338
Diagnosis
Serology FAT has been found useful for diagnosing Edw. tarda and determining
the presence of the pathogen in infected fi sh tissues (Amandi et al. 1982 ). Horiuchi
et al. ( 1980 ) considered FAT as being extremely valuable for fi eld diagnoses. iFAT
with monoclonal antibodies in an enzyme immunoassay is also useful for diagnosis
(Rogers 1981 ). An ELISA, which detects OMPs, particularly a common 44 kDa
protein, has been described (Kumar et al. 2007 ).
Molecular Methods PCR has been developed for the detection of Edw. tarda and
its differentiation from Edw. ictaluri (Williams and Lawrence 2009 ). LAMP has
been proposed for the rapid and sensitive detection of Edw. tarda (Savan et al. 2004 ;
Xie et al. 2013 ). Developed to detect the pathogen in Japanese fl ounder kidney and
spleen (and seawater), results were achieved in 45 min. An indication of sensitivity
is that the LAMP technique was positive for seawater containing 3.2 × 10
2 CFU of
Edw. tarda (Savan et al. 2004 ). Xie et al. ( 2013 ) targeted the upstream region of the
hlyb gene, and reported that their test was 100-times more sensitive than a conventional PCR, i.e. detected 10 compared to 10
3 CFU/test, could be recorded visually
or by the addition of a fl uorescent marker, and was specifi c to Edw. tarda . Individual
RT-PCRs were developed to detect Edw. tarda, Edw. piscicida and Edw. piscicida -
like organisms in channel catfi sh leading to sensitive (5 copies of the DNA in a 15 μl
reaction; 10
2 to 10
3 CFU in ~25 g amounts of kidney tissue, 35 ml of pond water, or
20 l of broth culture), specifi c and reproducible assays (Reichley et al. 2015 ).
Epizootiology
The precise source of infection is often unknown. However, between 2002 and
2004, Edw. tarda was recovered from seawater and the kidney and spleen of healthy
Japanese fl ounder when disease was occurring in farmed fi sh populations. It was
apparent that the antibody titre increased rapidly in the run-up to outbreaks of clinical disease. Interestingly, bacteriophage was found in the seawater at least 1 month
before the onset of and during the period of the disease outbreaks, but not afterwards. It was speculated that the presence of bacteriophage could be used as an
indicator for Edw. tarda cells (Matsuoka and Nakai 2004 ). Also, there has been
speculation that snakes or faecal contamination from humans or other animals may
have been involved in the fi rst documented outbreak (Meyer and Bullock 1973 ).
Certainly, experimentally infected fi sh (= Japanese fl ounder) were determined to
shed the pathogen 1–6 days before death, with the number of bacterial cells shed
from newly dead (and for several days afterwards) fi sh reaching 10
7 –10
8 CFU/min.
Of relevance these cells were more virulent than their counterparts from TSA. Such
bacteria, which are discharged from diseased/dead fi sh, may well have importance
in the spread of disease among fi sh populations (Matsuoka 2004 ). Environmental
6 Enterobacteriaceae Representatives
Diagnosis
Serology FAT has been found useful for diagnosing Edw. tarda and determining
the presence of the pathogen in infected fi sh tissues (Amandi et al. 1982 ). Horiuchi
et al. ( 1980 ) considered FAT as being extremely valuable for fi eld diagnoses. iFAT
with monoclonal antibodies in an enzyme immunoassay is also useful for diagnosis
(Rogers 1981 ). An ELISA, which detects OMPs, particularly a common 44 kDa
protein, has been described (Kumar et al. 2007 ).
Molecular Methods PCR has been developed for the detection of Edw. tarda and
its differentiation from Edw. ictaluri (Williams and Lawrence 2009 ). LAMP has
been proposed for the rapid and sensitive detection of Edw. tarda (Savan et al. 2004 ;
Xie et al. 2013 ). Developed to detect the pathogen in Japanese fl ounder kidney and
spleen (and seawater), results were achieved in 45 min. An indication of sensitivity
is that the LAMP technique was positive for seawater containing 3.2 × 10
2 CFU of
Edw. tarda (Savan et al. 2004 ). Xie et al. ( 2013 ) targeted the upstream region of the
hlyb gene, and reported that their test was 100-times more sensitive than a conventional PCR, i.e. detected 10 compared to 10
3 CFU/test, could be recorded visually
or by the addition of a fl uorescent marker, and was specifi c to Edw. tarda . Individual
RT-PCRs were developed to detect Edw. tarda, Edw. piscicida and Edw. piscicida -
like organisms in channel catfi sh leading to sensitive (5 copies of the DNA in a 15 μl
reaction; 10
2 to 10
3 CFU in ~25 g amounts of kidney tissue, 35 ml of pond water, or
20 l of broth culture), specifi c and reproducible assays (Reichley et al. 2015 ).
Epizootiology
The precise source of infection is often unknown. However, between 2002 and
2004, Edw. tarda was recovered from seawater and the kidney and spleen of healthy
Japanese fl ounder when disease was occurring in farmed fi sh populations. It was
apparent that the antibody titre increased rapidly in the run-up to outbreaks of clinical disease. Interestingly, bacteriophage was found in the seawater at least 1 month
before the onset of and during the period of the disease outbreaks, but not afterwards. It was speculated that the presence of bacteriophage could be used as an
indicator for Edw. tarda cells (Matsuoka and Nakai 2004 ). Also, there has been
speculation that snakes or faecal contamination from humans or other animals may
have been involved in the fi rst documented outbreak (Meyer and Bullock 1973 ).
Certainly, experimentally infected fi sh (= Japanese fl ounder) were determined to
shed the pathogen 1–6 days before death, with the number of bacterial cells shed
from newly dead (and for several days afterwards) fi sh reaching 10
7 –10
8 CFU/min.
Of relevance these cells were more virulent than their counterparts from TSA. Such
bacteria, which are discharged from diseased/dead fi sh, may well have importance
in the spread of disease among fi sh populations (Matsuoka 2004 ). Environmental
6 Enterobacteriaceae Representatives