with palmitic acid vinyl ester at 45 degrees C, whereas for the vinyl esters of lauric acid, capric acid, and caprylic acid, a lower reaction temperature (25 degrees C) was necessary to obtain solely the lysophospholipid products.

Two-enzyme system for the synthesis for 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate.

A combination of two enzymes, phospholipase D (PL D) and C (PL C), was investigated for the production of two lysophospholipids, 1-lauroyl-rac-glycerophosphate (1-LGP) and 1-lauroyl-dihydroxyacetonephosphate (1-LDHAP). The high transphosphatidylation ability of phospholipase D from Streptomyces sp. allowed the formation of 1-lauroyl-phosphatidylglycerol (1-LPG) and 1-lauroyl-phosphatidyldihydroxyacetone (1-LPDHA) from phosphatidylcholine (PC) and 1-monolauroyl-rac-glycerol (1-MLG) and 1-lauroyl-dihydroxyacetone (1-MDHA), respectively. A two-phase system, diethyl ether/water, was chosen for the convenience of the recovery of the water insoluble products. A similar two-phase system was used for hydrolysis of the complex phospholipids by phospholipase C form Bacillus cereus, which released both lysophospholipids. Only trace amounts of phosphatidic acid (PA) were detected showing that the enzyme is highly selective for the release of the diacylglycerol and 1-lauroyl-rac-glycerophosphate and 1-lauroyl-dihydroxyacetonephosphate.