Evaluation of Francisella orientalis ΔpdpA as a Live Attenuated Vaccine against Piscine Francisellosis in Nile Tilapia.

Francisella orientalis is an important bacterial pathogen of marine and freshwater fish with worldwide distribution. Fish francisellosis is a severe subacute to chronic granulomatous disease, with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this disease. In this study, we evaluated the efficacy of a defined F. orientalis pathogenicity determinant protein A (pdpA) mutant (ΔpdpA) as a live attenuated immersion vaccine against subsequent immersion challenge with the wild-type organism. Immunized Nile tilapia Oreochromis niloticus were protected (45% relative percent survival) from the lethal challenges and presented significantly lower mortality than nonvaccinated and challenged treatments. Although serum IgM was significantly higher in immunized fish, similar bacterial loads were detected in vaccinated and nonvaccinated survivors. In conclusion, although the F. orientalis ΔpdpA is attenuated and effectively stimulated an adaptive immune response, the low relative percent survival and high bacterial persistence in survivors of immunized and challenged treatments indicates low suitability of ΔpdpA as a mucosal vaccine for tilapia under conditions used in this study.

Prospecting of essential oils in combination with florfenicol against motile Aeromonas isolated from tambaqui (Colossoma macropomum).

Natural products with antimicrobial activity and their association with synthetic antimicrobials are a sustainable option in fish farming. The objective of this study was to determine antimicrobial activity, antibiofilm potential and synergism of five essential oils (EOs) with florfenicol against motile Aeromonas isolated from Amazonian Colossoma macropomum. As their major constituent, the EOs of the species of Aloysia triphylla, Croton cajucara (red and white morphotype), Cymbopongo citratus and Lippia gracilis present ß-pinene (22.1%), germacrene D (11.5%), linalool (23%), geranial (45.7%) and carvacrol (42.2%), respectively. The EOs of L. gracilis and C. citratus showed the best antimicrobial activities against the Aeromonas strains (5 mg mL-1). All EOs interfered with biofilm formation and consolidated biofilm. The EOs of A. triphylla, C. citratus and L. gracilis showed a synergistic effect with florfenicol, reducing the amount of the chemical into the water systems while treatment.

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare.

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.

Francisella marina sp. nov., Etiologic Agent of Systemic Disease in Cultured Spotted Rose Snapper (Lutjanus guttatus) in Central America.

Historically, piscine francisellosis in various warm-, temperate-, and cold-water fish hosts has been attributed to Francisella noatunensis From 2015 to 2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper (Lutjanus guttatus) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1,400-bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from seawater in the Gulf of Mexico, while sharing <99% similarity to other Fransicella spp. Biochemical analysis, multilocus sequence comparisons of select housekeeping genes, repetitive extragenic palindromic PCR fingerprinting, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and fatty acid methyl ester analysis revealed marked differences between these isolates and other described members of the genus. Koch's postulates were fulfilled by experimental intracoelomic injection and immersion trials using Nile (Oreochromis niloticus) and blue (Oreochromis aureus) tilapia. Based on observed phenotypic and genotypic differences from recognized Francisella spp., the name Francisellamarina sp. nov. (NRRL B-65518) is proposed to accommodate these novel strains.IMPORTANCE Finfish aquaculture is the fastest growing global food production sector. Infectious disease, particularly emergent pathogens, pose a significant threat to established and nascent aquaculture industries worldwide. Herein, we characterize a novel pathogen isolated from mortality events in cultured spotted rose snapper in Central America. The bacteria recovered from these outbreaks were genetically and phenotypically dissimilar from other known Francisella spp. from fish, representing a previously unrecognized member of the genus Francisella, for which the name Francisella marina sp. nov. is proposed.

Composition of Extracellular Polymeric Substances (EPS) produced by Flavobacterium columnare isolated from tropical fish in Brazil.

Thirty nine isolates of Flavobacterium columnare from Brazilian fish farms had their carbohydrate composition of EPS evaluated by high efficiency liquid chromatography, using the phenol-sulfuric acid method of EPS. The occurrence of capsules on F. columnare cells was not directly related to biofilm formation, and the predominant monosaccharide is glucose.