Investigations into the pathogenesis of aquatic Streptococcus agalactiae and Streptococcus iniae in Nile tilapia (Oreochromis niloticus)

Abstract

The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.