非哺乳期乳腺炎发病高危因素的病例-对照研究

目的:分析非哺乳期乳腺炎的临床特点及发病高危因素。方法:回顾性分析2011年1月至2016年1月秦皇岛市第一医院乳腺外科就诊,经病理确诊的非哺乳期乳腺炎患者110例(病例组)资料,另选取秦皇岛市第一医院体检中心的健康人群110例作为对照组。比较两组研究对象临床特征,并进行单因素及多因素分析。结果:病例组病人临床分型以肿块型(46.36%)为主,初诊主要症状为单纯肿块39例(35.45%),乳房红肿伴肿块37例(33.64%)。两组在生育次数、肥胖、初育年龄、平均哺乳时间、主动/被动吸烟、乳头内陷、失眠/焦虑、服用避孕药、乳头溢液病史方面比较差异有统计学意义(P0.05)。多因素分析显示乳头内陷(OR=5.29,95%CI 2.12~13.25,P=0.000)、失眠/焦虑(OR=4.20,95%CI 1.68~10.48,P=0.002)为非哺乳期乳腺炎发病高危因素,而生育次数(OR=0.42,95%CI 0.27~0.64,P=0.001)为该病的保护因素。结论:失眠、焦虑及乳头内陷增加该病发病风险,生育次数多可降低该病的发病风险。     

Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132^T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes.Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132^T (=DSM 111922^T, = CCOS 1972^T).     

Failure costs associated with mastitis in smallholder dairy farms keeping Holstein Friesian × Zebu crossbreed cows

Mastitis is a costly disease and in many areas of the world, these costs have been quantified to support farmers in their decision making with regard to prevention of mastitis. Although for subsaharan circumstances estimates have been made for the costs of subclinical mastitis (SCM), farm-specific cost estimations comprising both clinical mastitis (CM) and SCM are lacking. In this paper, we quantified failure costs of both CM and SCM on 150 Ethiopian market-oriented dairy farms keeping Holstein Friesian × Zebu breed cows. Data about CM were collected by face-to-face interviews and the prevalence of SCM was estimated for each farm using the California mastitis test. All other relevant information needed to calculate the failure costs, such as the consequences of mastitis and price levels, was collected during the farm visits, except for the parameter for milk production losses due to SCM, which was based on literature estimates and subjected to sensitivity analyses. The average total failure costs of mastitis was estimated to be 4 765 Ethiopian Birr (ETB) (1 ETB = 0.0449 USD) per farm per year of which SCM contributed 54% of the costs. The average total failure costs per lactating cow per farm per year were 1 961 ETB, with a large variation between farms (range 0 to 35 084 ETB). This large variation in failure costs between farms was mainly driven by variation in incidence of CM and prevalence of SCM. Milk production losses made the largest contribution (80%), whereas culling contributed 13% to 17% to the total failure costs. In our estimates, costs of veterinary services, drugs, discarded milk and labour made a minor contribution to the total failure costs of mastitis. Relative to the income of dairy farmers in North Western Ethiopia; the total failure costs of mastitis are high. In general, Ethiopian farmers are aware of the negative consequences of CM, but creating awareness of the high costs of SCM and showing large variation between farmers may be instrumental in motivating farmers to also take preventive measures for SCM.     

New approaches for risk assessment and management of bovine protothecosis

Protothecosis is a potential zoonosis related to bovine mastitis. In several countries, a higher incidence of protothecal bovine mastitis that is being recorded and the resistance of Prototheca species to various factors (chlorine, high temperatures, antimicrobial and antiseptic treatments, pH variations), make it difficult to control its spread among farms. The authors aim to describe the infection caused by microalgae, focusing on the problems within cattle farms and proposing new approaches to farm management, based on Regulation (EU) No 2016/429 on transmissible animal diseases. This new flexible approach, based on risk analysis, is a further tool in protecting against Prototheca species. The list of transmissible animal diseases under Regulation (EU) No 2016/429 includes those caused by microorganisms resistant to antimicrobials, which can have important implications for human and animal health, feed and food safety. This approach would involve a series of changes to the rules used for Official Controls (Regulation (EU) No 2017/625) moving from the concept of the food chain to that of the agri-food chain.     

Incidence of Subclinical Mastitis and Prevalence of Major Mastitis Pathogens in Organized Farms and Unorganized Sectors

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10⁵/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.     

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.     

A study on associaton between mastitis and serologically defined class I bovine lymphocyte antigens (BOLA-A) in Norwegian cows

A total of 102 cows was tested for class I antigens of the bovine major histocompatibility complex. Half of the animals (51) had completed at least four lactations without any veterinary treatment for mastitis. The distribution of BoLA-A antigens among these relative mastitis-resistant cows was compared to that in the other half of the material (51), which comprised animals with at least one recorded treatment for mastitis. There were no statistically significant differences in BoLA-A antigen frequency between cows with mastitis and cows without mastitis. The two most common antigens were A2 and w16. The frequency of these two antigens deviated from earlier estimates within the Norwegian cattle (NRF) population, the difference for w16 being statistically significant.     

Longitudinal random effects models for genetic analysis of binary data with application to mastitis in dairy cattle

A Bayesian analysis of longitudinal mastitis records obtained in the course of lactation was undertaken. Data were 3341 test-day binary records from 329 first lactation Holstein cows scored for mastitis at 14 and 30 days of lactation and every 30 days thereafter. First, the conditional probability of a sequence for a given cow was the product of the probabilities at each test-day. The probability of infection at time t for a cow was a normal integral, with its argument being a function of "fixed" and "random" effects and of time. Models for the latent normal variable included effects of: (1) year-month of test + a five-parameter linear regression function ("fixed", within age-season of calving) + genetic value of the cow + environmental effect peculiar to all records of the same cow + residual. (2) As in (1), but with five parameter random genetic regressions for each cow. (3) A hierarchical structure, where each of three parameters of the regression function for each cow followed a mixed effects linear model. Model 1 posterior mean of heritability was 0.05. Model 2 heritabilities were: 0.27, 0.05, 0.03 and 0.07 at days 14, 60, 120 and 305, respectively. Model 3 heritabilities were 0.57, 0.16, 0.06 and 0.18 at days 14, 60, 120 and 305, respectively. Bayes factors were: 0.011 (Model 1/Model 2), 0.017 (Model 1/Model 3) and 1.535 (Model 2/Model 3). The probability of mastitis for an "average" cow, using Model 2, was: 0.06, 0.05, 0.06 and 0.07 at days 14, 60, 120 and 305, respectively. Relaxing the conditional independence assumption via an autoregressive process (Model 2) improved the results slightly.     

我国奶牛乳房炎致病性金黄色葡萄球菌血清型分布

本试验采用玻片凝集法对从临床型乳房炎病乳中分离获得的56株金黄色葡萄球菌进行血清型分型。结果表明336型占67.9%(38/56),5型占7.1%(4/56),8型占3.6%(2/56)。     

Evaluation of the relative expression of genes associated with adherence after different hours of co-culture between Streptococcus uberis and MAC-T cells

Streptococcus uberis is an environmental pathogen associated with subclinical and clinical IMI in both lactating and non-lactating cows. RC19 strain was isolated from a cow with subclinical mastitis, qualitatively classified as moderate biofilm producer in Todd Hewitt medium (THB), and it showed a high value of the adhered bacteria (CFU/ml). Hence, the aims of this study were (a) to determine ability to adhere to and internalize into epithelial cells MAC-T for 1, 2 and 3 h, (b) to evaluate the relative expression of adherence-associated genes from co-cultures of S. uberis with MAC-T cells at 1, 2 and 3 h. We hypothesized that upon contact with bovine mammary epithelial cells, S. uberis upregulates adherence-associated genes encoding adhesins, which enable it a higher adherence to and/or internalization into host cells. Four to six genes increased their R with regard to the control after initial contact with MAC-T cells (group 1) at 1, 2 and 3 h. The highest value of R was observed at 2 h after co-culture between RC19 and MAC-T cells.     

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.     

Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections

AIMS: The aim of this study was to isolate and characterize bacteriophages against bovine Staphylococcus aureus associated with mastitis. METHODS AND RESULTS: We describe the isolation of two anti-staphylococcal phages namely DW2 and CS1 from farmyard slurry. Both phages were characterized by electron microscopy and restriction analysis and shown to belong to the Siphoviridae family. CS1 and DW2 were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. These phages were compared with the previously characterized Myoviridae phage K. Infusion of a cocktail of all three phages at 10(8) PFU ml(-1) into live cow teats resulted in no detectable increase in somatic cell counts in milks indicating that the phages did not irritate the animal. CONCLUSION: Two new anti-staphylococcal phages CS1 and DW2 were isolated and characterized and tested for immunogenicity in animal teats. SIGNIFICANCE AND IMPACT OF THE STUDY: The phages isolated in this study are active against pathogenic S. aureus and may be incorporated into teat-dips or teat-washes as a non-antibiotic prophylaxis against staphylococcal bovine mastitis.     

Experimental model of toxin-induced subclinical mastitis and its effect on disruption of follicular function in cows

This study establishes an experimental model for subclinical mastitis induced by Gram-positive (G+) exosecretions of Staphylococcus aureus origin or Gram-negative (G−) endotoxin of Escherichia coli origin to examine its effects on follicular growth and steroid concentrations in Holstein dairy cows. Cows were synchronized with the Ovsynch protocol followed by a series of follicular cycles that included GnRH and PGF_2α doses administered every 8 days. Cows received small intramammary doses of either G+ (10 μg, n = 10) or G− (0.5 μg, n = 6) toxin, or saline (n = 6; uninfected control) every 48 hours for 20 days. Follicular fluids were aspirated from preovulatory follicles before (aspiration one: control), at the end of (aspiration two: immediate effect), and 16 days after the end of (aspiration three: carryover effect) toxin exposure. During the 3 weeks of subclinical mastitis induced by G+ or G−, no local inflammatory signs were detected in the mammary gland and no systemic symptoms were noted: body temperatures of the treated cows did not differ from controls; plasma cortisol and haptoglobin concentrations were not elevated and did not differ among groups. Somatic cell count was higher in the treated groups than in controls, and higher in the G− versus G+ group. For analysis of reproductive responses, cows were further classified as nonaffected or affected based on an more than 20% decline in follicular androstenedione concentration in aspiration two or three relative to the first, control aspiration. Most G− (5/6) and 40% of G+ (4/10) cows were defined as affected by induced mastitis. An immediate decrease in the number of medium-size follicles was recorded on Day 4 of the induced cycle, toward the end of the 20-day mastitis induction, in the affected G+ compared with uninfected control group (1.0 ± 0.5 vs. 3.0 ± 0.4 follicles; P < 0.05); the affected G− and nonaffected G+ subgroups exhibited a similar numerical decline in the number of follicles. A carryover (but not immediate) decrease to 51% and 62% in follicular estradiol concentrations in G− affected group and G+ affected group was detected relative to controls (P < 0.05). The nonaffected G+ subgroup did not differ from its control counterparts. Based on the current experimental model, subclinical IMI induced by G+ or G− toxin disrupts follicular functions, and it seems that the ovarian pool of early antral follicles is susceptible to subclinical mastitis.     

microRNAs and the mammary gland: A new understanding of gene expression

MicroRNAs (miRNAs) have been identified in cells as well as in exosomes in biological fluids such as milk. In mammary gland, most of the miRNAs studied have functions related to immunity and show alterations in their pattern of expression during lactation. In mastitis, the inflammatory response caused by Streptococcus uberis alters the expression of miRNAs that may regulate the innate immune system. These small RNAs are stable at room temperature and are resistant to repeated freeze/thaw cycles, acidic conditions and degradation by RNAse, making them resistant to industrial procedures. These properties mean that miRNAs could have multiple applications in veterinary medicine and biotechnology. Indeed, lactoglobulin-free milk has been produced in transgenic cows expressing specific miRNAs. Although plant and animal miRNAs have undergone independent evolutionary adaptation recent studies have demonstrated a cross-kingdom passage in which rice miRNA was isolated from human serum. This finding raises questions about the possible effect that miRNAs present in foods consumed by humans could have on human gene regulation. Further studies are needed before applying miRNA biotechnology to the milk industry. New discoveries and a greater knowledge of gene expression will lead to a better understanding of the role of miRNAs in physiology, nutrition and evolution.     

Binding of bovine lactoferrin to Streptococcus agalactiae

Bovine lactoferrin is an iron-binding protein present in mammary gland secretions. The exposure of Streptococcus agalactiae to bovine lactoferrin resulted in the binding of this protein to all the 12 strains of bovine origin tested, and also, although to a lesser degree, to the five tested strains of human origin. The interaction of lactoferrin with one high-binding bovine strain (24/60, the prototype NT/X strain) was studied. Binding was time-dependent, dose-dependent, and saturable. The binding of lactoferrin was slightly affected by cultivation conditions, and appeared to be heat-stable. The binding of biotinylated lactoferrin was inhibited by unlabelled lactoferrin but not by bovine serum albumin.     

非哺乳期乳腺炎的临床治疗探讨

目的:观察抗生素联合泼尼松双联药物冲击方法治疗非哺乳期急性乳腺炎的临床疗效。方法:选取本院乳腺外科2016年6月至2017年6月收治的105例非哺乳期急性乳腺炎患者作为观察对象,依照诊治顺序将其随机分为观察1组、观察2组以及对照组,每组35例患者。对照组给予口服左氧氟沙星,0.5 g/次,qd;观察1组在对照组的治疗方法中加用醋酸泼尼松片强的松20mg,qd;观察2组在对照组的治疗方法中加用醋酸泼尼松片强的松40 mg,qd,三组均以14 d为一疗程。治疗1个疗程后,评价和比较各组治疗效果、血细胞数、好转时间、痊愈时、住院时间及不良反应的发生情况。结果:治疗后,观察1组、观察2组和对照组有效率分别为82.86%、97.14%和60.00%,观察2组有效率显著高于观察1组和对照组(P0.05)。三组患者治疗后白细胞,中性粒细胞,淋巴细胞均较治疗前明显下降(P0.05),且观察2组以上指标显著低于观察1组和对照组(P0.05)。观察2组的好转时间、痊愈时间及住院时间均明显短于观察1组和对照组(P0.05),而观察1组的好转时间、痊愈时间及住院时间明显短于对照组(P0.05)。观察1组出现1例轻微胃肠道反应,1例胸闷,不良反应总发生率为5.71%(2/35);观察2组出现1例轻微胃肠道情况,不良反应总发生率为2.86%(1/35);对照组2例患者发生胸闷,1例患者出现轻微胃肠道不适,1例患者出现眩晕,不良反应发生率为11.43%(4/35),三组不良反应发生率比较差异均无统计学意义(P0.05)。结论:口服抗生素联合泼尼松治疗非哺乳期急性乳腺炎患者能提高临床疗效,且加大泼尼松口服剂量可进一步提高临床有效率,控制炎性进展,缩短痊愈时间。     

Association of the M blood group system with bovine mastitis*

Associations of the 11 bovine blood group systems with mastitis were examined in Red Danish dairy cattle. The mastitis status was followed during three lactational periods. A significant effect of the M blood group system on mastitis incidence was observed in the first and second lactation periods and a lower frequency of mastitis is found among animals lacking the M' factor as compared to those having the M' blood group factor. The significance of these results are discussed in view of the close relation between the M blood group system and the bovine lymphocyte antigens (BoLA), and the expected effect of eliminating the M' gene from the breed is estimated. Among the remaining 10 blood group systems, the T' system was the only system showing an overall effect on mastitis, and only in first and third lactation. However, the T' system was inconsistent with regard to the effect of the T' gene on the various mastitis diagnoses.     

Isolation of <Emphasis Type="Italic">Candida</Emphasis> spp. from mastitic bovine milk in Brazil

The purpose of this study was to isolate yeast (Candida) from the quarter milk of cow udders from 37 dairy farms in Brazil and to identify the different species involved in mastitis. The samples were collected between October 2002 and February 2003. Two-hundred-and-sixty milk samples from cows with clinical and subclinical mastitis were examined. Milk samples were plated onto Blood agar, Mac Conkey agar and Sabouraud dextrose agar. Forty-five (17.3%) samples were positive for the genus Candida. The Candida species isolated were C. krusei (44.5%), C. rugosa (24.5%), C. albicans (8.9%), C. guilliermondii (8.9%), and others (13.2%). We also isolated Escherichia coli (26.5%), coagulase-positive Staphylococcus (25.0%), Streptococcus spp. (8.1%), Enterobacter spp. (8.1%), and other fungi (8.1%), among others.     

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk

AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. Significance and Impact of the Study: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk.     

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.