Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, were found dead from Karumba (Gulf of Carpentaria) in Northern Territory to Brisbane in southern Queensland, Australia. In twelve cases, Streptoccocus agalactiae (Group B Streptococcus, GBS) was isolated in pure culture from eyes and internal organs. GBS has been also isolated from other marine species (Javelin grunter, Giant catfish and Squaretail mullet) and caused an outbreak in three species of wild stingrays (Eastern shovelnose ray, Mangrove whipray and Estuary ray) in a public aquarium also in Queensland. These findings have raised public and industry concern over threats to human health from potential transmission between fish and humans, over environmental impacts on other aquatic animals, and over the potential threat posed to the growing Australian aquaculture industry in which, to date, there have been no reported cases of this disease in spite of the devastation caused to the industry overseas. Therefore, my thesis set out to determine whether GBS isolated from dead wild fish in Australia is indeed virulent in Queensland grouper and causative of mortality, to try to identify from where these infections in wild fish originated, to determine potential risk to human health and to other aquatic animals, and to begin to identify mechanisms by which these Australian isolates may cause fatal disease in Queensland grouper. This information may be employed in future risk assessment of potential human transfer, in mitigating further outbreaks in wild fish through understanding origin of infection, and in future development of preventative measures such as vaccination for farmed fish.
In order to confirm that S. agalactiae was the cause of the mortality in wild fish, experimental challenges were conducted by injection, immersion and through the oral route. To better understand why these strains of GBS are pathogenic in fish, we conducted a series of cellular immune assays using juvenile Queensland grouper head-kidney leucocytes in the presence of different piscine and terrestrial isolates. Whole-genome sequencing analysis of the piscine and terrestrial isolates provided information on potential origin and potential for transfer between species, and also informed us on critical virulence factors that could be used during pathogenesis of GBS. Moreover, genome analysis gave intriguing insight into the evolution and adaption of GBS to fish as a host and to life in the aquatic environment.
Based on results from infectivity trials, a local piscine isolate of Streptococcus agalactiae demonstrated high virulence in juvenile Queensland grouper. By injection it caused 100% morbidity and mortality regardless of challenge dose, and mortalities were significantly higher in fish challenged by immersion, suggesting that skin mucus plays an important role in the initial steps of the infection. Oral challenge by admixture with feed resulted in development of clinical symptoms in ~ 10 % of fish challenged with the highest dose, and a high level of asymptomatic carriage was detected by histology and PCR but no mortalities were recorded, suggesting a possible natural oral route of infection, but that other factors are required for overt disease. Clinical signs and pathologies typical of streptococcal infections were successfully reproduced and corresponded to what has been described in the wild cases in Australia and elsewhere.
To determine putative origin of GBS in wild fish in Australia, multilocus sequence typing (MLST) and capsular polysaccharide (CPS) were derived from whole-genome sequences and revealed that all piscine isolates from Australia were serotype Ib and fell into ST-261 lineage first identified in 1986 from Tilapia in Israel. Overseas, ST-261 has only been found in fish and poikilotherm animals, and has never been associated with humans. High similarity and identical point mutations in the capsular operon of the piscine isolates suggests a common source of infection and subsequent passage amongst these animals.
Mature macrophages and neutrophils from the head-kidney leucocytes are the major phagocytic cells of E. lanceolatus compared to the smaller and less complex monocytes. Quenching of reactive oxygen species (ROS) production coupled with high phagocytic indexes and elevated immune cell mortality rate caused by some terrestrial GBS isolates suggests potential for transmission from terrestrial animals to fish, in line with previous research in Brazil and the USA.
Whole-genome sequence analysis revealed a significant genome reduction in the piscine GBS isolates compared to the human and terrestrial strains, characterised by the loss of metabolic functions. Phenotypically, while piscine and terrestrial isolates shared the same enzymatic hydrolysis profile, their ability to ferment carbohydrates differed greatly, corroborative of substantial reduction in carbohydrate metabolic genes. Genomic mutations observed in the capsular polysaccharide were phenotypically relevant, with complete loss of the capsule in two of the piscine isolates. Marine GBS isolates grew more slowly than terrestrial isolates in rich medium, but this difference, though still significant, was less pronounced at lower temperature (at 28°C compared to 37°C). Most virulence factors found in mammalian isolates were missing from fish strains, but a few were conserved. As meningitis is common to GBS infection in both humans and fish, this thesis provides a basis to identify factors conserved between terrestrial and marine isolates of GBS that are critical to development of meningitis.