title: Detection and quantification of Streptococcus agalactiae from aquaculture with molecular means
Creators: name: Leigh, William James
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Aquaculture, fish vaccine, Streptococcus agalactiae, molecular qPCR.

description: The aim of this research was to improve the detection and diagnosis of Streptococcus agalactiae in aquaculture, as well as developing methods to improve the study of this bacteria in the laboratory. Difficulties in specifically detecting the presence of S. agalactiae from samples of an aquatic nature had been observed using primers developed for detecting S. agalactiae in other hosts. Cross reaction of the primers with closely related fish pathogens, or environmental bacteria had been observed when using these primers to identify aquaculture derived samples. A rapid, sensitive and specific Polymerase Chain Reaction (PCR) assay based on the S. agalactiae groEL gene was designed in order to quickly diagnose S. agalactiae from an infected or dead fish. It is important for rapid diagnosis of this disease as mortalities among the fish population can spread extremely rapidly. In the time taken to culture bacteria from a diseased fish in the lab, high levels of mortality among infected fish may already have occurred. If S. agalactiae can be detected in sick fish within 12 h it is possible to pre-emptively treat the remaining fish with an oral antibiotic before the disease begins to cause mass mortalities. A qPCR assay was also developed to target the S. agalactiae groEL gene in order to quantify the number of copies of S. agalactiae DNA from a sample. In the laboratory it is useful to detect the DNA copy number of bacteria within a sample. Knowing the copy number of DNA within the organs of experimentally infected fish is useful to researchers studying the progression of disease within a population. The results could help inform researchers on the portal of entry of S. agalactiae in fish and also the level of infection within fish. The DNA copy number is also useful to enumerate the number of bacteria within liquid cultures of bacteria, to measure the growth rates or finding the final concentration within a vaccine formulation for example. Both the PCR and qPCR primers were tested against the most closely genetically related species to S. agalactiae as well as other species, which are less closely related, but are likely to be found in the aquatic environment. This was to ensure that there was no cross reaction between the target and other species of bacteria which are likely to be found in the aquatic environment. The PCR primers were found to be specific to the target, with no cross reactions noted between the primers and the species of bacteria tested. The primers were found to have an analytical sensitivity of between 10-100 copies of DNA in a 25 µL reaction mixture and had high epidemiological sensitivity, all 11 CCs tested were detected by the primers. The qPCR was found to be capable of quantifying DNA copy number over 8 orders of magnitude, with the limit of detection being 1.7 copies of DNA µL-1 of reaction mixture. The qPCR assay was found to be sensitive to all 11 CCs of S. agalactiae tested. This qPCR assay is not 100% specific to S. agalactiae however, but as the qPCR was not designed as a diagnosis tool the lack of 100% specificity was not considered to impact on its intended use. The work contributes to the field of aquaculture and aquaculture research by giving researchers a new assay to detect S. agalactiae from aquatic environments which has been developed with the most likely occurring contaminants of aquatic samples in mind. This rapid and sensitive method could prove very useful in detecting low levels of S. agalactiae in aquaculture systems. The qPCR assay is useful to quantify DNA copy number in the laboratory, and has been used to help study the progression of disease in a vaccine study performed on tilapia against S. agalactiae. The assay has been used by scientists within the aquaculture research group at the Moredun Research Institute to quantify the DNA copy number of bacteria within the brains of dead fish and reported as part of a vaccine development study.