Agar culture of<i>Piscirickettsia salmonis</i>, a serious pathogen of farmed salmonid and marine fish

Mikalsen, Jarle; Skjærvik, Olaf; Wiik‐Nielsen, Jannicke; Wasmuth, M A; Colquhoun, Duncan J.

doi:10.1111/j.1574-6968.2007.00977.x

Cited by 79 publications

(91 citation statements)

References 13 publications

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“…During necropsy, samples of the liver, kidney and spleen were aseptically removed and the gDNA extracted for qPCR (Corbeil et al 2003) as described above. Samples were also obtained using sterile loops from the liver, kidney and spleen for direct inoculation onto BCG (Mauel et al 2008), CHAB (Mikalsen et al 2008) and the selectivelynegative sheep blood agar plates. Plates were incubated at 20°C, and qPCR (Corbeil et al 2003) was used to verify the identity of TRLO-suspected growth.…”

Section: Isolation Of Trlo In Culturementioning

confidence: 99%

“…Unfortunately, polyclonal antiserum raised against TSRV could not be used to negatively select against the virus as it was non-neutralising (Corbeil & Crane 2004). Rather than try to solve this technical challenge, we profited from specialised enriched agar media that support the growth of Piscirickettsia salmonis (Mauel et al 2008, Mikalsen et al 2008. Given that no outbreaks of disease with TRLO were occurring at the time attempts were made to first isolate the bacterium, active sampling on commercial marine farms in south-east Tasmania (Fig.…”

Section: Specialised Enriched Media Support the Growth Of Trlomentioning

confidence: 99%

“…The development of these new media provided an ideal opportunity to attempt to isolate TRLO in a cell culture-independent fashion. Therefore in this study, we adopted the infection strategy of Jones et al (1998) and took advantage of blood -cysteineglucose (BCG) agar (Mauel et al 2008) and cysteine heart agar with sheep blood (CHAB) (Mikalsen et al 2008) to isolate TRLO in culture. Data are presented showing evidence of multiple TRLO serotypes in Tasmania, all of which are pathogenic in juvenile Atlantic salmon.…”

Section: Introductionmentioning

confidence: 99%

“…It is also culturable in Xenopus laevis (Daudin) XTC-2 and fall army worm Spodoptera frugiperda Sf21 cells (Birkbeck et al 2004a). For many years, P. salmonis was thought to be obligately intracellular (Fryer et al 1992, Almendras & Fuentealba 1997, Mauel & Miller 2002, Fryer & Hedrick 2003, but using specialised enriched media (Mauel et al 2008, Mikalsen et al 2008, Gómez et al 2009, Otterlei et al 2016), the bacterium is now considered fastidious and facultatively intracellular. The development of these new media provided an ideal opportunity to attempt to isolate TRLO in a cell culture-independent fashion.…”

mentioning

confidence: 99%

“…This may be due to the technically challenging process of developing polyclonal antisera using cells propagated by cell culture, although methods to purify the bacterium from cell culture debris have been optimised (Kuzyk et al 1996). The establishment of agar (Mauel et al 2008, Mikalsen et al 2008) and liquid (Vera et al Yañez et al 2012, Henríquez et al 2013) media for P. salmonis may also assist in the production of highly specific antisera.As yet, the antigenic basis for the distinction between the 3 (or possibly 4) serotypes of TRLO is unknown. Serotypic diversity among many groups of Gram-negative bacteria is driven by variability in the heat-stable lipopolysaccharide O-antigens.…”

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confidence: 99%

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Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

Morrison¹,

Young²,

Knowles³

et al. 2016

Dis. Aquat. Org.

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Atlantic salmon Salmo salar L. farmed in south-east Tasmania, Australia, are susceptible to infection by the Tasmanian Rickettsia-like organism (TRLO), a Gram-negative bacterium. Here, we report the first isolation of TRLO from south-east Tasmania in pure culture and show that the bacterium is culturable on both specialised enriched agar and in cell culture using the CHSE-214 cell line. In vitro cultured TRLO was used to reproducibly elicit disease in Atlantic salmon parr held in fresh water. In inoculated fish, TRLO was observed intracytoplasmically in peripheral blood leucocytes, suggesting that these cells are responsible for haematogenous dispersal of the bacterium within the host. Fish with experimentally induced disease presented with gross and histopathological changes similar to TRLO-infected fish at commercial marine farms. TRLO was also isolated in culture from farmed Atlantic salmon in the Tamar River and Macquarie Harbour production areas in Tasmania, both of which have no history of TRLO-associated disease. These TRLO isolates appear to be serologically distinct from each other as well as from isolates obtained from south-east Tasmania, linking each serotype to a specific geographical location within Tasmania. Despite the lack of clinical evidence of TRLO-linked disease in fish grown in the Tamar River and Macquarie Harbour, experimental infection trials demonstrably showed the pathogenic potential of these TRLO serovars. Together, these data provide evidence that TRLO is a fastidious, facultative intracellular bacterium and confirm TRLO as a pathogen of Atlantic salmon, causing a disease designated Tasmanian salmonid rickettsiosis.KEY WORDS: Rickettsia-like organism · TRLO · Atlantic salmon · Salmo salar · Culture · Pathogenicity · Tasmania Resale or republication not permitted without written consent of the publisherDis Aquat Org 122: [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] 2016 RLO (Khoo et al. 1995, Athanassopoulou et al. 2004, Corbeil et al. 2005, Wu et al. 2005 or P. salmonis-like organism (Antonio et al. 2000, Chen et al. 2000a,b, Mauel et al. 2003 until their taxonomic status can be formally resolved. This includes the Tasmanian RLO (TRLO), a bacte rium detected in Atlantic salmon Salmo salar L. farmed on the south-east coast of Tasmania, Austra lia (Corbeil et al. 2005).TRLO is an intracellular, Gram-negative coccoid bacterium that emerged in the summer of 2001 (Department of Primary Industries, Parks, Water and Environment [DPIPWE] unpublished data). Since then, TRLO has been implicated in minor sporadic outbreaks of disease which have typically coincided with the annual peak in water temperature. The disease is characterised by a high morbidity rate which results in significant production loss through reduced feed intake but relatively low mortality, less than 10% of affected stock. During outbreaks, affected fish are commonly co-infected with the Tasmanian Atlantic salmon reovirus (TSRV; Zainathan et al. 2015), and it is not known wh...

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