Biochemical and genetic characterization of bacteria isolated from diseased rainbow trout (Oncorhynchus mykiss) farmed in Lesotho and Mpumalanga province of South Africa

Kutu, Vuyokazi (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Rainbow trout farms in Mpumalanga Province, South Africa and Lesotho, have periodically suffered significant losses from infections caused by Gram-positive bacteria. Such outbreaks have hampered the development of this industry in both South Africa and Lesotho. A total of 55 bacterial strains had been isolated between 2006-2012 from infected trout farmed in Lesotho and Mpumalanga Province and had been stored for long term by freeze drying. Some isolate identification had been performed and a few were used for vaccine development. Vaccines were however only effective for one or two seasons, highlighting the need to properly characterize these Gram-positive bacteria. The aims of the study were therefore to: (i) investigate the genetic diversity of these bacterial isolates by their phenotype; antimicrobial susceptibility and 16S ribosomal RNA (rRNA) sequencing and phylogenetic analysis, (ii) investigate the different antigenic epitopes that exist within this group of bacterial isolates by development of an enzyme-linked immunosorbent assay (ELISA) utilizing six rabbit produced polyclonal antibodies, produced against six selected bacterial isolates from the 55 isolates investigated in this study. Phenotypic analysis showed that fifty of the isolates were Gram-positive cocci and five were Gram-positive rods. Their growth characterists and antimicrobial susceptibility were extensively characterized. The 16S rRNA analysis indicated the following isolate composition: 49 Lactococcus garvieae, one Lactococcus lactis, three Carnobacterium maltaromaticum and two Weissella species, which is the first report of Weissella from diseased trout from South Africa. Antigenicity analysis showed that there were highly specific epitopes that were limited to very few isolates, but also common epitopes that were shared between isolates of the same genus, but even some epitopes that were shared between different bacterial genera. The patterns of epitope sharing broadly correlated with the 16S rRNA phylogeny, but not entirely which was not unexpected as phylogeny does not indicate the presence or absence of bacterial epitopes. These results address the importance and accuracy of molecular identification of disease causing species and the need to investigate the antigenic differences expressed by these pathogenic bacteria to assist in generating correct information needed for the development of vaccines of high efficacy.

AFRIKAANSE OPSOMMING: Reenboogforelplase in Mpumalanga, Suid-Afrika, en Lesotho het met tussenposes betekenisvolle verliese gelei as gevolg van infeksies veroorsaak deur Gram-positiewe bakterieë. Sulke uitbrake het die onwikkeling van die bedryf beperk in sowel Suid-Afrika asook Lesotho. ‘n Totaal van 55 bakteriese rasse is geïsoleer tussen 2006-2012 van geïnfekteered forelle waarmee geboer is in Lesotho en Mpumalanga en wat langtermyn deur vriesdroging opgeberg is. Party isolate is geïdentifiseer en ’paar is vir entstofontwikkeling gebruik. Entstowwe was egter slegs vir een of twee seisoene effektief, wat die belang van die karakterisering van die Gram-positiewe bakterieë beklemtoon het. Die doelwitte van hierdie studie was dus om: (i) die genetiese diversiteit van die bakteriele isolate deur fenotipe; antimikrobiese gevoeligheid en 16S ribosomale RNA (rRNA) volgordebepaling en filogenetiese analise te bepaal, (ii) die verskillende antigeniese epitope wat binne hierdie groep voorkom, deur middel van ensiem gekoppelde immunoessais (ELISA) wat ontwikkel is met ses konyn verwekte poliklonale antiliggame wat teen ses geselekteerde bakteriele isolate geproduseer is, te bepaal. Fenotipiese analise van vyftig van die isolate is gevind om Gram-positiewe cocci en vyf om Gram-positiewe stawe te wees. Hulle groei eienskappe en antimikrobiese gevoeligheid is deeglik gekarateriseer. Die 16S rRNA analise het die volgende isolaat samestelling aangedui: 49 Lactococcus garvieae, een Lactococcus lactis, drie Carnobacterium maltaromaticum en twee Weissella spesies, wat die eerste berig is van die voorkoms van Weissella in Suid-Afrikaanse forelle. Antigeniese analise het getoon dat hoogs spesifieke epitope beperk is tot sekere isolate, maar dat daar ook ‘n paar epitope voorkom wat deur isolate gedeel word tussen lede van dieselfde genus, en selfs tussen verskillende bakteriele genera. Die patrone van epitoop deling korreleer in die bree met die 16S rRNA filogenie, maar nie heeltemal nie, wat nie onverwag was nie, aangesien filogenie nie die teenwoordigheid of afwesigheid van epitope kan aandui nie. Hierdie resultate beklemtoon die belang en akkuraatheid van molekulêre identifikasie van siekteverwekkende spesies en die behoefte om die antigeniese verskille tussen hierdie patogeniese bakterieë te ondersoek om korrekte inligting te genereer vir die ontwikkeling van hoogs doeltreffende entstowwe.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/102988
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