Phenotype characterization of Staphylococcus species strains isolated from buffalo (Bubalus bubalis) milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

ORGANOCHLORINE PESTICIDE-RESIDUES IN HUMAN-MILK IN THE RIBEIRAO-PRETO REGION, STATE OF SAO-PAULO, BRAZIL

Thirty-seven samples of human milk (colostrum) from donors living in the Ribeirao Preto region were analyzed to determine the levels of organochlorine pesticide residues. Donors were classified into two groups, i.e., occupationally exposed and non-exposed to pesticides. Other factors such as age, previous lactations, race, smoking habit, occupation, family income and educational level were also considered. Analysis was performed by preliminary lipid extraction followed by fractional partition on a column and finally by gas chromatography with an electron capture detector. Lindane was found in 32% of the samples in amounts of less than 0.001 mg/kg; heptachlor was found in 65% of the samples at mean levels of 0.001 mg/kg, i.e., a level five-fold lower than that established by FAO/WHO (1970) for cow's milk. Aldrin and endrin were not detected in any of the samples. Dieldrin was detected in only one sample at a level of 0.038 mg/kg, which is considered high. DDT and DDE amounts are reported as total DDT and at least one of these compounds was present in every sample. Amounts detected in donors occupationally exposed to pesticides ranged from 0.008 to 0.455 mg/kg (mean, 0.149 mg/kg), i.e., three times the limit established by FAO/WHO (1970), while values for donors who had not been exposed ranged from 0.002 to 0.072 mg/kg (mean, 0.025 mg/kg), i.e., half the limit. Considering the level of acceptable daily intake proposed by FAO/WHO (1973), lactents ingested 1% of the acceptable intake of lindane (all donors), 30% of the acceptable intake of heptachlor (all donors), 60% of the acceptable intake of DDT (non-exposed donors), and 3.7 times the acceptable intake of DDT (exposed donors). Comparing the present results with those obtained 10 years ago, the total DDT level in human milk is decreasing in this part of the country. The mean amount of organochlorine residues in non-exposed women's milk was one of the lowest levels among those recorded in the literature. DDT levels of occupationally exposed women's milk were comparable with those reported for developed countries and lower than those detected in Latin American countries. When the results of this survey are considered in relation to the advantages of breast-feeding, the risk-benefit balance is still favorable to breast-feeding. However, given the lack of long-term epidemiological studies, undesirable or harmful long-lasting effects cannot be excluded.

On-line extraction and determination of carbofuran in raw milk by direct HPLC injection on an ISRP column

A method has been developed for extraction and determination of carbofuran in milk. The method involved direct injection of raw milk on to a human serum albumin dimethyloctyl-silica gel (HSA-Cs) column and the use of 80:20 (v/v) 0.01 M phosphate buffer pH 5.5 - acetonitrile as mobile phase. UV spectrophotometric detection was performed at 220 nm. Identification was based on retention time. Quantification was performed by automatic peak-area determination and was calibrated by use of an external standard.

Use of chemically modified silica with beta-diketoamine groups for separation of alpha-lactoalbumin from bovine milk whey by affinity chromatography

Silica gel surface was chemically modified with beta-diketoamine groups by reacting the silanol from the silica surface with 3-aminopropyl-triethoxysilane and 3-bromopentanedione, With this material, copper ions were adsorbed from aqueous solutions, the chemical analysis of the silica-gel-immobilized acetylacetone provided a quantity of 0.67 mmol g(-1) of organic groups attached to the support and 0.63 mmol g(-1) of copper, This material was used as a stationary phase in IMAC (immobilized metal affinity chromatography), to separate alpha-lactoalbumin from bovine milk whey, the results showed an efficient separation in the chromatographic column, the possibility of reutilization of the stationary phase was also investigated. (C) 1997 Academic Press

Internal standardization in graphite furnace atomic absorption spectrometry: Comparative use of As and Ge to minimize matrix effects on Se determination in milk

Arsenic and germanium have been evaluated as internal standards to minimize matrix effects on the direct determination of selenium in milk by graphite furnace atomic absorption spectrometry (GFAAS) using tubes with integrated platform, pre-treated with W together with I'd as chemical modifier. The efficiency of As and Ge as internal standards for 25 mu g L-1 Se plus 500 mu g (L)-1 As or Ge in diluted (1 + 9 v/v) milk plus 1.0% (v/v) HNO3 was evaluated by means of correlation graphs plotted from the normalized absorbance signals (n = 20) of internal standard (axis gamma) versus analyte (axis x). The equations that describe the linear regression were: A(As)= - 0.004 +/- 0.019 +/- 1.02 + 0.019 A(Se) (r=0.9967 +/- 0.005); A(Ge)= - 0.0 17 +/- 0.015 + 1.01 +/- 0.015 A(Se) (r = 0.9978 +/- 0.004). Samples and reference solutions were automatically spiked with 500 mu g L-1 Ge or As and 1.0% (v/v) HNO3 by the autosampler. For 20 mu L of aqueous standard solutions, analytical curves in the 5.00-40.0 mu g L-1 Se range were established using the ratio of Se absorbance to internal standard absorbance (A(Se)A(IS)) versus analyte concentration, and good linear correlations were obtained. The characteristic mass was 40 pg Se. Limits of detection were 0.55 and 0.40 mu g L-1 with As and Ge as the internal standard, respectively. Relative standard deviations (RSD) for a sample containing 25 mu g L-1 Se were 1.2% and 1.0% (n = 12) using As and Ge, respectively. The RSD without internal standardization was about 6%. The accuracy of the proposed method was evaluated by an addition-recovery experiment and all recovered values were in the 99-105% range with IS and in the 70-80% range without IS. Using Ge as the internal standard, results of analysis of standard reference materials were in agreement with certified values at a 95% confidence level. The selenium concentration for 10 analyzed milk samples varied from 5.0 to 20 mu g L-1. (c) 2005 Elsevier B.V. All rights reserved.

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein :true protein ratio were found.

Pyrethroid residues in milk and blood of dairy cows following single topical applications

The presence of the pyrethroid insecticides flumethrin, deltamethrin, cypermethrin and cyhalothrin in milk and blood of 10 cows was determined after single dermal applications of recommended doses. Milk and blood samples were collected every 7 days over a 35 d period and analyzed by high performance liquid chromatography. The highest residues in milk were found on day 28 for flumethrin and day 1 for deltamethrin, cypermethrin and cyhalothrin, while in blood the highest concentrations were present on day 28 for flumethrin and deltamethrin, the first day for cypermethrin, and day 14 for cyhalothrin.

HPLC determination of flumethrin, deltamethrin, cypermethrin, and cyhalothrin residues in the milk and blood of lactating dairy cows

A procedure to determine residue concentrations of synthetic pyrethroid insecticides (flumethrin, deltamethrin, cypermethrin and cyhalothrin) in the milk and blood of lactating dairy cows was developed. Extraction was performed with acetonitrile, n-hexane partitioning, and silica gel column cleanup with n-hexane and diethyl ether. Analysis was carried out by high- performance liquid chromatography and ultraviolet detection. Recovery of the four pyrethroids averaged 78 to 91% with a minimum detectable concentration of 0.001 mg/kg. The method was reproducible and sensitive.

Consistency of side choice in the milking parlour by holstein-friesian cows and its relationship with their reactivity and milk yield

Dairy cows often have to choose which of two sides to enter in the milking parlour. Some cows are very consistent in this choicė, and it is common to assume that when these cows are more disturbed are being milked in their non-preferred side. Such disturbance might involve significantly poor welfare. In order to assess this assumption, we decided to study the behaviour and milk yield of dairy cows and their relationships with side preference in the milking parlour. The study was carried out at Cambridge University Farm, in a two-sided tandem milking parlour. The data collection followed the daily management routine. We recorded the side chosen by each cow (left or right) during 40 milking sessions. Data from 70 cows, which were present in at least 25 milking sessions (mode = 36), were included in the statistical analysis. Cows' reactivity (CR) during premilking udder preparation, time spent fitting the milking cluster (FT), milk yield (MY) and duration of milking (DM) were measured. There was evident individual variation in the consistency of side choice. Individual differences (ANOVA, P < 0.001) were also found in CR, FT, MY and DM; although these variables were not significantly affected by the side or the interaction animal × side (ANOVA, P < 0.05). The comparison between left and right side means (paired t-test) of these variables did not show significant differences (P < 0.05). We concluded that there is no evidence that the cows were discomforted or stressed when milked in the non-preferred side of the milking parlour. © 2001 Elsevier Science B.V.

Variance heterogeneity for milk yield in Brazilian and Colombian Holstein herds

Heterogeneity of variances for milk yield (MY) was determined for Brazilian and Colombian herds. The herds were grouped as high or low variability within each country, using as criterion the phenotypic standard deviation (PSD) of MY in the contemporary groups of cows, from the first to the sixth calving. Brazilian and Colombian herds with PSD greater than 1,168 kg or 1,012 kg, respectively, were classified as high variability while the herd groups with values lower than those were classified as low variability. The genetic parameters for MY within each herd group were estimated using bivariate analysis in an animal model and the restricted maximum likelihood method with a derivative free algorithm, using 72,280 first lactations of cows, daughters of 1,880 sires. Heterogeneous variances were found, and Brazilian herds with high PSD had the greatest additive and residual genetic variances and heritability coefficients for MY. MY genetic correlation coefficients between herds of high and low variability within each country were 0.96 and 0.93 while between countries they varied from 0.72 to 0.81, suggesting that there was a reclassification of animals in the two countries and also heterogeneity of variances. This phenomenon leads to the questioning of the strategy of imported semen usage and the need to do genetic evaluations to identify sires with greater genetic potential for (sub) tropical environmental conditions.

Factors that cause genotype by environment interaction and use of a multiple-trait herd-cluster model for milk yield of Holstein cattle from Brazil and Colombia

Descriptive herd variables (DVHE) were used to explain genotype by environment interactions (G x E) for milk yield (MY) in Brazilian and Colombian production environments and to develop a herd-cluster model to estimate covariance components and genetic parameters for each herd environment group. Data consisted of 180,522 lactation records of 94,558 Holstein cows from 937 Brazilian and 400 Colombian herds. Herds in both countries were jointly grouped in thirds according to 8 DVHE: production level, phenotypic variability, age at first calving, calving interval, percentage of imported semen, lactation length, and herd size. For each DVHE, REML bivariate animal model analyses were used to estimate genetic correlations for MY between upper and lower thirds of the data. Based on estimates of genetic correlations, weights were assigned to each DVHE to group herds in a cluster analysis using the FASTCLUS procedure in SAS. Three clusters were defined, and genetic and residual variance components were heterogeneous among herd clusters. Estimates of heritability in clusters 1 and 3 were 0.28 and 0.29, respectively, but the estimate was larger (0.39) in Cluster 2. The genetic correlations of MY from different clusters ranged from 0.89 to 0.97. The herd-cluster model based on DVHE properly takes into account G x E by grouping similar environments accordingly and seems to be an alternative to simply considering country borders to distinguish between environments.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

The influence of sardine consumption on the omega-3 fatty acid content of mature human milk

Objectives: The purpose of this study was to investigate what effect the ingestion of sardines, rich in omega-3 series polyunsaturated fatty acids, has on the composition of breastmilk. Methods: This was a prospective study of 31 nursing mothers under observation at the Hospital Guilherme Álvaro. Each was given 2 kg of fresh sardines twice with a 15-day interval. Milk was sampled and a 24-hour dietary recall questionnaire was applied on days 0, 15 and 30. Milk was assayed for fatty acid content by gas chromatography. Statistical analysis of the results was performed using nonparametric tests with significance set at p < 0.05. Results: The results demonstrate that the nutritional intake of the nursing mothers was adequate at all three sample points. With regard to the omega-3 series fatty acid content of the breastmilk, it was observed that regular consumption and shorter intervals between ingestion and milk collection resulted in higher concentrations of docosapentaenoic acid and docosahexaenoic acid at 15 and 30 days into the study. Fatty acids from the omega-3 and omega-6 series exhibited a significant correlation, r 2 was 0.58 and 0.59 at 15 and 30 days, respectively. Conclusion: These results suggest that incorporating fish into the diets of nursing mother during lactation, in the form of 100 g of sardines two or three times a week, contributes to an increase in omega-3 series fatty acids. Copyright © 2006 by Sociedade Brasileira de Pediatria.

Subclinical mastitis caused by staphylococcus aureus: Analysis cost benefit of antibiotic therapy in lactating cows

The cost benefit analysis of treatment of bovine subclinical mastitis caused by S. aureus was evaluated. Two hundred and seventy udder quarters with subclinical mastitis and healthy were selected in four groups, in conformity to lactational stage and with the treatment or not. Group 1 included treated animals 10 to 60 days in milk; group 2 included treated animals 61 days in milk until two months before the end of lactation; group 3 included animals not treated 10 to 60 days in milk; group 4 included animals not treated from 61 days in milk until two months before the end of lactation. Treatment with gentamicin (150 mg) was accomplished by intramammary doses, once a day, after sensitivity tests. The mammary quarters were evaluated after 30 days again. The costs with the treatment were calculated considering a S. aureus prevalence of 5%, expenses with antibiotic, loss in milk, tests of sensitivity and workload. There was loss of income of 2% and 14% in the groups 1 and 2, respectively, when compared with the incomes before treatment. In such case, the treatment of bovine subclinical mastitis by S. aureus in the lactation was economically not practicable.