Biomedical subjects

W L Hurley

Publications and source records attributed to W L Hurley.

At least 19 recordsLinked to original sources

PubMed2003

Sequence and expression of the FcRn in the porcine mammary gland.

Transport of immunoglobulin G across epithelial cell barriers is thought to occur by a system involving the Fcgamma receptor called the neonatal Fc receptor (FcRn). The FcRn may also play a role in IgG transport in the mammary gland. To determine the presence of FcRn in the porcine mammary gland, biopsies were taken from glands 3 days prepartum and on the day of farrowing. The full length porcine FcRn cDNA sequence was obtained by rapid amplification of cDNA ends and determined to be 1557 base pairs in length that codes for a 359 amino acid peptide. Expression of FcRn mRNA in the porcine mammary gland was determined by reverse transcriptase-PCR and revealed that the mRNA is present prepartum and on the day of farrowing. These results indicate that the FcRn is expressed in porcine mammary tissue and are consistent with the hypothesis that FcRn may have a role in mammary gland immunoglobulin transport during colostrogenesis.

Animals↗

PubMed2002

Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk.

The goal of this study was to determine whether the presence of the bovine alpha-lactalbumin transgene in first-lactation gilts enhances lactational performance and litter growth. Transgenic and sibling nontransgenic gilts were bred to nontransgenic boars. Litters were standardized to 10 piglets within 24 h of farrowing. Milk production was measured by the weigh-suckle-weigh method on d 3, 6, 9, and 12 of lactation. Bovine alpha-lactalbumin was present in the colostrum and milk of transgenic gilts throughout lactation. The expression of the transgene was associated with alterations in composition of mammary secretions, especially in early lactation. Lactose concentrations were greater (P < 0.05) in mammary secretions of transgenic gilts during the first 12 h postpartum compared with controls. In contrast, total solids concentration in mammary secretions from transgenic gilts were lower (P < 0.05) relative to controls during the first 6 h postpartum. Transgenic gilts produced more milk than controls on d 3, 6, and 9 of lactation (P < 0.01). By d 12, differences in milk production between transgenic and control sows were no longer different. Lactose intake by transgenic-reared litters was greater than lactose intake by control-reared litters on d 6 of lactation (P < 0.05). Total solids intake was significantly greater (P < 0.05) by transgenic-reared litters on d 3 and 6 compared to control-reared litters. The day x genotype interaction on litter weight gain after birth was highly significant (P = 0.011), with transgenic-reared litters gaining weight at a greater rate than control-reared piglets. Expression of the transgene was associated with increased milk production in lactating gilts and increased growth of transgenic-reared piglets. Increased lactose synthesis in response to the presence of the transgene may result in increased milk production in early lactation, leading to increased milk component intake by transgenic litters, and ultimately to increased growth of litters reared by first-parity transgenic gilts.

Animals↗

PubMed2001

Cellular uptake of taurine by lactating porcine mammary tissue.

Milk taurine plays a critical role in neonatal development. Taurine uptake in lactating sow mammary tissue has not been characterized previously. The kinetic properties, ion dependence and substrate specificity of taurine uptake were characterized in mammary tissue collected from lactating sows at slaughter. Tissue explants were incubated in an isosmotic physiologic buffer with [3H]taurine tracer to measure taurine uptake. Taurine uptake was dependent upon the presence of extracellular sodium and chloride ions, which is consistent with the co-transport of sodium and chloride with taurine. Uptake was not dependent upon ion exchange mechanisms or upon furosemide-sensitive ion co-transport. Taurine uptake was saturable and exhibited an apparent Km of 20 microM and a V(max) of 386 micromol/kg cell water/30 min. Substrate specificity studies indicated a strong interaction of beta-amino acids with the taurine transport system. Taurine transport in lactating sow mammary tissue is therefore a high affinity, sodium-dependent mechanism specific for beta-amino acids, and is analogous to sodium-dependent taurine uptake in other tissues. The high affinity and high specificity of the taurine uptake system allows for concentration of taurine within the mammary cell and is ultimately responsible for provision of taurine required for neonatal development.

Animals↗

PubMed2001

The regression of unsuckled mammary glands during lactation in sows: the influence of lactation stage, dietary nutrients, and litter size.

During lactation in the sow, mammary glands that are not regularly suckled undergo regression. This study characterizes the regression of unsuckled mammary glands and how that regression is affected by dietary nutrients and litter size. Sixty-nine primiparous sows were fed one of four diets containing combinations of two protein levels (32 or 65 g lysine/d) and two energy levels (12 or 17.5 Mcal ME/d) during lactation. Litter size was adjusted to 10. Sows were killed on d 0, 5, 10, 14, 21, or 28 of lactation. In another experiment, twenty-eight primiparous sows were allotted to have different litter sizes and were killed on d 21 of lactation. The day before slaughter, teat order of each litter was observed. After death, mammary glands were removed and dissected. Skin and extraneous fat pads were removed from the mammary glands and individual glands were separated. Each gland was weighed, cut in half to measure cross-sectional area, and ground for chemical analysis. The amounts of dry tissue, protein, fat, ash, and DNA were measured. Only glands observed to be unsuckled were included in the results. Regression of unsuckled mammary glands occurred rapidly during the first 7 to 10 d of lactation, as indicated by a decline in wet weight, dry weight, protein, fat, DNA, and cross-sectional area. The rate of regression was slowed after the early lactation period. The rate of regression of unsuckled glands was affected by dietary nutrient levels. Dietary energy level affected (P < 0.05) the decline in wet and dry weights, protein, fat and DNA content, and cross-sectional area, whereas dietary protein level affected (P < 0.05) the decline in dry weight and fat content. At d 5 of lactation, the wet weight of unsuckled mammary glands in sows fed the high-energy high-protein diet was 91% greater (P < 0.05) than in sows fed the low-energy low-protein diet. Effects of litter size on size and composition of unsuckled glands were not significant by d 21 of lactation. Unsuckled mammary glands regress rapidly during early lactation, and the rate of regression is affected by dietary nutrient intake.

Animal Feed↗

PubMed2001

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk.

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

Animals↗

PubMed2000

Cellular uptake of valine by lactating porcine mammary tissue.

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.

Amino Acids, Branched-Chain↗

PubMed2000

Lysine uptake by mammary gland tissue from lactating sows.

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.

Animals↗

PubMed2000

Growth of nursing pigs related to the characteristics of nursed mammary glands.

The purpose of this study was to determine growth performance of nursing pigs in relationship to teat order and to observe teat preference by pigs. In the first experiment, litter size of 13 primiparous sows was adjusted to 9 (8.7 +/- 1.5) pigs and teat order of each litter was observed on the day before slaughter. Another group of eight sows was killed on d 0 (within 12 h after farrowing). In the second experiment, litter size was adjusted to 9 (8.9 +/- 1.4) pigs for 20 primiparous sows and teat order for each litter was observed 1 d before slaughter. The weights of sows and individual pigs were recorded at farrowing, weekly, and on the day before slaughter. Mammary glands were collected at slaughter on d 21 of lactation and trimmed of skin and the extraneous fat pad. Individual glands were separated, weighed, and ground for measurement of dry matter, dry fat-free tissue, protein, fat, ash, and DNA contents. Middle mammary glands had the greatest wet weight among glands obtained within 12 h after weaning (P < .05). For sows completing the 21 d lactation, only glands known to have been nursed were included in the data sets. Greater than 60% of the first four pairs of mammary glands were nursed, and less than 40% of the seventh and eighth glands were nursed by pigs during lactation. Pigs that nursed the first five pairs of anterior glands gained faster than pigs nursing the remaining glands. The first five pairs of anterior glands had greater wet and dry weights, and greater protein and DNA contents compared with the remaining glands. Pigs that nursed heavier glands gained weight faster (r = .68, P = .0001), and those heavier glands contained greater amounts of protein (r = .98, P = .0001) and DNA (r = .66, P = .0001). Variation in weight gain of pigs nursing the anterior and middle glands was not statistically significant. The functional superiority of anterior and middle glands was positively correlated with body weight gain of nursing pigs.

Animals↗

PubMed2000

Passive immunoglobin transfer in newborn calves fed colostrum or spray-dried serum protein alone or as a supplement to colostrum of varying quality.

Two experiments were conducted to investigate the effect of serum-derived immunoglobin (Ig) source and the effect of colostrum supplementation with serum-derived Ig on the attainment of passive immunity in newborn colostrum-deprived calves. In experiment 1, colostrum-deprived Holstein bull calves were fed pooled colostrum (PC, n = 9), spray-dried bovine serum (BS, n = 11), or spray- dried porcine serum (PS, n = 9). All treatments were balanced to provide 45 g of IgG in a 2-L volume at birth and again 12 h later. Calves receiving BS had higher 24-h serum IgG concentrations than did calves receiving PC or PS (8.3, 5.7, and 4.2 g of IgG/L for BS, PC, and PS, respectively). In experiment 2, the effect of supplementing bovine colostrum of varying quality with BS on Ig absorption was assessed. Thirty-two colostrum-deprived Holstein bull calves and four freemartin heifer calves were allotted by birth order to receive one of three treatments. Treatments consisted of 1) 2 L of pooled high quality colostrum (95.8 g of IgG, 0% from BS), 2) 2 L of pooled medium quality colostrum mixed with BS (95.2 g of IgG, 47% from BS), or 3) 2 L of low quality colostrum mixed with BS (98.8 g of IgG, 70% from BS). Serum IgG concentrations at 24 h after treatment were greater for calves receiving medium and low quality colostrum supplemented with BS (6.2, 9.6, and 9.6 g of IgG/L for high, medium, and low quality colostrum, respectively). Similarly, apparent efficiency of IgG absorption was greater for calves receiving medium and low quality colostrum supplemented with BS (25, 37, and 38% for high, medium, and low quality colostrum, respectively). The results of these studies suggest that dried BS contains a concentrated source of Ig, which is efficiently absorbed by newborn calves. Supplementation of marginal or low quality colostrum with dried BS is an effective means of improving passive transfer of IgG in newborn calves.

Animals↗

PubMed1999

Use of mammary gland and colostral characteristics for prediction of colostral IgG1 concentration and intramammary infection in Holstein cows.

OBJECTIVE: To determine whether mammary gland or colostral characteristics at calving could be used to predict colostral immunoglobulin G1 (IgG1) concentration or intramammary infection (IMI) and whether leakage of colostrum affects IgG1 concentration. DESIGN: Prospective study. ANIMALS: 113 multiparous Holstein cows. PROCEDURE: Cows were examined within 3 hours of calving, and mammary gland and colostral characteristics, colostral volume, somatic cell count, and concentrations of IgG1, fat, and protein were determined. Bacteriologic culture of mammary secretions was performed approximately 14 and 7 days before calving and at calving. Associations of gland and colostral characteristics with colostral IgG1 concentration, colostral volume, and IMI were examined. RESULTS: Thick or thin colostrum had higher IgG1 concentration than colostrum of intermediate viscosity. Colostrum from mammary glands that were firm had low IgG1 concentration. Colostral IgG1 concentration was weakly correlated with volume. Intramammary infection was likely to be detected if colostrum contained clots or blood or if the California Mastitis Test (CMT) score was > or = 2. Somatic cell count was higher for glands with IMI than for uninfected glands, and CMT score was correlated with cell count. CLINICAL IMPLICATIONS: Mammary gland and colostral characteristics were of little value in predicting IgG1 concentration. Our findings do not support recommendations that first milking colostrum that is thin (watery) or that is from cows producing large volumes not be fed to dairy calves. Colostral characteristics, particularly CMT score, were of value for predicting IMI.

Animals↗

PubMed1999

Effect of nutrient intake on mammary gland growth in lactating sows.

Sixty-one primiparous sows were used to determine the response of mammary gland growth to different energy and protein intakes during lactation. After birth, litter size was set to 9 or 10 pigs. Sows were slaughtered at selected times up to 30 d of lactation. Individual sows were fed one of four diets that were combinations of different amounts of energy and protein (3.0 Mcal ME and 8.0 g lysine/kg diet; 3.0 Mcal ME and 16.2 g lysine/kg diet; 3.5 Mcal ME and 6.4 g lysine/kg diet; or 3.5 Mcal ME and 13.0 g lysine/kg diet). Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, ground, and stored at -20 degrees C for chemical analysis. Frozen, ground tissue was used to determine dry matter, dry fat-free tissue (DFFT), total tissue protein, ash, and DNA content. Only glands known to have been suckled were included in this data set. Response surface regression was used for statistical analysis. The percentage of protein, fat, ash, and DNA in each suckled mammary gland was affected only by total energy intake (P<.05). The percentage of dry tissue and fat decreased as the total energy consumed during lactation increased, whereas the percentage of protein and DFFT increased as total energy intake increased. There were quadratic effects (P<.05) of both total energy and protein intake on wet weight, dry weight, protein amount, DFFT amount, and DNA amount of each suckled mammary gland during lactation. This study shows that mammary gland growth is affected by nutrient intake during lactation. The weight of suckled mammary glands and the amount of mammary tissue protein, DFFT, and total DNA were maximal on d 27.5 of lactation when sows had consumed an average of 16.9 Mcal of ME and 55 g of lysine per day during lactation. Provision of adequate amounts of nutrients to sows during lactation is important for achieving maximal growth of mammary glands and maximal milk production.

Animal Feed↗

PubMed1999

Mammary gland growth as influenced by litter size in lactating sows: impact on lysine requirement.

Twenty-eight primiparous sows were used to determine the effect of litter size on the growth of mammary glands and nursing pigs during lactation. Litter size was set to 6, 7, 8, 9, 10, 11, or 12 pigs by cross-fostering immediately after birth. Four sows were allotted to each litter-size group. Sows were allowed to consume a daily maximum of 13.6 Mcal ME and 46.3 g of lysine during lactation. Sows were slaughtered on d 21 (20.6+/-1.1) of lactation. Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was separated, weighed, and ground for chemical analysis. Dry matter, dry fat-free tissue (DFFT), crude protein, ash, and DNA contents were measured. Only glands known to have been nursed were included in the data set. Wet and dry weights and the amounts of DFFT, protein, DNA, ash, and fat in individual nursed mammary glands linearly decreased (P<.05) as litter size increased. Percentages of DFFT, protein, and DNA were quadratically affected (P<.05) by litter size on d 21 of lactation. Total mammary wet and dry weights and total DFFT, protein, DNA, fat, and ash amount of all nursed mammary glands of each sow were increased as litter size increased (P<.05). Changing litter size from 6 to 12 pigs resulted in 2,098, 432, 253, 227, 4.4, 178, and 20 g increases in the amounts of total mammary wet weight, dry weight, DFFT, protein, DNA, fat, and ash, respectively, on d 21 of lactation. Litter weight gain was 18.1 kg greater in sows with 12 pigs than in sows with 6 pigs. Sows with a larger litter size had a greater increase in total mass of mammary gland tissue and litter weight but had lower growth of individual nursed mammary glands and individual pigs than sows with the smaller litter size. The need for nutrients to support additional mammary gland and litter growth as litter size increases should be considered when estimating nutrient requirements for lactating sows. Sows need an additional .96 g lysine per day to account for mammary gland growth for each pig added to a litter.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Changes in tissue composition associated with mammary gland growth during lactation in sows.

Twenty-four primiparous sows were used to determine the extent of mammary gland growth during lactation. Litter size was set to nine or 10 pigs immediately after birth. Sows were slaughtered in groups representing d 0 (within 12 h after farrowing), 5, 10, 14, 21, and 28 of lactation. Sows were provided 17.5 Mcal ME and 65 g of lysine per day during lactation. Mammary glands were collected at slaughter and trimmed of skin and extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, and ground for chemical analysis. Dry matter content, dry fat-free tissue (DFFT) content, protein content, amino acids composition, ash content, and DNA content were measured. Only glands known to have been suckled were included in these data. Wet and dry tissue weight; cross-sectional area; and the amount of DFFT, tissue protein, and amino acids in each suckled mammary gland increased (P < .05) during lactation to a peak on d 21. Fat percentage of each suckled gland declined (P < .05) and the percentage of protein and DFFT increased (P < .05) as lactation progressed. These results suggest that hypertrophy occurred in the tissue during lactation. There was a linear increase in the amount and percentage of DNA during lactation (P < .05), suggesting hyperplasia of the mammary tissue. Mammary tissue growth continues in suckled glands during lactation in sows, with gland wet weight increased by 55% and total gland DNA increased by 100% between d 5 and 21 of lactation.

Adipose Tissue↗

PubMed1999

Effects of secretion removal on bovine mammary gland function following an extended milk stasis.

The objective of this study was to determine whether lactation function could be reinitiated after a period of extended milk stasis. Involution was induced by milk stasis in lactating Holstein cows for a period of 11 d. On d 11, one side of the mammary gland was milked twice daily for 3 d. The contralateral side remained unmilked for the 14-d experimental period. Cows were slaughtered, and mammary tissue was collected from both udder halves for further analysis. Mammary secretion volume was partially restored in the milked udder half, but reestablished milk yields were variable among cows. A partial recovery of lactation function was further indicated by elevated levels of lactose and protein profiles resembling milk in mammary secretions from the milked glands. Lactose and protein profiles from the unmilked glands were similar to those of glands undergoing involution. Lactoferrin levels were elevated in secretions from the milked and unmilked udder halves. Casein and lactoferrin synthesis by mammary explants and beta-casein and lactoferrin mRNA abundance in mammary tissues corresponded to protein profiles from milked and unmilked mammary secretions. alpha-Lactalbumin mRNA was variable but was more abundant in the milked glands compared with the unmilked glands. Lectin fluorescence microscopy for soybean agglutinin preferentially stained the apical surface of the mammary epithelial cells from the milked glands. Staining was absent in the unmilked glands and suggested resumption of lactation function in all such milked glands. These results suggest that mammary involution can be partially reversible after 11 d of milk stasis.

Animals↗

PubMed1998

Peptides generated from milk proteins in the bovine mammary gland during involution.

Proteolytic activity in mammary gland secretions is associated with hydrolysis of secretory proteins during involution. Peptides generated from hydrolysis of milk proteins were characterized in secretions from the bovine mammary gland during involution to understand the fate of the milk proteins better. Mass spectral analysis of mammary secretions showed numerous peptides ranging between 0.7 and 14 kDa during mammary involution, but these peptides were not observed in normal milk. Mass spectral profiles representing discrete peptide fragments were similar on d 7, 14, and 21 of involution, suggesting that milk proteins were only partially hydrolyzed during involution. N-Terminal amino acid sequences of four peptides indicated that they were produced by hydrolysis of beta-casein during involution and probably resulted from plasmin hydrolysis. A 20-kDa peptide was identified as a fragment of a 39-kDa protein that was previously identified in bovine mammary secretions during involution. Mass spectral analysis of lactoferrin isolated from mammary secretions during involution showed major hydrolytic products. Immunoblot analysis confirmed that lactoferrin that was isolated from mammary secretions during involution contained a number of hydrolytic products. Intramammary hydrolysis of milk proteins by plasmin probably leads to the generation of the discrete peptides observed in the mammary secretions and contributes to the fate of these proteins during involution of the bovine mammary gland.

Amino Acid Sequence↗

PubMed1998

Effects of mastitis on the volume and composition of colostrum produced by Holstein cows.

The effects of mastitis during the late nonlactating period on colostral volume and concentrations and total yields of immunoglobulin (Ig) G1, fat, and protein in colostrum were investigated using matched pairs of mammary glands from multiparous Holstein cows. Samples of mammary secretions were collected at approximately 14 and 7 d prepartum and within 3 h after calving. At each sampling time, the glands and secretions were examined for gross abnormalities, and the California Mastitis Test was performed. Duplicate secretion samples from each gland were cultured, and somatic cell count, pH, and fat and protein concentrations were determined. The volume of colostrum obtained at the first milking of each gland was quantified using a quarter milking device, and its IgG1 concentration was measured. Colostral volume from persistently infected mammary glands was lower than that from matched uninfected glands, as was the total mass of IgG1. However, infection did not alter IgG1 concentration in colostrum. Fat and protein percentages were lower in prepartum secretions but not in colostrum from infected glands. Persistent infection was associated with increased somatic cell count and pH of secretions at all sampling times, and California Mastitis Test scores were higher for colostrum from infected glands. The appearance of secretions was extremely variable, but the presence of flakes or clots in colostrum was associated with infection. We concluded that mastitis during the late nonlactating period alters mammary gland function but is unlikely to be an important contributor to the high rate of failure of passive transfer of immunoglobulins in calves.

Animals↗

PubMed1998

Identification of lactoferrin complexes in bovine mammary secretions during mammary gland involution.

Part of the antimicrobial activity of lactoferrin resides in its ability to bind to bacteria. The complexing of lactoferrin with other proteins could alter its activity. This study identified the presence of lactoferrin complexes in mammary secretions during mammary gland involution and determined the proportion of free and complexed lactoferrin in mammary secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution. Proteins were fractionated from defatted, filtered mammary secretions by sucrose density gradient ultracentrifugation and by gel filtration chromatography. Proteins contained in separated fractions were identified by SDS-PAGE. The presence of lactoferrin was confirmed by immunoblot analysis. Lactoferrin was present as complexed forms of high molecular mass in mammary secretions at each day of involution. The majority of lactoferrin was present in complexes of higher molecular mass rather than as monomers. A majority of lactoferrin existed in fractions of approximately 250 kDa, although peaks of lactoferrin at 150, 300, and 800 kDa were also found. The presence of lactoferrin complexes may result from interactions with casein or immunoglobulins or from the formation of lactoferrin multimers in the secretions. The interaction of lactoferrin with other proteins in mammary secretions during involution may affect the antimicrobial properties of lactoferrin.

Animals↗

PubMed1998

Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis.

OBJECTIVE: To evaluate polymerase chain reaction (PCR) analysis for detection of Staphylococcus aureus (nuc gene) in fresh and formalin-preserved milk. SAMPLE POPULATION: Samples from 80 lactating sheep and 100 lactating dairy cows. PROCEDURE: 4 lactating sheep were inoculated with S aureus by intramammary infusion. A set of primers specific for the nuc gene of S aureus was used to develop a PCR technique, and modification of the rapid boil method was used to isolate bacterial DNA. Milk was obtained from experimentally infected sheep before and after infusion with S aureus, and from the 100 cows and remaining 76 sheep. Samples were screened by bacteriologic culture and PCR. To validate the PCR assay, S aureus or other pathogens were added to distilled water and "normal" sheep milk samples, with and without formalin. RESULTS: The PCR assay was 100% specific for S aureus when known negative and positive samples were tested. Sensitivity was 100% for samples with added S aureus or other pathogens. Sensitivity was lower for samples obtained from experimentally infected sheep, but increased from 53% to 90% with increased washing of target DNA. CONCLUSIONS: The PCR technique based on the nuc gene is able to detect S aureus in sheep milk yields results faster than does traditional culturing, is highly specific, and is able to detect S aureus in formalin-fixed milk samples. CLINICAL RELEVANCE: The assay is particularly suitable for analysis of samples shipped or stored without refrigeration. Although antibiotics in milk may inhibit growth in culture, they should not affect the results of the PCR assay.

Animals↗