Date of Graduation

8-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Rhoads, Douglas D.

Committee Member

Rosenkrans, Charles F. Jr.

Second Committee Member

Kwon, Young Min

Third Committee Member

Ivey, D. Mack

Keywords

A reliable new genetic marker for Staphylococcus species identification; Air sampling system for screening the airborne pathogens; Bacterial Chondronecrosis with Osteomyelitis in Broiler; DNA electroporation to Staphylococcus agnetis; Genome comparison of cattle and broiler Staphylococcus agnetis isolates; Rapid DNA extraction using NaOH

Abstract

Lameness is a major issue in animal welfare and the broiler industry. Bacterial chondronecrosis with osteomyelitis (BCO) is one of the main causes of lameness. Many staphylococcal species, including Staphylococcus agnetis isolate 908, have been isolated from the bones and blood of lame broilers at the University of Arkansas. Staphylococcus agnetis is a coagulase-variable, Gram-positive bacterial species that has been previously associated with subclinical or mild clinical cases of mastitis in dairy cattle. The annotated complete genome of hypervirulent strain 908 was published at NCBI. In this study, it has been compared to nine genomes we assembled for hypervirulent isolates in dairy cattle. Phylogenomic analyses of chicken and cattle isolates of S. agnetis and Staphylococcus hyicus suggest a very close relationship between the cattle and chicken isolates. The hypervirulent chicken isolate, 908, clustered with two of the cattle isolates, including strain 1379. A catalogue of gene differences between the cattle and chicken isolates was constructed using reciprocal blast analyses at the nucleotide and polypeptide level. More than 40 genes and 3 plasmids from strain 908 are absent or poorly conserved in any of the cattle S. agnetis isolates. No transformation protocol has been described for S. agnetis. Subsequently, an electroporation procedure has been optimized for DNA transformation of Staphylococcus agnetis. Therefore, we have optimized an electroporation method for DNA transformation so that we regularly obtain 10 to 20 transformants per ng using a Gram+/Gram- shuttle vector. Moreover, among the BCO pathogens isolated from the lame broilers, there are a number of Staphylococcus species, such as, S. agnetis, S. hyicus, S. chromogenes, S. aureus, S. cohnii, S. saprophyticus, S. epidermidis, and S. capitis, which are hard to accurately identify based just on genes like 16S rDNA. Therefore, using pfbA gene, a novel PCR assay was optimized for Staphylococcus species discrimination and strain typing. Moreover, extraction of bacterial DNA for subsequent molecular diagnostic applications remains a costly and time-consuming operation. We developed a technique for rapidly extracting genomic DNA from the BCO pathogens and other environmental bacteria based on sodium hydroxide cell lysis with or without magnetic bead capture. Finally, the BCO pathogens are transmitted via air. Our efficient air sampling system was designed for the quick screening of these airborne BCO pathogens and is transferable to monitor agriculturally important pathogenic bacteria.

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