District-Wide Herd Sanitation and Eradication of Intramammary Staphylococcus aureus Genotype B Infection in Dairy Herds in Ticino, Switzerland.

The present study demonstrates successful herd sanitation and eradication of contagious mastitis caused by Staphylococcus aureus genotype B (S. aureus GTB) in an entire Swiss district (Ticino) including 3,364 dairy cows from 168 farms. Herd sanitation included testing of all cows using a highly GTB specific and sensitive qPCR assay, implementation of related on-farm measures, appropriate antibiotic therapy of GTB-positive cows and culling of therapy-resistant animals, respectively. A treatment index was used as an objective criterion to select GTB-positive cows eligible for culling and replacement payment. 62 herds (37%) were initially GTB-positive with a cow prevalence between 10% and 100% and were submitted to sanitation. Twenty mo after the start of the campaign, all these herds were free from S. aureus GTB, whereby 73% of them were sanitized during the first 7 mo. At the cow level, a total of 343 animals were infected. 50 of them were immediately culled and financially compensated based on their treatment index value. The remaining 293 cows were intramammarily treated with antibiotics either during lactation using the combination of cephalexin-kanamycin or penicillin-gentamicin or at dry-off using cloxacillin. Out of these cows, 275 (93.9%) were treated successfully meaning that their milk was twice GTB-negative by qPCR after therapy. For lactational treatment, control samples were taken ≥10 and ≥20 d after treatment, for dry off treatment ≥14 and ≥24 d after parturition. Neither lactation number nor SCC before treatment of the cow nor the type of therapy were associated with therapeutic cure. Using data of 30 GTB-positive and 71 GTB-negative herds (1855 observations), the impact of GTB sanitation on bulk tank milk SCC (BTSCC) was evaluated applying a linear mixed statistical model. In the year before sanitation, BTSCC was always higher in GTB positive than in GTB negative herds. After the start of the campaign, BTSCC declined rapidly in the herds under GTB sanitation and achieved values that no longer differed statistically from those of GTB-free herds after only 2 mo, remaining very similar for the rest of the campaign. The farmers were very satisfied with the outcome of the campaign as all GTB positive herds could be sanitized rapidly, sanitation was sustainable, and milk quality increased.

Penicillin resistance in bovine Staphylococcus aureus: Genomic evaluation of the discrepancy between phenotypic and molecular test methods.

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis, causing serious costs in the dairy industry. Early diagnosis and adequate therapy are therefore 2 key factors to deal with the problems caused by this bacterium, and benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains shows discrepant results depending on the test used; consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was therefore to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies. A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution [minimum inhibitory concentration (MIC)] and 2 different disk diffusion protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, and a subset of 45 strains was also subjected to whole genome sequencing (WGS). Discrepant results were obtained when penicillin resistance of bovine S. aureus was evaluated by disk diffusion, MIC, and PCR methods. The discrepancies, however, could be fully explained by WGS analysis. In fact, it turned out that penicillin resistance is highly dependent on the completeness of the bla operon promotor: when the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed. Further, WGS analyses showed that penicillin resistance is truly assessed by the MIC assay. In contrast, caution is required when interpreting disk diffusion and PCR results.

Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study.

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

Short communication: Lack of intramammary niche recolonization during a sanitation program for the contagious mastitis pathogen Staphylococcus aureus genotype B.

In Switzerland, sanitation programs of dairy herds infected with the contagious mastitis pathogen Staphylococcus aureus genotype B (GTB) have been established for several years. In recent years, Streptococcus uberis and non-aureus staphylococci have emerged as the bacteria most frequently isolated from bovine milk samples. The latter cause subclinical mastitis, and some species are more persistent or pathogenic than others. The present study aimed to investigate the developments in the intramammary colonization spectrum of 5 dairy herds undergoing a sanitation program for Staph. aureus GTB. We collected single-quarter milk samples aseptically from all lactating cows at 3-mo intervals during the sanitation period; after classical bacteriological analysis, MALDI-TOF mass spectrometry was used to identify the isolates to the species level. Non-aureus staphylococci were found to be the bacterial group most frequently occurring on the selected farms, with Staphylococcus chromogenes and Staphylococcus xylosus being predominant. The present study demonstrated that GTB-infected cows treated with antibiotics lacked systematic recolonization with other bacteria during herd sanitation for the contagious Staph. aureus GTB.

Sanitation of Staphylococcus aureus genotype B-positive dairy herds: A field study.

Staphylococcus aureus is one of the most widespread mastitis pathogens infecting dairy cattle worldwide. In Switzerland, different bovine genotypes of Staph. aureus have been identified, and genotype B (GTB) was demonstrated to be a highly contagious subtype, causing herd problems in cattle. As the efficacy of antibiotic therapy against Staph. aureus is not satisfactory, the most promising strategy for controlling this udder pathogen is the implementation of specific sanitation programs for affected farms. The aim of the present longitudinal study was the field evaluation of 2 analytical approaches for the sanitation of Staph. aureus GTB-positive dairy herds. We compared a new real-time quantitative PCR (qPCR) assay based on the detection of the unique target gene adlb with classical bacteriology. Sanitation was successfully achieved using both analytical methods, but the qPCR approach showed some main advantages, namely the use of clean (instead of aseptically collected) milk samples facilitates sample collection in terms of time and costs, enabling the sampling of entire herds during a regular milking procedure and by the farm staff. The high inclusivity and exclusivity of the new target gene adlb enable very specific detection of only the genotype of interest. Because of the very high diagnostic sensitivity of qPCR, each GTB-positive cow can be correctly identified at any time point during lactation, allowing farmers to continuously update milking groups to prevent transmission during milking. Milk sample analysis becomes more objective, faster, less expensive, and more suitable for routine application, enabling the sanitation of even big herds in short time.

Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk, targeting the new gene adlb.

The specific and reliable diagnosis of mastitis pathogens is essential for successful sanitation programs. The aim of the present study was to develop and evaluate a new real-time quantitative PCR (qPCR) assay for the very sensitive and specific detection of Staphylococcus aureus genotype B in cow milk samples. This mastitis pathogen is contagious and particularly prevalent in Switzerland and other central European countries. The new test is based on a rapid preparation of bacteria, followed by DNA isolation and qPCR for a unique target gene coding for the adhesion-like bovine protein (adlb). The inclusivity of the new target gene was 97% and the exclusivity 98%, meaning that other genotypes and bacterial species could be excluded with high reliability. The limit of detection of the new assay was 235 staphylococcal cell equivalents/mL of culture. The new test shows high intra- and interassay repeatability. Results are available within 2 d after sampling, allowing farmers and veterinarians to apply sanitation measures immediately. Based on the results of a preliminary field study, the diagnostic sensitivity and specificity of the new qPCR assay are 99 and 100%, respectively. The new analytical procedure is straightforward and can be applied for routine diagnostics.

Maternal mortality in Serbia.

Maternal mortality related to obstetric events is still high today. The main components of reproductive health are evaluated. Early diagnosis of the obstetric risks can significantly reduce maternal mortality.

Validation of liquid chromatographic method for analysis of lidocaine hydrochloride, dexamethasone acetate, calcium dobesilate, buthylhydroxyanisol and degradation product hydroquinone in suppositories and ointment.

In this paper, there was developed a sensitive, precise and accurate reversed-phase liquid chromatographic (RP-HPLC) method and validated for simultaneous determination of lidocaine hydrochloride, dexamethasone acetate (DA) and calcium dobesilate (CD) in suppositories and ointment. Also there was achieved a parallel analysis of buthylhydroxyanisol, as a preservative, and hydroquinone, as a degradation product of calcium dobesilate, present in these dosage forms. The relative standard deviation (RSD) values for all five compounds indicated a good precision and accuracy of the RP-HPLC method. Method is selective, sensitive and reproducible with good recovery values and can be applied in simultaneous determination of all mentioned compounds.

Lipoprotein(a) in patients with carotid atherosclerosis and ischemic cerebrovascular disorders.

The aim of this study was to evaluate the role of lipoprotein(a) in the carotid atherosclerosis and ischemic cerebrovascular disorders. Four groups of subjects were included: 49 patients with transient ischemic attacks, 61 with acute cerebral infarction, 48 with asymptomatic carotid atherosclerosis and a group of 50 healthy subjects. Lipoprotein(a) serum concentration and its correlation with various clinical parameters was analysed. The results revealed a significant difference between lipoprotein(a) serum levels in patients and healthy subjects and positive correlation between lipoprotein(a) serum levels and the degree of carotid atherosclerosis. Moreover, some other correlations are found and discussed. It was concluded that lipoprotein(a) is involved in the pathogenesis of carotid atherosclerosis, but it is not associated with the development of ischemic cerebrovascular disorders.

Application of experimental design to the development of an HPLC method for the analysis of ochratoxin A in Triticum aestivum grain.

Ochratoxin A is a mycotoxin, a natural product of Aspergillus and Penicillium species. It can be present in grain from Triticum aestivum, (Graminae) and other starch-abundant cereals. This paper describes the investigation of ochratoxin A in grain from Triticum aestivum using a statistically optimized HPLC method. The assay was developed using two mathematical statistical models: factorial design and response surface mapping. The final step was to optimize the values of variables by response surface design. The analysis of variance 'ANOVA' method was applied to the analytical results in order to construct an adequate model. The optimal experimental conditions obtained by the response surface diagram method were: pH = 2.5, composition of the mobile phase acetonitrile: water 55:45 v/v and flow rate 1.0 ml/min. with a C18 column. Retention time and capacity factor for ochratotoxin A were 7.46 min. and 1.19, respectively.

[Lumbar spine fractures]. / Prelomi slabinske kicme.

In Special Hospital for orthopaedy, neurosurgery and neurology "Vaso Cukovic" in Risan, during last 30 months, 51 patients with lumbar spinal fractures were treated. Surgical intervention was performed in 12% of the patients while 88% of them were treated by postural reduction. In the moment of arrival, neurological deficit was noticed in ten patients, and after the treatment three of them had a significant improvement of the function. During the treatment we had one exitus as a consequence of the polytrauma. The authors analyse the results of the treatment favorising early mobilisation in compressive lumbar spinal fractures and surgical intervention in the patients with neurological deficit.

[Changes in N-acetyl-beta-D-glucosaminidase in the urine of patients with pyelonephritis and glomerulonephritis]. / Promena aktivnosti N-acetil-beta-D glukozaminidaze u urinu pacijenata obolelih od pijelonefritisa i glomerulonefritisa.

The activity of N-acetyl-beta-D-glucosaminidase (NAGase) activity was determined in urine of patients with pyelonephritis and glomerulonephritis. Determination was done with the non-commercial "Boehringer-Mannheim" test reagents for research purposes only. The protein concentrations were measured in the same samples. The obtained results were compared with results obtained in the control group and the significant increase (p less than 0.001) in NAGase activity was found in both groups of patients. At the same time normal protein values were found in 33% of all cases. It can be concluded that NAGase is a more sensitive parameter for early detection of renal disease.

Determination of platelet monoamine oxidase by new continuous spectrophotometric method.

A simple, continuous spectrophotometric method for the determination of tissue monoamine oxidase based on the oxidation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) using peroxidase has already been described (Ivanovic, I. & Majkic-Singh, N. (1986) Fresenius Z. Anal. Chem. 324, 307). In the present study the method is optimised for platelet monoamine oxidase assay and applied to healthy persons and schizophrenic patients. The obtained data were statistically analysed. The continuous ABTS method is sensitive, precise (CV below 6.9%) and linear up to 83 U/g protein. Comparison with the end-point method of Szutowicz et al. (1984) Anal. Biochem. 138, 86-94) gave a good correlation (r = 0.983). The reference values for the activity of human platelet monoamine oxidase by the new continuous ABTS method are 25 to 42 U/g protein (means = 33.2 U/g protein, CV = 15.5%, n = 67). No differences were found between females and males, or between three age groups ranging from 21 to 52 years. The patients with chronic (n = 76) or acute (n = 17) schizophrenia had significantly lower monoamine oxidase activities compared with normal values (p less than 0.005), which indicates that platelet monoamine oxidase can be a possible marker for schizophrenic diseases.

[Hemoglobin components in normal and anemic b/b rats]. / Komponente hemoglobina u normalnih i anemicnih b/b pacova.

The analitical comparison of normal and anemic b/b rat hemoglobins was made by polyacrylamide gel electrophoresis (PGE) nad also by DEAE-cellulose chromatography. Six different hemoglobin components constitute total hemoglobin of normal rat. The component six of total hemoglobin (Hb VI) of controls was absent from the hemolysates of anemic animals. This could be demonstrated either by PGE or by DEAE-cellulose chromatography. Hb V was also missing to a verying degree in some of anemic animal.

Rat b/b anemia: translation of normal and anemic globin mRNA in wheat-germ cell-free system.

Globin mRNAs were isolated from circulating reticulocytes both from rats carrying a homozygous, recessive mutation causing a severe thalassemia-like syndrome and from normal rats. After first identifying the rat globin chains as alpha or beta chains, the translational products primed by both polysomal and nonpolysomal mRNAs in wheat germ 30000 x g supernatant were analyzed: the ratio of alpha to beta globin mRNAs found in polysomes isolated from mutant rats is identical to the ratio of their products synthesized in vivo while the ratio of these mRNAs is quite different in the nonpolysomal fraction, the latter being enriched in alpha globin mRNA. No difference is found in the ratio of alpha and beta globin mRNAs in the polysomal and nonpolysomal RNA isolated from normal rats, both being identical to the ratio of their products synthesized in vivo. One third of the total amount of mRNA found in mutant cells is not in polysomes as compared to only 6 percent for the mRNA from normal lysates. These results suggest that a translational control mechanism is involved although the decreased globin synthesis in b/b anemia can not be fully accounted for by its operation.

[Changes in serum enzyme values as possible indicators of the severity of blast injuries]. / Promene vrednosti enzima u serumu kao moguci pokazatelj tezine blast povrede.

In order to detect biochemical parameters in the peripheral blood which would serve as an indicator of severity of blast injuries, the enzyme dynamics (creatine kinase, aspartat transaminase, alanin transaminase, lactic dehydrogenase, alkaline phosphatase, angiotensin convertase) has been followed up in the serum of sheep. The significant increase was found in the values of creatine kinase, aspartat transaminase and alanine transaminase in the serum of the experimental animals an hour after effects of the blast wave. The authors have concluded that changes of the mentioned enzymes can serve as one of signs of damages of the lungs and gastrointestinal tract caused by blast wave in the early posttraumatic period.

Involvement of the central nervous system in the general response to pulmonary blast injury.

The local, general, and cerebral responses of rabbits exposed to pulmonary blasts were examined to define the role of vagal afferentation in cardiorespiratory as well as metabolic control after a blast injury. Two series of experiments were conducted on rabbits to analyze the general, local, and cerebral responses to pulmonary injury caused by blast overpressure, and to evaluate the effects of bilateral vagotomy on the general, local, and cerebral responses to local (pulmonary) blast injury. The blast wave was generated in laboratory conditions using an air-driven shock tube that was able to cause moderate pulmonary blast injury, i.e., four pulmonary contusions characterized as confluent ecchymoses involving 30 to 60% of the lungs. One group of animals was subjected to pulmonary deafferentation, performed by bilateral transections of the vagus, glossopharyngeal, and hypoglossal nerves. Numerous hemodynamic as well as biochemical parameters were observed in systemic circulation and in lung and brain (medulla oblongata) tissues. After observation during the early posttraumatic period, rabbits were sacrificed by decapitation 30 minutes after the blast injury. On the basis of obtained results, it was concluded that vagal afferents have an important role in the modification of general and local responses to a pulmonary blast injury. Furthermore, it was suggested that functional changes in medulla oblongata may be the consequences of afferent neural impulses from the injured region (lungs) rather than consequences of ischemia, energy transfer to the brain, or both.

Leukotrienes in the pathogenesis of pulmonary blast injury.

Our previous studies demonstrate a significant increase of sulfidopeptide leukotriene concentrations in animals exposed to a free air blast. The aim of this study was to analyze the role of leukotrienes in the local response of lung tissue as well as in the general response of organisms to blast overpressure. The study was conducted on adult rabbits exposed to moderate blast overpressure (four pulmonary contusions characterized as confluent ecchymoses involving 30 to 60% of the lungs), generated in laboratory conditions. One group of experimental animals was treated with 5-lipoxygenase (5-LO) inhibitor, diethylcarbamazine (DEC, Sigma, St. Louis, Missouri) (50 mg/kg, i.v.), immediately before blast. The early posttraumatic period was observed (30 minutes after blast). Hemodynamic parameters (mean arterial pressure, heart rate, blood gases) as well as arterial plasma levels of conjugated dienes were observed. The myeloperoxidase activity, lipid peroxidation products levels, and water contents were measured in the lung tissue of injured rabbits. We observed that 5-LO inhibition reduced edema formation, accumulation of neutrophils, and generation of lipid peroxidation products in injured lungs. In this study, we demonstrated that treatment with DEC inhibits the increased systemic generation of conjugated dienes after blast injury. Although DEC exerts local antioxidant activity with beneficial effects on lung tissue, this 5-LO inhibitor intensifies the blast overpressure caused hemodynamic insufficiency.