Novel chlamydia-like agents of epitheliocystis in wild and cultures Australian finfish

Epitheliocystis is a Chlamydia-like, obligate intracellular bacterial condition that affects the gills and skin of finfish. To date, epitheliocystis have occurred in both wild and farmed fish populations and have been described in over 90 fish species worldwide, including hosts from marine and freshwater environments. In Australia, the condition has been reported from economically important species, such as yellowtail kingfish (Seriola lalandi), barramundi (Lates calcarifer) and silver perch (Bidyanus bidyanus). It is characterised by the presence of membrane-enclosed basophilic inclusions in host epithelial cells and can cause a severe proliferative host response, including swollen gills, excessive mucus production and respiratory distress. Affected fish, especially those in commercial culture, exhibit lethargy, surface gaping and reduced growth. Epitheliocystis can cause mortalities of up to 100% in commercially cultured fish. While over ten species from Australian waters have been reported with epitheliocystis infections, there is however very little information available on the infection levels of this condition in both wild and farmed fish in Australia. In addition, the diversity of Chlamydia-like epitheliocystis agent(s) is not fully known and as it stands, no epitheliocystis agent has been characterised in the Southern Hemisphere. The overall objective of this thesis was to study the disease epitheliocystis in wild and farmed Australian finfish and to gain a greater understanding of the aetiological agent(s) involved. To address this objective the following specific aims were studied: a) Investigate and document infection levels in wild and farmed fish, and determine which factors affect epitheliocystis outbreaks b) Characterise epitheliocystis agents from at least two species of Australian fish c) Critically review the current understanding of the role of Chlamydia-like organisms found in association with epitheliocystis Much still needs to be learnt about the epidemiology of chlamydial infections in fish. Due to the inherent nature of intracellular bacteria requiring a host cell to survive, there is currently no in vitro method to culture these bacteria in pure form. As a result, Koch's postulates are not possible to fulfil at this time and the molecular postulates developed by Fredricks and Relman were used instead to prove the disease causation relationship. This thesis presents the results for epitheliocystis in six species of finfish of Australian origin. Three wild caught species, jack mackerel (Trachurus declivis), sand flathead (Platycephalus bassensis) and tiger flathead (Neoplatycephalus richardsoni), were surveyed to document the natural prevalence rates of epitheliocystis in Eastern Tasmanian waters. Histology of gill tissue revealed epitheliocystis to be present in all three species. In addition, there was a significant correlation of increasing prevalence of epitheliocystis with a decrease in seawater temperature. This was the first published report of epitheliocystis in both sand flathead and tiger flathead. Three commercially farmed species, yellowtail kingfish, striped trumpeter (Latris lineata) and barramundi were also studied. Gill tissue from yellowtail kingfish, striped trumpeter and barramundi were sampled for histology, 16S rRNA amplification and sequencing, in situ hybridisation and transmission electron microscopy. Epitheliocystis was present in all commercial species tested by histology. DNA analysis revealed the presence of novel Chlamydia-like 16S rRNA sequences from gill DNA of all three fish species, and were found to be only 87-88% similar to their nearest known relative, the previously published 'Candidatus Piscichlamydia salmonis' (AY462244) from Atlantic salmon (Salmo salar) and Arctic charr (Salvelinus alpinus). Supporting the Chlamydia-like result was the in situ hybridisation and transmission electron microscopy, revealing that the bacteria inside the cysts were in fact Chlamydia-like. From comprehensive phylogenetic analysis, it was found that a novel family lineage, two novel genera lineages and three novel species lineages within the Order Chlamydiales had been discovered and these were then described. These novel bacteria were named 'Ca. Parilichlamydia carangidicola', 'Ca. Similichlamydia latridicola' and 'Ca. Similichlamydia laticola', from yellowtail kingfish, striped trumpeter and barramundi, respectively. The most exciting find, a horizontal transmission route of the Chlamydia-like bacteria causing epitheliocystis infections in barramundi, is a world first. The molecular characterisation and description of these epitheliocystis aetiological agents reported in this thesis are the first of their kind of Chlamydia-like bacteria associated with epitheliocystis from any fish species found in the southern hemisphere. These chlamydial infections of fish are becoming more broadly recognised by researchers and farmers as an important cause of disease, especially within commercial aquaculture. Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the Order Chlamydiales. Remarkably, in most cases, each new piscine host studied has revealed the presence of a phylogenetically unique and novel chlamydial pathogen. The work presented in this thesis has made a significant contribution to the knowledge of epitheliocystis and its many aetiological agents.