508
Serology An ELISA was developed, which successfully identifi ed Ph. damselae
subsp. piscicida albeit in artifi cially infected fi sh tissue within 4 h. By visually
recording the ELISA the threshold for positivity was 10
5 cells/ml. However, use of
a reader cut this level to only 10
3 cells/ml (Bakopoulos et al. 1997a ). Magnetic
beads incorporating polyclonal antibodies with an enzyme immuno assay have
found success for the rapid diagnosis of pasteurellosis, in which the commercial kit
had a detection limit of 10
4 cells albeit with a problem of specifi city, i.e. other photobacteria namely Ph. damselae subsp. damselae and Ph. histaminum , were also
detected (Romalde et al. 1999 ).
Molecular Methods Specifi c DNA hybridisation probes for Ph. damselae subsp.
piscicida offer promise for the future. Already, initial studies with a
32 P-labelled
DNA probe indicated a minimum detection limit of 3.9 ng of DNA or 10
5 bacterial
cells (Zhao and Aoki 1989 ). Aoki et al. 1997 ) emphasised the value of a 629 base
pair DNA fragment from the subspecies specifi c plasmid pZP1 for PCR. Kvitt et al.
( 2002 ) reported a detection limit of 0.35 pg, which equated to ~40 bacterial cells. A
nested PCR detected 10 fg to 1 pg of DNA, which was considered to be equivalent
to 20 to 200 cells, being sensitive enough to detect the pathogen in asymptomatic
fi sh (Osorio et al. 1999 ). In comparison, PCR-RFLP approach detected <180 fg of
purifi ed DNA, and was useful for use with sea bream and sea bass (Zappulli et al.
2005 ). In one case, it was necessary to discriminate subspecies piscicida from damselae, which was achieved using TCBS on which the former did not grow (Rajan
et al. 2003 ). However, this 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 ).
Epizootiology
There is no doubt that pasteurellosis is a serious condition of both farmed and wild
fi sh populations (Snieszko et al. 1964 ; Kusuda and Yamaoka 1972 ; Ohnishi et al.
1982 ; Yasunaga et al. 1983 ). Heavy mortalities, in the range of 40–50 % of the stock,
have occurred during summer months. Unfortunately, the reasons for these outbreaks are largely unknown. Likewise, the precise nature of the pathogenicity
mechanism remains to be elucidated.
It is thought that infection takes place in sea water at temperatures of approximately 25 °C (Yasunaga et al. 1983 ). Toranzo et al. ( 1982 ) devised a series of survival experiments, and concluded that Ph. damselae subsp. piscicida was short-lived
in freshwater and estuarine conditions. Thus in freshwater, the organism could not
be cultured after 48 h at 20 °C. Survival in estuarine water (salinity = 12‰) was
10 Vibrios
Serology An ELISA was developed, which successfully identifi ed Ph. damselae
subsp. piscicida albeit in artifi cially infected fi sh tissue within 4 h. By visually
recording the ELISA the threshold for positivity was 10
5 cells/ml. However, use of
a reader cut this level to only 10
3 cells/ml (Bakopoulos et al. 1997a ). Magnetic
beads incorporating polyclonal antibodies with an enzyme immuno assay have
found success for the rapid diagnosis of pasteurellosis, in which the commercial kit
had a detection limit of 10
4 cells albeit with a problem of specifi city, i.e. other photobacteria namely Ph. damselae subsp. damselae and Ph. histaminum , were also
detected (Romalde et al. 1999 ).
Molecular Methods Specifi c DNA hybridisation probes for Ph. damselae subsp.
piscicida offer promise for the future. Already, initial studies with a
32 P-labelled
DNA probe indicated a minimum detection limit of 3.9 ng of DNA or 10
5 bacterial
cells (Zhao and Aoki 1989 ). Aoki et al. 1997 ) emphasised the value of a 629 base
pair DNA fragment from the subspecies specifi c plasmid pZP1 for PCR. Kvitt et al.
( 2002 ) reported a detection limit of 0.35 pg, which equated to ~40 bacterial cells. A
nested PCR detected 10 fg to 1 pg of DNA, which was considered to be equivalent
to 20 to 200 cells, being sensitive enough to detect the pathogen in asymptomatic
fi sh (Osorio et al. 1999 ). In comparison, PCR-RFLP approach detected <180 fg of
purifi ed DNA, and was useful for use with sea bream and sea bass (Zappulli et al.
2005 ). In one case, it was necessary to discriminate subspecies piscicida from damselae, which was achieved using TCBS on which the former did not grow (Rajan
et al. 2003 ). However, this 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 ).
Epizootiology
There is no doubt that pasteurellosis is a serious condition of both farmed and wild
fi sh populations (Snieszko et al. 1964 ; Kusuda and Yamaoka 1972 ; Ohnishi et al.
1982 ; Yasunaga et al. 1983 ). Heavy mortalities, in the range of 40–50 % of the stock,
have occurred during summer months. Unfortunately, the reasons for these outbreaks are largely unknown. Likewise, the precise nature of the pathogenicity
mechanism remains to be elucidated.
It is thought that infection takes place in sea water at temperatures of approximately 25 °C (Yasunaga et al. 1983 ). Toranzo et al. ( 1982 ) devised a series of survival experiments, and concluded that Ph. damselae subsp. piscicida was short-lived
in freshwater and estuarine conditions. Thus in freshwater, the organism could not
be cultured after 48 h at 20 °C. Survival in estuarine water (salinity = 12‰) was
10 Vibrios