43
whole cell proteins, concluding that En. seriolicida was closely related to
Lactococcus garvieae. Also, Eldar et al. ( 1996 ) reached the same conclusion after
studying the type strains phenotypically and by DNA:DNA hybridisation. The
taxon is certainly homogeneous (Kawanishi et al. 2006 ), as verifi ed by RFLP
(Eyngor et al. 2004 ), although three groupings along geographical lines were recognised by RAPD, the outcome of which should have value for epizootiology (Ravelo
et al. 2003 ). However, Lactococcus garvieae appears to be similar in terms of phenetic data, to Lactococcus lactis (Zlotkin et al. 1998 ). This similarity could result in
mis-identifi cation of fresh isolates.
Cultures were recovered from diseased yellowtail in Japan and although regarded
as Lactococcus garvieae did not agglutinate with antiserum raised against capsulated cells (Oinaka et al. 2015 ). Such isolates could confuse diagnosticians, and lead
to mis-diagnosis.
Diagnosis
Phenotypic Methods Presumptive identifi cation of Lactococcus garvieae -like
organisms has been made following growth on bile (40 %)-aesculin agar (see
Facklam and Moody 1970 ), with hydrolysis of aesculin and by the characteristic
growth on eosin-methylene blue agar (lactose is not fermented).
Molecular Methods A PCR using a 1100 bp fragment has distinguished
Lactococcus garvieae from Lactococcus lactis (Zlotkin et al. 1998 ). In terms of
sensitivity, the PCR detected Lactococcus garvieae in 1 μl of fi sh plasma. Another
publication reported a PCR based on a 709 bp fragment that was specifi c for
Lactococcus garvieae, enabling a positive result to be obtained in 4 h (Aoki et al.
2000 ). Two primers, namely ITSLg30F and ITSLg319R, from the sequence in the
16S-23S ITS region and incorporated into a PCR reportedly gave improved specifi city (Dang et al. 2012 ).
Multiplex PCR A multiplex PCR has been developed, and successfully recognised the fi sh pathogenic lactococci-streptococci, i.e. Lactococcus garvieae, Str. diffi cilis (= Str. agalactiae), Str. iniae and Str. parauberis from culture and fi sh tissues
with a sensitivity for the purifi ed DNA of 30 pg, 12.5 pg, 25 pg and 50 pg, respectively (Mata et al. 2004 ). The value of multiplex PCR was reinforced by Itsaro et al.
( 2013 ), who distinguished Lactococcus garvieae (sensitivity = 19.53 pg of DNA)
from Str. galactiae and Str. iniae.
RT-PCR RT-PCR proved successful for the recognition of Lactococcus garvieae
with a detection limited of 32 fg (Jung et al. 2010 ).
LAMP-PCR A LAMP PCR was specifi c and sensitive, detecting 300 CFU, which
was considered to be 100-fold more sensitive than conventional PCR (Tsai et al.
2013a ).
Streptococcaceae Representatives
whole cell proteins, concluding that En. seriolicida was closely related to
Lactococcus garvieae. Also, Eldar et al. ( 1996 ) reached the same conclusion after
studying the type strains phenotypically and by DNA:DNA hybridisation. The
taxon is certainly homogeneous (Kawanishi et al. 2006 ), as verifi ed by RFLP
(Eyngor et al. 2004 ), although three groupings along geographical lines were recognised by RAPD, the outcome of which should have value for epizootiology (Ravelo
et al. 2003 ). However, Lactococcus garvieae appears to be similar in terms of phenetic data, to Lactococcus lactis (Zlotkin et al. 1998 ). This similarity could result in
mis-identifi cation of fresh isolates.
Cultures were recovered from diseased yellowtail in Japan and although regarded
as Lactococcus garvieae did not agglutinate with antiserum raised against capsulated cells (Oinaka et al. 2015 ). Such isolates could confuse diagnosticians, and lead
to mis-diagnosis.
Diagnosis
Phenotypic Methods Presumptive identifi cation of Lactococcus garvieae -like
organisms has been made following growth on bile (40 %)-aesculin agar (see
Facklam and Moody 1970 ), with hydrolysis of aesculin and by the characteristic
growth on eosin-methylene blue agar (lactose is not fermented).
Molecular Methods A PCR using a 1100 bp fragment has distinguished
Lactococcus garvieae from Lactococcus lactis (Zlotkin et al. 1998 ). In terms of
sensitivity, the PCR detected Lactococcus garvieae in 1 μl of fi sh plasma. Another
publication reported a PCR based on a 709 bp fragment that was specifi c for
Lactococcus garvieae, enabling a positive result to be obtained in 4 h (Aoki et al.
2000 ). Two primers, namely ITSLg30F and ITSLg319R, from the sequence in the
16S-23S ITS region and incorporated into a PCR reportedly gave improved specifi city (Dang et al. 2012 ).
Multiplex PCR A multiplex PCR has been developed, and successfully recognised the fi sh pathogenic lactococci-streptococci, i.e. Lactococcus garvieae, Str. diffi cilis (= Str. agalactiae), Str. iniae and Str. parauberis from culture and fi sh tissues
with a sensitivity for the purifi ed DNA of 30 pg, 12.5 pg, 25 pg and 50 pg, respectively (Mata et al. 2004 ). The value of multiplex PCR was reinforced by Itsaro et al.
( 2013 ), who distinguished Lactococcus garvieae (sensitivity = 19.53 pg of DNA)
from Str. galactiae and Str. iniae.
RT-PCR RT-PCR proved successful for the recognition of Lactococcus garvieae
with a detection limited of 32 fg (Jung et al. 2010 ).
LAMP-PCR A LAMP PCR was specifi c and sensitive, detecting 300 CFU, which
was considered to be 100-fold more sensitive than conventional PCR (Tsai et al.
2013a ).
Streptococcaceae Representatives