Comparison of bacterial communities in faeces of beef cattle fed diets containing corn and wet distillers' grain with solubles.

AIM: The mammalian intestinal microflora has been shown to impact host physiology. In cattle, intestinal bacteria are also associated with faecal contamination of environmental sources and human illness via foodborne pathogens. Use of wet distillers' grains with solubles (WDGS) in cattle feed creates a gastrointestinal environment where some bacterial species are enriched. Here, we examine if a diet containing 40% WDGS results in fundamentally different microbial community structures. METHODS AND RESULTS: The 20,002 16S r-RNA gene sequences from 20 beef cattle were analysed using Sanger sequencing methods. At the genus level, Prevotella (Gram negative) and Anaerobacter (Gram positive) were the most frequently occurring bacteria in our beef cattle faecal samples. Diet-associated differences in prevalence were noted for Prevotella but not Anaerobacter. CONCLUSIONS: Diet affects community structure. Faecal communities of co-housed beef cattle are not identical. SIGNIFICANCE AND IMPACT OF THE STUDY: It is known that a diet of 40% corn-based WDGS increases the generic Escherichia coli in the faeces and enriches E. coli O157:H7. The results from the current study suggest that in addition to previously observed changes in E. coli, the entire bacterial community structure is different for animals fed 40% corn-based WDGS compared to a traditional corn-finishing diet.

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes.

AIMS: To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains. METHODS AND RESULTS: Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes. CONCLUSIONS: This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.

Study of magnetic anisotropy and magnetization reversal using the quadratic magnetooptical effect in epitaxial Co(x)Mn(y)Ge(z)(111) films.

Magnetic anisotropy, magnetization reversal and the magnetooptic Kerr effect in Co(x)Mn(y)Ge(z) have been studied over a range of compositions between 0 and 50 at.% of Ge and between 1 and 3 in the Co to Mn atomic ratio, including the Heusler alloy Co(2)MnGe. A strong quadratic magnetooptic Kerr effect has been observed within a narrow region of composition centered around the Co to Mn atomic ratio of 2, which has been used to probe and quantify the magnetic anisotropy and magnetization reversal of the system. The anisotropy is sixfold with a weak uniaxial component, and it exhibits sensitive dependence on composition, especially on the atomic ratio between Co and Mn. The magnetization reversal process is consistent with the single-domain Stoner-Wohlfarth model.

Evaluation of genome-derived amplicon length polymorphism PCR primers for the genetic evaluation of related strains of Salmonella.

AIMS: The intent of this study is to exploit both the genetic diversity and conservation demonstrated between the Salmonella enterica ssp. enterica serovar Typhi CT18 and Salm. enterica ssp. enterica serovar Typhimurium LT2 genomes by utilizing amplicon length polymorphism (ALP) to detect and differentiate various Salmonella strains. METHODS AND RESULTS: Methods of ALP-PCR analysis were developed based on identifying DNA sequence deletions within highly homologous regions of the Salm. Typhi CT18 and Salm. Typhimurium LT2 genomes. This study describes the application of genome-based ALP-PCR using primer pairs designed to detect genomic differences present within both Salmonella genomes and evaluated against a reference set of Salmonella strains. CONCLUSIONS: This study defines a collection of primer sequences broadly distributed along the Salmonella genome that can differentiate various Salmonella strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Genome-based ALP-PCR provides a useful and powerful analytical method to evaluate variability within a group of Salmonella strains independent of serological, phenotypic or other molecular approaches.

Impaired ability of the Na+/Ca2+ exchanger from the Dahl/Rapp salt-sensitive rat to regulate cytosolic calcium.

We previously cloned Na(+)/Ca(2+) exchanger (NCX1) from mesangial cells of salt-sensitive (SNCX = NCX1.7) and salt-resistant (RNCX = NCX1.3) Dahl/Rapp rats. The abilities of these isoforms to regulate cytosolic Ca(2+) concentration ([Ca(2+)](i)) were assessed in fura 2-loaded OK cells expressing the vector (VOK), RNCX (ROK), and SNCX (SOK). Baseline [Ca(2+)](i) was 98 +/- 20 nM (n = 12) in VOK and was significantly lower in ROK (44 +/- 5 nM; n = 12) and SOK (47 +/- 13 nM; n = 12) cells. ATP at 100 microM increased [Ca(2+)](i) by 189 +/- 55 nM (n = 12), 21 +/- 9 nM (n = 12), and 69 +/- 18 nM (n = 12) in VOK, ROK, and SOK cells, respectively. ATP (1 mM) or bradykinin (0.1 mM) caused large increases in [Ca(2+)](i) and ROK but not SOK cells were much more efficient in reducing [Ca(2+)](i) back to baseline levels. Parental Sprague-Dawley rat mesangial cells express both RNCX (SDRNCX) and SNCX (SDSNCX). SDRNCX and RNCX are identical at every amino acid residue, but SDSNCX and SNCX differ at amino acid 218 where it is isoleucine in SDSNCX and not phenylalanine. OK cells expressing SDSNCX (SDSOK) reduced ATP (1 mM)-induced [Ca(2+)](i) increase back to baseline at a rate equivalent to that for ROK cells. PKC downregulation significantly attenuated the rate at which ROK and SDSOK cells reduced ATP-induced [Ca(2+)](i) increase but had no effect in SOK cells. The reduced efficiency of SNCX to regulate [Ca(2+)](i) is attributed, in part, to the isoleucine-to-phenylalanine mutation at amino acid 218.

The influence of plant age on tolerance of rice to injury by the rice water weevil, Lissorhoptrus oryzophilus (Coleoptera: Curculionidae).

For most plant species, tolerance to many types of herbivory increases as plants age, but the applicability of this pattern to root herbivory has not been tested. Injury to roots of rice plants by larvae of the rice water weevil, Lissorhoptrus oryzophilus Kuschel, causes severe reductions in yields in the United States. It is generally thought that young rice plants, because their root systems are smaller, are less tolerant than older plants of root feeding by L. oryzophilus. Field experiments were conducted to test this hypothesis. Plots of rice (4.7 to 6.5 m2) were established and subjected to natural infestations of L. oryzophilus larvae. A soil insecticide was applied to plots at different times during the tillering phase of rice in order to manipulate the timing of weevil infestation. The impact of these treatments (timings of insecticide applications) was assessed by comparing relationships between yield loss and larval pressure for each treatment using analysis of covariance. Yield losses ranged from 13% to over 40% in plots not treated with insecticide. Patterns of yield losses from plots treated with insecticide at different times were best explained by the hypothesis that yield loss is determined both by the age of plants infested and by the size of larvae infesting plants. Young plants appear to be less tolerant than older plants, and feeding by large larvae appears to be more deleterious than feeding by smaller larvae. Management practices that delay infestation of rice by L. oryzophilus until plants are older may be an important component of management programmes for this pest.

Identification of rice cultivars resistant to Lissorhoptrus oryzophilus (Coleoptera: Curculionidae), and their use in an integrated management program.

The rice water weevil, Lissorhoptrus oryzophilus Kuschel, is the most destructive insect pest of rice in the United States and is a particularly severe pest in Louisiana. The current management program for this insect in Louisiana relies heavily on insecticides, most notably the seed treatment fipronil (Icon). Diversification of the management program by incorporation of alternative strategies is needed to improve the effectiveness and long-term stability of the program. In the three experiments reported here, three components of a diversified management program for the rice water weevil in Louisiana--host plant resistance, treatment of seeds with Icon, aid the cultural practice of delayed flooding--were investigated. Comparison of the densities of weevil larvae on the roots of several commercial cultivars indicated that the long-grain cultivar 'Jefferson' was more resistant to infestation by the rice water weevil than the other cultivars. The medium-grain cultivars 'Bengal', 'Earl' and 'Mars' and the long-grain variety 'Cocodrie' were the most susceptible to infestation. Comparison of yield data from untreated plots and plots treated with Icon indicated that the long-grain cultivars Cocodrie, Lemont, and Jefferson were more tolerant of weevil injury than the other cultivars. A 2-wk delay in flooding was associated with yield benefits in plots not treated with Icon. Treatment of seeds with Icon controlled weevils in all three screening experiments. The implications of these results for the development of an integrated management program for the rice water weevil are discussed.

Mediated pathogenicity of the baculovirus AcMNPV by the venom from Euplectrus comstockii Howard (Hymenoptera: Eulophidae).

The compatibility of the venom from the parasitic species Euplectrus comstockii Howard (Hymenoptera: Eulophidae) with the pathogenicity of Autographa californica (Speyer) (Lepidoptera: Noctuidae) MNPV baculovirus (AcMNPV) was tested in third and fourth instar larvae of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae). The presence of AcMNPV did not alter the ability of the venom to arrest ecdysis in T. ni larvae. The presence of the venom delayed the rate of viruses by AcMNPV but increased the total mortality rates from days 9 to 14 in both third and fourth instar T. ni larvae. The delay in viruses was minimized by administering the virus prior to envenomation. In the presence of the venom, the final LD50 values were lower for fourth instar larvae than for third instar larvae. Surface response equations were developed to visualize the effect of the venom on the viruses caused by AcMNPV.

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.

Expression of polyomavirus large T antigen by using a baculovirus vector.

A gene encoding the large T antigen of polyomavirus was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus so that gene expression was under the control of the strong, very late polyhedrin gene promoter. Significantly more large T antigen was produced in recombinant virus-infected insect cells than was observed in polyomavirus-transformed mouse cells. The insect-derived T antigen exhibited polyomavirus origin-specific DNA binding. The baculovirus expression system provides a convenient source of T antigen for in vitro studies.

Baculovirus transcription in the presence of inhibitors and in nonpermissive Drosophila cells.

Regulation of transcription within four well-defined regions of the DNA genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was studied in a permissive lepidopteran cell line. Spodoptera frugiperda IPLB-SF-21 and a nonpermissive dipteran cell line, Drosophila melanogaster DL-1. Cycloheximide, an inhibitor of protein synthesis, was used to identify immediate early transcripts and aphidicolin, an effective inhibitor of DNA replication, was used to distinguish early and late transcripts in S. frugiperda cells. Immediate early transcripts were identified in the HindIII-K (85 to 87.5%), the HindIII-I/EcoRI-F (I/F from 35 to 37%) and the HindIII-P/EcoRI-P (p10 from 88.5 to 89.5%) regions of the genome. Late RNAs, defined by their sensitivity to aphidicolin as well as cycloheximide, overlapped the immediate early RNAs. Specific viral transcripts were synthesized in nonpermissive D. melanogaster cells infected with AcNPV but late viral transcripts were not observed.

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.