Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Severe diarrhea outbreak in beef calves (Bos indicus) caused by G6P[11], an emergent genotype of bovine rotavirus group A

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study. The monitoring of the G and P genotypes of BoRVA strains involved in diarrhea outbreaks in calves is important for both animal and public health by allowing the identification of the most frequent genotypes, the characterization of novel genotypes and to identify reassortments with genotypes described in animal and human hosts. The results of this study show the importance of the monitoring of the genotypes of BoRVA strains involved in episodes of bovine neonatal diarrhea as for characterization of frequency of occurrence and pathogenic potential of uncommon genotypes as for monitoring of the emergency of different BoRVA genotypes not included in commercial vaccines.

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis

The objective of this study was to evaluate herd management practices and mastitis treatment procedures as risk factors associated with Staphylococcus aureus antimicrobial resistance. For this study, 13 herds were selected to participate in the study to evaluate the association between their management practices and mastitis treatment procedures and in vitro antimicrobial susceptibility. A total of 1069 composite milk samples were collected aseptically from the selected cows in four different periods over two years. The samples were used for microbiological culturing of S. aureus isolates and evaluation of their antimicrobial susceptibility. A total of 756 samples (70.7%) were culture-positive, and S. aureus comprised 27.77% (n=210) of the isolates. The S. aureus isolates were tested using the disk-diffusion susceptibility assay with the following antimicrobials: ampicillin 10mg; clindamycin 2μg; penicillin 1mg; ceftiofur 30μg; gentamicin 10mg; sulfa-trimethoprim 25μg; enrofloxacin 5μg; sulfonamide 300μg; tetracycline 30μg; oxacillin 1mg; cephalothin 30μg and erythromycin 5μg. The variables that were significantly associated with S. aureus resistance were as follows: the treatment of clinical mastitis for ampicillin (OR=2.18), dry cow treatment for enrofloxacin (OR=2.11) and not sending milk samples for microbiological culture and susceptibility tests, for ampicillin (OR=2.57) and penicillin (OR=4.69). In conclusion, the identification of risk factors for S. aureus resistance against various mastitis antimicrobials is an important information that may help in practical recommendations for prudent use of antimicrobial in milk production.

Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

Treponema denticola in microflora of bovine periodontitis

Periodontitis in cattle is an infectious purulent progressive disease associated with strict anaerobic subgingival biofilm and is epidemiologically related to soil management at several locations of Brazil. This study aimed to detect Treponema species in periodontal pockets of cattle with lesions deeper than 5mm in the gingival sulcus of 6 to 24-month-old animals considered periodontally healthy. We used paper cones to collect the materials, after removal of supragingival plaques, and kept frozen (at -80°C) up to DNA extraction and polymerase chain reaction (PCR) using T. amylovorum, T. denticola, T. maltophilum, T. medium and T. vincentii primers. In periodontal pocket, it was possible to identify by PCR directly, the presence of Treponema amylovorum in 73% of animals (19/26), T. denticola in 42.3% (11/26) and T. maltophilum in 54% (14/26). Among the 25 healthy sites, it was possible to identify T. amylovorum in 18 (72%), T. denticola in two (8%) and T. maltophilum in eight (32%). Treponema medium and T. vincentii were not detected over all 51 evaluated samples. The presence of Treponema amylovorum, T. maltophilum and, in particular, the widely recognized T. denticola in subgingival microflora brings an original and potencially important contribution in studies of the bovine periodontitis.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Morphoquantitative description of bovine digital cushion

Abstract The digital cushion is characterized as a modified subcutaneous tissue that absorbs the shock during gait, assists venous return of the hoof and supports a considerable part of body weight. Digital cushions have particular importance in the pathogenesis of the hoof, since they need to properly work in order to prevent compression and traumas in soft tissues. This study aimed to measure and determine how is the arrangement of these structures, and for this it was established the proportions of connective, adipose, vascular tissues and collagen fibers and collagen types found in palmar and plantar digital cushion of bovine using fore and hindlimbs of twelve adult zebu cattle of both sexes, 11 male and one female, with 269kg average carcass weight and without limb disorders. Fragments of cushions were subjected to conventional histology, cut to a thickness of 4µm and stained with Red Picrosirius. With digital optical microscope, the quantification of the connective tissue and differentiation of types of collagen used the Image Pro Plus® software, and of adipose and vascular tissue, the test point system. The mean and standard error were estimated with the GraphPad Prism 5.0 software, and then data were subjected to Kolmogorov-Smirnov normality test and Student's t-test with significance level set at 5% for determining the amount of different tissues between fore and hindlimbs of studied animals. In forelimbs the mean and standard error of the connective tissue proportion was 50.10%+1.54, of the adipose tissue was 21.34%+1.44, and of vascular tissue was 3.43%+0.28. Hindlimbs presented a proportion of connective tissue of 61.61%+1.47, 20.66%+1.53 of adipose tissue, and 3.06%+0.20 of vascular tissue. A significant difference (p<0.001) was detected in the connective tissue proportion between fore and hindlimbs. Types I and II collagen fibers have presented, respectively, a proportion of 31.89% and 3.9% in forelimbs and 34.05% and 1.78% in hindlimbs. According to the used methodology, digital cushions had a clear differentiation relative to adipose tissue between fore and hindlimbs.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Growth and mineral nutrition Of Ipomoea quamoclit

A greenhouse experiment was carried out from November 2006 to April 2007 at FCAV/UNESP, Brazil, aiming to study the dry mass production and the accumulation and distribution of macronutrients in Ipomoea quamoclit, an important weed for annual and perennial crops in Brazil. The plants were grown in seven liter pots with sand substrate, daily irrigated with Hoagland & Arnon nutrient solution. The experimental design was completely randomized with four replicates. The treatments corresponded to evaluation times at 14 day intervals, beginning 21 days after emergence (DAE). In each evaluation, the plants of four pots were analyzed in function of dry mass production and macronutrient content. I. quamoclit had a small dry mass and macronutrient accumulation at the beginning of the experimental stage, increasing after 77 DAE and reaching the maximum theoretical value at 146, 143, 140, 149, 142, 153, and 124 DAE, for dry mass, N, P, K, Ca, Mg, and S, respectively. K and N were the macronutrients most accumulated by I. quamoclit plants.

Growth and mineral nutrition of Solanum americanum

A greenhouse trial was carried out from November 1995 to April 1996 at FCAV/UNESP, Brazil, aiming to study the dry matter production and the accumulation and distribution of macronutrients in Solanum americanum, an important weed for annual and perennial crops in Brazil. The plants were grown in seven liter pots with sand substrate, irrigated daily with Hoagland & Arnon nutrient solution. The experimental design was completely randomized with four replicates. The treatments corresponded to evaluation times at 14 day intervals, beginning 21 days after emergence (DAE). In each evaluation, the plants of four pots were analyzed for dry matter production and macronutrient content. S. americanum had a small dry matter and macronutrient accumulation at the beginning of the experimental stage, increasing after 77 DAE and reaching the maximum theoretical value at 142, 142, 164, 149, 140, 149 and 152 DAE, for dry matter, N, P, K, Ca, Mg, and S, respectively. K and N were the most accumulated macronutrients for S. americanum plants.

Bovine serum albumin potentiates caffeine- or ATP-induced tension in human skinned skeletal muscle fibers

Human skinned muscle fibers were used to investigate the effects of bovine serum albumin (BSA) on the tension/pCa relationship and on the functional properties of the Ca2+-release channel of the sarcoplasmic reticulum (SR). In both fast- and slow-type fibers, identified by their tension response to pSr 5.0, BSA (0.7-15 µM) had no effect on the Ca2+ affinity of the contractile proteins and elicited no tension per se in Ca2+-loaded fibers. In contrast, BSA (>1.0 µM) potentiated the caffeine-induced tension in Ca2+-loaded fibers, this effect being more intense in slow-type fibers. Thus, BSA reduced the threshold caffeine concentration required for eliciting detectable tension, and increased the amplitude, the rate of rise and the area under the curve of caffeine-induced tension. BSA also potentiated the tension elicited in Ca2+-loaded fibers by low-Mgv solutions containing 1.0 mM free ATP. These results suggest that BSA modulates the response of the human skeletal muscle SR Ca2+-release channel to activators such as caffeine and ATP.

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Pre- and postnatal nutrition – target for allergy risk reduction

A rapid increase in allergic diseases in Western societies has led to the conclusion that our modern lifestyle is a risk factor for immune dysregulation. Potential culprits and benefactors are searched among early dietary and microbial exposures, which may act to program later allergic disease. The aim of this thesis was to investigate the role of early maternal and child nutrition in reducing the risk of child allergy. The study population comprised of 256 mother – child pairs from families with a history of allergy participating in a randomized controlled dietary counseling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) study from early pregnancy onwards. The dietary counseling aimed for a diet complying with dietary recommendations for pregnant and lactating women, with special attention to fat quality. Maternal dietary counseling was reflected in cord blood fatty acids suggesting better essential fatty acid status in infants in the counseling group. Dietary counseling with probiotics or placebo had no effect on child allergy risk, but associations between maternal diet during pregnancy and breastfeeding and child allergic outcomes were found in secondary analyses. During pregnancy, milk intake was related to decreased and cheese intake to increased risk of child atopic eczema. During breastfeeding, intake of vitamin C was related to increased risk of asthma and intake of egg was related to decreased risk of atopic eczema. The timing of introduction of complementary foods to infant’s diet was not associated with risk of atopic eczema, when adjusted with parental opinion of child allergic symptoms (i.e., potential reverse causality). In conclusion, the results demonstrate that infant fatty acid supply can be modified via maternal dietary changes. In addition, interesting associations of maternal diet with child allergy risk were discovered. However, no difference in the incidence of allergic diseases with dietary counseling was observed. This suggests that more potent dietary interventions might be necessitated to induce clinical risk reduction of allergy. Highrisk families can safely adhere to dietary recommendations for pregnant and lactating women, and the results support the current conception that no additional benefit is gained with delaying introduction of complementary feeding.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.