503
Molecular Methods It may be necessary to discriminate subspecies piscicida
from damselae, which has been achieved using TCBS on which the former did not
grow (Rajan et al. 2003 ). However, this situation regarding discrimination between
the two subspecies had been previously resolved by Osorio et al. ( 2000 ), who used
multiplex-PCR to detect and differentiate subsp. damselae and piscicida. The former produced two amplifi cation products, i.e. of 267 (fragment of 16S rRNA) and
448 bp (fragment of ureC gene) whereas the latter revealed only the 267 bp product.
This suggests – and was confi rmed by dot blot hybridisation – that subsp. piscicida
lacks the ureC gene. A multiplex PCR was developed with a detection limit of
500 fg, which equals 100 genome equivalents (Amagliani et al. 2009 ). RT-PCR and
PCR-DGGE of the toxR gene fragment were developed, and detected 1797.85 +/−
376.15 ( ca. 0.71 fg) and 2976.68 +/− 1253.63 (ca. 1.76 fg) gene copy numbers of
toxR genes in aquaculture samples (Martins et al. 2015 ).
Epizootiology
The organism was associated initially with ulcerative lesions along the fl ank of
blacksmith (Chromis punctipinnis), one of the damselfi sh. These ulcers were noted
in summer and autumn among fi sh populations in the coastal waters of southern
California. Surveys of wild fi sh populations led to a conclusion that the ulcers were
restricted to species of damselfi sh. Additional information pointed to a role in human
pathogenicity, insofar as the organism has been isolated from human wounds (Love
et al. 1981 ). Subsequent work demonstrated this organism in sharks (Grimes et al.
1984a , b ; Fujioka et al. 1988 ), turbot (Fouz et al. 1991 , 1992 ), yellowtail (Sakata
et al. 1989 ) and nurse sharks and rainbow trout [in Denmark] (Pedersen et al. 1997a ).
Ulcerated fi sh, presumably the result of disease caused by Ph. damselae, accounted
for 10–70 % of the population in King Harbor, Redondo Beach, California, during
August to October, and at a second site (Ship Rock, Catalina Island) during June to
October. This suggests a seasonal distribution in the incidence of disease, and possibly coincides with warmer water temperatures and lower resistance caused by
physiological changes in the host during sexual maturity. Conceivably, Ph. damselae
occurs normally in the marine environment, where it could pose a constant threat to
susceptible fi sh species. Indeed, seawater is the likely mode of transmission of virulent cells of the pathogen, with cells adhering to and resisting the bacteriocidal effects
of mucus. This suggests that skin is a site of entry into the host (Fouz et al. 2000 ).
Pathogenicity
The pathogen has been implicated with disease in sharks, turbot and yellowtail
(Fujioka et al. 1988 ; Sakata et al. 1989 ; Fouz et al. 1991 ) and rainbow trout in
marine sites in Denmark (Pedersen et al. 2009 ). Laboratory infections of C.
Photobacterium damselae subsp. damselae
Molecular Methods It may be necessary to discriminate subspecies piscicida
from damselae, which has been achieved using TCBS on which the former did not
grow (Rajan et al. 2003 ). However, this situation regarding discrimination between
the two subspecies had been previously resolved by Osorio et al. ( 2000 ), who used
multiplex-PCR to detect and differentiate subsp. damselae and piscicida. The former produced two amplifi cation products, i.e. of 267 (fragment of 16S rRNA) and
448 bp (fragment of ureC gene) whereas the latter revealed only the 267 bp product.
This suggests – and was confi rmed by dot blot hybridisation – that subsp. piscicida
lacks the ureC gene. A multiplex PCR was developed with a detection limit of
500 fg, which equals 100 genome equivalents (Amagliani et al. 2009 ). RT-PCR and
PCR-DGGE of the toxR gene fragment were developed, and detected 1797.85 +/−
376.15 ( ca. 0.71 fg) and 2976.68 +/− 1253.63 (ca. 1.76 fg) gene copy numbers of
toxR genes in aquaculture samples (Martins et al. 2015 ).
Epizootiology
The organism was associated initially with ulcerative lesions along the fl ank of
blacksmith (Chromis punctipinnis), one of the damselfi sh. These ulcers were noted
in summer and autumn among fi sh populations in the coastal waters of southern
California. Surveys of wild fi sh populations led to a conclusion that the ulcers were
restricted to species of damselfi sh. Additional information pointed to a role in human
pathogenicity, insofar as the organism has been isolated from human wounds (Love
et al. 1981 ). Subsequent work demonstrated this organism in sharks (Grimes et al.
1984a , b ; Fujioka et al. 1988 ), turbot (Fouz et al. 1991 , 1992 ), yellowtail (Sakata
et al. 1989 ) and nurse sharks and rainbow trout [in Denmark] (Pedersen et al. 1997a ).
Ulcerated fi sh, presumably the result of disease caused by Ph. damselae, accounted
for 10–70 % of the population in King Harbor, Redondo Beach, California, during
August to October, and at a second site (Ship Rock, Catalina Island) during June to
October. This suggests a seasonal distribution in the incidence of disease, and possibly coincides with warmer water temperatures and lower resistance caused by
physiological changes in the host during sexual maturity. Conceivably, Ph. damselae
occurs normally in the marine environment, where it could pose a constant threat to
susceptible fi sh species. Indeed, seawater is the likely mode of transmission of virulent cells of the pathogen, with cells adhering to and resisting the bacteriocidal effects
of mucus. This suggests that skin is a site of entry into the host (Fouz et al. 2000 ).
Pathogenicity
The pathogen has been implicated with disease in sharks, turbot and yellowtail
(Fujioka et al. 1988 ; Sakata et al. 1989 ; Fouz et al. 1991 ) and rainbow trout in
marine sites in Denmark (Pedersen et al. 2009 ). Laboratory infections of C.
Photobacterium damselae subsp. damselae