Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.     

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1?kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R2?=?0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.     

Effect of delayed colostrum collection on colostral IgG concentration in dairy cows

OBJECTIVE: To determine the effect of timing of first-milking colostrum collection on colostral IgG concentration. DESIGN: Prospective study. ANIMALS: 13 healthy Holstein cows. PROCEDURES: All calvings were observed. After parturition, calves were not allowed to suckle and were separated from the dam. Colostrum was collected from a single randomly selected quarter at 2, 6, 10, and 14 hours after parturition until all 4 quarters were sampled. Colostral IgG concentration was determined via radial immunodiffusion. RESULTS: Mean colostral IgG concentration was 113, 94, 82, and 76 g/L at 2, 6, 10, and 14 hours after calving, respectively. Colostrum collected 6, 10, and 14 hours after calving had significantly lower IgG concentrations than did colostrum collected 2 hours after calving. Mean colostral IgG concentration at 14 hours after calving was significantly lower than that at 6 hours after calving. Cows in their third or greater lactation had mean colostral IgG concentrations 2 hours after calving (132 g/L) that were greater than the first and second lactation cows (mean, 95 and 100 g/L, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that early or immediate colostrum collection from dairy cows will maximize colostral IgG concentration. Adjustment of routine dairy farm management procedures may be required to maximize colostrum quality and minimize prevalence of failure of passive transfer in dairy calves.     

Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)-polymerase chain reaction (PCR). In 9 of 12 roosters, Campylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.