Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk. (65/786)

Mycobacterium paratuberculosis is a recognized chronic enteric pathogen that can affect many different species of animals, including primates. It has been suggested that this organism is associated with Crohn's disease in humans, and that milk is a potential source of human exposure to this organism. The limit of the detection of M. paratuberculosis in milk samples by direct PCR was 10(5) cfu/mL if the traditional boiling method was used for template DNA preparation. In this study, an improved method for template DNA preparation was examined. The method involves the use of a bead beater, which breaks up bacterial cell wall mechanically by vibrating bacteria with microbeads at high speed. The effectiveness of this method for lysing M. paratuberculosis cells was compared to that of the freeze-thaw method, and use of commercial kits such as the InstaGene Matrix and the QIAamp Tissue Kit. The bead beater procedure was tested in combination with various cell lysis and template DNA preparation procedures to determine which of these steps improved the limit of detection of PCR assay that amplifies a 413 bp fragment of the IS900 gene. Results showed that the use of the bead beater, in combination with the use of lysis buffer, boiling, and isopropanol precipitation, decreased the limit of detection of M. paratuberculosis in milk by the PCR to 10(2) cfu/mL. The limit of detection was further decreased to 10 cfu/mL when 0.0037% bovine serum albumin was included in the PCR reaction mixtures. The improved assay was 10- to 10(4)-fold more sensitive than the PCR assays using template DNA prepared by other lysis procedures including boiling alone, freeze-thaw plus boiling, or use of commercial kits for lysis.  (+info)

Effect of milk sampling techniques on milk composition, bacterial contamination, viability and functions of resident cells in milk. (66/786)

Three different milk sampling techniques were evaluated during milk sampling: a direct aseptic collection from the udder through a sterile cannula was used as the reference technique, compared with either a manual or a mechanical sampling method. In this study 30 high-yielding Holstein-Friesian dairy cows at different stages of lactation and free of udder infection were used. For each milk sample, the influence of milk sampling techniques was determined for the following parameters: somatic cell count, milk composition, bacterial contamination, viability, in vitro phagocytosis and overall killing of Staphylococcus aureus Newbould 305, and cellular chemiluminescence. Because milk sampling occurred throughout lactation, the differences between early, mid- and late lactation were estimated. It was concluded that bacterial contamination was not significantly different in manual milking samples and the reference technique; bacterial contamination was, however, significantly (P < 0.001) higher in machine milking samples than in the reference technique. Among the different sampling techniques, no significant effects on SCC, milk composition, viability and functions of the cells isolated from milk were observed. It was found that viability, intracellular killing and cellular chemiluminescence of milk PMN were significantly lower (P < 0.05) in early lactation compared to mid-lactation. Phagocytosis was significantly (P < 0.05) higher in early lactation compared to mid- and late lactation, and no significant differences were observed between mid- and late lactation. From this study, it can be concluded that despite a higher bacterial contamination obtained with the mechanical sampling method, the 3 milk sampling techniques described in this study can be used for the evaluation of milk cell functions.  (+info)

The effects of cognitive behavioral intervention on the attitude and behavior of stockpersons and the behavior and productivity of commercial dairy cows. (67/786)

Two experiments, one involving 29 commercial farms and the other involving 94 commercial farms, were conducted to examine the effects of a training program targeting a number of attitudinal and behavioral variables in stockpeople. These stockperson variables had been previously shown to be related to fear of humans and productivity of commercial cows. In both experiments, two treatments were imposed: an Intervention treatment, consisting of a cognitive-behavioral intervention procedure designed to improve the attitude and behavior of stockpeople toward cows, and a Control treatment, in which no intervention was attempted. In Exp. 1, an analysis of covariance, using previous lactation variables as the covariate, revealed effects of the Intervention treatment on the attitude and behavior of the stockpeople and the behavior of cows. Stockpeople at Intervention farms showed more (P < 0.01) positive beliefs about handling cows and used a lower (P < 0.05) number and percentage of negative tactile interactions in handling cows than stockpeople at the Control farms. Cows at the Intervention farms showed a shorter (P < 0.05) flight distance to humans, indicating a lower level of fear of humans by these cows. However, 36% of the Intervention farms failed to show a reduction in average flight distance over the two lactations. Although there was no significant treatment effect on milk yield, the Intervention farms in which fear levels declined following the intervention had a higher (P < 0.05) milk yield than the other farms. In Exp. 2, a significant (P < 0.05) increase was found in the milk yield of cows following the Intervention treatment. Similar treatment effects were observed on both milk protein and milk fat. These results indicate that cognitive-behavioral interventions that successfully target the key attitudes and behavior of stockpeople that regulate the cow's fear of humans offer the industry good opportunities to improve the productivity of cows.  (+info)

A dynamic model of N metabolism in the lactating dairy cow and an assessment of impact of N excretion on the environment. (68/786)

Improving N utilization in dairy cows and especially reducing N output in excreta is desirable due to global concerns of agricultural contribution of N to environmental pollution, particularly as ammonia. Data from five N balance experiments were used to develop a dynamic model that was evaluated with independent data. Model predictions of feces, urine, and milk outputs were close to observed values. Statistical analysis showed that 96% of mean square prediction error for feces and urine N output predictions was due to random variation. However, the model tends to overpredict milk N output, especially at higher N intake levels. Evaluation of model predictions for independent experimental observations from Agricultural Development Advisory Service at Bridgets (U.K.) showed good agreement between predicted and observed urine N output (95% due to random variation). However, there was a slight underprediction for fecal N output (14% mean square prediction error due to bias) and overprediction of milk N output (22% of mean square prediction error due to bias). The model predictions of N outputs in excreta were sensitive to changes in energy concentration of the diet. Dietary protein degradability had only a small influence on predicted fecal N output. However, the model was sensitive in its predictions of urine N when protein degradability was varied. Application of the model to assess reduction in ammonia emissions from dairy cows showed that increasing the energy concentration could potentially reduce ammonia emissions by up to 25% per cow. Similarly, reducing CP concentration in the diet to about 16% could reduce ammonia production by 20% and lower degradability of CP to match microbial requirement by 19% per cow. The model is a first step toward a mechanistic approach of nutrient modeling, and it is a valuable method for predicting N excretions and estimating N emissions from dairy systems.  (+info)

Interpretation of the gamma interferon test for diagnosis of subclinical paratuberculosis in cattle. (69/786)

A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-gamma) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-gamma responses were significantly reduced (P < 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-gamma test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.  (+info)

Shedding of Escherichia coli O157:H7 in dairy cattle housed in a confined environment following waterborne inoculation. (70/786)

A study of Escherichia coli O157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration of E. coli O157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers of E. coli O157:H7 isolates shed in these calves varied from 60 to 10(5) CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by using XbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 10(3) to 10(4) CFU of E. coli O157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 10(2) to 10(6) CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 10(6) CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination of E. coli O157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.  (+info)

Effects of conjugated linoleic acids (CLA) on tissue response to homeostatic signals and plasma variables associated with lipid metabolism in lactating dairy cows. (71/786)

We conducted a series of experiments to evaluate the effects of conjugated linoleic acids (CLA) on lipid metabolism and energy homeostasis in lactating dairy cows. In all experiments, multiparous Holstein cows in mid to late lactation were abomasally infused with CLA for 5 d. The initial study established that trans-10, cis-12 CLA markedly reduced milk fat yield whereas cis-9, trans-11 CLA, the predominant CLA isomer in milk fat, had no effect. Across the three investigations, infusions of the pure trans-10, cis-12 CLA isomer (3.5 to 14.0 g/d) resulted in a 25 to 50% decrease in milk fat yield and this was energetically equivalent to 6 to 11% of net energy intake. Effects were specific for milk fat as there were little or no changes in feed intake and the yield of milk or milk protein. In Exp. 1, infusing trans-10, cis-12 CLA had no effect on circulating plasma concentrations of glucose, insulin, or leptin. Basal NEFA concentrations were also unaffected, but lipolytic response to an epinephrine challenge was reduced (33%) when cows received trans-10, cis-12 CLA; this minor change in lipolytic response would be consistent with the slightly more positive net energy balance when cows received trans-10, cis-12 CLA. In Exp. 2, infusing differing amounts of trans-10, cis-12 CLA had only minor effects on basal NEFA concentrations, but again cows receiving trans-10, cis-12 CLA tended to have reduced (24%) lipolytic response to trans-10, cis-12 CLA compared to the control period. In Exp. 3, infusing trans-10, cis-12 CLA had no effect on basal glucose concentrations or glucose response to an insulin challenge. The fractional rate of glucose clearance in response to insulin was also not altered by treatment. In summary, the effects of trans-10, cis-12 CLA in lactating dairy cows appear to be specific for the mammary gland, resulting in reduced milk fat synthesis; adipose tissue response to a homeostatic signal regulating lipolysis (epinephrine), whole-body response to a homeostatic signal regulating glucose homeostasis (insulin), and plasma variables associated with lipid metabolism and energy homeostasis were relatively unaffected by treatment with trans-10, cis-12 CLA.  (+info)

Duodenal and milk trans octadecenoic acid and conjugated linoleic acid (CLA) isomers indicate that postabsorptive synthesis is the predominant source of cis-9-containing CLA in lactating dairy cows. (72/786)

Duodenal and milk samples obtained from lactating cows in a previous study were analyzed to compare the content and isomer distribution of conjugated linoleic acids (CLA) and trans-18:1 fatty acids (tFA). Four diets containing either low [25 g/100 g dry matter (DM)] or high (60 g/100 g DM) forage were fed with or without 2% added buffer to four multiparous Holstein dairy cows in a 2 x 2 factorial, 4 x 4 Latin square design with 3-wk experimental periods. Duodenal flows of CLA were low (1.02-1.84 g/d), compared with that of tFA (57-120 g/d), regardless of diet. The greatest amounts of CLA and tFA, as well as the greatest proportions of trans-10-18:1 (P < 0.02), and cis-9, trans-11 (P < 0.01) and trans-10, cis-12 CLA (P < 0.01) were in the duodenal flow of cows fed the low forage unbuffered diet. In milk fat, tFA were increased by the low forage unbuffered diet and the trans-10-18:1 (P < 0.02) replaced trans-11-18:1 as the major 18:1 isomer. Milk CLA secretion (7.2-9.1 g/d) was greater (P < 0.001) than that in the duodenal flow with each diet. This was due to the increase in cis-9, trans-11-18:2 and trans-7, cis-9 CLA, resulting most likely from endogenous synthesis via Delta9-desaturation of ruminally derived tFA. For other CLA isomers, duodenal flow was always greater than milk secretion, suggesting that they essentially were produced in the rumen.  (+info)