123
Diagnosis
Chemical Method A simple chemical technique has been described, which may
readily delineate Nocardia from Mycobacterium (Kanetsuna and Bartoli 1972 ).
Assuming that pure cultures are available, the bacteria are saponifi ed in 2.5 % (w/v)
potassium hydroxide in a 1:1 (v/v) mixture of methanol and benzene at 37 °C for
24 h. Crude mycolic acids from bona fi de mycobacteria may be subsequently precipitated by addition of an equal volume of ethanol to an ethereal solution of the
extracted lipids. Mycobacteria give rise to copious quantities of white precipitate of
melting point between 45 and 70 °C, whereas nocardias produce negligible amounts,
which do not melt below 150 °C (Kanetsuna and Bartoli 1972 ).
Molecular Methods A noteworthy advance in diagnoses resulted from the use of
PCR technology to identify Mycobacterium spp. in sea bass (Knibb et al. 1993 ),
Myc. chelonei in a cichlid oscar (Astronotus ocellatus) (McCormick et al. 1995 ) and
Myc. marinum in goldfi sh ( Carassius auratus ) (Pourahmad et al. 2014 ). A PCR followed by reverse cross blot hybridisation identifi ed Mycobacterium sensitively (to
100 fg of DNA, which equated to 20 mycobacterial cells) to the species level
(Puttinaowarat et al. 2002 ). Myc. shottsii and Myc. pseudoshottsii were detected in
striped bass using a PCR-RFLP (Gauthier et al. 2010 , 2011a ). Real-time PCR, based
on the polymerase ß subunit gene ( rpoß ) was used to detect Mycobacterium spp.,
albeit with cross reaction from pure DNA from Noc. seriolae and Rhodococcus
erythropolis [these cross reactions were not recorded with formalin-fi xed, paraffi nembedded section] , with a detection limit of 10
2 CFU/g in the case of Myc. salmoniphilum infected tissues (Zerihun et al. 2011a ). High-resolution melting analysis
determined as a result of studying the melting temperature and melting profi le of the
16S–23S rRNA ITS was employed to differentiate reliably 12 species of
Mycobacterium with a detection limit of 10 genome equivalents/reaction (Phung
et al. 2013 ). A qPCR was advocated for the detection of Myc. marinum (Slany 2014 ).
Epizootiology
Very little is known about the epizootiology of fi sh pathogenic mycobacteria.
Undoubtedly, the reservoir for the organism is the aquatic environment (Beran et al.
2006 ), although the factors that lead to the development and spread of the disease
condition are unknown. To illustrate the potential spread of the disease, in Oregon
as many as 26 % of hatchery fi sh may be infected (Arakawa and Fryer 1984 ).
Possibly, transmission may be by ingesting contaminated food or debris (Dulin
1979 ). Infection, via the intra-ovarian route, has been demonstrated for the Mexican
platyfi sh ( Xiphophorus maculatus) (Conroy 1966 ). However, other investigators
have ruled out vertical (i.e. egg) transmission as a means of spreading the disease
(Ross and Johnson 1962 ; Wood 1974 ). Using real time PCR, Myc. pseudoshottsii
was found to be ubiquitous in water and sediment samples from the Chesapeake
Bay and Rappahannock River in Virginia, respectively [where the disease is occurring]. In contrast, Myc. shottsii has been found only in fi sh, suggesting that the
organism is an obligate pathogen (Gauthier et al. 2010 ).
Mycobacteriaceae Representatives
Diagnosis
Chemical Method A simple chemical technique has been described, which may
readily delineate Nocardia from Mycobacterium (Kanetsuna and Bartoli 1972 ).
Assuming that pure cultures are available, the bacteria are saponifi ed in 2.5 % (w/v)
potassium hydroxide in a 1:1 (v/v) mixture of methanol and benzene at 37 °C for
24 h. Crude mycolic acids from bona fi de mycobacteria may be subsequently precipitated by addition of an equal volume of ethanol to an ethereal solution of the
extracted lipids. Mycobacteria give rise to copious quantities of white precipitate of
melting point between 45 and 70 °C, whereas nocardias produce negligible amounts,
which do not melt below 150 °C (Kanetsuna and Bartoli 1972 ).
Molecular Methods A noteworthy advance in diagnoses resulted from the use of
PCR technology to identify Mycobacterium spp. in sea bass (Knibb et al. 1993 ),
Myc. chelonei in a cichlid oscar (Astronotus ocellatus) (McCormick et al. 1995 ) and
Myc. marinum in goldfi sh ( Carassius auratus ) (Pourahmad et al. 2014 ). A PCR followed by reverse cross blot hybridisation identifi ed Mycobacterium sensitively (to
100 fg of DNA, which equated to 20 mycobacterial cells) to the species level
(Puttinaowarat et al. 2002 ). Myc. shottsii and Myc. pseudoshottsii were detected in
striped bass using a PCR-RFLP (Gauthier et al. 2010 , 2011a ). Real-time PCR, based
on the polymerase ß subunit gene ( rpoß ) was used to detect Mycobacterium spp.,
albeit with cross reaction from pure DNA from Noc. seriolae and Rhodococcus
erythropolis [these cross reactions were not recorded with formalin-fi xed, paraffi nembedded section] , with a detection limit of 10
2 CFU/g in the case of Myc. salmoniphilum infected tissues (Zerihun et al. 2011a ). High-resolution melting analysis
determined as a result of studying the melting temperature and melting profi le of the
16S–23S rRNA ITS was employed to differentiate reliably 12 species of
Mycobacterium with a detection limit of 10 genome equivalents/reaction (Phung
et al. 2013 ). A qPCR was advocated for the detection of Myc. marinum (Slany 2014 ).
Epizootiology
Very little is known about the epizootiology of fi sh pathogenic mycobacteria.
Undoubtedly, the reservoir for the organism is the aquatic environment (Beran et al.
2006 ), although the factors that lead to the development and spread of the disease
condition are unknown. To illustrate the potential spread of the disease, in Oregon
as many as 26 % of hatchery fi sh may be infected (Arakawa and Fryer 1984 ).
Possibly, transmission may be by ingesting contaminated food or debris (Dulin
1979 ). Infection, via the intra-ovarian route, has been demonstrated for the Mexican
platyfi sh ( Xiphophorus maculatus) (Conroy 1966 ). However, other investigators
have ruled out vertical (i.e. egg) transmission as a means of spreading the disease
(Ross and Johnson 1962 ; Wood 1974 ). Using real time PCR, Myc. pseudoshottsii
was found to be ubiquitous in water and sediment samples from the Chesapeake
Bay and Rappahannock River in Virginia, respectively [where the disease is occurring]. In contrast, Myc. shottsii has been found only in fi sh, suggesting that the
organism is an obligate pathogen (Gauthier et al. 2010 ).
Mycobacteriaceae Representatives