临沂市奶牛乳房炎病原菌的分离鉴定与药敏试验

目的为弄清临沂市奶牛乳房炎主要病原菌种类及其药物敏感情况,为科学防治本病提供依据。方法采集患乳房炎奶牛的乳样进行细菌分离鉴定,并对主要病原菌做药敏试验。结果从115份乳样中,分离出6种127株病原菌,其中金黄色葡萄球菌46株,占36.22%,无乳链球菌33株,占25.98%,停乳链球菌21株,占16.53%,大肠埃希菌17株,占13.39%,乳房链球菌8株,占6.3%,沙门菌2株,占1.57%;主要病原菌均对头孢喹诺和左氧氟沙星高度敏感。结论临沂市奶牛乳房炎主要致病菌为金黄色葡萄球菌、链球菌和大肠埃希菌。首选药物为头孢喹诺和左氧氟沙星。因此,治疗奶牛乳房炎应通过药敏试验,合理地选择药物。     

泰州市奶牛乳房炎调查及其病原菌的分离

奶牛乳房炎是奶牛场的常发病和难以防治的疾病,为进一步了解引起奶牛乳房炎的病原微生物,本研究通过对泰州市3个奶牛场572头泌乳牛的乳房炎发病率进行了调查并对乳房炎主要病原菌进行分离和鉴定。结果表明,奶牛临床型乳房炎的发病率为7.69%,隐性型乳房炎发病率为56.64%,乳区阳性率为33.8%,其中临床型:隐性=1:7.36。从65份确定患有乳房炎的奶样中共分离获得8种细菌、156株分离株,且引起乳房炎的主要病原菌是金黄色葡萄球菌和表皮葡萄球菌,其次是停乳链球菌、大肠杆菌、乳房链球菌和无乳链球菌,其检出率分别为42.31%、23.08%、8.97%、6.41%、5.13%和1.28%。该项调查结果初步明确了泰州市乳房炎发病情况,同时为进一步综合防治奶牛乳房炎和研制乳房炎治疗药物提供了科学依据。     

海金沙提取物体外抑菌性能研究

用M／C和纸片法考查了海金沙提取物对藤黄球菌、枯草芽孢杆菌、金黄色葡萄球菌和乙型溶血性链球菌的抑菌性能。结果表明：海金沙对4种受试菌株都有抑菌活性;37℃时,醇提物对乙型溶血性链球菌的抑菌效果最好,最大抑菌圈为8．5mm;42℃时,对藤黄球菌、金黄色葡萄球菌和枯草杆菌的抑菌效果最好,最大抑菌圈分别为21、13．2和6．5mm。pH值为7．6时,海金沙醇提物在对藤黄球菌、金黄色葡萄球菌、枯草杆菌和乙型溶血性链球菌的最大抑菌圈分别为9．1、8．2、9mm和11．3mm。海金沙水提物和醇提物对藤黄球菌、乙型溶血性链球菌、枯草芽孢杆菌、金黄色葡萄球菌的最低抑菌质量分数分别为25％、12．5％、12．5％、25％和3．12％、1．56％、6．25％、3．12％．     

新型奶牛乳房炎多联苗的研制及免疫力试验

目的：筛选出毒力较强的金黄色葡萄球菌、大肠杆菌、无乳链球菌作为制苗菌株,制成多联菌苗,预防及治疗奶牛乳房炎。方法：通过菌株的分离、纯化、鉴定试验及毒力试验获得毒力较强的菌株,制成金黄色葡萄球菌类毒素及菌体蛋白、大肠杆菌和无乳链球菌灭活菌苗,合理配伍制成多联菌苗,进行安全、异常毒性及免疫力试验。结果：所选金黄色葡萄球菌、大肠杆菌、无乳链球菌的最小致死量分别是3．0×10^8／ml、1．5×10^8／ml、9．0×10^8／ml,多联苗安全、异常毒性试验合格,对免疫组小白鼠保护率为83．3％。结论：该多联菌苗具有安全、无毒、高效的特点,可以用来预防及治疗奶牛乳房炎。     

黄花夹竹桃内生真菌抗病原细菌的初步研究

从植物黄花夹竹桃 (Thevetiaperuviana)的根、茎、叶、果实中分离出内生真菌 10 1株。以鼠伤寒沙门氏菌 (Salmonellatyphimurium)、肺炎链球菌 (Streptococcuspneumoniae)、乙型溶血链球菌 (Streptococcushemolyticus)、金黄色葡萄球菌 (Staphyllococcusaureus) 4种人类病原菌为指示菌 ,对其进行抑菌活性筛选 ,结果有 17株内生真菌对 1株或 1株以上人类病原菌有抑菌活性 ,其中平板抑菌圈直径大于 2 0mm的菌株有9株。具有抗菌活性的内生真菌分别来自木霉属 (Trichoderma)、曲霉属 (Aspergillus)等 9个属。     

矾冰纳米乳对临床常见病原菌体外抗菌活性的研究

研究纳米化提高白矾与冰片复合物体外抗菌活性的效果。分别采用琼脂扩散法、体外杀菌试验及试管稀释法,测定白矾与冰片O/W型复合纳米乳对临床常见病原菌的体外抑菌、杀菌效果及最低抑菌浓度(MIC),实验中以等浓度矾冰液作为对照。结果显示,矾冰纳米乳对金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌、铜绿假单胞菌、白假丝酵母菌的抑制及杀灭活性均明显强于矾冰液(P0.05)。矾冰纳米乳对金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌临床菌株MIC90值分别为1.02、2.04和2.04 mg/mL,均明显低于矾冰液的MIC90值(P0.05)。上述实验结果提示,矾冰纳米乳与矾冰液均有广谱体外抑菌及杀菌活性,白矾及冰片复合物纳米化可提高抗菌效果。     

黄连内生真菌的分离鉴定及抑菌活性研究CSCD

为研究四川省峨眉地区黄连内生真菌多样性及菌株抑菌活性和机制,发掘黄连内生真菌资源,本研究从川黄连植株分离鉴定14株黄连内生真菌,分属于2纲,5目,7科,11属。根状茎、茎、叶和须根中的内生真菌定殖率分别是27.8%、56.7%、52.5%和39.6%。14株黄连内生真菌对金黄色葡萄球菌均有抑菌效果,Cop S01、Cop L01、Cop FR01的抑菌圈直径超过35 mm,纸片扩散法测得3株内生真菌发酵液对金黄色葡萄球菌均有效,Cop L01抑菌圈直径大于20 mm,抑菌效果最好,其发酵产物最低抑菌浓度(MIC)为10 mg/mL,最低杀菌浓度(MBC)为11 mg/mL。初步探究Cop L01抑菌机制发现该菌株发酵产物破坏金黄色葡萄球菌细胞膜,引起胞内核酸及蛋白外泄,造成金黄色葡萄球菌死亡。本研究表明川黄连内生真菌具有丰富的多样性,部分菌株及其发酵产物对金黄色葡萄球菌均表现出较好的抑菌效果,预示着黄连内生真菌Cop L01具有产生新型抗菌活性物质的潜力,为后续深入开发这一资源宝库提供了新的菌种和思路。     

奶牛乳房炎源金黄色葡萄球菌耐药性研究进展

金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是奶牛乳房炎(Bovine mastitis)最主要致病菌之一。抗菌药物在奶牛乳房炎治疗中的作用无可替代,但随着严重依赖甚至过度使用抗菌药物,S.aureus耐药性等问题日益凸显,我国现阶段尤为严重。近年来,耐药菌株、耐药谱逐渐变宽,耐药水平不断提高,使其耐药性越来越强,耐药机制变得复杂,耐药基因种类繁多,导致乳房炎源S.aureus广谱耐药性和多重耐药性现象已经十分严重,甚至出现超级耐药细菌-耐甲氧西林金黄色葡萄球菌(Methicillin-resistant staphylococcus aureus,MRSA),为奶牛乳房炎治疗带来极大困难。S.aureus耐药性不仅对奶牛养殖业和乳房炎防治造成影响,同时直接或间接威胁人类健康。因此,监测S.aureus耐药性及变迁情况,探明其具体耐药机制是解决问题的关键。目前,S.aureus可通过产生修饰酶、改变抗菌药物的作用靶点、降低细胞壁的通透性等不同作用机制,对β-内酰胺类、氨基糖胺类、四环素类、大环内酯类、林可胺类和喹诺酮类等抗菌药物产生不同程度的耐药,但基因编码的分子耐药机制尚未明确。综述了奶牛乳房炎中S. aureus耐药性及其分子耐药机制,讨论了明确S.aureu耐药机制的意义及存在的问题,以期为奶牛乳房炎临床合理用药及有效防治提供思路和依据。     

越南槐内生真菌JXRPH-24的抗菌活性及其鉴定

以2株病原细菌金黄色葡萄球菌ATCC25923和耐甲氧西林金黄色葡萄球菌临床分离株为靶标菌,采用琼脂块法和改良的微量肉汤稀释法测定越南槐内生真菌JXRPH-24的抗菌活性;根据形态和分子特征鉴定该菌株的分类学地位。结果表明,该菌株的活菌块对金黄色葡萄球菌ATCC25923的抑菌圈直径与阳性对照相当;其代谢产物对这2株病原细菌的最小抑菌浓度均为10μg/mL,仅为阳性对照的16倍;基于形态和ITS序列分析未能鉴定该菌株。菌株JXRPH-24可能是一个潜在的新内生真菌,对参试金黄色葡萄球菌均具有强的抗菌活性,具有较大的开发应用潜力。     

濒危植物七子花内生真菌的分离鉴定及抗菌活性筛选

首先从濒危植物七子花中分离到38株内生真菌,综合运用形态特征和分子生物学技术,明确其分类地位。38株植物内生真菌分属于6个属和1个无孢子群,其中交链孢属和刺盘孢属为优势菌群,葡萄座腔菌属、镰刀菌属、赤霉菌属、拟茎点霉属和无孢子群为常见属。以大肠埃希菌、白色念珠菌、金黄色葡萄球菌为靶标菌对获得菌株发酵液粗提物进行抑菌试验,发现95%以上的菌株对试测细菌具有一定的抗菌活性,表明七子花内生真菌的抗菌活性十分普遍;其中QZHⅡ10对大肠埃希菌具有较好的抑菌活性,抑菌圈直径为12.0 mm;而QZHⅡ07则对金黄色葡萄球菌的抑菌活性较强,抑菌圈达到15.5 mm。这表明七子花内生真菌具有开发为微生物源杀菌剂的价值。     

Association af Changes in the Bacterial Ecology of Bovine Mastitis with Changes in the use of Milking Machines and Antibacterial Drugs

The results of mastitis bacteriology made by the National Veterinary and Food Research Institute in Finland during the past 50 years (1.15 million samples) are viewed in relation to simultaneous changes in dairy cow management. Although intensive preventive measures have been applied for decades, the prevalence of bovine mastitis has not decreased. Instead, pathogenic bacteria are becoming progressively less susceptible to the available therapy. In part this must be due to the emergence of antibiotic resistant strains of bacteria, but it seems that the bacterial spectrum has also changed. The incidence of Streptococcus agalactiae (group B streptococci) has decreased, while in contrast, the incidence of staphylococci, initially Staphylococcus aureus and later coagulase-negative staphylococci, has increased. Results suggest that external pressure, like changes in animal husbandry, including antimicrobial treatments and introduction of modern milking machines, act as selective forces on the bacterial species which cause bovine mastitis.     

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.     

Relations Between Udder Infection and Somatic Cells in Camel (Camelus dromedarius) Milk

Quarter milk samples (n = 391) from 101 camels were examined to study the occurrence and causes of mastitis in traditionally managed camels in eastern Sudan and to evaluate the value of the California Mastitis Test (CMT), somatic cell count (SCC) and adenosine triphosphate (ATP) in the detection of subclinical mastitis in the camel. One hundred and seventy (43.5%) of the quarter milk samples yielded pathogenic bacteria. Streptococcus agalactiae, other Streptococcus spp., Staphylococcus aureus, coag–ulase–negative staphylococci, and Escherichia coli were isolated from milk. Thirty–two (8.2%) quarter milk samples yielded mixed cultures, and 189 (48.3%) yielded no growth. Mean values for CMT, SCC and ATP were higher for quarters infected with major pathogens. However, a significant number of quarter milk samples had elevated values in these tests but were from quarters from which no bacteria were isolated. The ability of the tests to predict a positive bacteriology increased slightly when 2 or 3 tests were combined. kw|Keywords|k]inflammation; k]diagnostic tests; k]Mastitis; k]CMT; k]ATP; k]bacteriology; k]Sudan     

ELISA检测奶牛乳房炎疫苗效果方法的建立

建立一种检测抗乳房炎疫苗抗体的酶联免疫吸附试验(ELISA)方法,用于免疫后具有保护性效果的抗体水平。通过对细胞抗原制备、最佳抗原包被浓度的确定、抗原包被和封闭最佳条件确定、酶标抗体的最佳工作浓度及作用时间等反应体系的筛选和确定,建立了检测抗乳房炎疫苗抗体的间接ELISA方法。结果表明,金黄色葡萄球菌(Staphylococcus aureus)、无乳链球菌(Streptococcus agalactiae)、停乳链球菌(Streptococcus dysgalactiae)抗原最佳包被质量浓度分别为5 mg.L-1、10 mg.L-1、10 mg.L-1;包被条件为4℃过夜;用2%人血白蛋白作为封闭剂最佳封闭条件是37℃封闭30 min;酶标抗体最佳作用时间为30 min。结论攻毒试验表明,疫苗免疫后,血清中抗体滴度达到或超过免疫前2倍时,奶牛即具有较好的乳腺保护效力。这一指标可作为疫苗效力检验的标准。     

Genetic parameters for pathogen-specific mastitis resistance in Danish Holstein Cattle

The objective of this study was to estimate heritabilities for and genetic correlations among different pathogen-specific mastitis traits. The traits were unspecific mastitis, which is all mastitis treatments regardless of the causative pathogen as well as mastitis caused by Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci (CNS), Staphylococcus aureus and Streptococcus uberis. Also groups of pathogens were investigated, Gram-negative v. Gram-positive and contagious v. environmental pathogens. Data from 168 158 Danish Holstein cows calving first time between 1998 and 2006 were used in the analyses. Variances and covariances were estimated using uni- and bivariate threshold models via Gibbs sampling. Posterior means of heritabilities of pathogen-specific mastitis were lower than the heritability of unspecific mastitis, ranging from 0.035 to 0.076 for S. aureus and S. uberis, respectively. The heritabilities of groups of pathogen ranged from 0.053 to 0.087. Genetic correlations among the pathogen-specific mastitis traits ranged from 0.45 to 0.77. These estimates tended to be lowest for bacteria eliciting very different immune responses, which can be considered as the overall pleiotropic effect of genes affecting resistance to a specific pathogen, and highest for bacteria sharing characteristics regarding immune response. The genetic correlations between the groups of pathogens were high, 0.73 and 0.83. Results showed that the pathogen-specific traits used in this study should be considered as different traits. Genetic evaluation for pathogen-specific mastitis resistance may be beneficial despite lower heritabilities than unspecific mastitis because a pathogen-specific mastitis trait is a direct measure of an udder infection, and because the cost of a mastitis case caused by different pathogens has been shown to differ greatly. Sampling bias may be present because there were not pathogen information on all mastitis treatments and because some farms do not record pathogen information. Therefore, improved recording of pathogen information and mastitis treatments in general is critical for a successful genetic evaluation of udder health. Also, economic values have to be specified for each pathogen-specific trait separately.     

Commensal bacilli inhibitory to mastitis pathogens isolated from the udder microbiota of healthy cows

AIMS: To isolate from the microbiota of the healthy cow udder commensal bacteria having antimicrobial activity against bovine mastitis pathogens, with a long-term view to their potential application as antimastitis probiotics. METHODS AND RESULTS: Bacterial isolates from four healthy cow udders were tested for inhibitory activity against three Gram-positive indicator bacteria. This led to the selection of nine broadly inhibitory strains. All were of the Bacillus genus and their antimicrobial activities, which appeared heterogeneous on the basis of their antibacterial spectra and heat susceptibilities, enabled grouping of the inhibitory bacilli into six different inhibitory profiles. All displayed strong in vitro activity against Gram-positive mastitis pathogens. Inhibitory bacilli were recovered from each of the 11 udder samples collected over 7 months from one of these cows and the isolates included representatives of all six inhibitory profiles. CONCLUSIONS: Bacilli present in the udder microbiota of healthy cows can produce a variety of broadly active inhibitors of Gram-positive bacteria, including potential mastitis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibitor-producing strains of commensal Bacillus species have been identified, which may have the potential for use as possible antimastitis probiotics.     

Accurate detection of dairy cow mastitis with deep learning technology: a new and comprehensive detection method based on infrared thermal images

Mastitis is one of the most common diseases in dairy cows and has a negative impact on their welfare and life, causing significant economic losses to the dairy industry. Many attempts have been made to develop a detection method for mastitis using thermal infrared thermography. However, the use of this detection technique to determine the health of the cow's udder is susceptible to external factors, resulting in inaccurate detection of dairy cow mastitis. Therefore, this study explored a new and comprehensive detection method of dairy cow mastitis based on infrared thermal images. This method combined the left and right udder skin surface temperature (USST) difference detection method with the ocular surface temperature and USST difference detection method with improvements. The effect of external factors on dairy cow USST was effectively reduced. In addition, after comparing different target localisation algorithms, this paper used the You Only Look Once v5 (YOLOv5) deep learning network model to obtain the temperature information of eyes and udders, and mastitis detection of dairy cows was performed. A total of 105 dairy cows passing through a passage were randomly selected from the thermal infrared video and detected by the new and comprehensive detection method, and the results of cow mastitis detection were compared with somatic cell count. The results showed that the accuracy, specificity, and sensitivity of mastitis detection were 87.62, 84.62, and 96.30%, respectively. Using the YOLOv5 deep learning network model to locate the key parts of the cow had a good effect, with an average accuracy of 96.1%, and an average frame rate of 116.3f/s. The detection accuracy of dairy cow mastitis by deep learning technology combined with the detection method in this paper reached 85.71%. The results showed that the new and comprehensive detection method based on infrared thermal images can be used for the detection of dairy cow mastitis with high detection accuracy. This method can reduce the influence of external factors and can be integrated into the automatic identification system of dairy mastitis based on YOLOv5 to realise on-site monitoring of dairy mastitis.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

α-溶血性链球菌在健康人群口咽部分布及 生物拮抗作用

目的通过采集健康人群口咽部分泌物,分析上呼吸道中α-溶血性链球菌的分布状况,并对革兰阳性化脓性球菌进行生物拮抗试验,为进一步研究上呼吸道益生菌提供理论基础。方法随机自愿原则,用无菌咽拭子采集沈阳市年龄在3～75岁的300名健康人群咽后壁分泌物,对α-溶血性链球菌进行鉴定和定量分析。对致病菌的生物拮抗试验采用小缸杯法。结果定量分析显示不同年龄人群咽后壁的α-溶血性链球菌检出率均较高。在咽后壁菌群中α-溶血性链球菌构成比最多的是幼儿组,达到60.3%。其中唾液链球菌群在幼儿组所占比重最大;老年组人群格氏链球菌占比较大;儿童、青年、成人以缓症链球菌和口腔链球菌为主。对革兰阳性化脓性球菌的生物拮抗试验显示,1株婴儿链球菌婴儿亚种能够拮抗8株致病菌;4株分离菌只能拮抗1株病原菌,提示不同的菌株拮抗病原菌的能力差异较大。结论α-溶血性链球菌在人群中分布广,数量多,不同年龄人群的菌群构成存在差异。并且某些菌株显示出对致病菌较强的生物拮抗作用,推测这些菌株在呼吸道黏膜保护中起到重要作用,可作为上呼吸道益生菌的备选菌株。