Development of a polyclonal antibody-based sensitive enzyme-linked immunosorbent assay for fumonisin B(4)

Polyclonal antibodies (PAb) against fumonisin B(4) (FmB(4)), which have good cross-reactivity with four major fumonisins, were produced by immunizing a rabbit with FmB(4)-keyhole limpet hemocyanin conjugate. A sensitive competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for fumonisins was developed. Because of the limited supply of FmB(4), both FmB(1)-horseradish peroxidase conjugate (HRP) and FmB(3)-HRP were tested as the toxin-enzyme markers in the CD-ELISA. In the FmB(1)-HRP-based CD-ELISA, the concentrations of FmB(1), FmB(2), FmB(3), and FmB(4) causing 50% inhibition of binding of enzyme marker (IC(50)) were 9.0, 2.1, 9.0, and 6.5 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 58.5, 309.5, 58.5, and 100%), respectively. In the FmB(3)-HRP-based CD-ELISA, the IC(50) values for FmB(1), FmB(2), FmB(3), and FmB(4) were 7.1, 1.9, 7.6, and 5.3 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 74, 280, 70, and 100%), respectively. The FmB(3)-HRP-based CD-ELISA was then used in a series of analytical recovery experiments using Fusarium moniliforme corn culture material spiked with FmB(1) and with clean corn spiked with a FmB(3)/FmB(4)-containing extract. The overall recovery of FmB(1) from culture material in the range of 10-100 ppm was 65%. The detection limit for FmB(1) with clean corn as matrix was between 100 and 500 ppb. F. moniliforme cultures were analyzed with the developed CD-ELISA and a well-established FmB(1) antibody-based ELISA, which is not sensitive to FmB(4). Differences in the fumonisin levels found by the two assays were used as an indication of the presence of FmB(4) in the culture material and, therefore, as a method to identify FmB(4)-producing strains. Using ELISA in combination with HPLC individual B-series fumonisins were quantified. The ELISA developed in the present study would be a useful supplement to FmB(1) antibody-based ELISA for screening of Fusarium strains for the production of major fumonisins.     

Yields of wheat and soil carbon and nitrogen contents following long-term incorporation of barley straw and ryegrass catch crops

Abstract. Three successive crops of winter wheat were grown on a sandy loam to test the residual effect of long‐term annual incorporation of spring barley straw at rates of 0, 4, 8 and 12 t ha^?1, and ryegrass catch crops with or without additions of pig slurry. Soil receiving 4, 8 and 12 t ha^?1 of straw annually for 18 years contained 12, 21 and 30% more carbon (C), respectively, than soil with straw removal, and soil C and nitrogen (N) contents increased linearly with straw rate. The soil retained 14% of the straw C and 37% of the straw N. Ryegrass catch‐cropping for 10 years also increased soil C and N concentrations, whereas the effect of pig slurry was insignificant. Grain yield in the first wheat crop showed an average dry matter (DM) increase of 0.7 t ha^?1 after treatment with 8 and 12 t straw ha^?1. In the two subsequent wheat crops, grain yield increased by 0.2–0.3 t DM ha^?1 after 8 and 12 t straw ha^?1. No grain yield increases were found after 4 t straw ha^?1 in any of the three years. Previous ryegrass catch crops increased yields of wheat grain, but effects in the third wheat crop were significant only where ryegrass had been combined with pig slurry. Straw incorporation increased the N offtake in the first wheat crop. In the second crop, only 8 and 12 t straw ha^?1 improved wheat N offtake, while the N offtake in the third wheat crop was unaffected. Ryegrass catch crops increased N offtake in the first and second wheat crop. Again, a positive effect in the third crop was seen only when ryegrass was combined with slurry. Long‐term, annual incorporation of straw and ryegrass catch crops provided a clear and relatively persistent increase in soil organic matter levels, whereas the positive effects on the yield of subsequent wheat crops were modest and transient.     

Nitrogen turnover rates in a riparian fen determined by 15N dilution

Summary Gross rates of N mineralization, assimilation, nitrification, and NO _in3 ^sup- reduction were determined in soil from a wet riparian fen by 1-day incubations of soil cores and slurries with ¹⁵N-labelled substrates. N mineralization transformed 0.1% of the total organic N pool daily in the soil cores, of which 25% was oxidized through autotrophic nitrification and 53%–70% was incorporated into microorganisms. N mineralization and nitrification were markedly inhibited below 5 cm in soil depth. At least 80% of the NO _in3 ^sup- reduction in aerated cores occurred through dissimilatory processes. Dissimilatory reduction to NH _in4 ^sup+ (DNRA) occurred only below 5 cm in depth. The results show that NH _in4 ^sup+ oxidation was limited by available substrate and was itself a strong regulator of NO _in3 ^sup- -reducing activity. NO _in3 ^sup- reduction was significantly increased when the soil was suspended under anaerobiosis; adding glucose to the soil slurries increased NO _in3 ^sup- reduction by 2.4–3.7 times. Between 3% and 9% (net) of the added NO _in3 ^sup- was reduced through DNRA in the soil slurries. The highest percentage was observed in soil samples from deeper layers that were pre-incubated anaerobically.     

Cadmium soil sorption at low concentrations: VIII. correlation with soil parameters

Cadmium distribution coefficients, K _d were determined at low Cd concentrations (solute: 0.2 to 3.0 μg Cd dm^?3, soil: 0.044 to 1.1 mg Cd kg^?1) for 63 Danish agricultural soils. The K _d values ranged from 15 to 2450 L kg^?1. About 40% of the soils had K _d values below 200 L kg^?1. The observed K _d values correlated very well with soil pH (r ² = 0.72). Introducing soil organic matter content as a second parameter improved the correlation some (r ² = 0.79). No further improvements were obtained by introducing traditional soil parameters as clay, silt, fine sand, coarse sand and CEC or ‘reactive’ parameters as oxyhydroxides of Mn, Fe and Al. The identified regression equation for predicting K _d values indicates that K _d approximately doubles for each 0.5 unit increase in pH or 2% increase (weight basis) in organic matter content.     

Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch

Background

Studies on transmission of Enterococcus faecalis among chickens during hatch have not been carried out so far. Information about vertical transmission and subsequent spreading and colonization of the cloacal mucosa through cloacal ''drinking'' during hatch are important to understand the epidemiology of E. faecalis infections. In the present investigation vertical transmission and subsequent spreading and colonization of the cloacal mucosa of chickens by E. faecalis through cloacal ''drinking'' were examined.

Methods

Two different batches of layer chickens originating from 45 weeks old Brown and White Lohmann parents, respectively from the same farm were sampled in the hatcher. Isolates were confirmed to be E. faecalis by polymerase chain reaction (PCR) and further by multilocus sequence typing (MLST) to state their population structure and comparison made to sequence types previously obtained from chicken.

Results

A total of 480 chickens were swabbed from the cloacae just after hatch and after 24 hours. A total of 101 isolates were confirmed as E. faecalis by a species specific PCR. The prevalence of E. faecalis increased from 14% at 0 h to 97% after 24 h for the Brown Lohmann chickens and from 0.5% to 23% for the White Lohmann flock. The 84 isolates analysed by MLST were distributed on 14 sequence types (ST). Three ST (401, 82 and 249) accounted for 64% of all isolates analysed by MLST after 24 h. ST 82 has previously been reported from amyloid arthropathy and other lesions in poultry.

Conclusions

The present findings demonstrated a high potential of a few contaminated eggs or embryos to rapidly facilitate the spread of E. faecalis to almost all chickens during hatch.     

Diversity and stability of Aleutian mink disease virus during bottleneck transitions resulting from eradication in domestic mink in Denmark

Aleutian mink disease (plasmacytosis) virus (AMDV) in domestic mink (Neovison vison) has been subject to eradication in Denmark since 1976. In 2001, approximately 5% of Danish mink farms were still infected and all were located in the northern part of the peninsula of Jutland. In the present study a total of 274 Danish isolates of AMDV collected during the two seasons of 2004 and 2005 were characterized by partial sequencing of the coding region of the non-structural (NS) proteins. Older AMDV isolates from Denmark, available, were also included. The Danish isolates represent a very homogenous cluster compared with Swedish, Finnish and Dutch isolates and seem to represent a minor fraction of the genetic diversity previously found in Denmark. Stability of nucleotide deviations reveals that the purifying selection of bottlenecks imposed on the AMDV population in Denmark by the stamping out policy for more than 6 years exceeds the rate of mutation driven diversity. Among the isolates from farms in northern Jutland two distinct types could be identified and within each of them a number of sub-types which were all useful in tracking spread of infections. Infection at a farm the preceding season was a predisposing risk parameter for disease outbreak at a farm, and strain identity substantiates the suggestion that inadequate disinfection is involved in the recurrence of outbreaks. In cases of new introductions to farms it is indicated that contact including transport between farms played a most significant role.     

The excretion of ammonium by enchytraeids (Cognettia sphagnetorum)

Enchytraeids are involved both directly and indirectly in decomposition processes and nitrogen mineralization in soil. Their influence is especially important in nitrogen poor ecosystems such as heathland where the enchytraeid species, Cognettia sphagnetorum, is often abundant and playing a significant role in the N-cycling. The objective of this study was to quantify NH₄⁺-N excretion of C. sphagnetorum at different temperatures. The results were combined with investigations of population dynamics during one year to estimate annual NH₄⁺-N excretion of the population of C. sphagnetorum in a dry Danish heath soil. C. sphagnetorum significantly increased its NH₄⁺-N excretion rate with increasing temperature. At 5 °C about 0.5 μg NH₄⁺-N mg dry weight⁻¹ day⁻¹ was excreted increasing to about 3.3 μg NH₄⁺-N mg dry weight⁻¹ day⁻¹ at 20 °C. Average enchytraeid biomass in the field was 2.5-3.5 g dry weight m⁻² during cool and wet periods. Dry and warm conditions in May and June, 2008, had a drastic and long-term negative impact on the enchytraeid community. The excretion of NH₄⁺-N by enchytraeids was therefore highest during the cool and moist months despite low temperatures (October 2007-May 2008) and amounted to about 2 mg NH₄⁺-N m⁻² day⁻¹ during this period. The estimated annual NH₄⁺-N excretion of the enchytraeid community was approximately 0.3 g N m⁻² year⁻¹. The results of the present study and the method described for estimation of N-excretion can increase our understanding of enchytraeids’ role in nitrogen mineralization.     

AFLP analysis of genetic diversity in leafy kale (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. convar. <Emphasis Type="Italic">acephala</Emphasis> (DC.) Alef.) landraces,cultivars and wild populations in Europe

AFLP markers were used to characterize diversity and asses the genetic structure among 17 accessions of kale landraces, cultivars and wild populations from Europe. The range of average gene diversity in accessions was 0.11–0.27. Several landraces showed higher levels of diversity than the wild populations and one cultivar had the lowest diversity measures. The landraces that were most genetically diverse were from areas where kales are known to be extensively grown, suggesting in situ conservation in these areas as a supplement to storage of seeds in gene banks. An analysis of molecular variance (AMOVA) showed that 62% of the total variation was found within accessions. For most accessions, genetic distance was not related to geographic distance. Similarities among accessions were probably not caused by recent gene flow since they were widely separated geographically; more likely the relationship among them is due to seed dispersal through human interactions. Our results indicate that a kale population found in a natural habitat in Denmark was probably not truly wild but most likely an escape from a cultivated Danish kale that had subsequently become naturalized.     

Training young horses to social separation: effect of a companion horse on training efficiency

Reasons for performing study: The intensity with which a horse responds to separation from its group and subsequently to being alone is relevant for both horse and handler safety. Identification of training methods that may reduce responses to separation would be useful in practice. Objectives: To investigate whether the initial presence of a familiar companion horse modifies responses to separation from the group, lowers stress levels (as measured by heart rate) and increases training efficiency. Hypothesis: Habituation to separation proceeds more quickly if the horse is first trained with a companion, and heart rate is lower when the horse is subsequently trained alone, compared to control horses trained individually from the start. Methods: Young mares (n = 32), kept in groups of 4 were exposed to social separation: 2 horses of the group were trained singly (S1, n = 16) and the remaining 2 horses (n = 16) were trained first with a companion (P2) and then alone (P1). The training comprised 3 steps whereby distance from the group was gradually increased. The final learning criterion was met when a horse fed calmly alone inside a test arena (Step 3). Horses that were trained in a pair had to succeed in Step 3 together before they repeated the steps alone. Feeding behaviour and heart rate were recorded. Results: There were no significant differences between S1 and P1 horses, indicating that the initial pair‐training did not reduce responses in P1 horses. However, heart rate was significantly lower when horses were trained in pairs (P2) compared to when the same horses were subsequently trained alone (P1). Conclusions and potential relevance: It may not be efficient to habituate naïve young horses to social separation initially with a partner as these horses appear to have to relearn being in the test situation alone when switching to the individual training.