Evaluation of the agreement between Brix refractometry and serum immunoglobulin concentration in neonatal piglets.

Neonatal piglets lack immunoglobulins at birth. Sufficient colostrum intake (CI) and immunoglobulin absorption are essential for an appropriate passive transfer of immunity via the colostrum. Most methods to measure immunoglobulins in serum of piglets are labour-intensive, expensive or imprecise and not designed for on-farm use. The present diagnostic test study evaluated digital Brix refractometry to measure immunoglobulins in serum of neonatal piglets and to suggest thresholds for different serum immunoglobulin concentration. Additionally, agreements between Brix refractometry and optical refractometer (serum total protein, STP) and between Brix refractometry and ELISA (immunoglobulin G, IgG) were also investigated. Forty-five sows and 269 piglets from three different farms were enrolled in the study. Piglets were weighed at birth and 24 h later to calculate the CI. Serum was collected at 24 h after birth and analysed for STP, γ-globulins (electrophoresis), % Brix and IgG. In piglets, median (interquartile range, IQR) CI was 412 (196) g per piglet. Median (IQR) STP, γ-globulin and % Brix concentrations in piglet serum were 60 (11) g/L, 35 (10) g/L and 8 (2) %, respectively. Average (±SD) IgG concentration was 49 ±â€¯23 g/L. Passing-Bablok regression revealed a strong concordance between % Brix and STP (Kendall's tau (Τ): 0.620, P < 0.0001, n = 267) and % Brix and γ-globulin concentration (Kendall's Τ: 0.575, P < 0.0001, n = 267). The agreement between the Brix refractometer and IgG concentration was poor (Kendall's Τ: 0.267, P < 0.0001, n = 269). Receiver operating characteristic curves were performed to evaluate test characteristics of Brix refractometry for three γ-globulin cut-off values, i.e. 10, 20 and 30 g/L. The % Brix cut-off values resulting in the optimal combination of sensitivity and specificity were 5.4 (100 and 98.5%), 7.0 (100 and 89.3%) and 7.9 (90.1 and 80.6%), respectively. In conclusion, digital Brix refractometry is a sufficiently fast and practical method to assess serum γ-globulin concentrations in neonatal piglets on-farm and to evaluate them by considering the thresholds found in this study. Further studies are needed to validate those thresholds regarding piglet's survival in the pre-weaning period.

Sow and piglet factors determining variation of colostrum intake between and within litters.

Colostrum intake has a short- and long-term beneficial impact on piglet performance and mortality. Sows' colostrum production and piglets' colostrum intake are limited and highly variable. The present study investigated sow and piglet factors explaining the variation of colostrum intake between and within litters. The CV for colostrum intake and birth weight (BWb) of all piglets within a litter was calculated to evaluate the variation of colostrum intake and BWb within a litter (colostrum and litter BWb heterogeneity, respectively). A total of 1937 live-born piglets from 135 litters from 10 commercial herds were included. Colostrum intake per piglet averaged 371±144 g and was affected by breed (P=0.02). It was lower when oxytocin was administered to the sow during parturition (P=0.001) and with increased litter size (P<0.001). It was higher when the interval between birth and first suckling decreased (t FS, P<0.001). Colostrum intake was positively influenced by BWb (P<0.001) and this association was more pronounced in piglets from Topigs (P=0.03) and Hypor (P=0.03) sows compared with piglets from Danbred sow breeds. The positive relationship between colostrum intake and BWb was more pronounced when t FS lasted longer (P=0.009). Heterogeneity in colostrum intake averaged 31±11%, it increased when oxytocin was applied during farrowing (P=0.004) and when stillbirth occurred (P=0.006). Colostrum heterogeneity was positively associated with litter size (P<0.001) and litter BWb heterogeneity (P=0.01). The positive relationship between colostrum and litter BWb heterogeneity was more pronounced when oxytocin was applied during farrowing (P=0.04). The present study demonstrated that oxytocin should be used cautiously in sows during farrowing. Farrowing and colostrum management should prevent or counteract the adverse influences of stillbirth, large and heterogeneous litters on colostrum intake and colostrum heterogeneity. The study also confirmed the expected association between BWb and colostrum intake and indicated that the impact of BWb on colostrum intake was different among breeds (Hypor v. Danbred) and dependent on piglets' latency to first suckling. Hence, colostrum management should focus on low birth weight piglets, especially in some breeds, and low colostrum intake in low birth weight piglets can be counteracted by shortening the t FS.

Effect of rubber flooring on group-housed sows' gait and claw and skin lesions.

This study evaluated the influence of floor type on sow welfare in terms of lameness, claw lesions, and skin lesions. In a 2 × 3 factorial design, we have investigated the effect of rubber coverings on concrete floors and the effect of 3 levels of dietary zinc supplementation on locomotion and claw and skin lesions in group-housed sows. Six groups of 21 ± 4 hybrid sows were monitored during 3 successive reproductive cycles. The sows were group housed from d 28 after insemination (d 0) until 1 wk before expected farrowing date (d 108) in pens with either exposed concrete floors or concrete floors covered with rubber in part of the lying area and the fully slatted area. During each reproductive cycle, locomotion and skin lesions were assessed 4 times (d 28, 50, 108, and 140) and claw lesions were assessed twice (d 50 and 140). Results are given as least squares means ± SE. Locomotion and claw scores were given in millimeters, on analog scales of 150 and 160 mm, respectively. Here, we report on the effect of floor type, which did not interact with dietary zinc concentration ( > 0.10 for all variables). At move to group (d 28) and mid gestation (d 50), no differences between floor treatments were seen in locomotion ( > 0.10). At the end of gestation (d 108), sows housed on rubber flooring scored 9.9 ± 4.1 mm better on gait ( < 0.001). Regarding claw disorders, both parameters "heel overgrowth and erosion" (difference of 4.6 ± 1.8 mm; = 0.01) and "heel-sole crack" (difference of 3.1 ± 1.5 mm; = 0.04) scores were better for sows on rubber flooring at mid gestation (d 50). However, sows on rubber flooring scored worse for "vertical cracks in the wall horn" (difference of 3.4 ± 1.7 mm; = 0.04). At the end of lactation (d 140), both "white line" (difference of 2.9 ± 1 mm; = 0.02) and "claw length" (difference of 4.7 ± 1.4 mm; < 0.001) had better scores on rubber flooring. No differences for skin lesions were observed between floor treatments. The improved scores for gait toward the end of gestation and some types of claw disorders at mid gestation suggest that rubber flooring in group housing has a beneficial effect on the overall leg health of sows. The documented increase in vertical cracks in the wall horn at d 50 requires further investigation.

Long-term effects of colostrum intake in piglet mortality and performance.

The present study investigated the long-term effects of colostrum intake on performance and mortality in pigs. A total of 1,455 live-born piglets in 10 commercial herds were followed from birth until 22 wk of age. Pigs were individually weighed at birth, at weaning, at onset (intermediate weight), and during the fattening period (finishing weight). Colostrum intake was calculated by the mechanistic model developed by Theil et al. (see text for citation). One linear mixed model was fitted to model the possible associations between colostrum intake and weight at the weaning, intermediate, and finishing periods. In addition to colostrum intake as the main predictor of interest, other predictor variables were also tested, namely birth weight, birth order, sex, breed, and the interval between birth and first suckling (t). Colostrum intake and birth weight were positively associated with weaning ( < 0.001), intermediate ( < 0.001), and finishing ( < 0.001) weights. Furthermore, higher colostrum intake is more beneficial to weaning ( < 0.001), intermediate ( < 0.001), and finishing ( = 0.02) weights in piglets with low versus high birth weights. Birth order was positively associated with weight at each measurement time ( = 0.01). Sex affected only finishing weight ( < 0.001). Some breeds differed in piglets' weight at onset or during the fattening period. The association between t and weaning weight differed by breed. Three generalized linear mixed models were performed to model the probability of dying during the suckling, the nursery, or the fattening period. Colostrum intake, birth weight, birth order, sex, breed, and t were tested. Preweaning mortality was negatively associated with colostrum intake ( < 0.001) and birth weight ( = 0.004) and positively associated with t ( < 0.001). Mortality during the nursery period was negatively associated with colostrum intake ( < 0.001) and birth weight ( = 0.002). The negative association between colostrum intake and mortality during the suckling ( < 0.001) and the nursery ( = 0.008) periods was more pronounced in small versus heavy piglets. Mortality during fattening was associated with weaning ( = 0.04) and intermediate ( = 0.006) weight. In conclusion, colostrum intake significantly influences piglets' short-term and long-term performance and mortality. As colostrum yield is reported to be independent of litter size, sufficient colostrum intake per piglet is crucial, especially in hyperprolific sows.

Sow and litter factors influencing colostrum yield and nutritional composition.

One of the main characteristics of colostrum intake (CI), colostrum yield (CY), and colostrum composition (CC) in pigs is its variability. The present observational study aimed to investigate factors influencing CY and CC in 10 commercial herds. In total, 100 sows of 5 different breeds and their 1,455 live-born piglets were included. Sows' CY was estimated by the CI of their suckling piglets. Colostrum composition was analyzed by LactoScope Fourier transform infrared spectroscopy. Colostrum yield averaged 3,500 ± 110 g and the percentage of colostral fat (CF), protein, and lactose in colostrum averaged 5.39 ± 0.12, 16.49 ± 0.14, and 2.02 ± 0.05 %, respectively. The effect of sow, litter, and parturition factors on CY and CC were evaluated with a linear mixed regression model with herd included as a random factor. Sows with a gestation length (GL) of 113 d had a higher CY (4,178 ± 506 g) than sows with a GL of 114 to 115 d (3,342 ± 107 g; = 0.04). An interaction was found between the litter birth weight of suckling piglets (LW) and GL ( = 0.03). In sows with a GL of 114 to 115 d, CY increased with higher LW ( = 0.009). A shorter interval between birth and first suckling of the litter was related to a higher CY ( < 0.01). The percentage of fat in colostrums was higher in Hypor sows (6.35 ± 0.51) than in PIC (4.98 ± 0.27; = 0.001), Topigs 20 (5.05 ± 0.14; < 0.001), and Danbred (5.34 ± 0.22; < 0.001) sows. The percentage of CF was negatively associated with parity ( = 0.02) and positively associated with the number of live-born piglets ( = 0.03). The percentages of colostral protein and lactose were not significantly associated with any factor in the multivariable model. In conclusion, this study demonstrated that CY and CF are affected by different sow and litter factors. Pig producers may implement these observations in their management to maximize production or reproduction potential by optimizing CI, CY, and CC.

Effect of peripartal feeding strategy on colostrum yield and composition in sows.

Research showed a positive association between back fat (BF) change the week before farrowing and colostrum yield (CY). This study tested the causality of this association, hence to optimize CY by altering the sows' peripartal feeding strategy. Sows were randomly divided into 2 treatment groups at d 108 of gestation. The first group (L, n = 28) received 1.5 kg feed·d(-1), the second group (H, n = 22) received 3 times 1.5 kg feed·d(-1) until farrowing. Daily feed intake and CY were measured. Colostrum was analyzed for nutrient composition, AA and fatty acids, IgG and IgA. Sow serum was obtained at d 108 of gestation and d 1 of lactation after overnight fasting and analyzed for NEFA, (iso)butyrylcarnitine (C4), creatinine, urea, 3-OH-butyrylcarnitine (3-OH-C4), IgG, and IgA. Based on BF at d 108, sows were divided into body condition (BC) groups: skinny (<17 mm, n = 15), moderate (17 to 23 mm, n = 21), fat (>23 mm, n = 14). We performed ANOVA with treatment and BC as fixed factors and Scheffé post-hoc test. The week before farrowing, the L group had the lowest daily feed intake (DFI; 1.5 kg), and within the H group, fat sows (3.8 kg) had a lower DFI than skinny sows (4.3 kg; p = 0.006). The H group tended to have a greater total CY (P = 0.074) and had a greater CY/kg liveborn piglet (P = 0.018) than the L group. Compared with sows in moderate BC, fat sows had a lower total CY (P = 0.044) and a lower CY/kg liveborn piglet (P = 0.005). The H group had a greater concentration of lactose (p = 0.009) and n-3 PUFA (p < 0.001) but a lower concentration of protein (p = 0.040) in colostrum than the L group. The concentration of IgG and IgA did not differ between treatment and BC groups. Serum parameters at d 108 were similar between the treatment groups and BC groups. At d 1, the H group mobilized less body fat (NEFA: p = 0.002) and protein (creatinine: p < 0.001, C4: p = 0.016) reserves but had a greater ratio urea:NEFA (p < 0.001) and less ketone bodies (3-OH-C4: p < 0.001) compared with the L group. This indicates a more balanced entry of metabolites in the citric acid cycle and thus a better support of the maternal peripartal metabolism in the H group. Serum parameters did not differ between BC groups. Both CY and composition can be influenced by the peripartal feeding strategy and BC. The highest CY and most beneficial colostrum composition were obtained when sows entered the farrowing unit in a moderate BC and were provided a high peripartal feeding strategy.

Changes in back fat thickness during late gestation predict colostrum yield in sows.

Directing protein and energy sources towards lactation is crucial to optimise milk production in sows but how this influences colostrum yield (CY) remains unknown. The aim of this study was to identify associations between CY and the sow's use of nutrient resources. We included 37 sows in the study that were all housed, fed and managed similarly. Parity, back fat change (ΔBF), CY and performance parameters were measured. We obtained sow serum samples 3 to 4 days before farrowing and at D1 of lactation following overnight fasting. These were analysed for non-esterified fatty acids (NEFA), urea, creatinine, (iso)butyrylcarnitine (C4) and immunoglobulins G (IgG) and A (IgA). The colostrum samples collected 3, 6 and 24 h after the birth of the first piglet were analysed for their nutrient and immunoglobulins content. The technical parameters associated with CY were parity group (a; parities 1 to 3=value 0 v. parities 4 to 7=value 1) and ΔBF D85-D109 of gestation (mm) (b): CY (g)=4290-842a-113b. (R 2=0.41, P<0.001). The gestation length (P<0.001) and the ΔBF between D109 and D1 of lactation (P=0.050) were identified as possible underlying factors of the parity group. The metabolic parameters associated with CY were C4 at 3 to 4 days before farrowing (a), and 10logC4 (b) and 10logNEFA (c) at D1 of lactation: CY (g)=3582-1604a+1007b-922c (R 2=0.39, P=0.001). The colostrum composition was independent of CY. The negative association between CY and ΔBF D85-D109 of gestation could not be further explained based on our data. Sows that were catabolic 1 week prior to farrowing seemed unable to produce colostrum to their full potential. This was especially the case for sows with parities 4 to 7, although they had a similar feed intake, litter birth weight and colostrum composition compared with parities 1 to 3 sows. In conclusion, this study showed that parity and the use of body fat and protein reserves during late gestation were associated with CY, indicating that proper management of the sow's body condition during late gestation could optimise the intrinsic capacity of the sow's CY.

Evaluation of three intervention strategies to reduce the transmission of Salmonella Typhimurium in pigs.

Despite current control measures, Salmonella in pigs remains a major public health concern. In this in vivo study, the effect of three intervention strategies on Salmonella Typhimurium transmission in pigs was evaluated. The first intervention was feed supplemented with coated calcium-butyrate (group A); the second comprised oral vaccination with a double-attenuated Salmonella Typhimurium strain (group B), and the third was acidification of drinking water with a mixture of organic acids (group C). After challenge at 8 weeks of age, animals were individually sampled for 6 weeks (blood once per week; faeces twice per week) and then were euthanased at 14 weeks of age. Post-mortem ileum, caecum, ileocaecal lymph nodes, and tonsils were sampled, along with ileal, caecal and rectal contents, and tested for the presence of Salmonella spp. Transmission was quantified by calculating an 'adjusted' reproduction ratio 'Ra' and its 95% confidence interval (CI). The proportion of pigs that excreted Salmonella spp. via the faeces was significantly higher in group C (58%, P<0.0001) and the positive control group (41%, P=0.03), compared to group B (15%), and the proportion in group C was also significantly higher than in group A (23%, P=0.01). Group A had the lowest proportion of positive post-mortem samples (18%), followed by group B (31%), the positive control group (41%) and group C (64%) (P<0.03). The highest transmission was seen in the positive control group and group C (Ra=+∞ with 95% CI [1.88; +∞]), followed by group B (Ra=2.61 [1.21; 9.45]) and A (Ra=1.76 [1.02; 9.01]). The results of this study suggest that vaccination and supplementation of the feed with coated calcium-butyrate limited Salmonella transmission in pigs and might be useful control measures.

Comparison of oral versus parenteral iron supplementation on the health and productivity of piglets.

The aim of this study was to compare oral iron supplementation in feed with a standard iron injection and assess the impact on the health and productivity of pigs. The reliability of a quick test (HemoCue) for the measurement of blood haemoglobin concentration was also investigated. Three pig herds, with a total of 88 sows and their piglets, were included in the study. Piglets in treatment group O were fed a special iron-rich feed on days 2 to 4, 5 to 7 and 8 to 12 of lactation using a specially designed feeding device. Piglets in group IM received 200 mg of an iron dextran complex intramuscularly at three days of age. Haemoglobin concentrations and performance of the piglets were compared between groups. The mean haemoglobin concentrations in pigs at weaning were 131.4 and 116.4 g/l for pigs in groups O and IM, respectively (P<0.01). Daily weight gain (253.9 v 248.8 g/day) and piglet mortality (11.4 v 12.2 per cent) were slightly better in group O than in group IM (P>0.05).

Effect of organic selenium in the diet on sperm quality of boars.

The effect of a diet supplemented with organic selenium (Se) on sperm production and quality of boars was investigated. Sixty mature boars from a commercial artificial insemination centre were randomly allocated at Day (D) 0 into Group A and B. Group A received the regular ration supplemented with inorganic Se (0.4 mg/kg feed as Na(2) SeO(3)) whereas Group B was switched to the same diet but with organic Se (0.4 mg/kg fed as Se-yeast). The sperm was investigated during 4 months (D0, D30, D60, D75, D90, D105 and D120). Sperm concentration and motility were objectively measured using a photometer and Computer Assisted Semen Analysis (CASA) respectively. Morphology of the sperm was assessed using eosin-nigrosin staining and the resistance to induction of oxidative stress (production of malonaldehyde, MDA) through thiobarbituric acid reagent species analysis. Additionally, the Se concentration in sperm and blood plasma were measured. Repeated measures analysis of variance (anova) from D60 to 120 (spermatogenesis of approximately 2 months) or anova at D120 (Se concentrations) were used for statistical analysis. The total number of ejaculated sperm was not significantly different between both groups, but boars of Group B had a significantly higher sperm concentration (434.6 vs 514.1 × 10(6) sperm/ml; p < 0.05). Small differences (p < 0.05) were observed between both groups for some CASA parameters, namely straight line velocity (µm/s) (Group A: 48.3, Group B: 45.1), straightness (%) (Group A: 65.6, Group B: 62.2) and linearity (%) (Group A: 32.2, Group B: 29.3). The sperm of Group B showed more oxidative stress (4.1 vs 4.9 µmol MDA/l; p < 0.05) compared with those of Group A. No significant differences (p > 0.05) were observed for the other parameters. Under the present study conditions, changing from inorganic Se to organic Se in the diet of boars increased sperm concentration but reduced some motility parameters and resistance to oxidative stress.

Cryotolerance of bovine blastocysts is affected by oocyte maturation in media containing palmitic or stearic acid.

In this study, non-esterified fatty acids (NEFAs) were added during in vitro maturation at concentrations measured previously in follicular fluid (FF) of high-producing dairy cows in a negative energy status to evaluate their subsequent effect on the embryos cryotolerance. Oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of NEFAs dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in synthetic oviduct fluid medium supplemented with 5% FCS. Embryos that had at least reached the blastocyst stage were vitrified by open pulled straw (OPS) vitrification. Addition of palmitic (C16 : 0) or stearic acid (C18 : 0) during oocyte maturation had significant negative effects on embryo cryotolerance, whereas ethanol or oleic acid (C18 : 1) had no effect. These in vitro results suggest that high NEFA concentrations in FF during a period of negative energy balance in high-yielding dairy cows can have carry-over effects on embryo quality.

Prevention of oxidative damage that contributes to the loss of bioenergetic capacity in ageing skin.

Skin ageing is a complex biological process related to a decline in physiological and biochemical performance. A decline in the mitochondrial energy production is a feature of ageing at the cellular level. This is partially attributed to excessive production of reactive oxygen species such as superoxide and hydrogen peroxide in aged individuals. We have investigated the effect of (glyc)oxidative stress on two biochemical targets relevant for the energy metabolism of the skin. First, we showed an age dependent decline in the activity of the hydrogen peroxide detoxifying antioxidant catalase in stratum corneum on a chronically sun-exposed site. Furthermore catalase was sensitive to peroxynitrite-induced in vitro inactivation. Catalase mimetics as well as peroxynitrite scavengers are proposed to maintain hydrogen peroxide detoxification pathways. The second target was creatine kinase, an enzyme that controls the creatine-creatine phosphate shuttle. Creatine kinase lost activity after in vitro glycation by methylglyoxal. This activity loss could be prevented by antiglycation actives. These data suggest that biomolecules involved in energy homeostasis become damaged by different sources of stress. Actives specifically selected for optimal protection against these stress situations will decrease skin vulnerability and prevent the premature loss of skin function.

Tetracycline-resistance in lactose-positive enteric coliforms originating from Belgian fattening pigs: degree of resistance, multiple resistance and risk factors.

Between March and October 2003 a field study was conducted in 50 randomly selected pig herds to assess the degree of tetracycline-resistance in lactose-positive enteric coliforms (LPEC) originating from fattening pigs and to evaluate the combined effects of various husbandry conditions on the development and persistence of antimicrobial-resistant bacteria. Data on housing, management and antimicrobial-drug consumption were collected, as well as faecal samples at three production stages: end of the nursery period (mean age: 72 days), end of the grower period (mean age: 125 days) and end of the finisher period (mean age: 186 days). The degree of tetracycline-resistant LPEC was determined by means of an agar dilution method. Tetracycline-resistant LPEC were found in every herd. The overall degree of tetracycline-resistance in LPEC was 56.8% (S.D. 22.4%). Only a very weak relation was found between the degrees of TETR in the different production stages within the same herd, indicating that the degree of TETR is mainly associated with the production stage rather than with the farm as a whole. The risk factor analysis showed that besides the antimicrobial-drug use, other factors like inside pen hygiene can influence the development and maintenance of antimicrobial-resistant bacteria in the gastrointestinal tracts of pigs. It was also observed that tetracycline-resistance in commensal Escherichia coli is often linked with resistance to other antimicrobial drugs like ampicillin and trimethoprim-sulphonamides. These results illustrate that the epidemiology of antimicrobial resistance is influenced by antimicrobial-drug use, cross-resistance development and non-antimicrobial risk factors.

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa.

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.

Rheumatic manifestations of scurvy. A report of two cases.

Bleeding into the muscles and joints can be the presenting manifestation of scurvy, as illustrated by two case-reports. One patient presented with hemarthrosis of the tibiotalar joint due to an insufficiency fracture and was suspected to have scurvy based on the presence of purpura and hypertrophy of the gums with loss of teeth. In the other patient, multiple hematomas in the lower limbs were found at presentation and the presence of coiled hairs suggested the diagnosis. Both patients had completely eliminated fruit and vegetables from their diet. Low levels of ascorbic acid were found in serum and urine. A full recovery was achieved in both cases under ascorbic acid supplementation.

Crystal structure determination and refinement at 2.3-A resolution of the lentil lectin.

We report on the X-ray structure determination of the orthorhombic crystal form of lentil lectin by molecular replacement using the pea lectin coordinates as a starting model. The structure was refined at 2.3-A resolution with a combination of molecular dynamics refinement and classical restrained least-squares refinement. The final R value for all data Fo > 1 sigma (Fo) between 7.0- and 2.3-A resolution is 0.164%, and deviations from ideal bond distances are 0.014 A. The C-terminus of the beta-chain proved to be 23 amino acids longer than found in previous studies. This together with several inconsistencies between the previously determined amino acid sequence and the observed electron density forced a redetermination of the amino acid sequence of the protein. The overall structure is very similar to that of pea lectin and isolectin I of Lathyrus ochrus, the most prominent deviations being confined to loop regions and the regions of intermolecular contact. The largest difference between the pea and lentil lectin monomers is situated in the loop region of amino acids 73-79 of the beta chain. There are no significant differences between the two crystallographic independent lentil lectin monomers in the asymmetric unit. The model includes 104 well-defined water molecules, of which a significant number have a counterpart in the pea lectin structure. As for the other legume lectins, each lentil lectin monomer contains one calcium ion in a highly conserved environment. On the contrary, the manganese binding sites are distorted with respect to the pea lectin and concanavalin A structures. The Asp beta 121 side chain apparently does not ligate the Mn2+ ion. This difference is consistent in both lentil lectin monomers and agrees with earlier solution studies. Possible implications for oligosaccharide binding are discussed.