Role of Staphylococcus aureus GapC and GapB in immunity and pathogenesis of bovine mastitis

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of S. aureus is a major economically important disease affecting the dairy industry worldwide. S. aureus is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure S. aureus intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against S. aureus mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of S. aureus to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. S. aureus also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against S. aureus mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of S. uberis and S. dysgalactiae have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from S. aureus isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of S. aureus as model antigens for mastitis and to determine the regulation of expression of gapB and gapC genes and their roles in the pathogenesis of bovine S. aureus mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of S. aureus as antigens will protect against bovine S. aureus mastitis. To test this hypothesis we took the approach of using the gapB and gapC genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by S. aureus. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of S. aureus mastitis using the S. aureus wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in S. aureus mastitis. In aim 6 we examined the role of sar and agr loci on the expression of gapC and gapB genes by qRT- PCR using S. aureus RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both gapB and gapC expression were down regulated in the mutant strains, indicating that the expression of the gapB and gapC genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of gapB and gapC using q-RT-PCR. Our results showed that the expression of gapB and gapC genes in different strains of S. aureus was not consistent under the above-mentioned environmental conditions

Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major Bacterial Bovine Mastitis Pathogens

Economic losses due to bovine mastitis is estimated to be $2 billion in the United States alone. Antimicrobials are used extensively in dairy farms for prevention and treatment of mastitis and other diseases of dairy cattle. The use of antimicrobials for treatment and prevention of diseases of dairy cattle needs to be prudent to slow down the development, persistence, and spread of antimicrobial-resistant bacteria from dairy farms to humans, animals, and farm environments. Because of public health and food safety concerns regarding antimicrobial resistance and antimicrobial residues in meat and milk, alternative approaches for disease control are required. These include vaccines, improvements in housing, management practices that reduce the likelihood and effect of infectious diseases, management systems and feed formulation, studies to gain a better understanding of animal behavior, and the development of more probiotics and competitive exclusion products. Monitoring antimicrobial resistance patterns of bacterial isolates from cases of mastitis and dairy farm environments is important for treatment decisions and proper design of antimicrobial-resistance mitigation measures. It also helps to determine emergence, persistence, and potential risk of the spread of antimicrobial-resistant bacteria and resistome from these reservoirs in dairy farms to humans, animals, and farm environments

PROTOTYPE RANGKAIAN LAMPU LED INFINITY UNTUK DESAIN INTERIOR RUMAH MENGGUNAKAN ARDUINO

Tujuan daripenelitian ini adalah untuk menghasilkan hasil nyata dalam memberikan kesan keindahan terhadap interior plafon rumah atau kemudahan menyalakan atau mematikan lampu yang menambah keindahan plafon rumah, Metode yang digunakan dalam pembuatan Prototype Rangkaian Lampu Led Infinity Untuk Desain Interior Plafon Rumah Menggunakan Arduino. Metode penelitian ini terdiri dari beberapa tahap, penelitian kepustakaan, metode analisis, metode perancangan dan implementasi. Hasil yang dicapai adalah menambah kesan mewah terhadap interior plafon rumah dan dapat dioperasikan menggunakan android sebagai alat menghidupkan, mematikan lampu led infinity, di mana serangkaian alat ini dapat bekerja setelah perangkat bluetooth tersedia di smartphone android. terhubung ke modul bluetooth yang terhubung ke mikrokontroler arduino. Pada dasarnya penggunaan prinsip dari alat ini pengguna harus dapat mengoperasikan smartphone berdasarkan sistem operasi Android. Kesimpulannya adalah peralatan ini sudah teruji dan dapat digunakan sebagai pengontrol cahaya smartphone android ringan melalui koneksi Bluetooth tanpa harus menekan tombol lampu

Fructose separation from sorghum syrup by using HPLC approach: a review

Liquid sugar available today is usually the result of dissolving granulated sugar using hot water. Sorghum syrup can be obtained from the concentrate without involving crystallization, centrifugation, sieving and drying and dissolving processes. However, the sorghum syrup produced from the concentrate still containing complex sugar components such as sucrose, sugar, fructose and others. This review was examined the separation of fructose from sorghum syrup using the HPLC approach. Compared with artificial sweeteners or sugar derivative products such as dextrose, maltodextrin, sorbitol, saccharin, sucralose, and xylitol, sorghum syrup still has lower economic value. The manufacture of these sugar derivatives generally uses chemical processes such as chlorination of sucralose, hydrogenation of xylitol or enzymatic processes and fermentation of fructose. Chemical processes in general can pose a danger to consumer health, while enzymatic and biological processes require high operational costs and complex processes of enzyme and cell separation such corn fructose production. Chromatography is a technology for separating complex mixtures such as sorghum concentrates to obtain separate components, such as fructose syrup and byproducts. On a laboratory scale, sugar fractionation or fructose purification is generally carried out using High Performance Liquid Chromatography (HPLC) with the Carbopac ion exchange column as the stationary phase and ultrapure water as the mobile phase. The industrial scale fractionation in the food sector is still applied to palm oil processing. This is a great opportunity to conduct research related to the components of sorghum concentrates using chromatography column fractionation technology to obtain pure fructose with greater process efficiency and economics

Bovine Mastitis: Part I

Bovine mastitis is one of the most important bacterial diseases of dairy cattle throughout the world. Mastitis is responsible for great economic losses to the dairy producer and to the milk processing industry resulting from reduced milk production, alterations in milk composition, discarded milk, increased replacement costs, extra labor, treatment costs, and veterinary services. Economic losses due to bovine mastitis are estimated to be $2 billion in the United States,$400 million in Canada (Canadian Bovine Mastitis and Milk Quality Research Network-CBMQRN) and $130 million in Australia per year. Many factors can influence the development of mastitis; however, inflammation of the mammary gland is usually a consequence of adhesion, invasion, and colonization of the mammary gland by one or more mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, and Escherichia coli

PENERAPAN MODEL PEMBELAJARAN CONTEXTUAL TEACHING AND LEARNING MENGGUNAKAN METODE EKSPERIMEN UNTUK MENINGKATKAN AKTIVITAS DAN HASIL BELAJAR IPA SISWA KELAS VII SMPS KELIMUTU ENDE

Penelitian ini dilakukan dengan tujuan meningkatkan aktivitas dan hasil belajar IPA siswa kelas VII SMPS Kelimutu Ende dengan menerapkan model pembelajaran Contextual Teaching and Learning menggunakan metode eksperimen.Penelitian ini merupakan penelitian tindakan kelas (Classroom Action Research) dengan menggunakan model Kemmis & Mc. Taggart. Penelitian ini terdiri dari 2 siklus dengan masing-masing dilaksanakan dalam empat tahapan yaitu; perencanaan, pelaksanaan, observasi dan refleksi. Penelitian ini dilakukan sampai memperoleh target ketuntasan hasil belajar dan aktivitas belajar yaitu ? 70% dari 23 siswa yang menjadi subjek penelitian. Pengambilan data menggunakan instrumen lembar observasi untuk mengukur aktivitas belajar siswa, dan instrumen soal tes bentuk uraian untuk mengukur hasil belajar siswa. Analisi data menggunakan analisis deskriptif kualitatif.Hasil penelitian menunjukkan bahwa ada peningkatan aktivitas belajar siswa kelas VII SMPS Kelimutu Ende dengan menerapkan model pembelajaran Contextual Teaching Learning yaitu pada siklus I jumlah siswa yang mendapat skor ? 6 (34,78%) meningkat menjadi 73,91% pada siklus II . Ada peningkatan hasil belajar siswa kelas VII SMPS Kelimutu Ende dengan menerapkan model pembelajaran Contextual Teaching Learning yaitu pada siklus I jumlah siswa yang mencapai nilai ? 70 sebanyak 10 orang siswa (43,47%), pada siklus II meningkat menjadi 78,26% atau mencapai 18 orang sisw

The properties of Sweet Sorghum Syrup Produced by Combined Vacuum Falling Film and Rotary Evaporation

The combination of Vacuum Falling Film Evaporator(FFE) and Rotary Evaporator (RE) was conducted in producing sweet sorghum syrup. The aim of this research was to evaluate the performance of single FFE and combined FFE-RE on sorghum syrup concentration. Single FFE was studied at the temperature of 70, 80, and 90°C. The best single FFE treatment was continued by RE at 60, 70, and 80°C. Sweet sorghum that were concentrated using single FFE(90°C) and combined FFE(90°C)-RE(80°C) had the highest Total Soluble Solid(TSS) of 44.2°Brix and 87.53°Brix, also the acceptable lightness(L*) of 30.13 and 25.83 respectively. That combined FFE-RE produced sorghum syrup had the highest overall Hedonic score 3.34 within the taste parameter value of 2.89; color of 3,75; aroma of 3,29; and texture of 3.42. It was also accomplished with the redness(a*) of -2.11, yellowness(b*) of 5.13, turbidity of 387.66 NTU, viscosity of 2036.67cP, and reducing sugar of 52.54%

Analisis Kompetensi Pedagogik Guru IPS di SMP Negeri 1 Kabupaten Pulau Morotai

Permasalahan dalam penelitian ini adalah kompetensi pedagogik guru IPS di SMP Negeri 1 Pulau Morotai selama pelaksanaan proses pembelajaran di kelas. Tujuan dari penelitian ini adalah untuk menganalisis kemampuan kompetensi pedagogik guru sosial ketika menerapkan pembelajaran. Metode penelitian yang digunakan dalam penelitian ini adalah metode kualitatif, dengan teknik pengumpulan data observasi langsung (observasi), wawancara, dan dokumentasi. Sedangkan teknik analisis data menggunakan teknik analisis interaktif yaitu reduksi data, penyajian data, dan penarikan kesimpulan (Miles dan Huberman). Hasil penelitian menunjukkan bahwa kompetensi pedagogik guru IPS di SMP Negeri 1 Kabupaten Pulau Morotai dalam melaksanakan pembelajaran di kelas cukup baik, dari lima indikator kompetensi pedagogik, hanya satu indikator yang belum sepenuhnya dikuasai oleh guru IPS yaitu pemahaman terhadap peserta didik, guru tidak benar-benar memahami karakteristik peserta siswa dan juga manajemen kelas yang perlu ditingkatkan lagi, sedangkan dalam indikator perancangan pembelajaran, menerapkan pembelajaran, mengevaluasi hasil belajar, mengembangkan siswa untuk mengaktualisasikan berbagai potensi yang dimiliki, guru telah memenuhi keempat indikator dengan cukup baik