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.     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

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 lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis

All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth.     

Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus

Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Identification and analysis of putative promoter motifs in bovine herpes virus

The purpose of this study is to identify and analyse the putative promoter motifs in the bovine herpes virus which causes severaldiseases in cattle worldwide including bovine mastitis with large economic impact on dairy industry. Bovine mastitis caused dueto virus is often neglected as bacterial infections are held mainly responsible for the disease. Therefore, in this in silico investigationwith all the existing experimental data a total of 147 promoter were identified along with their sequences from three genome vizbovine herpes virus 1 (BHV), bovine herpes virus 4 and bovine herpes virus 5, out of which 39 promoters were from bovine herpesvirus 4 (BHV 4), 95 from BHV1 and 13 from BHV5 and it was observed that BHV1 and BHV5 have a close evolutionary history.However, they belong to the same subfamily and size of the genome and GC% of BHV1 and BHV5 was almost equal and very highcompare to that of BHV4. This analysis may help in designing the live attenuated vaccine against BHV causing bovine mastitis thatreduces the incidence of bovine mastitis. Identification of promoters may also help in designing of expression vectors which help inbetter understanding of the regulation of gene expression. In the era of large genomics and proteomics prediction of promoters inthe whole genome is crucial for the advancement of drug discovery and gene therapy.     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

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。     

In silico characterization of putative drug targets in Staphylococcus saprophyticus,causing bovine mastitis

The bovine mastitis caused by coagulase negative staphylococci (CNS) has increased in many herds of urban and rural areas ofIndia. Emergence of multi drug resistant bacteria has further made its management more complex and serious. Therefore,innovation of novel specific drug for the treatment of disease caused by particular organism remained to be a challenge. Hence, inthe present study a bacterium was isolated from milk of the cow with bovine mastitis and was identified as S. saprophyticus, 44pathways of S. saprophyticus retrieved (KEGG) from web server were found to be non homologous to the host Bos taurus, out ofwhich 39 pathways were found to be in cytoplasm, 2 in cell wall and 3 in the cell membrane. The knowledge of the present studycould make the drug discovery easier which have high affinity to the target site of the causative organism.     

Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae)

Echinococcosis is a serious helminthic zoonosis in humans, livestock and wildlife. The pathogenic organisms are members of the genus Echinococcus (Cestoda: Taeniidae). Life cycles of Echinococcus spp. are consistently dependent on predator–prey association between two obligate mammalian hosts. Carnivores (canids and felids) serve as definitive hosts for adult tapeworms and their herbivore prey (ungulates, rodents and lagomorphs) as intermediate hosts for metacestode larvae. Humans are involved as an accidental host for metacestode infections. The metacestodes develop in various internal organs, particularly in liver and lungs. Each metacestode of Echinococcus spp. has an organotropism and a characteristic form known as an unilocular (cystic), alveolar or polycystic hydatid. Recent molecular phylogenetic studies have demonstrated that the type species, Echinococcus granulosus, causing cystic echinococcosis is a cryptic species complex. Therefore, the orthodox taxonomy of Echinococcus established from morphological criteria has been revised from the standpoint of phylogenetic systematics. Nine valid species including newly resurrected taxa are recognised as a result of the revision. This review summarises the recent advances in the phylogenetic systematics of Echinococcus, together with the historical backgrounds and molecular epidemiological aspects of each species. A new phylogenetic tree inferred from the mitochondrial genomes of all valid Echinococcus spp. is also presented. The taxonomic nomenclature for Echinococcus oligarthrus is shown to be incorrect and this name should be replaced with Echinococcus oligarthra.     

Analysis of rhizobial endosymbionts of Vicia,Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain)

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.