Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol. 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I. It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding.     

Net portal appearance of volatile fatty acids in sheep intraruminally infused with mixtures of acetate, propionate, isobutyrate, butyrate, and valerate

The net portal appearance of volatile fatty acids (VFA) was investigated in four ruminally fistulated and multicatheterized sheep. During the experiments, the sheep were fed once every hour for 14 h and intraruminally infused with mixtures of VFA for the 12 h commencing 2 h after the initiation of the hourly feeding protocol. Paired arterial and portal blood samples were obtained hourly during the last 6 h of the experiments. In the control treatment (1), only water was infused intraruminally. In Treatments 2 through 4, the intraruminal infusion rates of propionate (40 mmol/h), isobutyrate (5 mmol/h), and valerate (5 mmol/h) were unchanged. In Treatments 2, 3, and 4, the acetate infusion rate was 100, 60, and 20 mmol/h, respectively, and the butyrate infusion rate was 10, 30, and 50 mmol/h, respectively. Thus, the infusion rate of VFA carbon was constant across Treatments 2 through 4. Portal recovery estimated from the increased net portal appearance in Treatments 2 through 4 compared to the control treatment was 85% for propionate and 60% for isobutyrate, and these recoveries were unaffected by treatment. The portal recovery of butyrate increased (from 21 to 32%) with increasing infusion rate of butyrate and decreasing infusion rate of acetate, as did the portal recovery of valerate (from 14 to 31%). The portal recovery of acetate was 55%, when measured as net portal appearance. Thus, it seems that the capacity for beta-oxidation in ruminal epithelium is limited, which would explain the increasing portal recovery of butyrate and valerate with increasing infusion rate of butyrate, when infusion rate of VFA carbon is unchanged.     

Development and validation of an improved enzyme-linked immunosorbent assay for the detection of thyroglobulin autoantibodies in canine serum samples

An enzyme-linked immunosorbent assay to detect thyroglobulin autoantibodies (TGAB) in canine serum was developed and validated. The test result for each sample was derived from the optical density readings (OD) and expressed as an Ab-score(%) calculated from three in-house calibrators. The assay specifically detected TGAB as judged from lack of response in the assay after samples had been incubated with specific antigen. Intra- and interassay coefficients of variation ranged from 2.0–4.9% and 4.6–9.9%, respectively. The detection limit, an Ab-score of 5.6%, was close to the median Ab-score of 10% observed in healthy dogs (n = 132). The median Ab-score of dogs with primary hypothyroidism and lymphocytic thyroiditis (n = 11), skin diseases (n = 35), and non-thyroidal diseases (n = 63) was 340%, 12%, and 8%, respectively. The prevalence of TGAB in hypothyroid dogs with lymphocytic thyroiditis (sensitivity) was 91% (95% confidence limits: 59%–99%). In dogs with dermatological diseases without lymphocytic thyroiditis the prevalence of TGAB was 3% corresponding to a specificity of 97% (95% confidence limit: 85%–100%). In dogs with non-thyroidal diseases and healthy dogs the prevalence of TGAB was 5% and 6%, respectively. The diagnostic accuracy of serum TGAB was evaluated by subjecting the data from 11 dogs with lymphocytic thyroiditis and 35 control dogs without lymphocytic thyroiditis to receiver-operating characteristic curve analysis. The area under the receiver-operating characteristic curve (W = 0.966; 95% confidence limit 87%–100%) was significantly higher than that of a worthless test (0.5) (P < 0.0001), thereby indicating that serum TGAB measurements distinguished between dogs with and without lymphocytic thyroiditis.     

Effects of vertical distribution of soil inorganic nitrogen on root growth and subsequent nitrogen uptake by field vegetable crops

Information is needed about root growth and N uptake of crops under different soil conditions to increase nitrogen use efficiency in horticultural production. The purpose of this study was to investigate if differences in vertical distribution of soil nitrogen (N_inorg) affected root growth and N uptake of a variety of horticultural crops. Two field experiments were performed each over 2 years with shallow or deep placement of soil N_inorg obtained by management of cover crops. Vegetable crops of leek, potato, Chinese cabbage, beetroot, summer squash and white cabbage reached root depths of 0.5, 0.7, 1.3, 1.9, 1.9 and more than 2.4 m, respectively, at harvest, and showed rates of root depth penetration from 0.2 to 1.5 mm day^?1 °C^?1. Shallow placement of soil N_inorg resulted in greater N uptake in the shallow‐rooted leek and potato. Deep placement of soil N_inorg resulted in greater rates of root depth penetration in the deep‐rooted Chinese cabbage, summer squash and white cabbage, which increased their depth by 0.2–0.4 m. The root frequency was decreased in shallow soil layers (white cabbage) and increased in deep soil layers (Chinese cabbage, summer squash and white cabbage). The influence of vertical distribution of soil N_inorg on root distribution and capacity for depletion of soil N_inorg was much less than the effect of inherent differences between species. Thus, knowledge about differences in root growth between species should be used when designing crop rotations with high N use efficiency.     

Effects of long-term contrasting lime and phosphorus applications on barley grain yield,root growth and abundance of mycorrhiza

Lime and phosphorus (P) applications are common agricultural management practices. Our aim was to quantify the effects of long-term application practices on root growth and abundance of arbuscular mycorrhizal fungi (AMF) under field conditions. We assessed the effects of lime and P fertilizer applications on barley yield, root growth and AMF abundance in 2016. Treatments were no, low, medium and high liming rate corresponding to application of 0, 4, 8 and 12 Mg lime ha⁻¹ every 5–9 years since 1942 combined with no or yearly application of 15.6 kg P ha⁻¹ since 1944. At harvest, grain yield, root intensity (core-break) and AMF abundance at different soil depths were estimated. Root development was monitored during early growth with minirhizotrons in treatments receiving low, medium and high liming rates and P fertilization. A quadratic model relating grain yield to liming rate estimated yields to peak at 6.4 Mg lime ha⁻¹ with yields of 4.2 and 3.2 Mg grain ha⁻¹ with and without P fertilization, respectively. Low and medium liming rates resulted in greater AMF abundance, especially in the no P treatments. During early growth in P-fertilized treatments, 77% and 65% more roots developed in the soil profile when treated with medium and high liming rate, respectively, compared to low liming rate. We conclude that long-term application of lime in soils receiving yearly P fertilization improved conditions for root growth in soil layers below 30 cm, but at the high liming rate, this did not translate into higher yield.     

Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis

Two approaches to shift the acylglycerol equilibrium were tested as follows: addition of monoacylglycerols and lowering of the temperature. None of these approaches were able to shift the equilibrium toward higher diacylglycerol (DAG) contents. The glycerolysis reaction was optimized with five factors using response surface methodology. Evaluation of the resulting model enabled the determination of optimal reaction conditions for glycerolysis aiming at high DAG yield. However, verification of the model showed that the model was unable to take the molecular equilibrium into account but it provided good insight in how process settings can be chosen to, for example, minimize production costs. Optimal conditions were found to be the following: no extra water, low content of glycerol (molar ratio of 2), temperature of 60-65 degrees C, 4-5 h reaction time, and only 5 wt % lipases. Up scaling of the glycerolysis process was performed and revealed that scale-up to a 20 kg production in a pilot plant batch reactor was possible with a similar DAG yield (60 wt %) as in lab scale. Purification of DAG oil using batch deodorization and short path distillation yielded 93 wt % pure DAG oil.     

Danish dairy farmers' perception of biosecurity

To implement biosecurity measures at farm-level is a motivational challenge to dairy farmers as emerging diseases and their consequences largely are unpredictable. One of the reasons for this challenge is that outcomes are more likely to benefit society than the individual farmer. From the individual farmer's point of view the impacts of zoonotic risk, international trade and welfare concerns appear less obvious than the direct costs at farm-level. Consequently, a social dilemma may arise where collective interests are at odds with private interests. To improve biosecurity at farm-level farmers must be motivated to change behavior in the 'right' direction which could provide selfish farmers with unintended possibilities to exploit the level of biosecurity provided by other dairy farmers' collective actions. Farmers' perception of risk of disease introduction into a dairy herd was explored by means of Q-methodology. Participating farmers owned very large dairy herds and were selected for this study because Danish legislation since 2008 has required that larger farms develop and implement a farm specific biosecurity plan. However, a year from introduction of this requirement, none of the participating farmers had developed a biosecurity plan. Farmers' perception of biosecurity could meaningfully be described by four families of perspectives, labeled: cooperatives; confused; defectors, and introvert. Interestingly, all families of perspectives agreed that sourcing of animals from established dealers represented the highest risk to biosecurity at farm-level. Farmers and policy-makers are faced with important questions about biosecurity at farm-level related to the sanctioning system within the contextual framework of social dilemmas. To solve these challenges we propose the development of a market-mediated system to (1) reduce the risk of free-riders, and (2) provide farmers with incentives to improve biosecurity at farm-level.     

Seawater tolerance in Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., and S. salar?×?S. trutta hybrids smolt

High levels of hybridization between Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) have been reported in the Gyrodactylus salaris infected Rivers Vefsna and Driva in Norway. The survival and behaviour during the sea phase of such hybrids is unknown. The reported work documents ionoregulatory status after 24 h seawater challenge tests (24hSW) and gill Na⁺/K⁺-ATPase (NKA) activity of migrating wild smolts of Atlantic salmon, brown trout and hybrids at two sampling dates during the 2006 smolt run in River Driva. Salmon, trout and hybrids contributed to 27, 52 and 21% of the catches, respectively. The large contribution of hybrids suggests both a high hybridization rate and a high survival rate from fry to smolt. Both salmon and hybrids had a well-developed seawater tolerance at the time of downstream migration, revealed by small ionoregulatory effects and no or low mortality rates during the 24hSW tests. The trout were not fully adapted to seawater, and high mortality rates were observed (71 and 92%) during the 24hSW tests. The NKA activity was not significantly different between salmon and hybrids. Most of the hybrids were physiologically capable of direct entry to full strength seawater. The incomplete seawater tolerance in trout compared to salmon corresponds well with differences in life-history patterns between these two species. The life history strategy of the hybrids during the sea phase is not known, and further investigations on the marine behaviour and survival is needed to evaluate the role of hybrids in the risk of spreading G. salaris to nearby river systems.     

Breed‐specific variation of hematologic and biochemical analytes in healthy adult Bernese Mountain dogs

Background: Hematology and serum biochemistry reference intervals in dogs may be affected by internal factors, such as breed and age, and external factors, such as the environment, diet, and lifestyle. In humans, it is well established that geographic origin and age may have an impact on reference intervals and, therefore, more specific reference intervals are sought for subpopulations. Objective: The objective of this study was to validate and transfer standard laboratory reference intervals for healthy Bernese Mountain dogs and to create new intervals for analytes where the established laboratory reference intervals were rejected. Methods: The procedure was performed using the human Clinical and Laboratory Standards Institute‐approved model modified for veterinary use. Thirty‐two dogs were included in the study using a direct a priori method, as recommended. Results: While 23 of the standard laboratory reference intervals were readily validated, 7 of the analytes (eosinophils, MCHC, alkaline phosphatase [ALP], γ‐glutamyltransferase, total bilirubin, amylase, and cholesterol) required new reference intervals according to the standard. These were calculated using the robust method. In particular, the new reference range for ALP was wide compared with the established laboratory reference interval. No clinical causes were found for differences in the results of these analytes. Conclusion: We found significant differences in 7 hematologic and serum biochemical analytes for which a breed‐specific variation appears to be the most plausible explanation. Breed‐specific reference intervals for Bernese Mountain dogs will help avoid misinterpretation of laboratory results in the diagnostic process.