http://hdl.handle.net/1957/18388 2013-05-22T06:19:09Z http://hdl.handle.net/1957/37954 Detection and transmission of Mycobacterium marinum and Mycobacterium chelonae in zebrafish (Danio rerio) Peterson, Tracy Shawn Mycobacteriosis is a common disease of laboratory zebrafish (Danio rerio). Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more wide spread and not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture, followed by molecular or biochemical identification is the traditional approach for species identification, but recently it has been shown that DNA of diagnostic value can be retrieved from paraffin blocks. Type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection were investigated as parameters to determine if the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21 and 45 days. Subsequently, fish were evaluated by H&E and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective, and obtained PCR product from 75% and 88% of the M. chelonae and M. marinum infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test. Regarding natural transmission, zebrafish are capable of contracting mycobacterial infections by feeding on infected fish tissue, but other natural routes have not been clearly elucidated. Free living amoebae have been shown to be vectors for mycobacteria and their virulence is enhanced when residing in these protozoans. Paramecium caudatum are commonly used as a first food for zebrafish, and I investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 days post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 days post hatch) showed 31% incidence (14/45). Adult fish fed gelatin diets containing bacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure: M. marinum OSU 214; in paramecia 47% (21/45; 3.5 x 10⁵ dose/fish/day), M. marinum CH in paramecia 47% (9/19; 3.6 x 10⁵ dose/fish/day), M. chelonae in paramecia 38% (5/13; 3.5 x 10⁵ dose/fish/day). I investigated the ability of mycobacteria to persist within paramecia, as this has previously been demonstrated in amoebae. Gram negative bacteria ingested by paramecia were processed within an hour. In contrast, I determined using GFP-labeled Mycobacterium marinum that mycobacteria can persist within paramecia digestive vacuoles. The concentration of M. marinum at 1 hour was similar to that at the time of ingestion. Twenty-four hours post-ingestion and later there was significant decline in M. marinum concentrations compared to time of ingestion, but M. marinum continued to persist inside digestive vacuoles for up to one week. My results demonstrate for the first time that Paramecium caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures. Graduation date: 2013; Access restricted to the OSU Community at author's request from April 2, 2013 - April 2, 2015 2013-02-27T00:00:00Z http://hdl.handle.net/1957/37622 Growth of lactococci relative to antibiotic and quaternary ammonium compounds Valladao, Marilin The work presented in this thesis is concerned with the effect of several antibiotics and quaternary ammonium sanitizers upon growth of lactic acid bacteria. Section I reports the purification of beta-lactamase from Lactococcus cremoris PR-108, by ion exchange chromatography, using the chromogenic substrate pyridine-2-azo-p-dimethylaniline (PADAC) as the enzymatic indicator. Section II reports a study of the influence of antibiotics on lactococcal growth, where the effects of incubation time, culture dilution and the use of seeded and spread agar plate techniques are investigated. These studies were extended, in section III, to include investigations of the effect of quaternary ammonium base sanitizer (Ster-bac) on lactic starters. In addition, this section describes an reverse phase high performance liquid chromatography assay for the detection of quaternary ammonium compounds in milk. Graduation date: 1991 1990-06-13T00:00:00Z http://hdl.handle.net/1957/37422 Ecology of a soil population of Rhizobium leguminosarum bv. trifolii Leung, Kam-tin Graduation date: 1993 1992-09-01T00:00:00Z http://hdl.handle.net/1957/37408 Symbiotic and saprophytic characteristics of a soil population of Rhizobium leguminosarum bv. trifolii Yap, Kathryn H. M. Although much has been learned about the comparative nodulating behavior of simple mixtures of rhizobial strains under non-soil situations, it is unclear how these findings relate to the factors influencing nodulation success by the complex mixtures of strains found within soil-borne rhizobial populations. Information on the structure and physiological behavior of soil populations is almost nonexistent. To achieve a better understanding of the situation in soil, studies were carried out with the following objectives. (i) To delineate by serological analysis the population composition of nodule occupants of Rhizobium leguminosarum bv.trifolii recovered from a variety of annual and perennial clover (Trifolium) species planted into Abiqua soil. (ii) To further the development of an assay to evaluate the substrate responsiveness of specific indigenous serotypes of R. leguminosarum bv. trifolii while they reside within the soil microbial community. Immunodiffusional analysis of isolates recovered from nodules of five annual (T. subterraneum, T. incarnatum, T. vesiculosum, T. parviflorum, T. patens) and three perennial (T. pratense, T. repens, T. hybridum) species of clover revealed that the serotypic composition of the natural population of R. leguminosarum bv. trifolii in Abiqua soil is almost completely known. With antisera to 14 antigenically distinct serotypes at our disposal, only 19 of 272 isolates recovered from these eight clover species were antigenically unknown. While the perennial species showed no pronounced preference for particular serotypes, a substantial proportion (37-75%) of nodule occupants from each of the annual clovers (with the exception of T. vesiculosum) reacted with antiserum AS6. These isolates could be subdivided by their serological reactions of non-identity with either antisera AS6, AS27, or both antisera AS21 and AS27. Using multi-locus allozyme electrophoresis (MLAE) to analyze population structure within serotypes, isolates representing serocluster AS6 were found to be rooted at a similarity of 0.82 and clustered with the other three serotypes (AG4, AS21, and AS27) only at a similarity of 0.37. In contrast to AS6, MLAE analysis revealed that "genotypic distances" between the 7 ETs representing AG4 could be large. The chapter on the nalidixic acid cell-elongation assay only represents the second report of its use on soil microbial populations. Nalidixic acid was found to be the most suitable DNA gyrase inhibitor for rhizobial studies since norfloxacin and ciprofloxacin at extremely low concentrations (2.0 and 0.5 mg/l, respectively) reduced the proportion of elongating cells significantly. In contrast to other indigenous serotypes, the majority of members of serotype AR23 did not elongate in response to yeast extract (YE). Regardless of nutrient type, or concentration, the percentage of elongated cells of AR23 remained low (<16%) even after 24 h of incubation. While the cell elongation response of serotype AS6 occured more rapidly to YE than did AR23, a less vigorous response by AS6 was observed when other nutrient sources were used. The appearance of elongated cells was delayed and the final percentage of elongated cells was reduced. Graduation date: 1992 1991-07-12T00:00:00Z