ǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚǰȱŜř
Francisellosis in ornamental ȱȱȱ E. Lewisch*, A. Dressler, S. Menanteau-Ledouble, M. Saleh and M. El-Matbouli Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria
Abstract ȱȱȱȱȱFrancisellaȱȱȱȱȱ¢ȱ¢ȱȱȱ ȱȱ ȱȱȱȱęǰȱ¢ȱȱ¢ȱȱȱȱȱȱȱȱȱęǯȱ ǰȱ ȱȱȱȱ Francisella noatunensis subsp. orientalisȱȱ ȱȱȱȱȱ ȱȱȱȱȱȱȱȱ¢ȱȱȱȱ ȱȱ ȱȱȱȱ¢ǯȱȱęȱ¢ȱęȱ¡ȱȱȱȱ ¡ǰȱ¢ǰȱȱȱǰȱ¢ȱ ȱ¢ȱǯȱȱȱ histological examination revealed an enlarged spleen, pale viscera and ascites, and, in some cases, ǰȱ¢ȱȱȱǯȱȱ¡ȱȱěȱȱ ȱȱȱ ȱȱȱ ȱȱşşƖȱȱȱ¢ȱ ȱF. noatunensis subsp. orientalis. ȱȱȱȱȱ ȱȱȱȱęȱ¢ȱȱǯȱȱ ǰȱ F. noatunensis subsp. orientalis had not been described in relationship with disease and mortality in ȱęǯȱȱȱ ȱȱ ȱȱȱȱȱȱęȱȱȱȱȱ¢ȱȱ¡ǰȱȱȱ¢ȱȱȱȱ be widely distributed and its prevalence underestimated.
Introduction In the last 20 years, granulomatous diseases ȱ¢ȱĴȬȱȱǻǼȱȱ
ȱ ȱȱȱ ȱȱȱ
been recognised as a serious threat in aquacul-
ǻ ȱȱǯǰȱŘŖŖŜDzȱȱȱǯǰȱŘŖŖŜǼǯȱȱ
ture (Chen et al., 1994; Chern and Chao 1994; ȱȱǯǰȱŗşşśDzȱĴȱȱǯǰȱ
ȱȱȱ¢ȱȱȱȱ
2004; Corbeil et al., 2005; Birbeck at al., 2011; Colquhoun and Duodu, 2011). Recently, thanks
ȱ ȱȱȱȱȱȱ
the spleen, kidney and liver, but have been ȱȱȱ¢ȱ¢ȱȱȱǻ ȱ ȱǯǰȱŘŖŖŜǰȱȱȱǯǰȱŘŖŖŜǼǯȱ ¢ǰȱȱ
ȱȱȱȱȱȱȱ-
ȱȱȱȱȱȱ ȱȱ
ȱȱǰȱȱȱȱȱFran-
¢ȱȱȱǯȱǻŘŖŖşǼȱȱȱ-
cisella could be revealed as the causative agent ȱ¢ȱȱȱǯ Francisella is a strictly ǰȱȱǰȱȱǰȱ
ȱȱȱ ȱȱȱȱFrancisella
gram negative coccobacilli. It is mostly associ-
kidney and mortalities occurring within 48
ǯȱȱ ȱȱ¢ȱȱȱȱ bloody ascites, slightly enlarged spleen and
* Corresponding author’s e-mail:
[email protected]
ŜŚǰȱǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚ
hours. Francisellosis has been associated with ȱ¢ȱǰȱȱȱ ȱȱ ¢ȱȱȱǻȱȱǯǰȱŘŖŖŜȱǼȱȱŚŖȱ ƖȱȱȱȱǻȱȱǯǰȱŘŖŖŜǼȱȱşśȱƖȱ
Boulenger 1908 , Nimbochromis linni Burgess and Axelrod 1975, Aulonocara stuartgranti “maleri” Meyer and Riehl 1985, Placidochromis sp. “blue hongi”, Protomelas sp. “Steveni Taiwan”,
in tilapia (Chern et al., 1994). Environmental
Naevochromis chryosogaster Trewavas 1935, Copa-
ǰȱ¢ȱ ȱǰȱȱȱ ȱȱĚȱȱȱȱ ȱȱ
dichromis mlotoȱ ȱŗşŜŖǰȱOtopharynx tetrastigma ûȱŗŞşŚǼȱ ȱȱȱȱȱ
¢ȱȱǻȱȱǯǰȱŘŖŖŜDzȱȱǯȱǯǰȱ
ȱ¢ǯȱ
ŘŖŗŘDzȱ¢ȱȱǯǰȱŘŖŖŜǼǯȱ ȱ¢ȱ occurs horizontally via the oral route (Hsieh
ȱ¢ȱȱ ȱęȱȱȱ
et al., 2007; Mikalsen et al., 2009), however,
breeders as well as wild caught specimens on a
various authors used cohabitation and immerȱ¢ȱȱ ȱȱȱȱȱ
regular basis to renew the breeding stock. None ȱȱ ¢ȱȱęȱ ȱǰȱ
water and the gills was also possible (Nylund et
although metronidazole was sporadically used
ǯǰȱŘŖŖŜDzȱȱȱǯǰȱŘŖŖşDzȱȱȱǯǰȱŘŖŖşǼǯȱ
ȱȱȱȱ¢ȱĚǯȱȱ
ȱȱȱȱȱȱȱȱ
¢ȱȱ ȱȱ DZȱȱ
grunt Parapristipoma trilineatum (Kamaishi et ǯǰȱŘŖŖśǼǰȱȱȱȱ Francisella have ȱȱȱȱȱȱȱȱ ȱ¢ȱȱȱęǰȱȱȱȱ,
24°C, alkalinity 82,2 ppm measured as CaCO3, ȱŖǯŘŜȱǯ-1 , ammoniac 0.00104 mg.L-1 ,nitrite 0.0 mg.L-1, nitrate 10 mg.L-1, phosphate 0.1 mg.L-1 .
Gadus morhuaȱǻȱ¢ȱȱǯǰȱŘŖŖŜDzȱȱȱǯǰȱ ŘŖŖŜDzȱĴȱȱǯǰȱŘŖŖŝDzȱȱȱǯǰȱŘŖŖŝǼ Nile tilapia, Oreochromis ǯȱǻ ȱȱǯǰȱŘŖŖŜDzȱ
ěȱęȱ ȱęȱȱȱȱ such as anorexia, tachypnea and abnormal
ȱȱǯǰȱŘŖŖŝDzȱȱȱǯǰȱŘŖŖşDzȱ ě¢ȱȱǯǰȱ
ȱȱ ȱ¢ȱȱ ȱ ȱ ȱȱȱȱȱȱȱǯ
2010), Atlantic salmon, Salmo salar (Birbeck et al., 2007; Bohle et al., 2009) and even molluscs (Kamaishi et al. 2010). More recently F. noatun-
ȱ ȱȱȱȱ¡ǰȱ
ensis subsp. orientalisȱȱȱȱȱ Ȭęȱȱęȱȱȱȱȱȱ
histopathology, bacteriology and molecular investigation.
ȱȱȱȱȱęȱȱȱ been discussed by Camus et al. (2013). Finally,
For histopathology, tissues (spleen, intestines)
in one case, molecular genetic investigation
ȱę¡ȱȱŗŖƖȱȱěȱǰȱ
ȱȱȱ¢ȱȱǰȱȬ ę¡ǰȱĜȮȱȱȱ
subjected to routine histological procedures and stained with haematoxylin and eosin (H&E) and
ȱȱȱFrancisella sp.in ȱȱ ȱȱȱȱ ȱǻ ȱ
Ziehl-Neelsen (ZN) stain, respectively.
et al., 2007).
ȱȱ¡ȱȱǰȱ
Materials and methods
liver and kidney were conducted by Gramand Zieh-Neelsen stains as well as by inocu-
Various Malawi cichlids (Nimbochromis venustus
ȱȱȱȱȱȬȱǻȱ
ǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚǰȱŜś
ȱęǰȱ¡ȱǰȱǰȱ UK) and cystein heart agar (CHAB). CHAB
ȱȱŗşȱęȱǻŗś¡ȱǰȱŞ¡ȱȱ tract, 2x brain, 7x liver, 1x kidney) was extracted
ȱȱ ȱŘȱƖȱȱȱȱ
using a QIAamp DNA Mini Kit (QIAGEN,
ǻȱȱęǰȱ¡ȱǰȱ-
Hilden, Germany). PCR was conducted using FrancisellaȬȱȱǻǼȬęȱȱ
ǰȱ ǼȱȱśŖȱΐǯȱ-1 ampicillin (SigmaAldrich Handels GmbH, Vienna, Austria), as described by Soto et al. (2009). Prior to streaking, the tissues were homogenized in cystein
ȱȱȱȱ¢ȱ ȱȱǯȱ ǻŘŖŖŝǼǯȱęȱȱ ȱȱ
ȱȱȱ ȱŘȱƖȱȱȱ
sequences where subjected to Basic Local Align-
ǰȱȱȱȱŘŚȱȱŚŞȱȱȱŘŜǚǯȱ
ȱȱȱǻǼȱȱȱę-
¢ǰȱȱȱ ȱȱȱ
tion based on sequence homology.
these broths and incubated under identical conditions. Cultures were controlled daily, colo-
¢ǰȱȱȱęȱȱȱŗŜȱȱȱ
ȱ ȱȱȱǰȱȱǰȱȱ negative coccobacilli were subcultured under
by LGC Genomics, Berlin, Germany, and the
Mycobacterium spp., was conducted, according to the protocol described by Talaat et al. (1997).
identical conditions.
Results ¢ȱȱȱȱȱing Francisella’s appearance, their identity was
ȱȱęȱȱęȱ
ęȱ¢ȱȱȱȱȱȱ
ȱȱȱȱȱȱȱŗşȱęȱ (Figure 1). Additionally, granulomas could be
identical to the one described below.
was an enlarged spleen with multiple white
Figure 1. Aulonocara stuartgrantiǯȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ spleen.
ŜŜǰȱǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚ
Figure 2.ȱȱȱȱǰȱ ȱȱ ȱȱǰȱȱȱ bacteria (arrows) H&E.
ȱȱȱȱǻŗŖȦŗşǼȱȱȱȱ¢ȱ ǻŗȦŗşǼȱȦȱȱȱǻŘȦŗşǼǯȱȱęȱȱȱ display the typical granulomas, but presented ȱȱȱǻŝȦŗşǼǰȱȱȱȱȱ
ȱȱȱȱ¢ȱȱȱ such as Citrobacter freundii, Aeromonas caviae or ȱĚǯ
ǻŚȦŗşǼǯȱ¢ȱȱȱȱ
ȱ ǰȱ ȱȱǰȱǰȱ ǰȱ
in the kidney as well as haemorrhage in the
slightly transparent colonies could be observed
ǰȱ¢ȱȱȱȱǰȱ ȱȱȱȱĚȱȱȱ ȱ¢ȱǻśȦŗşǼȱ ȱȱǯ
ȱȱȱęȱ¢ȱȱȱȱŘŜǚȱǻȱ řǼǯȱ ȱȱȱȱȱȱȱ
HE-and ZN stained histopathological sections
ȱȱȱȱȱǻśȦŗśǼǰȱȱǻŗȦŘǼȱ
ȱȱȱȱ ȱȱ ȱȱȱǰȱ¢ȱȱ
ȱȱǻŗȦŝǼǰȱȱ ȱȱȱȱȱȱ ȱ ȱȱȱȱȱŘŞŜȱȱȱ
ȱȱ ȱȱȱȱȱ
size. When sequenced, these products showed
macrophages (Figure 2).
ȱşşȱƖȱ¢ȱ ȱȱȱF. noatunensis
gram negative coccobacilli.
subsp. orientalis. ȱȱȱȱȱǰȱ liver and kidney on blood agar either did not result in any bacterial growth or only resulted
PCR targeting mycobacteria, on the other hand, ȱȱȱȱȱęȱǯ
ǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚǰȱŜŝ
Figure 3.ȱ¢ȱ ȱȱF. noatunensis subsp. orientalis on CHAB.
Discussion ȱȱȱȱȱȱȱęȱ among breeders and hobbyists. Wild caught
ȱȱȱȱǯȱ ȱȱlids, diseases are mostly diagnosed as the so
ȱęȱȱȱȱȱȱȱ
ȱȱǰȱȱ¡ȱȱȱ¢ȱ
ȱ ȱȱȱęȱȱȱ
anorexia, dyspnoea, abdominal distension and
ȱȱ¢ȱȱǻ ǰȱŗşşŖǼǯ
ȱȱǻǰȱŘŖŖŜǼǯȱȱ
¢ȱȱȱęȱ¢ȱȱȱȱ-
there is no consensus about the causative agent,
ȱȱęȱȱȱȱ¢ȱȱ ȱ¡ȱęȱȱǻ £ȱȱ
ȱȱȱȱȱ ȱ Cryptobiaȱǯȱȱ¢ȱȱȱȱ
ǯǰȱŗşşŘǼǯ¢ȱǰȱȱȱȱ
ȱǻǰȱŗşŞŜDzȱȱȱǯǰȱŗşŞśǼǯȱ
ȱȱȱęȱȱěȱȱȱ
ȱęȱȱȱȱȱ-
¡ȱȱȱǯȱ¢ȱ¢ǰȱȱȱȱ
ent with the alterations observed during acute
ȱȱ Ȭęȱȱęȱȱ ȱȱȱȱȱȱ ȱȱ
ȱȱȱ ȱǯȱ ǰȱ ȱȱȱȱȱȱȱ-
ǻȱȱǯǰȱŘŖŗřǼǯȱȱěȱęȱ ȱnosed while still being quarantined and spread
ȱ ȱȱĚȱȱȱ ȱȱǻǰȱŘŖŖŜǼǰȱȱ
ȱȱęȱȱȱȱ ȱǯȱ
ȱȱȱȱȱȱ¢ǯȱ -
¢ǰȱ¢ȱȬȱ-
¢ǰȱȱȱȱȱęȱǰȱȱ ¢ȱĚȱȱǯȱǰȱȱ
ers do not seek veterinary advice when health
investigations investigation could detect the
ŜŞǰȱǯȱǯȱǯȱȱǯǰȱřŚǻŘǼȱŘŖŗŚ
ȱȱF. noatunensis subsp. orientalis even ȱęȱȱȱȱȱȱȱ ȱ¢ȱ¢ȱęȱȱȱȱ
Acknowledgments ȱ¢ȱ ȱȱ¢ȱȱ¢ȱȱ ¢ȱǰȱȱǻŘşŗŗŗŘŜŝǼǯ
such as a slightly swollen spleen, bleedings in the abdominal cavity or sanguineous ascites.
References
ȱ ȱȱȱǰȱȱȱ¢ȱȱ ȱȱȱȱȱȱĴȱȱȱ
Abd H, Johansson T, Golovliov I, Sandstrom G and Forsman M (2003). Survival and growth ȱ Francisella tularensis in Acanthamoeba castellanii. Applied and environmental microbiology 69ǰȱŜŖŖȮŜŖŜǯ
ȱȱȱ¢ȱęȱȱ with Francisella sp. ȱȱ ȱȱȱȱȱȱF. noatunensisȱ ȱęȱȱȱȱ¢ȱȱȱȱEuplotes raicovi (Schrallhammer et al., 2011). In this context and considering that F. tularensisȱȱ ȱȱȱĴȱ by protists and arthropods (Abd et al., 2003; ȱȱǯǰȱŘŖŖşǼǰȱȱȱȱȱǰȱ ȱĚǰȱȱȱȱȱ ȱȱȱǯ Although Francisella sp. has been demonstrated in ȱȱȱȱ (Hsieh et al., 2007), it had not been described as the causaȱȱȱȱȱȱ¢ȱȱȱęȱȱǯȱȱȱȱȱȱȱȱ that, especially when not presenting the typical ȱǰȱȱȱȱ¢ȱ be overlooked in routine examinations because ȱȱȱȱȱȱȱȱȱ Ĝ¢ȱȱȱȱǻȱȱǯǰȱŘŖŖŚǼǯȱ Furthermore, mycobacteriosis is an important ěȱȱȱȱȱȱ ȱȱȱęȱȱȱ¢ȱȱ ȱ ȱFrancisella sp. In Europe, there ȱȱȱȱȱ ȱFrancisella sp. other than F. noatunensis subsp. orientalis in ȱ ȱǻ ě¢ȱȱǯǰȱŘŖŗŖǼȱȱ ¢ȱǻȱȱ ǯǰȱŘŖŖŜDzȱ¢ȱȱǯǰȱŘŖŖŜDzȱĴȱȱǯǰȱŘŖŖŝǼǯȱ However, until now, Francisellosis has not been described in Central Europe.
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