Protection and antibody reactivity in lumpsucker (Cyclopterus lumpus L.) following vaccination against Pasteurella sp.

Two monovalent vaccines against pasteurellosis were developed and tested for efficacy using a previously established bath challenge model. High levels of specific antibodies were detected following vaccination. While the vaccine efficacy trial indicated that some level of protection was obtained, high mortality was still observed. qPCR analysis of head kidney tissues from surviving fish post challenge showed no difference in bacterial numbers in vaccinated and non-vaccinated fish. Clinical symptoms observed in moribund and diseased fish included white spots on the skin and around the eyes, frayed fins and redness around the mouth and fin bases. Despite production of specific antibodies, the protection against experimental challenge was relatively weak. A reason for this could potentially be that the specific antibodies produced are not alone enough to provide complete protection against pasteurellosis in lumpsuckers. Confocal and scanning electron microscopy of head kidney leucocytes exposed to Pasteurella sp. in vitro gave indications of the interactions between the pathogen and leucocytes. The results indicate that parts of the immune system other than humoral antibodies could be important for protection against pasteurellosis. Our combined results highlight the need for further work on host-pathogen interaction between Pasteurella sp. and lumpsuckers.

Francisella noatunensis subspecies noatunensis clpB deletion mutant impairs development of francisellosis in a zebrafish model.

BACKGROUND: Francisella noatunensis ssp. noatunensis (F.n.n.) is the causative agent of francisellosis in Atlantic cod and constitutes one of the main challenges for future aquaculture on this species. A facultative intracellular bacterium like F.n.n. exert an immunologic challenge against which live attenuated vaccines in general are most effective. Thus, we constructed a deletion in the F.n.n. clpB gene as ΔclpB mutants are among the most promising vaccine candidates in human pathogenic Francisella. PURPOSE: Characterization of F.n.n. ΔclpB using primary Atlantic cod head kidney leukocytes, the zebrafish embryo and adult zebrafish model with focus on potential attenuation, relevant immune responses and immunogenic potential. MAIN RESULTS: Interleukin 1 beta transcription in Atlantic cod leukocytes was significantly elevated from 24 to 96 h post infection with F.n.n. ΔclpB compared to F.n.n. wild-type (wt). Growth attenuation of the deletion mutant in zebrafish embryos was observed by fluorescence microscopy and confirmed by genome quantification by qPCR. In the immunization experiment, adult zebrafish were immunized with 7 × 106 CFU of F.n.n. ΔclpB before challenge four weeks later with 6 × 108 CFU of F.n.n. wt. One day after challenge, immunized zebrafish responded with significantly lower interleukin 8 levels compared to the non-immunized control. Immunized fish were protected against the acute mortality observed in non-immunized zebrafish after challenge and bacterial genomes quantified by qPCR were reduced to a minimum 28 days post challenge, indicating protective immunity stimulated by F.n.n. ΔclpB. CONCLUSION: Deletion mutation of clpB in F.n.n. causes in vitro and in vivo attenuation and elicits a protective immune response in adult zebrafish against a lethal dose of F.n.n. wt. Taken together, the results presented increases the knowledge on protective immune responses against F.n.n.

Protection and antibody reactivity following vaccination of lumpfish (Cyclopterus lumpus L.) against atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida is frequently associated with disease and mortality in farmed lumpfish (Cyclopterus lumpus L). Challenge experiments using different modes of exposure identified both high and low pathogenic isolates. Intraperitoneal vaccination induced production of high levels of specific antibodies particularly in fish given multiple injections. The immune sera contained antibodies cross reactive with both high and low pathogenic isolates. SDS-PAGE and LC/MSMS analyses showed that the highly virulent isolate expressed the virulence array protein (A-layer) while the less virulent isolate did not. Vaccines, containing the highly virulent isolate, formulated as a monovalent or as a trivalent vaccine, provided 73 and 60 relative percent survival (RPS) respectively, following intraperitoneal challenge. The detection of high levels of specific antibodies in immune sera and the protection provided by the test vaccines strongly indicate that it is possible to vaccinate lumpfish against atypical A. salmonicida and most probably also against other infectious bacterial diseases.

Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model.

Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gram-negative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium F. noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IglC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish.

Establishment of three Francisella infections in zebrafish embryos at different temperatures.

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells.

Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions.

BACKGROUND: Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species. RESULTS: Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene. CONCLUSION: Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.

Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar).

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development.

First reported isolation of Neisseria canis from a deep facial wound infection in a dog.

Neisseria canis was isolated in pure culture from a mandibular abscess in a dog. Ultrasound-guided fine-needle aspiration was used to obtain a sample from the abscess. Conventional bacteriological examination techniques followed by 16S rRNA gene sequencing from pure subculture and construction of a phylogenetic tree verified the isolate as N. canis. 16S rRNA sequence analysis revealed that a broader phylogenetic platform is needed in the part of the phylogenetic tree where the canine pathogenic N. canis isolate is located. The canine pathogenic isolate was found to be resistant to cephalexin and trimethoprim.