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

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 β, 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

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 fulfilment 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 8 housekeeping genes and the 16S rRNA-ITS-23S rRNA sequence. The phenotypic profiling indicated a high degree of similarity between the Fno taxon as all were able to metabolise 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 analysed. In all the phylogenetic trees the Fno strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the individual trees i.e mutS, putA, rpoB, the concatenated sequence and 16S rRNA-ITS-23S rRNA genes the two Francisella noatunensis ssp. diverged more from each other than from the closely related human pathogen Francisella philomiragia (Fp). The phenotypic and genetic characterisation 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 characterise strains of Fnn and Fp and verify their current taxonomic rank of Fno

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

Investigating the involvement of a Midichloria -like organism (MLO) in red mark syndrome in rainbow trout Oncorhynchus mykiss

Red mark syndrome (RMS) is a skin condition in Rainbow trout Oncorhynchus mykiss that has been reported worldwide but was first seen in the United Kingdom (UK) in 2003. The current study was conducted to examine if there was an association between a Midichloria-like organism (MLO) and RMS using a statistically appropriate sample set, whilst determining if there is a lack of association with Flavobacterium psychrophilum implicated in disease in previous studies. Fish in this study were obtained from three sites positive for RMS in the UK and United States (US), and three sites in the UK and the Netherlands that had no previous history of this condition. Samples taken from RMS-affected sites were found to show typical RMS pathology. Analysis of the major organs of affected fish by quantitative polymerase chain reaction (qPCR) demonstrated a significantly higher presence of the MLO in the RMS-affected tissues. Although most of the tissues were positive for the MLO, the highest correlation was seen in the skin, whilst the tissues from the unaffected fish were all negative. Thus, a strong positive correlation was found between the MLO and RMS-affected fish, whilst no association was found between the RMS-affected fish and F. psychrophilum other than superficial presence in the skin. The use of immunohistochemistry showed positive staining of what was considered to be MLO-related antigens in the internal organs of most RMS-affected fish. Attempts were made to culture the MLO, but no MLO was isolated

Increased robustness of postlarvae and juveniles from non-ablated Pacific whiteleg shrimp, Penaeus vannamei, broodstock post-challenged with pathogenic isolates of Vibrio parahaemolyticus (VpAHPND) and white spot disease (WSD)

The maturation and reproduction of Pacific whiteleg shrimp, Penaeus vannamei, through the practice of unilateral eyestalk ablation though common is an animal welfare concern. This study assessed the resilience of offspring from non-ablated P. vannamei when challenged with an isolate of Vibrio parahaemolyticus (Vp) causing acute hepatopancreatic necrosis disease (VpAHPND), and with white spot syndrome virus (WSSV). VpAHPND and WSSV challenges were conducted using PL and juveniles under controlled conditions, with both trials using four groups (i.e. shrimp from either ablated or non-ablated females and then either challenged with the pathogen or not challenged). For the VpAHPND challenge, ten replicate 20 L tanks (five replicates for each population) each containing 100 PL 17 (average weight 14 mg) in 15 ppt, 29.05 ± 0.13 °C water were challenged with 2 mL of 2.0 × 108 CFU mL−1 culture of V. parahaemolyticus. A further ten replicate tanks (five per population) served as the corresponding non-challenged controls. The shrimp mortalities were assessed every 3 h over the following 96 h. For the WSSV challenge, individual 1.4 g (average weight) shrimp (50 individuals per population) were housed in 1 L tanks and fed 0.1 g WSSV infected tissue (av. 2.02 × 109 WSSV). A further 50 shrimp per population served as non-challenged controls. The shrimp were maintained at 15 ppt, 26.3 ± 0.71 °C water and assessed every 3 h post-infection over the subsequent 168 h and mortalities at each time point noted. Postlarvae from non-ablated females had significantly (p = 2.4E-23) better survival (70.4%) than those from ablated females (38.8%) at 96 h post-challenge with VpAHPND. Both challenged populations had significantly (p ≤1.3E-36) lower survival than the control groups. The survival of the juveniles from non-ablated females (62%) at 168 h post-infection with WSSV was not significantly higher than that of the juveniles from ablated female (48%) although the difference was significantly different at 65 to 75 h. Both challenged populations also had significantly (p ≤1.0E-5) lower survival rates than the control groups. The study demonstrates that postlarvae and juveniles from non-ablated females are more resilient to typical pathogens (VpAHPND and WSSV) and may show higher survival rates during a disease outbreak

Oral vaccination of Nile tilapia (Oreochromis niloticus) against francisellosis elevates specific antibody titres in serum and mucus

Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed

Differential characterization of emerging skin diseases of rainbow trout - a standardized approach to capturing disease characteristics and development of case definitions

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example

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