1. Hawaiian Rickettsia –like Organism (HRLO) Allen C. Riggs DVM, MS Aquaculture Development Program- Disease Prevention (ADP-DP) Hawaii Department of Agriculture

2. Rickettsia characterisitics Non-motile, Gram negative, non- sporeforming bacteria Non-motile, Gram negative, non- sporeforming bacteria Pleomorphic shapes( cocci, rods or thread- like) Pleomorphic shapes( cocci, rods or thread- like) Obligate intracellular parasite – must live inside cytoplasm of host cell Obligate intracellular parasite – must live inside cytoplasm of host cell

3. Rickettsia characterisitics Cannot live in artificial nutrient environments – need tissue or embryo cultures Cannot live in artificial nutrient environments – need tissue or embryo cultures In the past positioned between viruses and bacteria In the past positioned between viruses and bacteria

4. RLO History Piscirickettsia salmonis – first RLO fish pathogen from coho salmon from Chile in the 1980s Piscirickettsia salmonis – first RLO fish pathogen from coho salmon from Chile in the 1980s Now numerous freshwater and saltwater species affected ( salmon, seabass, grouper, tilapia and several ornamentals)) Now numerous freshwater and saltwater species affected ( salmon, seabass, grouper, tilapia and several ornamentals))

5. RLO in tilapia First reported case in Taiwan - 1992 First reported case in Taiwan - 1992 Epizootic affecting six species of tilapia Epizootic affecting six species of tilapia FW and SW FW and SW Originated in 1 farm and eventually spread to 37 facilities Originated in 1 farm and eventually spread to 37 facilities Mortality 75%+ in severe cases – 30% average Mortality 75%+ in severe cases – 30% average

6. RLO in Hawaii Initial cases in mid 1990s on Oahu Initial cases in mid 1990s on Oahu Oreochromis mossambicus and Sarotherodon melanotheron (black chinned tilapia) Oreochromis mossambicus and Sarotherodon melanotheron (black chinned tilapia) Farmed and wild populations affected Farmed and wild populations affected Farmed mortalities exceeding 60% resulting in major financial losses Farmed mortalities exceeding 60% resulting in major financial losses

7. New name and culture conditions for RLO In 2007 researchers determined that the agent responsible for RLO is a Francisella sp. bacteria – not a rickettsia In 2007 researchers determined that the agent responsible for RLO is a Francisella sp. bacteria – not a rickettsia They were also able to culture the bacteria on modified artificial media – but difficult and not always successful They were also able to culture the bacteria on modified artificial media – but difficult and not always successful PCR assay was developed which is now the preferred method of confirmation PCR assay was developed which is now the preferred method of confirmation

8. Clinical signs of RLO/Francisella sp. Dark in color Dark in color Emaciation Emaciation Abnormal swimming behavior Abnormal swimming behavior Eye lesions Eye lesions Enlarged spleen Enlarged spleen White nodules in many visceral organs but NOT liver White nodules in many visceral organs but NOT liver

14. What are these white nodules? Granulomas – groups of inflammatory cells around a focus of infection Granulomas – groups of inflammatory cells around a focus of infection Typically chronic or slow, occurring over a long period of time Typically chronic or slow, occurring over a long period of time NOT specific to any one disease – other steps needed to ID cause NOT specific to any one disease – other steps needed to ID cause

17. How to ID cause of granulomas? Routine H & E histology to confirm that the structure is a granuloma Routine H & E histology to confirm that the structure is a granuloma Special stain to rule-out acid fast bacteria ( Mycobacterium sp.) Special stain to rule-out acid fast bacteria ( Mycobacterium sp.) Lowenstein-Jensen culture to rule-out Mycobacterium sp. Lowenstein-Jensen culture to rule-out Mycobacterium sp. PCR test for specific agent - Francisella PCR test for specific agent - Francisella

22. What factors lead to clinical outbreaks? Low water temperatures for longer than 7 days – most Hawaii outbreaks occur October - April Low water temperatures for longer than 7 days – most Hawaii outbreaks occur October - April Optimum temperature for tilapia is 29 – 31C or 85-88F Optimum temperature for tilapia is 29 – 31C or 85-88F Increased mortalities from all infectious diseases including RLO at temperatures below 19C or 66F Increased mortalities from all infectious diseases including RLO at temperatures below 19C or 66F Tilapia often stop feeding below 17C or 63F Tilapia often stop feeding below 17C or 63F Lower lethal temperature in normal non-infected tilapia typically 10 -11C or 50-52F Lower lethal temperature in normal non-infected tilapia typically 10 -11C or 50-52F

23. Secondary stressors: Overcrowding Overcrowding Sub-optimum water quality Sub-optimum water quality Inadequate nutrition Inadequate nutrition Presence of ectoparasites Presence of ectoparasites Combination of many or all is worst case scenario Combination of many or all is worst case scenario

24. Can it be treated ? Mortalities decreased with oxytetracycline medicated foods during the early cases in the 1990s Mortalities decreased with oxytetracycline medicated foods during the early cases in the 1990s Alternative treatment methods are being used/developed internationally but not available yet in the US Alternative treatment methods are being used/developed internationally but not available yet in the US

25. Questions to be considered? Is RLO/Francisella present on all of the Hawaiian islands? Is RLO/Francisella present on all of the Hawaiian islands? Is it present in both wild and farmed stocks? Is it present in both wild and farmed stocks? Can other important Hawaiian aquaculture species be affected? Can other important Hawaiian aquaculture species be affected? What can we do with infected stocks? What can we do with infected stocks? Is the bacteria endemic in the environment? Is the bacteria endemic in the environment? Is there a industry need for clean stocks? Is there a industry need for clean stocks?