L. F. P. Silva

Identification of Coagulase-Negative Staphylococci from Bovine Intramammary Infection by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Coagulase-negative staphylococci (CoNS) are among the main pathogens causing bovine intramammary infection (IMI) in many countries. However, one of the limitations related to the specific diagnosis of CoNS is the lack of an accurate, rapid, and convenient method that can differentiate the bacterial species comprising this group. The aim of this study was to evaluate the ability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to accurately identify CoNS species in dairy cow IMI. In addition, the study aimed to determine the frequency of CoNS species causing bovine IMI. A total of 108 bacterial isolates were diagnosed as CoNS by microbiological cultures from two milk samples collected from 21 dairy herds; the first sample was collected at the cow level (i.e., 1,242 composite samples from all quarters), while the second sample was collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows). After CoNS isolation was confirmed by microbiological culture for both samples, all CoNS isolates (n = 108) were genotypically differentiated by PCR restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and subjected to the MALDI-TOF MS identification procedure. MALDI-TOF MS correctly identified 103 (95.4%) of the CoNS isolates identified by PCR-RFLP at the species level. Eleven CoNS species isolated from bovine IMI were identified by PCR-RFLP, and the most prevalent species was Staphylococcus chromogenes (n = 80; 74.1%). In conclusion, MALDI-TOF MS may be a reliable alternative method for differentiating CoNS species causing bovine IMI.

Efeito do nível de células somáticas sobre os constituintes do leite II-lactose e sólidos totais

In laboratory, electronic determination of somatic cell count (SCC) and concentration of lactose and total solids of 1,361 milk samples were carried out in order to evaluate the effects of somatic cell count (SCC) on the concentrations of milk lactose and total solids. All the SCC data were transformed to a linear score. Increases in the SCC score were associated with decreased lactose concentration. This increase was highly significant (p < 0.0001), and about 34% of the lactose concentration variation could be accounted for variation of the SCC score. Total solids were not influenced by the SCC score.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 microg/ quarter per d with or without 10 microg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered approximately 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Effect of the kappa-casein gene polymorphism, breed and seasonality on physicochemical characteristics, composition and stability of bovine milk

The objective of this study was to evaluate the effect of genetic polymorphism of kappa-casein, breed and seasonality on the physicochemical characteristics, composition and stability of milk in commercial dairy herds. A total of 879 milk and blood samples were collected from 603 Holstein and 276 Girolando cows, obtained during rainy and dry seasons. Milk samples were analyzed to determine the physicochemical characteristics, composition and ethanol stability, while blood samples were subjected to polymerase chain reaction to identify the kappa-casein genotype. The frequencies of genotypes AA, AB and BB of k-casein were respectively, 66.83, 31.84 and 1.33% for Holstein, and 71.38, 27.90 and 0.72% for the Girolando cows, respectively. The A allele was more frequent than the B allele, both for Holstein (0.827 and 0.173) and Girolando cows (0.853 and 0.147), respectively. Cows of AB and BB genotypes showed a higher milk fat content compared to the AA genotype. There was an interaction between breed and seasonality on the concentration of milk urea with higher values for Holstein and Girolando cows in the rainy and dry season, respectively. The levels of lactose, total solids, crude protein, true protein, casein and the casein:true protein ratio were higher during the dry season, while during the rainy season, the somatic cell count and milk urea concentration were higher. There was no association between milk stability and k-casein genotypes, but Holstein cows showed higher milk stability than Girolando cows, and milk was more stable during the rainy season than during the dry season.

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction.

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10(1) cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.

Somatic cells in milk of brazilian herds

A survey of data of the Lactation Physiology Laboratory of ESALQ - USP, Piracicaba, SP, Brazil was performed to characterize the somatic cell count (SCC) of herds. Descriptive statistics was used to analyze results of 7941 records collected between December 1996 and July 1998. The SCC mean was 641 thousand cells mL-1, standart deviation 767 thousand cells mL-1. The log2 transformation was also applied to SCC values, a new mean was calculated and then again transformed into SCC (307 thousand cells mL-1). This was the best way to present the real SCC values of the herds. The population of herds are distributed according to SCC count, considering the SCC limits of 250, 500, 750, 1000, 1250 e 1500 thousand cells mL-1, in the following percentages: 26, 53, 71, 83, 89, 93%. The quartiles of the samples were 25, 50, 75, 90, 95, 99 e 100% showing 244, 468, 815, 1302, 1797, 4099, 9223 thousand cells mL-1 respectively.

Staphylococcus aureus intramammary infection affects milk yield and SCC of dairy cows

Staphylococcus aureus (S. aureus) is the most prevalent infectious microorganism affecting dairy cattle worldwide, and its pathogenic characteristics facilitate its spread in dairy herds. S. aureus intramammary infections (IMI) are mainly subclinical, and associated losses can exceed average herd losses where the pathogen is not isolated. However, the extent it affects milk composition at udder and quarter levels is still unknown, and cow composite milk losses may be underestimated due to the dilution effect. The aim of this study was to investigate the effects of S. aureus subclinical mastitis on mammary quarter milk yield and composition. In order to determine the effects of the pathogen on milk yield and composition at quarter level, a pairwise comparison of infected and non-infected mammary quarters (n = 28) from two dairy herds was carried out. Quarters were individually milked, and milk production and composition were assessed. S. aureus has increased somatic cell counts at quarter level; however, no effect of S. aureus IMI on milk lactose, fat, and protein contents was observed. Fat yield from infected quarters decreased, but losses due to the infection caused by S. aureus were not associated with quarter positioning in cows.

Organic and inorganic sources of zinc, copper and selenium in diets for dairy cows: intake, blood metabolic profile, milk yield and composition

The present study was carried out with the objective of evaluating the effects of feeding dairy cows with organic or inorganic sources of zinc (Zn), copper (Cu) and selenium (Se) on blood concentrations of these minerals, blood metabolic profiles, nutrient intake and milk yield and composition. Nineteen Holstein cows were selected and randomly assigned to two groups for receiving organic (n = 9) or inorganic (n = 10) sources of Zn, Cu and Se from 60 days before the expected date of calving to 80 days of lactation. Samples of feed, orts and milk were collected for analysis. Body condition score (BCS) was determined and blood samples were collected for analysis of Zn, Cu and Se concentrations, as well as for metabolic profile. Supplying organic or inorganic sources of Zn, Cu, and Se did not affect dry matter and nutrient intake, blood metabolic profile, milk yield and composition, plasma concentration of these minerals, and BCS or change the BCS in cows from 60 days before the expected date of calving to 80 days of lactation. An effect of time was observed on all feed intake variables, plasma concentrations of Zn and Se, milk yield, milk protein content, BCS and change in BCS.

In situ degradability of corn stover and elephant-grass harvested at four stages of maturity

Among tropical forages, corn silage is largely used by farmers trying to explore the maximum genetic potential from the animals. However, other tropical forages, such as elephant-grass (Pennisetum purpureum), are more productive and therefore cheaper to use than corn silage. Our objective was to compare the in situ degradability of elephant-grass with that from corn hybrids, all harvested at four stages of maturity. The experimental design followed a randomized block design with nested subplots. Two corn hybrids: AG5011, ZN8392 were harvested with 25, 30, 35, and 40% dry matter (DM) in the whole plant, and separated in stem + leaf sheath + leaf blade (stover), and cobs. Elephant-grass was harvested with 30, 40, 50 and 60 days after a leveling cut. Dried and ground samples were incubated in nylon bags inside the rumen for 0, 6, 12, 24, 48 and 72 h to estimate the kinetics of ruminal DM and neutral detergent fiber (NDF) degradation. The advance of maturity increased the NDF and acid detergent fiber (ADF) content in elephant-grass, and reduced its DM degradability. However, maturity had little or no effect on fiber content and DM degradability of corn stover. Elephant-grass had a higher NDF degradability than corn stover, and there was no effect of maturity on NDF degradability of either elephant-grass or corn stover. Fiber degradability of elephant-grass was not worse than that of corn stover, and therefore the choice of forage should be made on economical analysis rather than assuming an intrinsic low production potential for elephant-grass based diets.

Leptin and hypothalamic gene expression in early- and late-maturing Bos indicus Nellore heifers

We investigated wether early-maturing or late-maturing Bos indicus Nellore heifers produced more leptin mRNA in adipose tissues and altered expression of hypothalamic genes related to leptin signaling. Six prepubertal and six pubertal heifers aged about 34 months and weighing 280 kg to 300 kg each were selected from a population of 100 Nellore heifers. Real-time PCR was used to quantify the expression of the leptin gene (LEP) in adipose tissues and the long isoform of the leptin receptor gene (Ob-Rb), the NK2 homeobox 1 hypothalamic marker gene NKX2-1, the suppressor of cytokine signaling 3 gene (SOCS-3), the neuropeptide Y genes (NPY) and the NPY G-protein coupled receptor genes NPY-Y1 and NPY-Y4 in the hypothalamus. Heifers attaining puberty earlier showed significantly greater LEP expression in adipose tissues (p < 0.05) and there was tissue interaction (p < 0.05). Hypothalamic expression of Ob-Rb, NKX2-1, NPY and SOCS-3 did not differ between groups, but in early-maturing heifers there was a tendency for lower expression of NPY-Y1 (8.3-fold less) and NPY-Y4 (14.3-fold less) compared to late-maturing heifers (p = 0.1). These results suggest that a combination of higher LEP expression, lower NPY-Y1 and NPY-Y4 expression could be a factor in regulating puberty in early-maturing B. indicus heifers.