Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium ssp. paratuberculosis, milk production, and longevity in Holstein dairy cows.

In summer 2007, a randomized controlled field trial was initiated on 6 large Midwest commercial dairy farms to investigate the effect of feeding heat-treated (HT) colostrum on transmission of Mycobacterium avium ssp. paratuberculosis (MAP) and on future milk production and longevity within the herd. On each farm, colostrum was collected daily from fresh cows, pooled, divided into 2 aliquots, and then 1 aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60min. A sample from each batch of colostrum was collected for PCR testing (MAP-positive vs. MAP-negative). Newborn heifer calves were removed from the dam within 30 to 60min of birth and systematically assigned to be fed 3.8 L of either fresh (FR; n=434) or heat-treated (HT; n=490) colostrum within 2h of birth. After reaching adulthood (>2 yr old), study animals were tested once annually for 3 yr (2010, 2011, 2012) for infection with MAP using serum ELISA and fecal culture. Lactation records describing milk production data and death or culling events were collected during the 3-yr testing period. Multivariable model logistic and linear regression was used to investigate the effect of feeding HT colostrum on risk for testing positive to MAP during the 3-yr testing period (positive/negative; logistic regression) and on first and second lactation milk yield (kg/cow; linear regression), respectively. Cox proportional hazards regression was used to investigate the effect of feeding HT colostrum on risk and time to removal from the herd. Fifteen percent of all study animals were fed PCR-positive colostrum. By the end of the 3-yr testing period, no difference was noted in the proportion of animals testing positive for MAP, with either serum ELISA or fecal culture, when comparing the HT group (10.5%) versus the FR group (8.1%). There was no effect of treatment on first- (HT=11.797kg; FR=11,671kg) or second-lactation (HT=11,013kg; FR=11,235kg) milk production. The proportion of cows leaving the herd by study conclusion was not different for animals originally fed HT (68.0%) versus FR (71.7%) colostrum. Although a previous study showed that feeding HT colostrum (60°C for 60min) produces short-term benefits, including improved passive transfer of IgG and reduced morbidity in the preweaning period, the current study found no benefit of feeding HT colostrum on long-term outcomes including risk for transmission of Mycobacterium avium ssp. paratuberculosis, milk production in the first and second lactation, and longevity within the herd.

Effects of weekly regrouping of prepartum dairy cows on metabolic, health, reproductive, and productive parameters.

The objectives of the current experiment were to determine the effect of 2 prepartum grouping strategies on the health, metabolic, reproductive, and productive parameters of dairy cows. Jersey cows enrolled in the experiment at 253±3 d of gestation (d 0=calving) were balanced for parity and projected 305-d mature equivalent and assigned to 1 of 2 treatments. Cows assigned to the traditional (TRD; n=6 replicates with a total of 308 cows) treatment were moved to the study pen as a group of 44 cows and weekly thereafter groups of 2 to 15 cows were moved to the study pen to reestablish stocking density. Cows assigned to the all-in-all-out (AIAO; n=6 replicates with a total of 259 cows) treatment were moved to the study pen in groups of 44 cows, but no new cows entered the AIAO pen until the end of the replicate. At the end of each replicate, a new TRD and AIAO group started but pens were switched. Cows were milked thrice daily and monthly milk yield, fat and protein contents, and somatic cell count data were recorded up to 305 d postpartum. Plasma nonesterified fatty acid concentration was measured weekly from d -18±3 to 24±3 and plasma ß-hydroxybutyrate was measured weekly from d 3±3 to 24±3. Cows were examined on d 1, 4±1, 7±1, 10±1, and 13±1 for diagnosis of uterine diseases and had their ovaries scanned by ultrasound on d 39±3 and 53±3 to determine resumption of ovarian cycles. Average stocking density was reduced for the AIAO (71.9%) treatment compared with the TRD (86.9%) treatment. Treatment did not affect the incidences of retained fetal membranes (TRD=10.9, AIAO=11.6%), metritis (TRD=16.7, AIAO=19.8%), and acute metritis (TRD=1.7, AIAO=3.6%). Concentrations of nonesterified fatty acids (TRD=80.4±8.2, AIAO=62.9±8.5 µmol/L) and ß-hydroxybutyrate (TRD=454.4±10.9, AIAO=446.1±11.1 µmol/L) were not different between treatments. Percentages of cows that resumed ovarian cycles by d 39±3 (TRD=70.8, AIAO=63.1%) and 53±3 (TRD=90.1, AIAO=90.2%) were not different between treatments. Similarly, treatment had no effect on rate of removal from the herd {TRD=referent, AIAO [(adjusted hazard ratio (95% confidence interval)]=0.85 (0.63, 1.15)} or rate of pregnancy [TRD=referent, AIAO=1.07 (0.88, 1.30)]. Finally, treatment did not affect energy-corrected milk yield (TRD=34.4±0.6, AIAO=34.3±0.7 kg/d). In conditions of adequate feed bunk space, the AIAO treatment did not improve health, metabolic, reproductive, or productive parameters compared with the TRD treatment.

Heat treatment of colostrum on commercial dairy farms decreases colostrum microbial counts while maintaining colostrum immunoglobulin G concentrations.

This study was conducted on 6 commercial dairy farms in Minnesota and Wisconsin to describe the effect of heat treatment (at 60°C for 60 min) on colostrum, on colostrum bacteria counts, and immunoglobulin G concentrations. First-milking colostrum was collected each day, pooled, divided into 2 aliquots, and then 1 aliquot was heat treated in a commercial batch pasteurizer at 60°C for 60 min. Frozen samples of pre- and post- heat-treated colostrum were submitted for standard microbial culture (total plate count and total coliform count, cfu/mL) and testing for immunoglobulin G concentrations (mg/mL). Data were analyzed from 266 unique batches of colostrum. Linear regression showed that heat treatment decreased colostrum total plate counts (-2.25 log(10)) and coliform counts (-2.49 log(10)), but, overall, did not affect colostrum IgG concentration. Though higher-quality batches of colostrum did experience a greater magnitude of loss of IgG as a result of heat treatment as compared with lower- or intermediate-quality batches of colostrum, the colostral IgG concentrations in these batches remained high overall, and within acceptable limits for feeding. This study demonstrates that batch heat treatment of colostrum at 60°C for 60 min can be successfully conducted on commercial dairy farms by farm staff to decrease colostrum microbial counts while maintaining colostrum IgG concentrations.

Heat-treated colostrum and reduced morbidity in preweaned dairy calves: results of a randomized trial and examination of mechanisms of effectiveness.

A randomized controlled clinical trial was conducted using 1,071 newborn calves from 6 commercial dairy farms in Minnesota and Wisconsin, with the primary objective being to describe the effects of feeding heat-treated colostrum on serum immunoglobulin G concentration and health in the preweaning period. A secondary objective was to complete a path analysis to identify intermediate factors that may explain how feeding heat-treated colostrum reduced the risk for illness. On each farm, colostrum was collected each day, pooled, and divided into 2 aliquots; then, one aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60 min. Samples of fresh and heat-treated colostrum were collected for standard microbial culture (total plate count and total coliform count, cfu/mL) and for measurement of immunoglobulin G concentrations (mg/mL). Newborn calves were removed from the dam, generally within 30 to 60 min of birth, and systematically assigned to be fed 3.8L of either fresh (FR, n=518) or heat-treated colostrum (HT, n=553) within 2h of birth. Venous blood samples were collected from calves between 1 and 7d of age for measurement of serum IgG concentrations (mg/mL). All treatment and mortality events were recorded by farm staff between birth and weaning. Regression models found that serum IgG concentrations were significantly higher in calves fed HT colostrum (18.0 ± 1.5 mg/mL) compared with calves fed FR colostrum (15.4 ± 1.5 mg/ml). Survival analysis using Cox proportional hazards regression indicated a significant increase in risk for a treatment event (any cause) in calves fed FR colostrum (36.5%, hazard ratio=1.25) compared with calves fed HT colostrum (30.9%). In addition, we observed a significant increase in risk for treatment for scours in calves fed FR colostrum (20.7%, hazard ratio=1.32) compared with calves fed HT colostrum (16.5%). Path analysis suggested that calves fed HT colostrum were at lower risk for illness because the heat-treatment process caused a significant reduction in colostrum total coliform count, which was associated with a reduced risk for illness as a function of improved serum IgG concentrations.

Animal welfare in cross-ventilated, compost-bedded pack, and naturally ventilated dairy barns in the upper Midwest.

The objective of this cohort study was to investigate animal welfare in 2 newer dairy housing options in the upper Midwest, cross-ventilated freestall barns (CV) and compost-bedded-pack barns (CB), compared with conventional, naturally ventilated freestall barns (NV). The study was conducted on 18 commercial dairy farms, 6 of each housing type, in Minnesota and eastern South Dakota. The primary breed in all farms was Holstein; 1 CV and 1 NV herd had approximately 30% Jersey-Holstein crossbreds. All freestall herds used sand for bedding. Farms were visited 4 times (once in each season) between January and November 2008, and approximately 93% of all animals in each pen were visually scored on each visit. Outcome-based measurements of welfare (locomotion, hock lesions, body condition score, hygiene, respiration rates, mortality, and mastitis prevalence) were collected on each farm. Lameness prevalence (proportion of cows with locomotion score ≥3 on a 1 to 5 scale, where 1=normal and 5=severely lame) in CB barns (4.4%) was lower than that in NV (15.9%) and CV (13.1%) barns. Lameness prevalence was similar between CV and NV barns. Hock lesion prevalence (proportion of cows with a lesion score ≥2 on a 1 to 3 scale, where 1=normal, 2=hair loss, and 3=swelling) was lower in CB barns (3.8%) than in CV (31.2%) and NV barns (23.9%). Hygiene scores (1 to 5 scale, where 1=clean and 5=very dirty) were higher for CB (3.18) than CV (2.83) and NV (2.77) barns, with no differences between CV and NV barns. Body condition scores, respiration rates, mastitis prevalence, culling, and mortality rates did not differ among housing systems. The CV and NV barns were evaluated using the cow comfort index (proportion of cows lying down in a stall divided by all animals touching a stall) and the stall usage index (proportion of cows lying divided by all animals in the pen not eating). The CV barns tended to have greater cow comfort index (85.9%) than the NV barns (81.4%) and had greater stall usage index (76.8% and 71.5%, respectively). Dairy cattle housed in CB barns had reduced lameness and hock lesions compared with those housed in freestall barns and had no adverse associations with body condition, respiration rates, mastitis prevalence, culling, or mortality. When comparing the 2 freestall housing options, CV barns had improved cow comfort indices compared with NV barns. Although cows in CB barns had better feet and leg health, as indicated by the reduced lameness and hock lesion prevalence, acquiring bedding and managing the bedded pack could limit their use.

Sex-sorted semen for dairy heifers: effects on reproductive and lactational performances.

The objectives of this study were to evaluate the effects of using sex-sorted semen for first AI of heifers on health and productivity during first lactation. Holstein heifers (herd A=227 and herd B=1,144) received first artificial insemination (AI) with sex-sorted semen (SX; n=343) or conventional semen (CS; n=1,028), and all heifers that displayed estrus after first AI were reinseminated with conventional semen up to 11 times before being culled. Age at first AI was 13.1+/-0.1 and 13.8+/-0.1 mo for SX and CS heifers, respectively, in herd A and 12.9+/-0.1 mo for both SX and CS heifers in herd B. Pregnancy per AI after first AI was greater for CS heifers than for SX heifers (51.8 vs. 40.2%). From heifers initially enrolled, 70.2% calved in herds A (n=188) or B (n=774) and first-lactation data were collected. Interval from first AI to calving was greater for SX heifers than for CS heifers (10.2+/-0.1 vs. 9.9+/-0.1 mo). Among heifers conceiving to first AI, SX heifers were more likely than CS heifers to deliver a female calf (85.7 vs. 47.7%), but because SX heifers were more likely to deliver a dead calf (8.8 vs. 3.4%), the difference in proportion of SX and CS heifers delivering a live female calf was smaller than expected (SX=79.1%; CS=47.2%). Rearing cost from first AI to calving was greater for SX heifers than for CS heifers ($775.3+/-6.7 vs. $750.0+/-5.9), but calf revenue tended to be greater for SX heifers ($142.0+/-7.2 vs. $126.7+/-6.4) and cost per female calf produced was smaller for SX heifers than for CS heifers ($-809.4+/-10.8 vs. $-1,249.7+/-10.9). Treatment did not affect calving difficulty, proportion of heifers needing assistance, and incidence of retained fetal membranes or metritis. Among heifers that conceived to first AI, however, SX heifers were more likely to be culled within 30 DIM (3.3 vs. 1.6%) and tended to be more likely to be culled within 60 DIM (5.5 vs. 3.4%) than CS heifers, but overall replacement cost was not different ($136.8+/-13.4). Total milk yield (9,245.5+/-84.7 kg) and income over feed cost ($554.7+/-5.1) were not different. Overall economic return was greater for SX heifers than CS heifers ($-83.7+/-36.7 vs. -175.3+/-33.4). Use of sex-sorted semen for first insemination of virgin heifers reduced the cost per female calf produced and increased the economic return during the first lactation.

Loss of income from cows shedding Mycobacterium avium subspecies paratuberculosis prior to calving compared with cows not shedding the organism on two Minnesota dairy farms.

Quantification of the financial effect of Mycobacterium paratuberculosis infection on lactation performance is essential to encourage participation of dairy cattle producers in Johne's disease (JD) control programs. The objective of this study was to evaluate the differences in net income per lactation of cows shedding Mycobacterium paratuberculosis before calving compared with test-negative cows. Two Minnesota dairies were enrolled in the study and fecal samples were collected from 1,048 cows during the close-up period. Milk production, clinical diseases (other than clinical JD), and reproductive performance data were recorded for each cow. Overall, fecal-culture-positive (FCP) cows produced 1,355 kg less than fecal-culture-negative (FCN) cows. Fecal-culture-positive cows that survived their current lactation produced $276 less in milk income than cows that were FCN ($1,956 vs. $1,680; SD $526, $570). Fecal-culture-positive cows were 3.0 (95% confidence interval: 1.6-5.8) times more likely to be culled than FCN cows. The mean days open (number of days from calving to conception) was not statistically significant and the cost differences for clinical disease other than JD were small and neither statistically nor economically significant between FCP and FCN cows. Among all FCP cows, income over feed costs losses were $366 per cow per lactation compared with FCN cows. Among FCP nonculled cows, income over feed costs losses were $276 more compared with FCN cows and this difference was statistically significant. There was a total loss of $155 per lactation for nonculled FCP cows retained in the herd compared with FCN cows retained in the herd. Among culled cows, FCP cow losses were $50 less because of age at culling and $120 for reduced beef value. This totaled a loss of $441 for culled FCP cows compared with culled FCN cows. The losses as a result of lower lactation performance and early culling from the herd should alarm dairy producers and motivate them to implement the appropriate control measures for the disease.

Exploring the impact of sexed semen on the structure of the dairy industry.

Widespread commercial application of sexed semen is expected within the next decade because of continued improvements in fertility of sexed semen and sorting capacity. The objective of this study was to explore the potential impact of widespread application of sexed semen on the structure of the dairy industry in the United States. Historically, female offspring from all heifers and cows were needed to produce enough dairy replacement heifers to replace culled cows. The use of sexed semen allows for a decoupling of breeding decisions necessary to obtain an adequate supply of dairy replacement heifers from those needed to achieve pregnancies needed to start new lactations. Application of sexed semen allows dairy producers to select among their herds' potential dams and produce dairy replacement heifers from only the genetically superior animals. The rate of genetic progress is expected to increase, but not more than 15% of the rate of gain accomplished through sire selection achieved through conventional (nonsexed) artificial insemination breeding. The supply of dairy replacement heifers is expected to grow to meet and temporarily exceed current demand, resulting in reduced prices for dairy replacement heifers. Consequently, herd turnover rates are expected to increase slightly, and herd expansions may accelerate. The rate of consolidation of dairy farms is expected to increase. Widespread application of sexed semen may temporarily increase the supply of milk, which would result in lower milk prices. The cost of milk production will be reduced as well. Many breeding options exist for the genetically poorer cows in the herd. The optimal breeding mix depends on the value of the various kinds of calves that could be produced. More crossbred calves for beef production may be produced; however, a market for these crossbred calves is not well established. Increased specialization is expected with more dairy producers deciding not to raise their own heifers but to purchase replacements. Other dairy farms might specialize in producing genetically superior dairy replacement heifers for sale. Depending on the value of calves not raised for replacements, artificial insemination organizations might market beef conventional semen or beef male sexed semen to dairy farms. The use of sexed semen should lower the cost of progeny-testing programs and embryo transfer and enhance the value of genetic markers. Eventually, the economic benefits from the use of sexed semen will be passed on to consumers.

Structural genomics and its importance for gene function analysis.

Structural genomics projects aim to solve the experimental structures of all possible protein folds. Such projects entail a conceptual shift from traditional structural biology in which structural information is obtained on known proteins to one in which the structure of a protein is determined first and the function assigned only later. Whereas the goal of converting protein structure into function can be accomplished by traditional sequence motif-based approaches, recent studies have shown that assignment of a protein's biochemical function can also be achieved by scanning its structure for a match to the geometry and chemical identity of a known active site. Importantly, this approach can use low-resolution structures provided by contemporary structure prediction methods. When applied to genomes, structural information (either experimental or predicted) is likely to play an important role in high-throughput function assignment.

Passive transfer of immunoglobulin G and preweaning health in Holstein calves fed a commercial colostrum replacer.

The objective of this study was to describe passive transfer of IgG and preweaning health in newborn calves fed a commercially available plasma-derived colostrum replacement (CR) product or maternal colostrum (MC). Twelve commercial Holstein dairy farms enrolled singleton newborn heifer calves to be fed fresh MC (n = 239 calves) or one dose of CR containing 125 g of Ig (n = 218 calves) as the first colostrum feeding. For 7 of these farms that routinely provided a second feeding of 1.9 L of MC to their calves 8 to 12 h after the first colostrum feeding, calves assigned to the CR treatment group were offered a second feeding consisting of 1.9 L of commercial milk replacer supplemented with one dose of a commercially available plasma-derived colostrum supplement, containing 45 g of Ig per dose, 8 to 12 h after the first colostrum feeding. A blood sample was collected from all calves between 1 to 8 d of age for serum IgG and total protein (TP) determination, and records of all treatment and mortality events were collected until weaning. Serum IgG and TP concentrations were significantly higher in calves fed MC (IgG = 14.8 +/- 7.0 mg/mL; TP = 5.5 +/- 0.7 g/dL) compared with calves fed CR (IgG = 5.8 +/- 3.2 mg/mL; TP = 4.6 +/- 0.5 g/dL). The proportion of calves with failure of passive transfer (serum IgG <10.0 mg/mL) was 28.0 and 93.1% in the MC and CR treatment groups, respectively. Though a trend was present, the proportion of calves treated for illness was not statistically different for calves fed MC (51.9%) vs. CR (59.6%). Total number of days treated per calf (MC = 1.7; CR = 2.0), treatment costs per calf (MC = $10.84; CR = $11.88), and proportion of calves dying (MC = 10.0%; CR = 12.4%) was not different between the 2 colostrum treatment groups. The mean serum total protein concentration predictive of successful passive transfer (serum IgG = 10 mg/mL) was 5.0 g/dL in calves fed MC or CR. Long-term follow-up of these calves (to maturity) is ongoing to describe the effects of feeding CR on longevity, productivity, risk for Johne's disease, and economics.

Evaluation of the Dairy Comp 305 module "Cow Value" in two Ontario dairy herds.

The study was conducted to evaluate how the "Cow Value" module of Dairy Comp 305 (Valley Agricultural Software, Tulare, CA) performed under commercial conditions. The "Cow Value" module, COWVAL, computes a farm-specific net present value relative to an average replacement heifer for each cow in the milking and dry herd, which allows a ranking of the cows on the farm compared with replacing her with a typical replacement heifer on that farm. The average replacement heifer is used as the baseline for comparison and has a COWVAL of $0. Retaining a cow with a negative COWVAL is projected to be less profitable than replacing that cow with a new heifer. The objectives of the study were to explore trends in COWVAL over and during multiple lactations for the same cows; to describe factors that influence changes in COWVAL from one monthly Dairy Herd Improvement test to the next; and to evaluate the behavior of COWVAL after it drops below a baseline of $0 during the lifetime of a cow. Monthly Dairy Comp 305 backup cow files from 2 On-tario dairy herds between December 1999 and Decem-ber 2005 were used to generate COWVAL and list production, reproduction, and disease data for the milking cows. In total, 1,463 cows and 20,071 tests were analyzed. Within the first 60 d in milk (DIM), COWVAL was unstable and showed large fluctuations over a range of several thousand Canadian dollars (Can$). After 60 DIM COWVAL was relatively stable. The variability from month to month became less as the lactation progressed and the risk of a change in reproductive status decreased. The reproductive status of the cow influ-enced COWVAL: fresh, open, and pregnant cows had a greater COWVAL than cows declared "do not breed." As parity increased, there was a tendency toward lower COWVAL and smaller monthly changes in COWVAL. The COWVAL of 170 cows dropped below the baseline of $0 after 60 DIM. The COWVAL of 54% of those cows remained below $0, whereas 31.6% had a subsequent COWVAL > $500 (Can$). Farm management should not rely exclusively on COWVAL for culling decisions, particularly for cows that have not had at least 3 milk tests.

Invited review: Culling: nomenclature, definitions, and recommendations.

Replacing cows on a dairy is a major cost of operation. There is a need for the industry to adopt a more standardized approach to reporting the rate at which cows exit from the dairy, and to reporting the reasons why cows are replaced and their destination as they exit the dairy. Herd turnover rate is recommended as the preferred term for characterizing the cows exiting a dairy, in preference to herd replacement rate, culling rate, or percent exiting, all of which have served as synonyms. Herd turnover rate should be calculated as the number of cows that exit in a defined period divided by the animal time at risk for the population being characterized. The terms voluntary and involuntary culling suffer from problems of definition and their use should be discouraged. Destination should be recorded for all cows that exit the dairy and opportunities to record one or more reasons for exiting should be provided by management systems. Comparing reported reasons between dairies requires considerable caution because of differences in case definitions and recording methods. Relying upon culling records to monitor disease has been and will always be an ineffective management strategy. Dairies are encouraged to record and monitor disease events and reproductive performance and use this information as the basis for management efforts aimed at reducing the need to replace cows.

Heat-treatment of bovine colostrum. II: effects of heating duration on pathogen viability and immunoglobulin G.

Batches (30-L) of first-milking bovine colostrum, inoculated with Mycoplasma bovis (10(8) cfu/mL), Listeria monocytogenes (10(6) cfu/mL), Escherichia coli O157:H7 (10(6) cfu/mL), Salmonella enteritidis (10(6) cfu/mL), and Mycobacterium avium subsp. paratuberculosis (Map; 10(3) cfu/mL), were heat-treated at 60 degrees C for 120 min in a commercial on-farm batch pasteurizer system. Duplicate 50-mL subsamples of colostrum were collected at 15-min intervals throughout the heat-treatment process for the purpose of bacterial culture and for measurement of IgG concentration (mg/mL) and antibody activity [log2(bovine viral diarrhea virus type 1 serum neutralization titer)]. Four replicate batches of colostrum were run for each of the 5 pathogens studied. There was no effect of heating moderate- to high-quality colostrum at 60 degrees C for at least 120 min on mean IgG concentration (pre = 60.5 mg/mL; post = 59.1 mg/mL). Similarly, there was no effect of heat-treatment on the mean log2 bovine viral diarrhea virus type 1 serum neutralization titer (pre = 12.3; post = 12.0). Viable M. bovis, L. monocytogenes, E. coli O157:H7, and S. enteritidis added to colostrum could not be detected after the colostrum was heat-treated at 60 degrees C for 30 min. Average bacteria counts showed that Map was not detected when batches were heated at 60 degrees C for 60 min. Although the authors believe that heat-treating colostrum at 60 degrees C for 60 min should be sufficient to eliminate Map from colostrum in most situations, further research is needed to determine whether these findings may be replicated, given that variability was observed in Map culture results.

Heat treatment of bovine colostrum. I: effects of temperature on viscosity and immunoglobulin G level.

The objective of this study was to identify the critical temperature, at or below which heat-treatment of bovine colostrum would produce no significant changes in viscosity, IgG concentration, or Ig activity. Results of preliminary work, using a Rapid Visco Analyzer (RVA) to heat 50-mL aliquots from 6 unique batches of bovine colostrum at 59, 60, 61, 62, and 63 degrees C, suggested that colostrum could be heated to 60 degrees C for up to 120 min without changing viscosity or IgG concentration. This finding was confirmed by heating 50-mL aliquots from 30 unique batches of colostrum in an RVA for 120 min at 60 and 63 degrees C. Heating colostrum to 63 degrees C resulted in an estimated 34% decrease in IgG concentration and 33% increase in viscosity. However, there was no difference in IgG concentration between preheat-treated (73.4 +/- 26.5 mg/mL) and post-heat-treated (74.5 +/- 24.3 mg/mL) samples after heating colostrum to 60 degrees C in an RVA for 120 min. Similarly, viscosity was unaffected after heating colostrum to 60 degrees C in an RVA for 120 min. High quality colostrum (> or =73.0 mg/mL) suffered greater losses of IgG and greater viscosity changes when heated to 63 degrees C than did moderate quality colostrum (<73.0 mg/mL). However, the effects of colostrum quality were minor if high quality colostrum was only heated to 60 degrees C. The results of a bovine viral diarrhea serum neutralization assay suggested that antibody activity was unchanged after heating colostrum to either 60 or 63 degrees C. However, these results were interpreted as being inconclusive due to a high proportion of missing results because of the congealing of many samples after heat treatment. The results of this study indicate that 50-mL volumes of bovine colostrum can be heat treated at 60 degrees C for up to 120 min in an RVA without affecting IgG concentration or viscosity.

Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases.

The practical exploitation of the vast numbers of sequences in the genome sequence databases is crucially dependent on the ability to identify the function of each sequence. Unfortunately, current methods, including global sequence alignment and local sequence motif identification, are limited by the extent of sequence similarity between sequences of unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence identity. To address this problem, a novel method for identification of protein function based directly on the sequence-to-structure-to-function paradigm is described. Descriptors of protein active sites, termed "fuzzy functional forms" or FFFs, are created based on the geometry and conformation of the active site. By way of illustration, the active sites responsible for the disulfide oxidoreductase activity of the glutaredoxin/thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented. First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the specified activity. Next, these FFFs are used to screen for active sites in low-to-moderate resolution models produced by ab initio folding or threading prediction algorithms. Again, the FFFs can specifically identify the functional sites of these proteins from their predicted structures. The results demonstrate that low-to-moderate resolution models as produced by state-of-the-art tertiary structure prediction algorithms are sufficient to identify protein active sites. Prediction of a novel function for the gamma subunit of a yeast glycosyl transferase and prediction of the function of two hypothetical yeast proteins whose models were produced via threading are presented. This work suggests a means for the large-scale functional screening of genomic sequence databases based on the prediction of structure from sequence, then on the identification of functional active sites in the predicted structure.

Functional analysis of the Escherichia coli genome using the sequence-to-structure-to-function paradigm: identification of proteins exhibiting the glutaredoxin/thioredoxin disulfide oxidoreductase activity.

The application of an automated method for the screening of protein activity based on the sequence-to-structure-to-function paradigm is presented for the complete Escherichia coli genome. First, the structure of the protein is identified from its sequence using a threading algorithm, which aligns the sequences to the best matching structure in a structural database and extends sequence analysis well beyond the limits of local sequence identity. Then, the active site is identified in the resulting sequence-to-structure alignment using a "fuzzy functional form" (FFF), a three-dimensional descriptor of the active site of a protein. Here, this sequence-to-structure-to-function concept is applied to analysis of the complete E. coli genome, i.e. all E. coli open reading frames (ORFs) are screened for the thiol-disulfide oxidoreductase activity of the glutaredoxin/thioredoxin protein family. We show that the method can identify the active sites in ten sequences that are known to or proposed to exhibit this activity. Furthermore, oxidoreductase activity is predicted in two other sequences that have not been identified previously. This method distinguishes protein pairs with similar active sites from proteins pairs that are just topological cousins, i.e. those having similar global folds, but not necessarily similar active sites. Thus, this method provides a novel approach for extraction of active site and functional information based on three-dimensional structures, rather than simple sequence analysis. Prediction of protein activity is fully automated and easily extendible to new functions. Finally, it is demonstrated here that the method can be applied to complete genome database analysis.

Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the complexes in the synapse and in protecting them from proteolysis during prolonged T-cell engagement.

Preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum.

The objectives of this study were to identify control points for bacterial contamination of bovine colostrum during the harvesting and feeding processes, and to describe the effects of refrigeration and use of potassium sorbate preservative on bacteria counts in stored fresh colostrum. For objective 1, first-milking colostrum samples were collected aseptically directly from the mammary glands of 39 cows, from the milking bucket, and from the esophageal feeder tube. For objective 2, 15-mL aliquots of colostrum were collected from the milking bucket and allocated to 1 of 4 treatment groups: 1) refrigeration, 2) ambient temperature, 3) refrigeration with potassium sorbate preservative, and 4) ambient temperature with potassium sorbate preservative. Subsamples from each treatment group were collected after 24, 48, and 96 h of storage. All samples underwent bacteriological culture for total plate count and coliform count. Bacteria counts were generally low or zero in colostrum collected directly from the gland [mean (SD) log10 cfu/mL(udder) = 1.44 (1.45)]. However, significant bacterial contamination occurred during the harvest process [mean (SD) log10 cfu/mL(bucket) = 4.99 (1.95)]. No additional bacterial contamination occurred between the bucket and the esophageal feeder tube. Storing colostrum at warm ambient temperatures resulted in the most rapid increase in bacteria counts, followed by intermediate rates of growth in nonpreserved refrigerated samples or preserved samples stored at ambient temperature. The most effective treatment studied was the use of potassium sorbate preservative in refrigerated samples, for which total plate count and total coliform counts dropped significantly and then remained constant during the 96-h storage period.

From genes to protein structure and function: novel applications of computational approaches in the genomic era.

The genome-sequencing projects are providing a detailed 'parts list' of life. A key to comprehending this list is understanding the function of each gene and each protein at various levels. Sequence-based methods for function prediction are inadequate because of the multifunctional nature of proteins. However, just knowing the structure of the protein is also insufficient for prediction of multiple functional sites. Structural descriptors for protein functional sites are crucial for unlocking the secrets in both the sequence and structural-genomics projects.

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.