Carbon and nitrogen mineralization after maize harvest between and within maize rows: a microcosm study using 13C natural abundance

The sequestration of carbon in soil is not completely understood, and quantitative information about the rates of soil organic carbon (SOC) turnover could improve understanding. We analyzed the effects of the uneven distribution of crop residues after harvest of silage maize on C and N losses (CO₂‐C, dissolved organic carbon (DOC) and nitrogen (DON), and NO₃^–) from a Haplic Phaeozem and on the occurrence of priming effects induced by the decomposition of accumulated maize residues. Soil columns were taken from a continuous maize (since 1961) field after harvest i) between maize stalk rows (M_bare), ii) within the maize rows including a standing maize stalk (M_stalk), and iii) from a continuous rye (since 1878) field after tillage (rye stalk and roots were mixed into the Ap horizon). The soil columns were incubated for 230 days at 8 °C with an irrigation rate of 2 mm 10^–2 M CaCl₂ per day. Natural ¹³C abundance was used to distinguish between maize‐derived C (in SOC and maize residues) and older C originating from former C₃ vegetation. The uneven distribution of maize residues resulted in a considerably increased heterotrophic activity within the maize rows as compared with soil between seed rows. Cumulative CO₂ production was 53.1 g CO₂‐C m^–2 for M_stalk and 23.3 g CO₂‐C m^–2 for M_bare. The contribution of maize‐derived C to the total CO₂ emission was 83 % (M_stalk) and 67 % (M_bare). Calculated as difference between CO₂‐C release from M_stalk and M_bare, 19 % of the maize residues (roots and stalk) in M_stalk were mineralized during the incubation period. There was no or only a marginal effect of the accumulation of maize residues in M_stalk on leaching of DOC, DON, and NO₃^–. Total DOC and DON leaching amounted to 2.5 g C m^–2 and 0.16 g N m^–2 for M_stalk and to 2.1 g C m^–2 and 0.12 g N m^–2 for M_bare. The contribution of maize‐derived C to DOC leaching was about 25 % for M_stalk and M_bare. Nitrate leaching amounted to 3.9 g NO₃^–‐N m^–2 for M_stalk and to 3.5 g NO₃^–‐N m^–2 for M_bare. There was no priming effect induced by the decomposition of fresh maize residues with respect to CO₂ or DOC production from indigenous soil organic carbon derived from C₃ vegetation.     

Prediction of deoxynivalenol and zearalenone concentrations in Fusarium graminearum inoculated backcross populations of maize by symptom rating and near‐infrared spectroscopy

Gibberella ear rot (GER) caused by Fusarium graminearum is a destructive disease in maize of temperate regions resulting in yield reduction and contamination by the mycotoxins deoxynivalenol (DON) and zearalenone (ZON). We wanted to analyse whether prediction of DON and ZON concentrations is feasible either by GER severity ratings or by near‐infrared spectroscopy (NIRS). We analysed 80 and 102 lines developed by backcrossing doubled‐haploid lines from segregating populations to the resistant and susceptible parent, respectively, by artificial infection at three locations in Germany and France. Both backcross (BC) populations differed substantially in their means for all traits with significant (P < 0.01) genotypic variances. DON and ZON concentrations measured by immunotests were significantly (P < 0.01) correlated with each other and with GER severity within each BC population (0.6 ≤ r ≤ 0.9, P < 0.01). DON concentration measured by immunotest and NIRS significantly correlated (r ≈ 0.9, P < 0.01). In conclusion, DON and ZON concentrations could be reliably predicted by GER severity. Additional NIRS analysis of DON concentration might be useful for the positively selected fraction.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0 kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48 h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.     

Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods

Lyme disease is a zoonotic, vector-borne disease and occurs in mammals including horses. The disease is induced by infection with spirochetes of the Borrelia burgdorferi sensu lato group. Infection of mammalian hosts requires transmission of spirochetes by infected ticks during tick bites. Lyme disease diagnosis is based on clinical signs, possible exposure to infected ticks, and antibody testing which is traditionally performed by ELISA and Western blotting (WB). This report describes the development and validation of a new fluorescent bead-based multiplex assay for the detection of antibodies to B. burgdorferi outer surface protein A (OspA), OspC and OspF antigens in horse serum. Testing of 562 equine sera was performed blindly and in parallel by using WB and the new multiplex assay. Because a true gold standard is missing for Lyme antibody testing, we performed and compared different statistical approaches to validate the new Lyme multiplex assay. One approach was to use WB results as a 'relative gold standard' in ROC-curve and likelihood-ratio analyses of the new test. Cut-off values and interpretation ranges of the multiplex assay were established by the analysis. The second statistical approach used a Bayesian model for the calculation of diagnostic sensitivities and specificities of the multiplex assay. The Bayesian analysis takes into consideration that no true gold standard exists for detecting antibodies to B. burgdorferi and estimated sensitivities and specificities of both tests that were compared. Therefore, the Bayesian analysis also resulted in an evaluation of diagnostic sensitivity and specificity of WB. Overall, the new assay was characterized by low background values and a wide dynamic quantification range for the detection of antibodies to OspA, OspC and OspF antigens of B. burgdorferi. The diagnostic sensitivity and specificity for the OspA bead-based assay were calculated as 49% and 85%, respectively, and by a standard ROC curve analysis only because the Bayesian model could not be run on this parameter. The Bayesian-derived diagnostic sensitivities of the OspC and OspF assays were 80% and 86%, respectively. For comparison, the Bayesian-derived estimates for WB resulted in sensitivities of 72% for OspC and 80% for OspF. The Bayesian diagnostic specificities of the multiplex assay were 79% and 69% for OspC and OspF, respectively. WB analysis had specificities of 92% for OspC and 77% for OspF. Although the analysis of a new assay in the absence of a true gold standard remains challenging, the approach used here can help to address this problem when new technologies and traditionally used test standards differ significantly in their analytical sensitivities, which consequently causes problems in the calculation of diagnostic sensitivity and sensitivity values for the new assay. In summary, the new multiplex assay for the detection of antibodies to B. burgdorferi OspA, OspC and OspF antigens in horse serum has improved analytical and diagnostic sensitivities compared to WB analysis. Multiplex analysis is a valuable quantitative tool that simultaneously detects antibodies indicative for natural infection with and/or vaccination against the Lyme pathogen.     

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25)

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.     

Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells

Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu–Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th₁ and CD8+ Tc₁ adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production.     

A comparison of intradermal testing and detection of allergen-specific immunoglobulin E in serum by enzyme-linked immunosorbent assay in horses affected with skin hypersensitivity

Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.     

Application of the ASVCP guidelines for the establishment of haematologic and biochemical reference intervals in Icelandic horses in Austria

Background

Despite the increasing popularity of Icelandic horses, published reference intervals (RIs) in this breed are rare. Due to their isolation and their small gene pool, alterations in some variables are likely and some possible breed-specific peculiarities have been described. The purpose of the present study was the establishment of comprehensive RIs in Icelandic horses according to recently published guidelines.In a prospective observational study, blood samples were collected from the jugular vein of 142 Icelandic horses into EDTA and serum tubes. Reference intervals were established for haematologic and biochemical analytes on the Advia 2120i™ and the Dimension ExL™ by established methods. RIs were defined as central 95 % intervals bounded by the 2.5th and 97.5th percentiles with their 90 % confidence intervals, calculated according to recently published ASVCP guidelines. An inhouse-developed quality control system using observed total allowable error was used for the surveillance of the internal quality control preceding the measurements.

Results

The RIs were as follows: haematocrit: 0.29–0.39, RBC: 5.79–8.63 T/l, haemoglobin: 102.0–142.3 g/l, MCV: 42–51 fl, platelets: 146–263 G/l, WBC: 4.13–8.57 G/l, segs: 1.98–4.73 G/l, lymphocytes: 1.25–3.49 G/l, monocytes: 0.06–0.31 G/l, eosinophils: 0.04–0.50 G/l, glucose: 4.0–5.7 mmol/l, urea: 3.2–6.4 mmol/l, creatinine: 79.6–141.4 μmol/l, total protein: 54.4–72.9 g/l, albumin: 27.7–36.8 g/l, total bilirubin: 8.1–21.1 μmol/l, triglycerides: 0.03–0.44 mmol/l, cholesterol: 1.75–2.90 mmol/l, ALP: 1.35–3.55 μkat/l, AST: 4.52–8.80 μkat/l, GLDH: 0.0–0.18 μkat/l, GGT: 0.11–0.39 μkat/l, CK: 2.53–6.52 μkat/l, LDH: 3.32–7.95 μkat/l, iron: 16.4–39.9 μmol/l, calcium: 2.69–3.19 mmol/l, phosphate: 0.5–1.3 mmol/l, magnesium: 0.6–0.9 mmol/l, sodium: 134–141 mmol/l, potassium: 3.6–4.7 mmol/l, chloride: 100–105 mmol/l.

Conclusions

Reference intervals of several haematologic and biochemical analytes differed from the transferred historical reference intervals applied to equine samples in the authors’ laboratory. These might be of clinical importance in some analytes such as creatine kinase.

Electronic supplementary material

The online version of this article (doi:10.1186/s13028-015-0120-4) contains supplementary material, which is available to authorized users.     

Stability and inactivation of vesicular stomatitis virus,a prototype rhabdovirus

Viruses may remain infectious outside the host cell for considerable time and represent a source of accidental infection if not properly inactivated. In this study, the survival of vesicular stomatitis virus (VSV) in suspension and dried on surfaces was analyzed. In addition, the sensitivity of VSV to disinfectants and physicochemical changes was investigated. VSV showed a notable stability in suspension at 4 °C with virus titers remaining high over several weeks. The presence of serum proteins had a stabilizing effect on virus infectivity, whereas elevated temperatures reduced survival times. VSV dried on polystyrene, glass or stainless steel surfaces remained infectious for at least 6 days at ambient temperature. VSV showed a remarkable resistance to extreme pH in particular in the alkaline range, but could be rapidly inactivated by heating at 55 °C or higher. The virus was highly sensitive to inactivation by commonly used disinfectants such as aldehydes, alcohols, and detergents. The high stability of VSV on surfaces and in suspension may facilitate dissemination of the virus in livestock by contaminated feeding and water troughs, hands, and milking equipment. This knowledge on the sensitivity of VSV to disinfectants will help to set up appropriate hygiene measures.