Made available in DSpace on 2016-03-28T12:45:45Z (GMT). No. of bitstreams: 2 vitor_ferreira_ioc_mest_2015.pdf: 1647160 bytes, checksum: 772b0b0b513c782442c3b14ee0949075 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-03-14 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Musca domestica é um díptero que apresenta sérios riscos à saúde pública e ambiental. Essa espécie transmite diversos patógenos, é causadora de miíases em humanos e animais além de causar prejuízos econômicos. O uso de inseticidas químicos causa danos ambientais e possui eficácia questionável em M. domestica. Inseticidas biológicos de origem bacteriana apresentam uma boa alternativa aos inseticidas químicos em relação aos impactos ambientais, as espécies Brevibacillus laterosporus, Bacillus thuringiensis e Lysinibacillus sphaericus demonstram ação inseticida em diversas ordens de insetos e podem apresentar atividade inseticida efetiva em M. domestica. Nove estirpes de bactérias foram testadas em M. domestica e destas três estirpes: BL102, BTI193A e BTK176 apresentaram valores de mortalidade maiores que 50% e por isso tiveram os efeitos subletais investigados. Em relação a massa das pupas, nenhuma das estirpes apresentou variação significativa na média da massa pupal, quando comparadas às médias dos controles puro e água O período de desenvolvimento também não se diferenciou de forma estatisticamente relevante dos dois controles em nenhum tratamento. As três estirpes testadas apresentaram mortalidade estatisticamente superior às mortalidades dos dois controles com LC50 nas concentrações de 12.40x108 UFC/mL para BTI193, 4.76x108 UFC/mL para BTK176 e 4.16x108 UFC/mL para BL102. As micrografias de transmissão demonstraram que todas as estirpes apresentam o perfil de danos celulares já descritos para essas espécies de bactérias entomopatogênicas, como elevada vacuolização do citoplasma, além de desorganização das organelas celulares, porém somente as estirpes de B. thuringiensis apresentaram deformação e interrupção das microvilosidades e conteúdo citoplasmático extravasado para o lúmen intestinal. Os resultados obtidos com de B. laterosporus e B. thuringiensis sugerem que essas estirpes são um agentes de controle biológico promissores para M. domestica Abstract: Musca domestica is a dipteran who presentes a serious risk to public and environmental health. This species carries diseases, causes myiasis in humans and animals and may cause economic losses. The use of chemical insecticides, beyond causing environmental damage, have questionable efficacy in M. domestica. Biologic insecticides of bacterial origin presents a good choice over chemical insecticides regarding environmental impact. Brevibacillus laterosporus, Bacillus thuringiensis and Lysinibacillus sphaericus species show insecticide action in several insect orders and may presente insecticide effectiveness in M. domestica. Nine strains of bacteria were tested for mortality where three strains: BL102, BTI193 and BTK176 showed values above 50%. These strains were submitted to sub lethal effects experiments. In relation to pupal weight, none of the strains showed statistical variation when compared to pure and water controls Development time was statistically undifferentiated from both controls in every treatment tested. All the tested strains showed statistically higher mortality when compared to both controls, showing LC50 concentrations of 12.40x108 CFU/mL for BTI193, 4.76x108 CFU/mL for BTK176 and 4.16x108 CFU/mL for BL102. Transmission micrographs showed that every strain presented cell damage profile established for these species of entomopathogenic bacteria, such as the high level of cytoplasm vacuolization and disorganization of cell organelles, however, only B. thuringiensis strains showed microvilli deformation and disruption and cytoplasmic content extruded into intestinal lumen. All these observations suggest that tested B. laterosporus and B. thuringiensis strains are promising agents to the biological control of M. domestica / Musca domestica is a dipteran who presentes a serious risk to public and environmental health. This species carries diseases, causes myiasis in humans and animals and may cause economic losses. The use of chemical insecticides, beyond causing environmental damage, have questionable efficacy in M. domestica . Biologic insecticides of bacterial origin presents a good choice over chemical insecticides regarding environmental impact . Brevibacillus laterosporus , Bacillus thuringiensis and Ly sinibacillus sphaericus species show insecticide action in several insect orders and may presente insecticide effectiveness in M. domestica . Nine strains of bacteria were tested for mortality where three strains: BL102, BTI193 and BTK176 showed values above 50% . These strains were submitted to sub lethal effects experiments . In relation to pupal weight , none of the strains showed statis ti cal variation when compared to pure and water controls. Development time was statis ti cally undifferentiated from bot h cont rols in every treatment tested. All the tested strains showed statis ti cally higher mortality when compared to both controls, showing LC50 concentrations of 12.40 x 10 8 CFU /mL for BTI193, 4.76 x 10 8 CFU /mL for BTK176 and 4.16 x 10 8 CFU /mL for BL102. Transmission microg raphs s howed that every strain presented cell damage profile established for these species of entomopathogenic bacteria, such as the high level of cytoplasm vacuolization and dis organization of ce ll organelles, however, only B. thurin giensis strains showed microvilli deformation and disruption and cytoplasmic content extruded into intestinal lumen. All these observations suggest that tested B. laterosporus and B. thuringiensis strain s are promising agent s to the biological control of M. domestica

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the causative agents of botulism and represents a family of seven structurally similar but antigenically different serotypes (A to G). The BoNTs are expressed in C. botulinum as a single polypeptide chain and then posttranslationally nicked, forming a di-chain polypeptide chain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a disulfide bond. Topologically, the neurotoxins are composed of three domains, a binding domain (HC), a translocation domain (HN) and a catalytic domain. The BoNTs act preferentially on cholinergic nerve endings in both humans and animals and thus produce a flaccid paralysis that may result in death. In southern Africa, BoNT types C and D have been associated with botulism in cattle. To combat the disease, a bivalent vaccine consisting of formalin-inactivated type C and D holotoxins is currently available, and although it is efficacious, several concerns regarding its production has been raised, most notably its cost. The development of efficacious recombinant subunit vaccines may provide a means whereby many of the production problems may be eliminated or minimized. Consequently, the aim of this investigation was to produce a recombinant botulinum neurotoxin serotype D binding domain [BoNT/D(HC)] vaccine candidate for preventing BoNT/D intoxication. Towards this end, the gene fragment for the heavy chain (HC) of the BoNT produced by the C. botulinum type D vaccine strain D-50 was amplified, cloned in Escherichia coli and characterized by nucleotide sequence analyses. An alignment of the deduced amino acid sequence with that of characterized clostridial type C and D neurotoxins demonstrated that the heavy chains are composed of highly conserved domains interceded with tracts of amino acids exhibiting little overall relatedness, although considerable identity between the components ofa specific pair is apparent in certain of the regions. The deduced amino acid sequence exhibited 99, 66 and 73% identity with the reported amino acid sequences of BoNT/D-SA, BoNT/D and BoNT/C1, respectively. Attempts at expressing the native gene sequence for the HC from BoNT/D-50 in Brevibacillus brevis 47-5Q were unsuccessful. This may have been due to differences in codon bias between the heterologous gene and B. brevis. Consequently, a completely synthetic codonoptimized gene encoding the HC of BoNT/D-SA was constructed and expressed using a B. brevis 47-5Q mutant as expression host, obtained on mutagenesis with N-methyl-N’-nitro-Nnitrosoguanidine (NTG). Extracellular expression of the 48-kDa recombinant protein was verified by Western blot analyses with anti-BoNT/D antibodies. The recombinant BoNT/DSA(HC) protein was purified from the culture supernatant and used to vaccinate mice, after which their survival against challenge with active toxin was evaluated. Mice given two subcutaneous vaccinations were protected against intraperitoneal administration of 4 X 102 mouse lethal dosages (MLDs) of 16S BoNT/D-50 toxin. Antibody levels in mice surviving challenge were determined by enzyme-linked immunosorbent assays and confirmed that BoNT/D-SA(HC) was successful in evoking a protective immune response, whilst Western blot analyses indicated the presence of anti-16S BoNT/D-50 toxin antibodies in the serum. From these results it could be concluded that the recombinant BoNT/D-SA(HC) protein is an effective immunogen, able to protect against a high challenge dose of BoNT/D-50 neurotoxin. / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

A leptospirose é causada por bactérias do gênero Leptospira e constitui um problema de saúde pública mundial, por acometer humanos e animais. Sua patogênese ainda é pouco esclarecida, especialmente quanto aos processos de invasão, adesão e colonização dos hospedeiros. Recentemente, proteínas secretadas por leptospiras foram identificadas por proteômica e análises in silico, algumas das quais apresentam possível envolvimento no desenvolvimento de quadros hemorrágicos, bem como podem ser possíveis antígenos para diagnóstico. Sendo assim, o presente trabalho propôs a clonagem em vetor de expressão heteróloga bacteriano das sequências codificantes das proteínas Sph2, LipA, ColA e LipL32 de L. interrogans sorovar Copenhageni, caracterização funcional das proteínas recombinantes purificadas e estudo do seu potencial uso no diagnóstico para a leptospirose. Essas proteínas foram escolhidas por possivelmente estarem envolvidas na patogênese de leptospiras. Para tanto, as sequências correspondentes aos genes das proteínas foram clonadas nos vetores de expressão em Brevibacillus choshinensis e Escherichia coli. A produção de soro policlonal e técnicas de ELISA, western-blotting e imuno-histoquímica foram realizadas para avaliar a funcionalidade das proteínas recombinantes purificadas. Também foram realizados testes de comprovação e caracterização da atividade enzimática para proteína LipA, que se apresenta como uma provável lipase na análise in silico. Os resultados obtidos mostraram que o sistema de expressão em Brevibacillus foi eficiente na expressão das proteínas, porém com baixo rendimento na purificação das proteínas Sph2, ColA e LipA. A proteína recombinante LipL32 purificada não apresentou diferença na sua atividade antigênica, em relação aos dois sistemas de expressão utilizados. Experimentos de Western - blotting demonstraram a presença de proteína LipA em diferentes sorovares patogênicos de Leptospira spp. A proteína LipA apresentou atividade lipásica sobre ésteres graxos de p-nitrofenil, sendo uma provável lipase euritérmica. Os dados obtidos com a análise de imuno-histoquímica sugerem que esta proteína possa participar dos eventos que levam a lesões na membrana celular no tecido hepático. Resultados de ELISA com soro de pacientes mostraram que as proteínas ColA e Sph2 são potenciais antígenos para diagnóstico da leptospirose. Apenas a proteína ColA apresentou ação hemorrágica na pele de camundongos. Estes resultados indicam que as proteínas LipA, ColA e Sph2 podem estar envolvidas em mecanismos patogênicos na leptospirose. / Leptospirosis is caused by bacteria of the genus Leptospira and it is a public health problem worldwide, for affecting humans and animals. Its pathogenesis is still unclear, especially regarding the processes of invasion, adhesion and colonization of hosts. Recently, proteins secreted by leptospires were identified by proteomics and in silico analyzes, some of which present possible involvement in the development of hemorrhagic conditions, as well they could be possible antigens for the diagnosis. Thus, the present work proposed the cloning into bacterial heterologous expression vectors of the coding sequences of the Sph2, LipA, ColA and LipL32 proteins of L. interrogans serovar Copenhageni, the functional characterization of the purified recombinant proteins and the study of their potential use in the diagnosis for the Leptospirosis. These proteins were chosen because they may be involved in the pathogenesis of leptospires. To that end, the coding sequences were cloned into the expression vectors in Brevibacillus choshinensis and Escherichia coli. The production of polyclonal serum and ELISA, Western-blotting and immunohistochemistry techniques were performed to evaluate the functionality of the purified recombinant proteins. Also, tests were carried out to prove and characterize the enzymatic activity of LipA protein, which presents as a probable lipase in the in silico analysis. The obtained results showed that the expression in the Brevibacillus system was efficient, but with low yield in the purification of Sph2, ColA and LipA proteins. The purified recombinant LipL32 protein showed no difference in its antigenic activity, in relation to the two expression systems used. Western-blotting experiments demonstrated the presence of LipA protein in different pathogenic serovars of Leptospira spp. The LipA protein showed lipase activity on p-nitrophenyl fatty esters, being a probable eurythermic lipase. The data obtained with the immunohistochemical analysis suggest that this protein can participate in the events that lead to cell membrane lesions in the hepatic tissue. ELISA results using serum from patients showed that ColA and Sph2 proteins are potential antigens for the diagnosis of leptospirosis. Only the ColA protein presented hemorrhagic action on the mice skin. These results indicate that LipA, ColA and Sph2 proteins may be involved in pathogenic mechanisms in leptospirosis.

A leptospirose é causada por bactérias do gênero Leptospira e constitui um problema de saúde pública mundial, por acometer humanos e animais. Sua patogênese ainda é pouco esclarecida, especialmente quanto aos processos de invasão, adesão e colonização dos hospedeiros. Recentemente, proteínas secretadas por leptospiras foram identificadas por proteômica e análises in silico, algumas das quais apresentam possível envolvimento no desenvolvimento de quadros hemorrágicos, bem como podem ser possíveis antígenos para diagnóstico. Sendo assim, o presente trabalho propôs a clonagem em vetor de expressão heteróloga bacteriano das sequências codificantes das proteínas Sph2, LipA, ColA e LipL32 de L. interrogans sorovar Copenhageni, caracterização funcional das proteínas recombinantes purificadas e estudo do seu potencial uso no diagnóstico para a leptospirose. Essas proteínas foram escolhidas por possivelmente estarem envolvidas na patogênese de leptospiras. Para tanto, as sequências correspondentes aos genes das proteínas foram clonadas nos vetores de expressão em Brevibacillus choshinensis e Escherichia coli. A produção de soro policlonal e técnicas de ELISA, western-blotting e imuno-histoquímica foram realizadas para avaliar a funcionalidade das proteínas recombinantes purificadas. Também foram realizados testes de comprovação e caracterização da atividade enzimática para proteína LipA, que se apresenta como uma provável lipase na análise in silico. Os resultados obtidos mostraram que o sistema de expressão em Brevibacillus foi eficiente na expressão das proteínas, porém com baixo rendimento na purificação das proteínas Sph2, ColA e LipA. A proteína recombinante LipL32 purificada não apresentou diferença na sua atividade antigênica, em relação aos dois sistemas de expressão utilizados. Experimentos de Western - blotting demonstraram a presença de proteína LipA em diferentes sorovares patogênicos de Leptospira spp. A proteína LipA apresentou atividade lipásica sobre ésteres graxos de p-nitrofenil, sendo uma provável lipase euritérmica. Os dados obtidos com a análise de imuno-histoquímica sugerem que esta proteína possa participar dos eventos que levam a lesões na membrana celular no tecido hepático. Resultados de ELISA com soro de pacientes mostraram que as proteínas ColA e Sph2 são potenciais antígenos para diagnóstico da leptospirose. Apenas a proteína ColA apresentou ação hemorrágica na pele de camundongos. Estes resultados indicam que as proteínas LipA, ColA e Sph2 podem estar envolvidas em mecanismos patogênicos na leptospirose. / Leptospirosis is caused by bacteria of the genus Leptospira and it is a public health problem worldwide, for affecting humans and animals. Its pathogenesis is still unclear, especially regarding the processes of invasion, adhesion and colonization of hosts. Recently, proteins secreted by leptospires were identified by proteomics and in silico analyzes, some of which present possible involvement in the development of hemorrhagic conditions, as well they could be possible antigens for the diagnosis. Thus, the present work proposed the cloning into bacterial heterologous expression vectors of the coding sequences of the Sph2, LipA, ColA and LipL32 proteins of L. interrogans serovar Copenhageni, the functional characterization of the purified recombinant proteins and the study of their potential use in the diagnosis for the Leptospirosis. These proteins were chosen because they may be involved in the pathogenesis of leptospires. To that end, the coding sequences were cloned into the expression vectors in Brevibacillus choshinensis and Escherichia coli. The production of polyclonal serum and ELISA, Western-blotting and immunohistochemistry techniques were performed to evaluate the functionality of the purified recombinant proteins. Also, tests were carried out to prove and characterize the enzymatic activity of LipA protein, which presents as a probable lipase in the in silico analysis. The obtained results showed that the expression in the Brevibacillus system was efficient, but with low yield in the purification of Sph2, ColA and LipA proteins. The purified recombinant LipL32 protein showed no difference in its antigenic activity, in relation to the two expression systems used. Western-blotting experiments demonstrated the presence of LipA protein in different pathogenic serovars of Leptospira spp. The LipA protein showed lipase activity on p-nitrophenyl fatty esters, being a probable eurythermic lipase. The data obtained with the immunohistochemical analysis suggest that this protein can participate in the events that lead to cell membrane lesions in the hepatic tissue. ELISA results using serum from patients showed that ColA and Sph2 proteins are potential antigens for the diagnosis of leptospirosis. Only the ColA protein presented hemorrhagic action on the mice skin. These results indicate that LipA, ColA and Sph2 proteins may be involved in pathogenic mechanisms in leptospirosis.

The first part of the dissertation presented a potentially novel aerobic, thermophilic, and cellulolytic bacterium identified as Brevibacillus sp. Strain JXL which was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between the effects caused by these two simple sugars. After the end of the 24 hour period, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also observed when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and optimal pH range of 6-8. These cellulases also had high thermotolerance as demonstrated by retaining more than 50% activity after 1 h at 100°C. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose. In the second part of the dissertation, the effect of Cellulose- and Organic-Solvent based Lignocellulose Fractionation (COSLIF) (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng.2007, 97 (2), 214−223) pretreatment conditions on sweet sorghum bagasse (SSB) feedstock was studied using Response Surface Methodology (RSM). Batch experimental matrix was set up based on response surface method's central composite design in two factors to determine the effects of reaction time and temperature on the yield of simple sugars after a sequential pretreatment-enzyme hydrolysis process. Accordingly, changes in delignification, total reducing sugar (TRS) yield, glucan retention, digestibility and overall sugar yields resulting from various combinations of reaction times and temperatures were determined. The results suggested that both pretreatment temperature and reaction time were significant factors, although temperature was more so than reaction time. COSLIF pretreatment conditions of 50°C and 40 min were found to be the optimum pretreatment conditions for the saccharification of SSB. At the end of pretreatment and enzymatic hydrolysis, maximum values of 51.4% delignification, 85% overall glucose yield, and 44% overall xylose yield at an ACCELERASE®1500 loading of 0.25 mL/g sweet sorghum bagasse were achieved. Optimum ACCELERASE®1500 dosage of 0.1 mL/g of sweet sorghum bagasse was identified which resulted in an overall glucose yield of 82.2%±1.05. An effort has also been made to prescribe predictive models which represented the correlation between independent variables (reaction time and temperature), and dependent variables (delignification, and overall glucose yield) using RSM. The significance of the correlations and adequacy of these models were statistically tested for the selected objective functions. The outcomes suggested very competent and statistically adequate regression models which provided quantitative information both for delignification and overall glucose yield for the batch experiments studied.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

Brevibacillus laterosporus (B. laterosporus) is a pathogen difficult to distinguish from Paenibacillus larvae (P. larvae), and contributes to Colony Collapse Disorder (CCD) of honeybees. To develop a biocontrol agent to limit its presence, bacteriophages were isolated from Utah County soil samples and used to infect B. laterosporus isolated from Utah County honey and larvae samples. Since CCD is prevalent in Utah beehives, bacteriophage that infect and lyse B. laterosporus may be isolated and characterized. Pathogens were isolated from soil samples, and 16S rRNA gene tests initially identified the strains as P. larvae. Bacteriophages were isolated, purified, and amplified sufficiently to obtain images by electron microscope and genome sequencing by 454 pyrosequencing. Genomes were annotated with DNA Master, a Multiple Document Interface (MDI) program. Open reading frames (ORF's) were compared to the National Center for Biotechnology Information's (NCBI) database of primary biological sequence information via the Basic Local Alignment Search Tool (BLAST) algorithm. Later testing determined the pathogen to actually be B. laterosporus. Plaques demonstrated lytic activity, and electron microscopy revealed bacteriophages of the myoviridae family. The five sequenced genomes were composed of linear dsDNA ranging from 45,552 to 58,572 base pairs in length, 92 to 100 genes per genome, and a 38.10% to 41.44% range of G + C content. Discovering and describing new bacteriophages is a reasonably reproducible process and contributes to appreciating the diverse relationships between bacteriophage, bacteria, and eukaryota. Scientific facilitation of the bacteriophages role in limiting detrimental bacteria may contribute as an adjunctive therapy for CCD.

No description available.

The Paenibacillaceae family of bacteria includes two species known to infect the hives of honeybees, Paenibacillus larvae and Brevibacillus laterosporus. P. larvae, the causative agent of American Foulbrood (AFB) causes a lethal infection of honeybee larvae, while B. laterosporus is a secondary invader following European Foulbrood (EFB) infection. Increasing antibiotic resistance of P. larvae bacteria has prompted a search for alternative treatment methods for this disease. Bacteriophages are the most diverse life forms on earth and can provide important insights about the bacterial hosts they infect. However, few Paenibacillaceae phages have been isolated or characterized. In this study, the first B. laterosporus phages are characterized with respect to host range, structural morphology, and sequence similarity. The isolation and characterization of many P. larvae field isolates together with 38 novel P. larvae phages made possible the first broad phage typing study of P. larvae. Phage typing data indicated that P. larvae strains tested could be categorized into one of two groups. Comparative genomics of bacteriophages was made easier by modifying Phamerator to make it broadly accessible and usable to phage researchers throughout the world. Additionally, raw sequencing data can now be used to identify phage DNA packaging strategies that are indicative of a phage’s physical ends. Using these data, phage genomes can be published in an orientation and complementarity that reflects the physical structure of the phage chromosome, providing order and consistency that will benefit all future phage researchers.

This study investigated the effects of Brevibacillus laterosporus and live yeast (LY) on rumen fermentation, nutrient digestibility and microbial protein synthesis. The basal diet was a total mixed ration formulated to fulfil the minimum nutrient requirement of early lactating 600 kg Holstein cow producing 40kg of milk with 3.5 % fat and 3.3 % protein using CPM-dairy software (NRC, 2001). Treatments were: T1 (Control: basal diet with no additive), T2 (Basal diet + Brevibacillus laterosporus), T3 (Basal diet + Live yeast), and T4 (Basal diet + Brevibacillus laterosporus + Live yeast). In situ degradation, in vitro batch fermentation were performed. Data obtained were subjected to analysis of variance (ANOVA) using PROC GLM (SAS Institute, 2009). The effective dry matter (DM) degradability evaluated at low (0.02) and medium (0.05) ruminal passage rate (ED1 and ED2) were higher (p<0.05) in T1 compared to T2 and T3, but did not differ (p>0.05) between T2, T3 and T4, and between T1 and T4. When evaluated at fast passage rate (0.08) the effective DM degradability (ED3) was higher (p<0.05) in T1 compared to T3 and T4, but did not differ (p>0.05) between T1 and T2. The difference in ammonia nitrogen production was observed only between T1 and T2, and was higher (p<0.05) in T1. The total VFA’s concentration was higher (p<0.05) in T3 compared to the control. All additives decreased the molar percentage of acetate (P<0.05). The concentration of acetate was lower (p<0.05) in T3 and T4 compared to control. Propionate concentration was higher (p<0.05) in T3 and T4 compared to other treatments and lower (p<0.05) in the control compared to the rest of treatments. Butyrate concentration was higher (p<0.05) in T2 and T4 compared to the rest of the treatments, and lower (p<0.05) in T3 than other treatments. The microbial protein synthesis measured as purine derivate done on residues was higher (p<0.05) for T3 compared to T1 and T2, but did not differ between T1, T2 and T4, and between T3 and T4. These results showed that the two additives have different individual effects on DM and CP degradability, but also associative effects in some fermentation parameters such as propionate production. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)