550
Pathogenicity
Infected (i.p. injection) dogfi sh ( Squalus acanthias) died within 18 h. Lemon
sharks were more resistant to infection, although internal damage followed the
injection of 5 × 10
7 cells. Yii et al. ( 1997 ) reported an LD 50 dose for grouper of
2.53 × 10
7 colony forming units (CFU)/g of body weight, with disease signs reminiscent of the natural infection, i.e. swollen intestine full of a yellowish fl uid. This
was higher than the LD 50 value of 1.5 × 10
5 to 1.6 × 10
6 CFU/fi sh reported for sea
bass (Pujalte et al. 2003 ). For comparison, Won and Park ( 2008 ) determined the
LD 50 dose to olive fl ounder ( Paralichthys olivaceus ) and black rockfi sh ( Sebastes
schlegeli ) as 2.48 × ~10
5 – 8.76 × ~10
7 and 2.0 × ~10
4 – 2.52 × ~10
6 CFU/g of fi sh,
respectively. Although the pathogenic mechanism remains to be fully elucidated, it
is relevant to note that the organism is slightly cytotoxic and produces ECPs
(Zhang and Austin 2000 ; Zorrilla et al. 2003 ; Won and Park 2008 ) containing haemolysins (Zhang and Austin 2000 ; Zhu et al. 2006 ; Li et al. 2011 ), caseinase, gelatinase, lipase and phospholipase (Zhang and Austin 2000 ). A possible relationship
between bioluminescence, quorum sensing and pathogenicity is unclear (Defoirdt
et al. 2008 ). Of relevance, Zhang et al. ( 2001 ) correlated virulence to salmonids
with the possession of duplicate haemolysin genes, termed vhhA and vhhB. The
VHH haemolysin protein demonstrated homology to the lecithinase of other vibrios, namely V. mimicus and V. cholerae. vhhA was overexpressed in Esch. coli, and
the purifi ed protein was characterized, and determined to be cytotoxic to fl ounder
gill cells and lethal to fl ounder with an LD 50 dose of 18.4 μg of protein/fi sh (Zhong
et al. 2006 ). Three strains, VJ1, VJ2 and VJ3, recovered from diseased Japanese
fl ounder in China demonstrated signifi cantly increased resistance to the bacteriocidal effects of host serum and induced greater levels of respiratory burst activity
in head kidney macrophages although there was an inability to replicate in the
macrophages. In cultured cells, apoptosis was induced as observed by DNA fragmentation, apoptotic bodies and elevated caspase 3 activity (Li et al. 2011 ). Using
black sea bream (Mylio macrocephalus) and silver sea bream (Sparus sarba) fi brobast cell lines, recombinant VHH induced apoptosis involving a decrease in mitochondrial membrane potential, and thence an increase in caspase 3 activity within
2–8 h, although there was not observed effect on ROS. However, HSP70 levels
were increased followed by 6 h of recovery. The reduction in mitochondrial membrane potential was suppressed when cells had a 6 h heat shock recovery period.
Moreover, the protective effect of HSP70 was annulled if cells were exposed to the
HSP70 inhibitor, quercetin. Thus, HSP70 may be attributed with an anti-apoptotic
role. Overall, the research highlighted that haemolysin leads to cell death by induction of apoptosis (Deane et al. 2012 ).
Parvathi et al. ( 2009 ) pointed to the heterogenicity of the vhh gene in Indian
isolates, albeit from shrimp. Sun et al. ( 2001 ) reported that the loss of a single residue change in haemolysin resulted in loss of haemolytic and phospholipase activities and thus pathogenicity to turbot. Also, siderophores are produced (Owens et al.
1996 ). More recently, research has highlighted a role for bacteriophage (Oakey and
10 Vibrios
Pathogenicity
Infected (i.p. injection) dogfi sh ( Squalus acanthias) died within 18 h. Lemon
sharks were more resistant to infection, although internal damage followed the
injection of 5 × 10
7 cells. Yii et al. ( 1997 ) reported an LD 50 dose for grouper of
2.53 × 10
7 colony forming units (CFU)/g of body weight, with disease signs reminiscent of the natural infection, i.e. swollen intestine full of a yellowish fl uid. This
was higher than the LD 50 value of 1.5 × 10
5 to 1.6 × 10
6 CFU/fi sh reported for sea
bass (Pujalte et al. 2003 ). For comparison, Won and Park ( 2008 ) determined the
LD 50 dose to olive fl ounder ( Paralichthys olivaceus ) and black rockfi sh ( Sebastes
schlegeli ) as 2.48 × ~10
5 – 8.76 × ~10
7 and 2.0 × ~10
4 – 2.52 × ~10
6 CFU/g of fi sh,
respectively. Although the pathogenic mechanism remains to be fully elucidated, it
is relevant to note that the organism is slightly cytotoxic and produces ECPs
(Zhang and Austin 2000 ; Zorrilla et al. 2003 ; Won and Park 2008 ) containing haemolysins (Zhang and Austin 2000 ; Zhu et al. 2006 ; Li et al. 2011 ), caseinase, gelatinase, lipase and phospholipase (Zhang and Austin 2000 ). A possible relationship
between bioluminescence, quorum sensing and pathogenicity is unclear (Defoirdt
et al. 2008 ). Of relevance, Zhang et al. ( 2001 ) correlated virulence to salmonids
with the possession of duplicate haemolysin genes, termed vhhA and vhhB. The
VHH haemolysin protein demonstrated homology to the lecithinase of other vibrios, namely V. mimicus and V. cholerae. vhhA was overexpressed in Esch. coli, and
the purifi ed protein was characterized, and determined to be cytotoxic to fl ounder
gill cells and lethal to fl ounder with an LD 50 dose of 18.4 μg of protein/fi sh (Zhong
et al. 2006 ). Three strains, VJ1, VJ2 and VJ3, recovered from diseased Japanese
fl ounder in China demonstrated signifi cantly increased resistance to the bacteriocidal effects of host serum and induced greater levels of respiratory burst activity
in head kidney macrophages although there was an inability to replicate in the
macrophages. In cultured cells, apoptosis was induced as observed by DNA fragmentation, apoptotic bodies and elevated caspase 3 activity (Li et al. 2011 ). Using
black sea bream (Mylio macrocephalus) and silver sea bream (Sparus sarba) fi brobast cell lines, recombinant VHH induced apoptosis involving a decrease in mitochondrial membrane potential, and thence an increase in caspase 3 activity within
2–8 h, although there was not observed effect on ROS. However, HSP70 levels
were increased followed by 6 h of recovery. The reduction in mitochondrial membrane potential was suppressed when cells had a 6 h heat shock recovery period.
Moreover, the protective effect of HSP70 was annulled if cells were exposed to the
HSP70 inhibitor, quercetin. Thus, HSP70 may be attributed with an anti-apoptotic
role. Overall, the research highlighted that haemolysin leads to cell death by induction of apoptosis (Deane et al. 2012 ).
Parvathi et al. ( 2009 ) pointed to the heterogenicity of the vhh gene in Indian
isolates, albeit from shrimp. Sun et al. ( 2001 ) reported that the loss of a single residue change in haemolysin resulted in loss of haemolytic and phospholipase activities and thus pathogenicity to turbot. Also, siderophores are produced (Owens et al.
1996 ). More recently, research has highlighted a role for bacteriophage (Oakey and
10 Vibrios