Reduced specificity of Erns antibody ELISAs for samples from piglets with maternally derived antibodies induced by vaccination of sows with classical swine fever marker vaccine CP7_E2alf.

Successful implementation of marker vaccines against classical swine fever virus is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA) as well as the development of a testing scheme during emergency vaccination. In this context, special attention needs to be paid to breeding farms, because the offspring of marker vaccinated sows possess maternally derived antibodies (MDAs). So far, limited information is available on the influence of MDAs on serological testing in the context of a DIVA strategy. Therefore, two commercially available Erns antibody ELISAs were compared, using serum samples of piglets with a high-to-moderate titre of MDAs against marker vaccine CP7_E2alf. False-positive results were detected by both Erns antibody ELISAs for serum samples of piglets with an age of up to 4 weeks. Interestingly, most samples tested false-positive in the first Erns antibody ELISA were identified correctly by the other Erns antibody ELISA and vice versa. In conclusion, in case of emergency vaccination of sows, the specificity of both ELISAs in newborn piglets younger than 4 weeks may be relatively low. This could be addressed in a testing strategy by either not sampling piglets up to the age of 4 weeks or using both ELISAs in a screening-confirmation set-up.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

Effects of growth-promoting implants on morphology of Longissimus and Semitendinosus muscles in finishing steers.

Growth-promoting implants lead to increased muscle accretion in ruminants. To elucidate the effects at a cellular level, muscle fiber distribution and cross-sectional area (CSA) of longissimus (LM) and semitendinosus (ST) muscles were compared in implanted and control steers. Sixty-four Charolais steers were assigned to one of four treatments (16 steers/treatment): (1) no implant, (2) Synovex-S® (estradiol benzoate+progesterone), (3) Ralgro® (zeranol) or (4) Revalor-S® (trenbolone acetate+estradiol-17ß). The experiment was carried out using four slaughter groups (SGRP). Sixteen steers each were slaughtered after 48, 104, 160 and 175 days (four steers/treatment) on trial. Steers on an implant treatment were first implanted at 15 months of age (day 0) and re-implanted at 56 and 112 days. Muscle fibers in the LM and ST (for both live biopsy and post-mortem samples) were characterized as either slow-twitch oxidative (SO), fast-twitch oxidative-glycolytic (FOG) and fast-twitch glycolytic (FG) fibers. Fiber distribution was minimally affected by SGRP in these physiologically mature steers. Implantation with Synovex did not alter fiber distribution in either muscle compared with control steers. Both Synovex-implanted and control steers showed a decrease of FG and an increase of FOG fibers in the LM from day 0 to SGRP 2 followed by an increase of FG and a decrease of FOG fibers. Ralgro- and Revalor-implanted steers had an almost constant fiber distribution in the LM throughout the experiment resulting in higher precentages of FG fibers in SGRP 2 (P<0.05) than SYN or CON steers. Biopsy samples of the LM muscle which were excised 51 days (SGRP 1-3) or 65 days (SGRP 4) before slaughter proved to be suitable for the determination of fiber distribution in live animals. Fiber area increased in post-mortem samples of both muscles from SGRP 1-3 in all treatment groups followed by a plateau. Implantation with Revalor led to an additional increase in fiber area from SGRP 3 and 4 (P<0.05). Synovex did not affect fiber area compared with control steers whereas Ralgro and Revalor implants led to larger fibers in SGRP 3 and 4, respectively. It can be concluded that some growth-promoting implants result in noticeable differences in muscle hypertrophic responses which coincide with their different effectiveness to enhance lean mass accretion.

Differences in natural steroid hormone patterns of beef from bulls and steers.

This investigation gives an overview of the concentrations of naturally occurring androgens, progestogens, corticosteroids, and their precursors and metabolites in meat from bulls and steers. A recently developed gas chromatography-mass spectrometry IGC-MS) method with improved sensitivity for steroid analysis was used. Eighty-two beef samples were analyzed using the GC-MS method. Beef from bulls contained higher concentrations of testosterone, which is an anabolic androgen, and its metabolite epitestosterone (P < .01) and the androgen precursor dehydroepiandrosterone (P < .05) than beef from steers. Beef from steers contained higher (P < .05) concentrations of the basic hormone precursor pregnenolone and cortisol, which is a catabolic corticosteroid, than beef from bulls. A classification of an unknown beef sample to one of the categories (bull or steer) was possible in most cases (>90%) using a masculinity index (MI) that was calculated using the concentrations of testosterone, epitestosterone, and pregnenolone. Because the hormonal status of beef cattle is related to meat quality characteristics, such as tenderness or fat and protein distribution, the MI may contribute to meat quality assessment and meat quality control.

Influence of sampling on steroid hormone patterns of beef from bulls and steers.

Three different muscles (Longissimus dorsi, Semitendinosus, Extensor carpi ulnaris) of bulls and steers, which represent different parts of the carcass and which have differing properties (function, proportions of fat and connective tissue), were analysed with GC-MS for their contents of testosterone, cortisol, cortisone, pregnenolone, dehydroepiandrosterone, progesterone, hydroxyprogesterone, androstenedione, dihydrotestosterone, epitestosterone and androsterone. No difference in the hormone patterns could be detected between the three muscles. However, the enrichment of beef samples with inter- and intramuscular fat decreased the levels of the polar corticosteroids, whereas the levels of lipophilic steroids were increased. The patterns of the lipophilic sex steroids, their precursors and metabolites, which can be used to determine the sexual origin of beef and which might prove useful in evaluating residues of administered steroid hormones, seem to be less affected by the beef sample's fat content, however.

Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN).

Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida. Both B. japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant. B. japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes. In symbiosis both rpoN genes could replace each other functionally. The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely. Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally. By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ. Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant. The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.

Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein.

A special sequence motif in the Bradyrhizobium japonicum NifA protein, consisting of two functionally essential cysteines separated by four other amino acids (Cys-aa4-Cys), has been proposed to be part of a potential metal-binding site [(1988) Nucleic Acids Res. 16, 2207-2224]. Using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function. The deletion of one amino acid to give a Cys-aa3-Cys motif renders the protein inactive whereas the creation of a Cys-aa5-Cys motif (one amino acid inserted) still leads to a partially active NifA protein.