Arcanobacterium haemolyticum, a Gram-positive bacterium, is an under-reportedagent of disease, causing pharyngitis, wound infections and a variety of invasive diseases.This work characterized a known A. haemolyticum toxin, phospholipase D (PLD), anddetermined its possible role in bacterial virulence. In addition, a novel toxin, arcanolysin(ALN), was identified and characterized. A draft genome sequence was determined andseveral additional virulence factors that may aid in disease pathogenesis were identified.PLD was present in all strains of A. haemolyticum tested, and was expressedmaximally during logarithmic growth. Recombinant PLD caused lipid raftrearrangement on the surface of HeLa cells in a dose-dependent manner. Thisrearrangement allowed maximal bacterial adhesion to the host, with a pld knockoutadhering only 39.7% to HeLa cells as compared to wildtype. Loss of production of PLDdid not affect bacterial invasion. However, PLD expressed by intracellular bacteria wascytotoxic to host cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate(MTS/PMS) viability assays. PLD caused host cell death via necrosis as determined bytransmission electron microscopy. PLD did not induce apoptosis, as caspases 3/7 and 9were not elevated in HeLa cells infected with wildtype A. haemolyticum.A. haemolyticum also expresses a Cholesterol-Dependent Cytolysin (CDC), ALN.Like pld, aln was present in all strains tested. ALN displays a variant undecapeptide andan unusual N-terminal extension not found in most other CDCs. Recombinant ALN11shows significantly increased activity against cultured cells and erythrocytes of humanorigin, compared with intermediate activity on rabbit and hamster cells, and low to noactivity on bovine and ovine cells as measured by hemolysis, cytotoxicity and membranebinding assays. ALN was less inhibited by free cholesterol when compared with otherCDCs, indicating the possibility of alternative receptor binding.The A. haemolyticum genome was sequenced to >20X coverage, and assembled to50 contigs covering ~95% of the genome. The genome is ~1.95Mb with a mol %G+C of53.1% and contained no plasmids. pld and aln have a reduced mol %G+C of 47.2% and46.5%, respectively, indicating the possibility of gene acquisition by horizontal transfer.Initial bioinformatics analysis identified genes encoding a protease, an extracellularDNase, two neuraminidases and three fimbrial biosynthetic operons were also identifiedwithin the genome.

Master of Science / Department of Animal Sciences and Industry / Terry A. Houser / Nursery age pigs (n=198) were used to evaluate the difference in the occurrence of injection site abscesses between needle-free jet injection and conventional needle-and-syringe injection systems. Pigs were fed for 21 d prior to treatment administration to acclimate the pigs to the environment of the Kansas State University Segregated Early Weaning (SEW) unit. On d 21 each pig was injected with aluminum hydroxide adjuvant in the neck and ham with needle-free jet injection (Pulse Needle-Free Systems, Lenexa, KS) and conventional needle-and-syringe injection. Needle-free and conventional needle-and-syringe injections were randomly assigned to pig side yielding a total of 396 injections per treatment with a total of 792 injections sites. Immediately prior to injection, the external surface of the injection sites were contaminated with an inoculum of Arcanobacterium pyogenes, a bacterium commonly associated with livestock abscesses. The pigs were then fed for a period of 27 or 28 d. On d 27 or d 28 the pigs were humanely euthanized and sent to the Kansas State Veterinary Diagnostics Laboratory where necropsies were performed and the injection sites harvested for histopathological evaluation. The needle-free jet injection system was associated with more injection site abscesses than the conventional needle-and-syringe injection method for both neck (P=0.0625) and ham (P=0.0313) injection sites. Twelve abscesses were found at injection sites administered via needle-free jet injection method while only 1 abscess was found with the conventional needle-and-syringe injection method. 5 abscesses were found at the neck injection sites and 8 abscesses were found at ham injection sites. There were no significant differences seen in tissue granulation resulting from reaction to the adjuvant. In summary, the implementation of needle-free jet injection systems in market hog production will be beneficial to eliminate needles and needle fragments in meat products but, when in the presence of Arcanobacterium pyogenes, it may increase the occurrence of injection site abscesses in pork carcasses that will need to be trimmed in pork processing plants. Although more abscesses were associated with needle-free jet injection, their occurrence was observed at a very low rate given that all injection sites were intentionally contaminated prior to injection.