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.

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)