Evaluation of quality changes in udder quarter milk from cows with low-to-moderate somatic cell counts

Much emphasis has been put on evaluating alterations in milk composition caused by clinical and subclinical mastitis. However, little is known about changes in milk composition during subclinical mastitis in individual udder quarters with a low-to-moderate increase in milk somatic cell count (SCC). This information is needed to decide whether milk from individual udder quarters with a moderate-to-high increase in milk SCC should be separated or not. The aim of this study was to determine how milk composition in separate udder quarters is affected when cow composite milk has low or moderately increased SCC levels. Udder quarter and cow composite milk samples were collected from 17 cows on one occasion. Milk yield was registered and samples were analyzed for SCC, fat, total protein, whey proteins, lactose, citric acid, non-protein nitrogen (NPN), lactoferrin, protein profile, free fatty acids (FFAs), lactate dehydrogenase (LDH), proteolysis, sodium and potassium. Bacteriological samples were collected twice from all four quarters of all cows. The cows were divided into three groups depending on their SCC at udder quarter level. The first group comprised healthy cows with four udder quarters with low SCC, <50 000 cells/ml; composition was equal when opposite rear and front quarters were compared. In the second and the third groups, cows had one udder quarter with 101 000 cells/ml < SCC < 600 000 cells/ml and SCC > 700 000 cells/ml, respectively. The remaining udder quarters of these cows had low SCC (<100 000 cells/ml). Despite the relatively low average cow composite SCC = 100 000 cells/ml of Group 2, milk from affected udder quarters exhibited lower casein number, content of lactose and β-casein (β-CN), while the content of whey protein, sodium, LDH and α-lactoalbumin (α-la) were higher compared to healthy opposite quarters. In addition to these changes, milk from affected udder quarters in Group 3 also exhibited lower values of potassium and αs1-casein (αs1-CN) and higher values of lactoferrin when compared to milk from opposite healthy quarters. This indicates that even when the SCC in cow composite milk is low, there might exist individual quarters for which milk composition is changed and milk quality impaired.     

An analysis of the genetic relationship between udder health and udder conformation traits in South African Jersey cows

A multi-trait animal model was used to estimate genetic parameters among lactation somatic cell score (SCS) and udder-type traits in South African Jersey cattle, through restricted maximum likelihood (REML) procedures. Data comprised records on 18 321 Jersey cows in 470 herds, collected through the National Milk Recording Scheme from 1996 to 2002. Average SCS in the first three lactations (SCS1, SCS2 and SCS3) were considered as different traits and the udder-type traits were fore udder attachment (FUA), rear udder height (RUH), rear udder width (RUW), udder cleft (UC), udder depth (UD), fore teat placement (FTP), rear teat placement (RTP) and fore teat length (FTL). Heritability estimates for the respective lactation SCS were 0.07 ± 0.01, 0.11 ± 0.01 and 0.11 ± 0.02. Udder-type traits had heritability estimates ranging from 0.14 ± 0.01 for UD to 0.30 ± 0.02 for FTL. Genetic correlations between SCS and udder-type traits ranged from -0.003 ± 0.07 between FUA and SCS3 to -0.50 ± 0.07 between UD and SCS3. Slow genetic progress is expected when selection is applied independently on SCS and udder-type traits, due to the generally low heritabilities. Tightly attached shallow udders with narrowly placed rear teats are associated with low SCS in the Jersey population.     

Kill rate of mastitis pathogens by a combination of cefalexin and kanamycin

Aim: To assess the bacterial killing rate produced by a combination of cefalexin and kanamycin at two different concentration ratios. Methods and Results: Time–kill kinetics of cefalexin and kanamycin, individually and in combination, were determined against one strain each of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. The combination was tested using two fixed ratios (cefalexin : kanamycin ratios of 1·25 : 1 and 1 : 2·3) and two concentrations of each ratio. Time–kill curves produced with either ratio were quite similar. Against most bacterial species, higher concentrations produced faster kill. In all cases, the combination of cefalexin and kanamycin showed faster and greater kill at lower antibiotic concentrations than those observed with either drug alone. Conclusions: The combination of cefalexin and kanamycin results in a fast initial killing of major mastitis pathogens at both concentration ratios. Significance and Impact of the Study: The combination of cefalexin and kanamycin achieved rapid bacterial kill at concentrations and ratios that can be achieved in vivo following intramammary infusion of a mastitis treatment.     

Molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Egypt

The aim of this study was to characterize the genetic basis of multidrug resistance in Gram-negative bacteria isolated from bovine mastitis cases in Egypt. Multidrug resistance phenotypes were found in 34 of 112 (30.4%) Gram-negative bacterial isolates, which harbored at least one antimicrobial resistance gene. The most prevalent multidrug-resistant (MDR) species were Enterobacter cloacae (8 isolates, 7.1%), Klebsiella pneumoniae (7 isolates, 6.3%), Klebsiella oxytoca (7 isolates, 6.3%), Escherichia coli (5 isolates, 4.5%), and Citrobacter freundii (3 isolates, 2.7%). The most commonly observed resistance phenotypes were against ampicillin (97.0%), streptomycin (94.1%), tetracycline (91.2%), trimethoprim-sulfamethoxazole (88.2%), nalidixic acid (85.3%), and chloramphenicol (76.5%). Class 1 integrons were detected in 28 (25.0%) isolates. The gene cassettes within class 1 integrons included those encoding resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17, and dfrA25), aminoglycosides (aadA1, aadA2, aadA5, aadA7, aadA12, aadA22, and aac(3)-Id), chloramphenicol (cmlA), erythromycin (ereA2), and rifampicin (arr-3). Class 2 integrons were identified in 6 isolates (5.4%) with three different profiles. Furthermore, the β-lactamase encoding genes, bla(TEM), bla(SHV), bla(CTX-M), and bla(OXA), the plasmid-mediated quinolone resistance genes, qnr and aac(6)-Ib-cr, and the florfenicol resistance gene, floR, were also identified. To the best of our knowledge, the results identified class 2 integrons, qnr and aac(6)-Ib-cr from cases of mastitis for the first time. This is the first report of molecular characterization for antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Africa.     

The effect of out-wintering pad design on dirtiness score, somatic cell score and mastitis incidence in dairy cows

This study aimed to compare three woodchip out-wintering pad (OWP) designs, and indoor cubicle housing with regard to cow dirtiness scores during the winter housing period, and udder health during both the winter period and the following lactation, for spring-calving dairy cows. The treatments were: an uncovered (UP) and covered (CP) OWP with a concrete feed apron; an uncovered OWP with self-feed silage pit provided directly on the woodchips (SP); and indoor cubicle housing (IC). Data were compared during 2 years: year 1 was a case study while year 2 was an experimental study. In year 1, treatments were UP (space allowance = 12 m2/cow), CP (6 m2/cow) and IC. In year 2, all three OWP designs (12 m2/cow) were compared with IC. Animals were assigned to treatments at the end of lactation in the autumn, and remained there while dry until calving the following spring. Subsequently, all cows were at pasture during lactation. Outcome measures for analysis were cow dirtiness score, somatic cell score (SCS) and incidence of clinical mastitis during the dry period and during lactation. Quarter milk samples were also taken at drying off, calving and 3 weeks post partum both years, and at approximately 113 days in milk in year 2. Samples were analysed for presence of mastitis-causing agents and SCS was determined. Sub-clinical mastitis was diagnosed when cows had an SCS greater than 200 000, or California mastitis test greater than 1 in at least one quarter. In year 1, cows in CP were dirtier than cows in the other two treatments. These animals also had the highest SCS during lactation and tended to have more mastitis-causing agents isolated from quarter milk samples. In year 2, when all cows were stocked at the same density, cows in the sheltered OWP (i.e. CP) had similar dirtiness scores to cows in cubicles and significantly lower dirtiness scores than cows in the unsheltered OWP designs, i.e. UP and SP. However, there were no effects on SCS or quarter sample results. Cleaning of OWP's stocked at 12 m2/cow reduced cow dirtiness scores. However, cleaning of CP in year 1 when cows were stocked at 6 m2/cow had no effect on dirtiness scores. We conclude that dry cows stocked at 12 m2/cow on OWP's are unlikely to have udder health problems in the subsequent lactation. Furthermore, provision of shelter and cleaning of the woodchips are management factors that help to keep cows clean on OWP's.     

Impact of subclinical and clinical mastitis on sensitivity to pain of dairy cows

A total of 90 cows from three commercial farms were used to evaluate the relationship between subclinical mastitis and clinical mastitis and thermal nociceptive threshold. Milk strips from all udder quarters were tested for clinical mastitis with visual inspection of milk and udder alterations and for subclinical mastitis using California Mastitis Test. Milk yield was recorded, milk was sampled and further analyzed for somatic cells count (SCC). Cows were considered healthy when SCC<200 000 cells/ml and no visual alterations in milk and/or udder, with mild subclinical mastitis when SCC>200 000 cells/ml and no visual alterations in milk and/or udder, with moderate subclinical mastitis when SCC>500 000 cells/ml and no visual alterations in milk and/or udder and with clinical mastitis when visual alterations in milk and/or udder were detected. Nociceptive threshold was evaluated with the thermal threshold meter apparatus applied to the rear legs. Thermal threshold (TT) decreased when we compared healthy cows with cows presenting clinical mastitis and tended to decrease when we compare healthy cows with those with moderate subclinical mastitis. TT was lower at the ipsilateral rear leg compared with the contralateral leg to the infected mammary gland. TT linearly decreases as _log10SCC increased and it showed sharp decrease as _log10SCC exceed the value of 6.4. Increase in one unit of _log10SCC increased the odds of low thermal threshold (lower than 55.8°C). Subclinical mastitis might be a welfare issue as it tended to decrease nociceptive thermal threshold.     

临床分离4544株革兰阴性杆菌的耐药分布与变迁

目的了解玉溪市革兰阴性杆菌的耐药分布与变迁,为临床用药提供依据。方法对玉溪市人民医院1999—2004年临床各科送检的各类标本中培养分离出的4544株革兰阴性杆菌作回顾性分析。结果9种（属）细菌对25种药物的药敏结果耐药率〉50％者72种次（40．9％）,〈20％者44种次（25．0％）。总的耐药情况为不动杆菌和阴沟肠杆菌最高,铜绿假单胞菌和大肠埃希菌次之,甲型副伤寒沙门菌和福氏志贺菌最低。亚胺培南对多种细菌有较高的敏感覆盖率。有4种细菌对10种抗菌药物耐药率的上升具有临床意义（P〈0.01或P〈0．05）,其中以甲型副伤寒沙门菌对氟喹诺酮类上升最为显著。结论临床分离革兰阴性杆菌的耐药形势十分严峻,应定期监测区域内细菌的耐药变化．指导临床合坪用药。     

Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis

In the present study 79 streptococcal cultures isolated from subclinical mastitis of 54 cows from seven dairy farms (A-G) in Hesse, Germany, were comparatively investigated using conventional and molecular methods. The isolates could be identified as Streptococcus agalactiae, belonging to Lancefield's serological group B by determination of cultural, biochemical and serological properties and by polymerase chain reaction (PCR)-mediated amplification of species-specific parts of the 16S ribosomal DNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor gene cfb. The investigated group B streptococci were further characterized serologically for specific polysaccharide and protein antigens. Serotyping the isolates revealed a predominance of surface protein antigen X, either alone or in combination with polysaccharide antigen Ia. This could be observed for 39 isolates of farms A, B and C. Six group B streptococci from farm E displayed the serotype pattern III/Rib, two isolates from farm G showed the serotype pattern Ib/calpha. The remaining cultures from farms D and F (n=32) were non-typable. The occurrence of protein Rib could be confirmed by PCR amplification of the gene rib. The two isolates with serotype pattern Ib/calpha also reacted positively for the cbeta-encoding gene bag. Additional properties which allowed a phenotypic characterization of the S. agalactiae were the degree of pigmentation, growth properties in fluid media and soft agar, the surface hydrophobicity, the ability to hemagglutinate rabbit erythrocytes and their resistance reactions to tetracycline and minocycline. The isolates of the seven farms showed identical or almost identical characteristics. The 79 group B streptococci were additionally investigated by macrorestriction analysis of their chromosomal DNA using the restriction endonucleases SmaI, ApaI and SalI. The restriction patterns obtained by pulsed-field gel electrophoresis displayed identical or closely related patterns for the cultures of the various farms. The pheno- and genotypic characteristics of the 79 group B streptococci of the present study revealed that a single S. agalactiae strain or at least closely related subtypes of this strain were responsible for the mastitis situation of the seven farms.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows’ mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down‐regulation and location of bta‐miR‐26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real‐time PCR, in situ hybridization and dual‐luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta‐miR‐26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.     

Lipopolysaccharide challenge of the mammary gland in cows induces nitrosative stress that impairs milk oxidative stability

The aim of this work was to study the effects of mastitis induced by intramammary lipopolysaccharide (LPS) challenge on milk oxidative stability, as well as to understand the underlying biochemical processes that cause such changes. LPS challenge was associated with nitric oxide burst from the surrounding mammary epithelial cells and consequently induced nitrosative stress that was induced by the formation of NO₂• from nitrite by lactoperoxidase. This response was associated with an ∼3-fold increased formation of hazardous compounds: nitrotyrosines, carbonyls and lipid peroxides. We sustained the involvement of xanthine oxidase as a major source of hydrogen peroxide. In consistent with previous findings, catalase has been shown to play a major role in modulating the nitrosative stress by oxidizing nitrite to nitrate. The current hygienic quality criteria cannot detect mixing of low-quality milk, such as milk with high somatic cells, and nitrite with high-quality milk. Thus, development of an improved quality control methodology may be important for the production of high-quality milk.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

Developmental trajectory of the healthy human gut microbiota during the first 5 years of life

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亚胺培南临床用药与非发酵菌群分离率和耐药性的关系

目的了解亚胺培南临床用药和非发酵菌群分离率与耐药性变迁的关系,有利于非发酵菌感染的预防与治疗。方法统计分析浙江省人民医院2000～2003年临床分离非发酵菌的菌株分布、与亚胺培南的临床用药的相关性及耐药率。结果4年来非发酵菌的分离以铜绿假单胞菌(30．7％)、乙酸钙-鲍曼不动复合杆菌(19．％)及嗜麦芽糖寡养单胞菌(19．1％)为主．其分离率与亚胺培南用药存在相关性。对各菌属的主要菌种进行耐药性分析,嗜麦芽糖寡养单胞菌、脑膜脓毒性金黄杆菌和洋葱伯克霍尔德菌的耐药率极高,铜绿假单胞菌和乙酸钙-鲍曼不动复合杆菌的耐药率呈现明显升高的趋势。结论非发酵菌的检出率与亚胺培南使用关系密切,耐亚胺培南的菌株迅速升高。临床应合理使用抗生素,并加强对耐药菌株的监控。     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

山西省健康人群的肠道菌群组成特征

目的探索山西省健康人群的肠道菌群组成特征及性别和年龄对肠道菌群组成的影响。方法应用16S rRNA基因测序技术,对山西省99名健康个体的粪便细菌DNA进行测序分析。结果山西省健康人群的肠道菌群在属水平分为两个集群,相对丰度最高的分别是拟杆菌属和普氏菌属;在区分这两个集群中,拟杆菌属与普氏菌属的曲线下面积（AUC）分别是0.97、1.00。男性组与女性组的物种丰富度（richness）和多样性（diversity）差异都无统计学意义（均P>0.05）,基于Bray-Curtis距离的主坐标分析（PCoA）图显示两组人群的样本分布没有明显分离（相似性分析：r=-0.0296,P>0.05）,LEfSe分析显示与性别分组有关的细菌很少。30-39岁、40-49岁和50-59岁三组人群的物种丰富度和多样性差异都无统计学意义（均P>0.05）,基于Bray-Curtis距离的PCoA图显示三组人群的样本分布没有明显分离（相似性分析：r=0.0109,P>0.05）,LEfSe分析显示几乎没有与年龄分组有关的细菌。结论山西省健康人群的肠道菌群更倾向分为两种肠型（拟杆菌型和普氏菌型）。性别对肠道菌群组成可能没有显著影响,30-59岁人群的肠道菌群组成比较稳定