118
2009 and 1010 in China, mycobacterial cultures were obtained, and equated with
M yc. arupense, Myc. chelonae, Myc. gordonae, Myc. fortuitum, Myc. marinum
(most common), Myc. porcinum and Myc. szulgai (Zhang et al. 2015 ). Again, the
list included some newcomers to fi sh pathology. In their excellent review, Kaattari
et al. ( 2006 ) discuss all the currently recognized mycobacterial fi sh pathogens,
including the new but not formally named species, Myc. “chesapeaki”.
Isolation
With many cases of mycobacteriosis, there is no attempt made at isolation of the
pathogen. Yet, great scientifi c conclusions seem to result from the examination of
only histological material. Nevertheless, attempts at isolating the aetiological agent
often fail, indicating a fastidiousness on the part of the pathogen. Some success
occurs by inoculating pieces of infected tissue (especially kidney, liver or spleen) on
standard mycobacterial media, including Petragnani, Löwenstein-Jensen,
Middlebrook 7H10 and Dorset egg media (see Appendix in Chap. 12 ), or even
blood agar, BHIA or TSA, whereupon growth may occur in 2–28 days at incubation
temperatures of 15–30 °C in aerobic or microaerophilic, i.e. 3–5 % carbon dioxide,
conditions (Dulin 1979 ; Lansdell et al. 1993 ). Most diffi culty surrounds the recovery of mycobacteria from marine fi sh species. Clearly, more effort is required to
understand the precise nutritional requirements of these organisms. Myc. abscessus
was not isolated on Löwenstein-Jensen medium. Instead, Middlebrook 7H10
medium was modifi ed by the addition of 10 μg/ml of amphotericin B, 500 μg/ml of
chloramphenicol, 5 μg/ml of gentamicin or 30 μg/ml of cephalothin - either singly
or in combination. Diseased fi sh were immersed in the modifi ed Middlebrook 7H10
broth for 1 h at room temperature, before homogenisation, and inoculation of modifi ed Middlebrook 7H10 agar with incubation at 25 °C for 14–28 days (Teska et al.
1997 ). Middlebrook 7H10 agar with incubation a 23 °C for 4–6 weeks but not
Löwenstein-Jensen medium permitted the recovery of Myc. shottsii (Rhodes et al.
2003 ). In comparison, Myc. montefi orense was isolated on blood agar and
Middlebrook medium after incubation at 25 °C for up to 20 weeks (Levi et al. 2003 ).
To recover Myc. gordonae, Sakai et al. ( 2005 ) dipped the spleen and liver of infected
guppies into 2 % (w/v) sodium hydroxide for 20 min, and then inoculated 1 %
Ogawa-egg medium with incubation at 30 °C for 1 month.
There has been one account of the organism associated with inappetance and
granulomas on a captive epaulette shark ( Hemiscyllium ocellatum ) in The
Netherlands (Janse and Kik 2012 ).
Characteristics of the Pathogens
The aetiological agents have been classifi ed, at various times, into a wide assortment of species including Myc. anabanti, Myc. aurum, Myc. chelonei, Myc. chelonei subsp. piscarium, Myc. fortuitum, Myc. marinum, Myc. parafortuitum, Myc.
piscium, Myc. platypoecilus, Myc. poriferae, Myc. ranae, Myc. salmoniphilum,
Myc. simiae, Myc. scrofulaceum , Myc. simiae and Myc. triplex . In addition, Myc.
3 Aerobic Gram-Positive Rods and Cocci
2009 and 1010 in China, mycobacterial cultures were obtained, and equated with
M yc. arupense, Myc. chelonae, Myc. gordonae, Myc. fortuitum, Myc. marinum
(most common), Myc. porcinum and Myc. szulgai (Zhang et al. 2015 ). Again, the
list included some newcomers to fi sh pathology. In their excellent review, Kaattari
et al. ( 2006 ) discuss all the currently recognized mycobacterial fi sh pathogens,
including the new but not formally named species, Myc. “chesapeaki”.
Isolation
With many cases of mycobacteriosis, there is no attempt made at isolation of the
pathogen. Yet, great scientifi c conclusions seem to result from the examination of
only histological material. Nevertheless, attempts at isolating the aetiological agent
often fail, indicating a fastidiousness on the part of the pathogen. Some success
occurs by inoculating pieces of infected tissue (especially kidney, liver or spleen) on
standard mycobacterial media, including Petragnani, Löwenstein-Jensen,
Middlebrook 7H10 and Dorset egg media (see Appendix in Chap. 12 ), or even
blood agar, BHIA or TSA, whereupon growth may occur in 2–28 days at incubation
temperatures of 15–30 °C in aerobic or microaerophilic, i.e. 3–5 % carbon dioxide,
conditions (Dulin 1979 ; Lansdell et al. 1993 ). Most diffi culty surrounds the recovery of mycobacteria from marine fi sh species. Clearly, more effort is required to
understand the precise nutritional requirements of these organisms. Myc. abscessus
was not isolated on Löwenstein-Jensen medium. Instead, Middlebrook 7H10
medium was modifi ed by the addition of 10 μg/ml of amphotericin B, 500 μg/ml of
chloramphenicol, 5 μg/ml of gentamicin or 30 μg/ml of cephalothin - either singly
or in combination. Diseased fi sh were immersed in the modifi ed Middlebrook 7H10
broth for 1 h at room temperature, before homogenisation, and inoculation of modifi ed Middlebrook 7H10 agar with incubation at 25 °C for 14–28 days (Teska et al.
1997 ). Middlebrook 7H10 agar with incubation a 23 °C for 4–6 weeks but not
Löwenstein-Jensen medium permitted the recovery of Myc. shottsii (Rhodes et al.
2003 ). In comparison, Myc. montefi orense was isolated on blood agar and
Middlebrook medium after incubation at 25 °C for up to 20 weeks (Levi et al. 2003 ).
To recover Myc. gordonae, Sakai et al. ( 2005 ) dipped the spleen and liver of infected
guppies into 2 % (w/v) sodium hydroxide for 20 min, and then inoculated 1 %
Ogawa-egg medium with incubation at 30 °C for 1 month.
There has been one account of the organism associated with inappetance and
granulomas on a captive epaulette shark ( Hemiscyllium ocellatum ) in The
Netherlands (Janse and Kik 2012 ).
Characteristics of the Pathogens
The aetiological agents have been classifi ed, at various times, into a wide assortment of species including Myc. anabanti, Myc. aurum, Myc. chelonei, Myc. chelonei subsp. piscarium, Myc. fortuitum, Myc. marinum, Myc. parafortuitum, Myc.
piscium, Myc. platypoecilus, Myc. poriferae, Myc. ranae, Myc. salmoniphilum,
Myc. simiae, Myc. scrofulaceum , Myc. simiae and Myc. triplex . In addition, Myc.
3 Aerobic Gram-Positive Rods and Cocci