A 'must-have' book for vets working with aquatic species.

Reviewed by Matthijs Metselaar, a specialist in fish health and production.

Anaesthesia of laboratory, aquaculture and ornamental fish: Proceedings of the first LASA-FVS Symposium.

Following on from the Annual Fish Veterinary Society Conference, this symposium was organised with the Laboratory Animal Science Association and brought together experts from ornamental (pond and aquarium) fish practice, aquaculture and aquatic-research facilities to discuss good practice of anaesthesia. This proceedings paper gives an overview of relevant experiences involving a range of immersion drugs including tricaine, benzocaine and isoeugenol, as well as a summary of the main topics of discussion. While fish anaesthesia is commonplace, administration methods, drugs and monitoring procedures may often be regarded as antiquated when compared with mammalian practice. These limitations notwithstanding, individual fish will benefit from good anaesthetic monitoring. Although the most common anaesthetic drugs may be perceived as equally efficacious and therefore interchangeable for different settings, challenges are different for the anaesthesia of grouped fish, when determining species-dependent anaesthetic dosing in a multi-species tank, or adapting to farming requirements, nationally licensed products, costs and withdrawal periods. The fish anaesthetic arsenal fails to address premedication, analgesia and issues of averseness. The two latter factors should be part of the evaluation of anaesthetic protocols; therefore, instructions for the analgesic provision of lidocaine to fin clipped zebrafish are proposed. Euthanasia practices could sometimes be refined too. Alternative physical methods such as electrical stunning are options to be considered.

Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis.

BACKGROUND: Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence. RESULTS: Larvae were killed by Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology (haematoxylin and eosin staining, Gram Twort and immunohistochemistry) and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts. CONCLUSIONS: This study shows that an alternative host, G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions.

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 ß, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.

Draft Genome Sequence of Francisella noatunensis subsp. orientalis STIR-GUS-F2f7, a Highly Virulent Strain Recovered from Diseased Red Nile Tilapia Farmed in Europe.

A highly virulent strain of Francisella noatunensis subsp. orientalis, STIR-GUS-F2f7, was isolated from moribund red Nile tilapia (Oreochromis niloticus) farmed in Europe. In this communication, the complete genome sequencing of this bacterium is reported.

A Polyphasic Approach for Phenotypic and Genetic Characterization of the Fastidious Aquatic Pathogen Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis, an emerging infectious disease in Asia and Latin America. In this study two outbreaks of francisellosis were diagnosed in the UK on the basis of histopathology, electron microscopy, PCR, bacterial isolation and fulfillment of Koch's postulates. Furthermore, a phenotypic fingerprint based on biochemical analyses, metabolic activity, chemotaxonomic composition, and antimicrobial assays was generated for the novel isolates, the Fno type strain Ehime-1 from Asia and other Fno from Latin America. The genetic relatedness between the novel Fno and other Francisellaceae species was investigated by sequencing and comparing the 16SrRNA gene, 8 housekeeping genes (individually and concatenated) and the 16SrRNA-ITS-23SrRNA sequence. The phenotypic profiling indicated a high degree of similarity among the Fno strains as all were able to metabolize dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate, and glucose-6-phosphate. The chemotaxonomic analyses indicated that 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%), and 18:0 (5.5%) were the predominant structural fatty acids in Fno. The antimicrobial assays showed little variation between the isolates and high susceptibility to enrofloxacin, gentamicin, neomycin, streptomycin, amikacin, ciprofloxacin, gatifloxacin, nitrofurantoin, tobramycin, kanamycin, tetracycline, oxytetracycline, florfenicol, oxolinic acid, and streptomycin in all the Fno analyzed. In all the phylogenetic trees the Fno strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the 11 trees i.e., mutS, putA, rpoB, 16SrRNA-ITS-23SrRNA, and concatenated sequence the two Francisella noatunensis ssp. diverged more from each other than from the closely related Francisella philomiragia (Fp). The phenotypic and genetic characterization confirmed the Fno isolates represent a solid phylo-phenetic taxon that in the current context of the genus seems to be misplaced within the species Fn. We propose the use of the present polyphasic approach in future studies to characterize strains of Fnn and Fp and verify their current taxonomic rank of Fno and other aquatic Francisella spp.