Factors affecting N-acetyl-ß-D-glucosaminidase as an indicator for mastitis detection in dairy sheep.

The study of new indirect methods for mastitis detection is of great relevance both at the economic level of the farm and dairies, and in terms of consumer health, and animal welfare. These methods help us to monitor the disease and speed up the decision-making process on treatment of the affected animal and the destination of the milk. The main aim of this work was to study the effect of intramammary infection and other non-infectious factors on the activity of the enzyme N-acetyl-ß-D-glucosaminidase (NAGase) in milk, in order to evaluate its use as an indicator for the early diagnosis of mastitis in sheep that could be less expensive, easier to measure and a better marker of inflammation or complementary to existing methods such as somatic cell count (SCC). Seven biweekly samplings were carried out, in which NAGase activity, SCC and milk were analyzed. Glands were classified according to their sanitary status based on the results of the SCC and bacteriological analysis. Non-infectious factors such as lactation stage, parity number and milking session had a statistically significant effect on NAGase values, finding the highest NAGase values at the onset and end of the study, in infectious mastitic glands of multiparous females and at morning milking. However, among the NAGase variation factors studied, the health status of the gland was the factor that caused the highest variation in enzyme levels, with infectious mastitic glands showing higher values than healthy glands. The predictive ability of NAGase was also studied by means of several logistic regression models, with the one that included NAGase together with lactation stage and parity obtaining the best results if sensitivity is to be prioritized, or the model that included NAGase, lactation stage, parity, milking and production if specificity is to be prioritized. From the results obtained, it can be concluded that the use of NAGase as an intramammary infection detection method in sheep can be useful when non-infectious factors that cause changes in the concentration of the enzyme are also considered.

Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Soil enzyme responses to land use change in the tropical rainforest of the Colombian Amazon region.

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.

Intramammary antibiotics with complementary acupuncture decreases milk serum N-acetyl-beta-D-glucosaminidase concentrations in dairy cattle with subclinical mastitis.

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.

Protective effects of probiotic Lactobacillus rhamnosus IMC501 in mice treated with PhIP.

The aim of the present study was to investigate the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC501; DNA damage was induced by one representative food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Mice were treated orally with suspensions of lactobacilli for 10 days before administration of food mutagen. During the treatment, the abundance of lactobacilli in feces, as assessed by qPCR analysis, increased, whereas ß-glucuronidase and N-acetyl-ß-glucosaminidase activities decreased. The extent of DNA damage was measured in colon and liver cells by comet assay. In colonocytes, diet supplementation with IMC501 resulted in a significant inhibition of DNA damage induced by PhIP. The results obtained in this in vitro study suggest that Lactobacillus rhamnosus IMC501 used as a dietary supplement can provide a useful integration of antimutagen food components of the normal diet, which are generally lower than the protective level.

Efficacy of parenteral administration of three antimicrobial agents in treatment of clinical mastitis in lactating cows: 487 cases (1989-1995).

OBJECTIVE: To evaluate the efficacy of parenteral administration of procaine penicillin G, spiramycin, or enrofloxacin in the treatment of clinical mastitis in lactating cows. DESIGN: Noncontrolled, clinical retrospective study. ANIMALS: 487 cows with mastitis involving 543 quarters. PROCEDURE: Clinical signs, histories, and results of bacteriologic examination, somatic cell count, and N-acetyl-beta-D-glucosaminidase activity of milk samples taken before and 3 to 4 weeks after treatment were retrieved from hospital records. Cows treated parenterally with procaine penicillin G, spiramycin, or enrofloxacin for 3 to 5 days were included. Supportive treatment alone was given to 35 cows infected with Escherichia coli. Factors possibly affecting outcome were analyzed, using ANOVA, correlation analyses, and the Mann-Whitney test. chi 2 Test was used to compare bacteriologic cure rates. RESULTS: Bacteriologic cure rates for mastitis caused by Staphylococcus aureus, coagulase-negative staphylococci, and streptococci were 34, 76, and 65%, respectively. Cure rates in cows in their first lactation and infected with S aureus and coagulase-negative staphylococci were significantly higher than those for older cows. In cows with mastitis caused by E coli, the cure rate was 74% for those treated with penicillin G and 71% for those not treated with antimicrobials. High N-acetyl-beta-D-glucosaminidase activity in milk samples obtained at initial examination indicated a poor outcome in S aureus and streptococcal mastitis. Cows infected in the early lactation period had more severe inflammatory responses and clinical signs if infected with coagulase-negative staphylococci and coliforms. CLINICAL IMPLICATIONS: 3 to 5 days of treatment with parenterally administered penicillin G for clinical mastitis caused by penicillin-susceptible S aureus strains is efficacious in young cows. Parenteral administration of spiramycin or enrofloxacin does not give satisfactory results in mastitis caused by penicillin-resistant S aureus. Use of antimicrobials in the treatment of mastitis caused by coliform bacteria is questionable.

Accuracy of methods using somatic cell count and N-acetyl-beta-D-glucosaminidase activity in milk to assess the bacteriological cure of bovine clinical mastitis.

This study examined the capability of milk somatic cell count (SCC) and NAGase activity to discriminate between quarters that had been cured versus those that had not been cured at 4 wk after antimicrobial therapy for clinical mastitis. The distribution of microorganisms that were isolated before therapy from 630 quarters with mastitis was as follows: 225 strains of Staphylococcus aureus, 96 strains of coagulase-negative staphylococci, 152 strains of streptococci (Streptococcus dysgalactiae and Streptococcus uberis), and 157 strains of coliform bacteria. Bacteriological cure rates were 35% for mastitis caused by Staph. aureus, 75% for mastitis caused by coagulase-negative staphylococci, 66% for mastitis caused by streptococci, and 72% for mastitis caused by coliforms. Diagnostic accuracy of milk SCC and NAGase and their interquarter ratios for predicting bacteriological status of the control samples was assessed by calculating sensitivity, specificity, and accuracy and by means of receiver operating characteristic analysis. The efficiency of milk SCC and NAGase for predicting bacteriological cure was greatest for cows that had been infected with Staph. aureus. The main problem in detecting coagulase-negative staphylococci was low sensitivity, and the main problem in detecting streptococci and coliforms was low specificity. Receiver operating characteristic analysis is not completely suitable for the detection of mastitis because reference method bacteriology and indirect tests can never fully agree. To assess the recovery of cows from mastitis caused by Staph. aureus, bacteriology should be supplemented with an examination of milk SCC or NAGase activity at threshold values such as those presented here.

Predicting injection site muscle damage. I: Evaluation of immediate release parenteral formulations in animal models.

PURPOSE: The current animal model generally accepted by the pharmaceutical industry and the FDA for assessment of muscle damage following intramuscular injection (IM) is the rabbit lesion volume model (RbLV). However, this model is resource intensive. The goal of this study was to find a resource sparing alternative to the rabbit lesion model for assessing injection site toleration in IM formulation screening. METHODS: Short term animal model alternatives to RbLV for evaluating IM formulations were examined. In addition to RbLV, myeloperoxidase (MPO), p-nitrophenyl N-acetyl-beta-glucosaminide (NA beta G) and/or plasma creatine phosphokinase (CK) activities were determined in rabbits (Rb) and rats (Rt) after injection of formulations (digoxin, azithromycin and danofloxacin). The edema from these formulations 24 hr after subcutaneous injection into the rat footpad (RFE) was also determined. RESULTS: MPO and NA beta G were not considered very useful as biochemical predictors of muscle damage for these formulations. Histology generally correlated with RbLV values. Compared to saline, RbLV was marked for all formulations within 1-3 days of injection. After day 3, lesions quickly resolved, and no significant differences were found. For these formulations, all CK animal models and RFE were generally predictive of RbLV. A formulation with RtCK > 1000 U/L or RbCK > 3000 U/L, was predicted to be poorly, tolerated. CONCLUSIONS: Due to ease, number of animals, time and intrinsic mechanism, we concluded that for most formulations, 2 and 4 hr RtCK data alone should be reasonably predictive of muscle damage.

[The role of tubulointerstitial changes in progression of kidney function failure in patients with chronic glomerulonephritis (GN)]. / Rola zmian cewkowo-sródmiazszowych w postepujacym uposledzeniu funkcji nerek u chorych na przewlekle klebuszkowe zapalenie nerek (KZN).

In most cases of glomerulonephritis (GN) long-term course lead to chronic renal failure. The cause of inevitably gradually progress of GN to end-stage renal disease (ESRD) is unclear. The histological abnormalities seen in patients with progressive renal failure consist of focal and segmental glomerulosclerosis and tubulointerstitial nephritis. At present it is considered that tubulointerstitial changes attends almost all forms of progressive glomerular and vascular injury. It was known that chronic tubulointerstitial nephritis is characterized morphologically by tubular atrophy, interstitial fibrosis and interstitial inflammation of variable severity. The pathomechanism of this changes is complicated. Tubular ischaemia results from obliteration of peritubular capillaries, adaptation of tubular function with increased oxygen consumption and increased glomerular capillary permeability to macromolecules are reasons of chronic tubular damage. Injured tubules release growth factors and cytokines, which induce interstitial fibroblast proliferation, chemo-attraction and proliferation of infiltrating cells, and disruption of the balance between synthesis and degradation of cellular constituents. The consequences of these processes are tubular atrophy and interstitial fibrosis. Because of many studies concurred that tubulointerstitial changes determinant the progression of GN, tubular injury markers were searched for. Although over 50 enzymes were detected in human urine, only a few have been used for diagnosis in renal disease. The most widely used are lysosomal enzyme N acetyl-beta-D-glucosaminidase (NAG) and brush border enzymes alanine-aminopeptidase (AAP) and gamma-glutamyltransferase (GGT). tubular damage in hypertension, diabetes and in diagnostics of renal disease. AAP and GGT, brush border enzymes seem to be sensitive markers of renal injury too. Pathological value of GGT was observed even in the early stage of disease. Measurement of urinary excretion of low molecular weight proteins was valuable supplement in estimation of tubulointerstitial system malfunction. These proteins are readily filtered by normal glomeruli and virtually completely reabsorbed by normal proximal tubules. Favour are alpha-1-microglobulin (alpha-1-m) and retinol-binding protein (RBP) because they are less affected than beta-2-microglobulin (beta-2-m) by low urine pH. Above presented review confirm that further research in correlation between activity of disease, histological picture, deterioration in renal function and changes in urinary excretion of markers proteins (for example alpha-1-m, AAP, NAG, GGT) is advisable, and can contribute to use in clinic diagnostics of GN.

Comparisons of selenite and selenium yeast feed supplements on Se-incorporation, mastitis and leucocyte function in Se-deficient dairy cows.

The dairy cows at the Estonian Agricultural University appeared to have an extremely low selenium status. The selenium level was 5.6 micrograms/l in whole blood and 3.2 micrograms/l in milk, on average. The blood glutathione peroxidase was consequently extremely low. The effects of organic selenium (selenized yeast) and sodium selenite were compared in a feeding experiment on 100 dairy cows. Selenium incorporation, udder health and the in vitro function of blood neutrophils were monitored. Supplementation of the feed either with 0.2 ppm organic selenium or sodium selenite for 8 weeks, increased the blood selenium level (geometric mean) within this period from the back-ground level (about 5.6 micrograms/l) to 167 (Se-yeast) and to 91 micrograms/l (selenite). The respective change in whole blood glutathione peroxidase (GSH-PX) was from 0.22 to 3.0 (Se-yeast) and to 2.3 (selenite) microKat/g Hb. Blood GSH-PX continued to increase up to 10 weeks after the supplementation was stopped. The bioavailability of yeast selenium was superior to selenite: the relative bioavailability (selenite = 1) of yeast selenium was 1.4 if blood GSH-PX, 1.9 if blood selenium, and 2.7 if milk selenium was used as the response criterion. Selenium-supplementation showed a positive effect on udder health. The percentage of quarters harbouring mastitis pathogens dropped from 22.9 to 13.0 in the Se-yeast group and from 18.4 to 7.4 in the selenite group during the supplementation period. The effect of selenium on mastitis was also reflected as a decrease in the output of milk somatic cells and N-acetyl-beta-D-glucosaminidase (NAGase). The time-luminescence profile of zymosan-induced activity of blood neutrophils became skewed to the left in Se-supplemented cows.

[Different effects of homeopathic potencies and conventional dilutions on specific liver enzymes of rats--an in vivo study]. / Unterschiedliche Wirkungen von homöopathischen Potenzen und konventionellen Verdünnungen auf spezifische Leberenzyme der Ratte--ein in vivo Versuch.

It was searched for differing effects of homeopathic potencies and equally concentrated conventional dilutions. Activities of enzymes from three different subcellular compartments of the rat liver served as parameters for the evaluation. Especially in the D15/10(-15) range differences proved to be statistically relevant. The series with potentiated carrier substance, necessary from heuristic reasons and related to the homeopathic potencies, resulted in hitherto not understandable findings.

The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage.

Analysis of bronchoalveolar lavage fluid (BALF) appears to be a sensitive approach to characterizing an acute inflammatory response within the lung. More work, however, is needed to determine if analyses of BALF endpoints can predict chronic responses (i.e., fibrosis). The objective of the present study was to compare the dose and temporal pulmonary response of a known fibrogenic agent, silica, and two known nonfibrogenic agents, aluminum oxide and titanium dioxide. Animals were instilled with silica (0, 0.2, 1.0, or 5.0 mg/100 g body wt), titanium dioxide (1.0 or 5 mg/100 g body wt), aluminium oxide (1.0 or 5.0 mg/100 g body wt) or saline. Animals (n = 5/group) were terminated 1, 7, 14, 28, and 63 days following instillation, and the BALF was characterized by biochemical and cellular assays. Histopathological changes were determined at 60 days after exposure. The biochemical results demonstrated BALF levels of lactate dehydrogenase (LDH), beta-glucuronidase (BG), N-acetylglucosaminidase (NAG), and total protein (TP) increased in a dose-related fashion at the earlier time points for all test materials, with the magnitude of change being greatest for silica. The temporal response for these parameters was significantly different for the two classes of materials. With time, the response for the fibrogenic dust steadily increased, while the levels for the nonfibrogenic dusts decreased toward normal values during the 2-month study period. Of the cellular changes, total cell numbers, neutrophils, and lymphocyte numbers were the most sensitive markers of the pulmonary response. As shown with the biochemical parameters, the cellular response to silica increased with time while that of the nuisance dusts did not. It was also found that, similar to inhalation studies, high doses of a nuisance dust may result in toxicity/inflammation. This toxicity at high dose levels emphasizes the importance of choosing relevant doses when comparing potentially fibrogenic and nonfibrogenic dusts. In conclusion, the persistent and progressive changes seen in the biochemical (LDH, TP, BG, NAG) and cellular parameters (total cells, neutrophils and lymphocytes) following silica administration correlated with the fibrotic response which occurred after exposure to this material. The less dramatic and transient changes seen with aluminum oxide and titanium dioxide correlated with the inert nature of these nuisance dusts. The results of this study indicate evaluation of BALF may provide a means to predict the chronic pulmonary response to a material.

Effect of topical anesthesia on secretion of lysozyme and lysosomal enzymes in human tears.

Unilateral topical anesthesia affected the secretion of human tear fluid and its concentrations of lysozyme and lysosomal enzymes. Results of a Schirmer's test with 0.4% oxybuprocaine showed that topical anesthesia reduced the mean test value by 47% and the secretion of protein, lysozyme, acid phosphatase, and N-acetyl-beta-D-glucosaminidase by 30%, 45%, 31%, and 33%, respectively. These results indicated that the paper strip induced reflex secretion from only the stimulated eye. The enzyme activity of lysozyme per fluid volume in tears from anesthetized eyes was as high as that from eyes without anesthesia, while acid phosphatase and N-acetyl-beta-D-glucosaminidase had higher activities. The amount of protein in the tear fluid was higher in anesthetized eyes than in unanesthetized eyes. Enzyme activity of lysozyme per protein of the tear fluid in the anesthetized eyes was lower than in the eyes without anesthesia, while acid phosphatase and N-acetyl-beta-D-glucosaminidase had higher activities per protein in the eyes with anesthesia. These findings disclosed that the concentrations of total protein, acid phosphatase, and N-acetyl-beta-D-glucosaminidase increased, while lysozyme value was constant when the tear secretion decreased.