444
Diagnosis
Serology Whole cell agglutination was effective for the detection of T. maritimum
(Wakabayashi et al. 1984 ). Also, FAT is useful for detecting (=T. maritimum) in fi sh
tissues (Baxa et al. 1988a ).
Molecular Methods PCR targeted 16S rRNA has been successful with Fla. branchiophilum, Fla. columnare and T. maritimum (Toyama et al. 1994 , 1996 ; AvendañoHerrera et al. 2004e ; Yeh et al. 2006 ) although freezing-thawing destroyed the cells
leading to the DNA being undetectable by PCR (Suomalainen et al. 2006a , b , c ).
However, a detection limit of 10
2
–10
4 cells/reaction tube was reported (AvendañoHerrera et al. 2004b ). Nested-PCR gave even greater sensitivity, detecting 1–250
cells/PCR reaction particularly in the skin and mucus (Avendaño-Herrera et al.
2004b ). This is compatible with the sensitivity of 75 CFU/g reported for the nestedPCR by Cepeda et al. ( 2003 ). A nested-PCR increased sensitivity to 10 bacterial
cells in asymptomatic fi sh (Bader et al. 2003a ). A quantitative real-time PCR, which
incorporated primers amplifying a 155-bp fragment of the 16S rRNA gene, had a
detection limit of 4.8 DNA copy number/μl and was applicable to use with formalinfi xed paraffi n-embedded samples from the fi eld (Fringuelli et al. 2012 ). DNA from
the pathogen was detected in most (89 %) of the tissue blocks, which did not have
clear sign of gill disease, and in 95 % of the blocks from fi sh with mild-to-severe gill
pathology (Fringuelli et al. 2012 ). Similarly, PCR technology has proven to be successful for the detection of Fla. psychrophilum and T. maritimum , and was considered to be more sensitive than culturing (Wiklund et al. 2000 ; Avendaño-Herrera
et al. 2004b ).
Epizootiology
T. maritimum may also be responsible for heavy losses, i.e. 20–30 % of the population, among juvenile red sea bream and black sea bream (Hikida et al. 1979 ). In
common with many organisms, extended survival of T. maritimum occurs in sterile
but not in natural seawater (~5 days), i.e. with a resident microfl ora (AvendañoHerrera et al. 2006 ).
Pathogenicity
In laboratory-based experiments, black sea bream were more susceptible to T. maritimum than red sea bream following i.m. injection of 0.02 ml of culture per fi sh, or
infection by bathing or direct application of cultures to the tail or mouth. Mortalities,
of up to 10 %, occurred in 3 days (Wakabayashi et al. 1984 ). An infection model has
also been established for turbot in which the fi sh were immersed for 18 h with the
7 Flavobacteria and Cytophagas
Diagnosis
Serology Whole cell agglutination was effective for the detection of T. maritimum
(Wakabayashi et al. 1984 ). Also, FAT is useful for detecting (=T. maritimum) in fi sh
tissues (Baxa et al. 1988a ).
Molecular Methods PCR targeted 16S rRNA has been successful with Fla. branchiophilum, Fla. columnare and T. maritimum (Toyama et al. 1994 , 1996 ; AvendañoHerrera et al. 2004e ; Yeh et al. 2006 ) although freezing-thawing destroyed the cells
leading to the DNA being undetectable by PCR (Suomalainen et al. 2006a , b , c ).
However, a detection limit of 10
2
–10
4 cells/reaction tube was reported (AvendañoHerrera et al. 2004b ). Nested-PCR gave even greater sensitivity, detecting 1–250
cells/PCR reaction particularly in the skin and mucus (Avendaño-Herrera et al.
2004b ). This is compatible with the sensitivity of 75 CFU/g reported for the nestedPCR by Cepeda et al. ( 2003 ). A nested-PCR increased sensitivity to 10 bacterial
cells in asymptomatic fi sh (Bader et al. 2003a ). A quantitative real-time PCR, which
incorporated primers amplifying a 155-bp fragment of the 16S rRNA gene, had a
detection limit of 4.8 DNA copy number/μl and was applicable to use with formalinfi xed paraffi n-embedded samples from the fi eld (Fringuelli et al. 2012 ). DNA from
the pathogen was detected in most (89 %) of the tissue blocks, which did not have
clear sign of gill disease, and in 95 % of the blocks from fi sh with mild-to-severe gill
pathology (Fringuelli et al. 2012 ). Similarly, PCR technology has proven to be successful for the detection of Fla. psychrophilum and T. maritimum , and was considered to be more sensitive than culturing (Wiklund et al. 2000 ; Avendaño-Herrera
et al. 2004b ).
Epizootiology
T. maritimum may also be responsible for heavy losses, i.e. 20–30 % of the population, among juvenile red sea bream and black sea bream (Hikida et al. 1979 ). In
common with many organisms, extended survival of T. maritimum occurs in sterile
but not in natural seawater (~5 days), i.e. with a resident microfl ora (AvendañoHerrera et al. 2006 ).
Pathogenicity
In laboratory-based experiments, black sea bream were more susceptible to T. maritimum than red sea bream following i.m. injection of 0.02 ml of culture per fi sh, or
infection by bathing or direct application of cultures to the tail or mouth. Mortalities,
of up to 10 %, occurred in 3 days (Wakabayashi et al. 1984 ). An infection model has
also been established for turbot in which the fi sh were immersed for 18 h with the
7 Flavobacteria and Cytophagas