Genetics of canine anal furunculosis in the German shepherd dog.

Canine anal furunculosis (AF) is characterised by ulceration and fistulation of perianal tissue and is a disease that particularly affects German shepherd dogs (GSDs). There are some similarities between AF and perianal Crohn's disease (CD) in man. An immune-mediated aetiopathogenesis for AF has been suggested due to tissue pathology, a major histocompatibility complex (MHC) association and clinical response to ciclosporin. Genome-wide association studies (GWAS) can be conducted in dogs with fewer markers and individuals than would be required in a human study. A discovery GWAS was performed on 21 affected and 25 control GSDs from the UK. No SNPs reached genome-wide significance levels at this stage. However, 127 nominally associated SNPs were genotyped in further 76 cases and 191 controls from the UK and Finland. Sequencing of these regions was undertaken to discover novel genetic variation. Association testing of these variants in the UK and Finnish cohorts revealed nine significantly associated SNPs, six of which cause non-synonymous changes in protein sequence. The ADAMTS16 and CTNND2 gene regions were most significantly associated with disease. Members of the butyrophilin protein family, important in intestinal inflammatory regulation, were also associated with disease, but their independence from the MHC region remains to be established. The CTNND2 gene region is also interesting as this locus was implicated in human ulcerative colitis and CD, albeit at a different candidate gene: DAP. We suggest that this represents a common association between inflammatory bowel disease-related conditions in both species and believe that future studies will strengthen this link.

Virulence, genomic features, and plasticity of Aeromonas salmonicida subsp. salmonicida, the causative agent of fish furunculosis.

The bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a systemic disease of fish in the salmonid family. Furunculosis is a ubiquitous disease that affects aquaculture operations worldwide and is characterized by high mortality and morbidity. A better understanding of the bacterium is required to find a cure. Thereby, this review centers on A. salmonicida subsp. salmonicida, its major virulence factors, and its genome. The classification and characteristics of A. salmonicida subsp. salmonicida, the virulence factors, such as the A-layer, extracellular molecules, and type three secretion system as well as the characteristics and plasticity of its genome are described.

Long-lasting protection induced by bath vaccination against Aeromonas salmonicida subsp. salmonicida in rainbow trout.

For decades Aeromonas salmonicida subsp. salmonicida (from here referred to as A. salmonicida) has been recognized as the causative agent of typical furunculosis. This disease has had a major impact on aquaculture worldwide, making it a target for international research, particularly within the field of immunoprohylaxis. Initial studies attempted vaccination via oral route and immersion. However, these vaccination methods proved insufficient when compared to intraperitoneally (i.p.) injected vaccines. The focus of vaccine research regarding A. salmonicida shifted towards the i.p.-injected vaccines during the 1980's and -90's, resulting in oil-adjuvanted vaccines providing high levels of protection over longer periods of time. The majority of this research has been conducted using salmon, while rainbow trout, which is also a commercially important species, has played a much less central role. In this study, we have examined the effect of a bath vaccination using an experimental A. salmonicida bacterin. Rainbow trout were vaccinated by a 5 min bath in a formalin-inactivated bacterin. Half of these fish was booster vaccinated using 50% of the initial vaccine dose 10 weeks post primary immunization. Along with an un-vaccinated control group, the fish were challenged by waterborne infection 24 weeks post primary immunization. Both vaccinated groups showed a significantly increased survival (>93% survival) compared to a 70% survival in the un-vaccinated control group (P = 0.005 and P = 0.019 for single and dual immunizations, respectively). When comparing the survival of the single and dual immunization groups, there was no significant difference (P = 0.531). ELISA showed no significant induction of specific circulating antibodies in either vaccinated group. These results are interesting with regard to the protective mechanisms, seen in the light of previous results obtained using bath as well as i.p. vaccination against furunculosis in salmonid fishes.

The improved PCR of the fstA (ferric siderophore receptor) gene differentiates the fish pathogen Aeromonas salmonicida from other Aeromonas species.

The members of the genus Aeromonas are autochthonous of aquatic ecosystems and several species have been associated to septicaemia, ulcerative and haemorrhagic diseases in fish, causing significant mortality in both wild and farmed, freshwater and marine fish species. The species Aeromonas salmonicida is generally recognized as the most important fish pathogen responsible for epidemic outbreaks of furunculosis in salmonids, also being able to produce infections in other cultured fish such as turbot, halibut, sea bream or goldfish. New species, i.e. Aeromonas aquariorum, Aeromonas tecta and Aeromonas piscicola, have recently been discovered and isolated from diseased fish. The species A. piscicola and Aeromonas bestiarum are practically impossible to differentiate phenotypically and genetically (when using the 16S rRNA gene) from each other and from A. salmonicida. In the present study, two previously described PCR protocols, based on the fstA and gyrB genes, for the specific detection of A. salmonicida were re-evaluated with the type strains of all Aeromonas species and with a set of A. piscicola and A. bestiarum strains. Contrary to what had been published previously it was demonstrated that the gyrB-PCR is not specific for A. salmonicida because of cross-reactions with other Aeromonas species. However, in agreement with previous results, A. salmonicida was detected on the basis of the fstA-PCR, for which an improved protocol was proposed.

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon, Oncorhynchus kisutch.

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non-salmonid fish.

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery-propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment-producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.

Antibody responses correlate with antigen dose and in vivo protection for oil-adjuvanted, experimental furunculosis (Aeromonas salmonicida subsp. salmonicida) vaccines in Atlantic salmon (Salmo salar L.) and can be used for batch potency testing of vaccines.

Salmon farming has increased dramatically over last thirty years and a key to the success is the introduction of protective vaccines. In Norway, almost 100% of all Atlantic salmon are vaccinated prior to sea transfer. This extensive use of vaccines demands use of a lot of resources in production and quality control of vaccines, and fish are now one of the most widely used laboratory animal species in Norway, since all batch testing today is performed by challenge experiments. With an increasing focus on the 3 R's (Replacement, Reduction and Refinement), new methods are needed. The aim of this study was to assess the use of different vaccine evaluation methods to identify furunculosis vaccines of different "potency", using ELISA as in vitro assay and intraperitoneal and cohabitation challenge as in vivo assays. Eleven vaccines with different antigen content (0, 2, 5, 10, 20, 40, 80, 100 and 200%) and different antigen qualities were included in the study. Challenge and blood sampling for the ELISA assay were conducted 9 weeks post vaccination. The results from this study indicated that there is a close correlation between the antigen dose in the vaccine and the antibody response against Aeromonas salmonicida as measured by ELISA. There is also a close correlation between the antibody response and protection for both i.p. and cohabitation challenge models. The ELISA method identified sub-potent batches better than currently used in vivo assay (i.p challenge) and seems to be the best method of performing a batch potency test of furunculosis vaccines particularly when taking the 3R's principles into account.

Detection of Aeromonas salmonicida by reverse transcription-multiplex polymerase chain reaction.

Aeromonas salmonicida is one of the major fish pathogens causing economically devastating losses in aquaculture. A. salmonicida subsp. salmonicida is a typical A. salmonicida causing furunculosis, while the other subspecies are atypical strains causing ulcer diseases. PCR-based methods of detecting A. salmonicida suffer from the drawback that they do not distinguish living (pathogenic) from dead cells. In this study, a method of detecting A. salmonicida was developed based on reverse transcription-multiplex PCR (RT-MPCR) using two sets of primers, SV1/SV2 and SF1/SF2, specific to the vapA gene and the fstB gene of A. salmonicida respectively. This method was found to detect A. salmonicida specifically with detection limits of 10 CFU in pure culture and 30 CFU in the presence of tissue debris. It was also found distinguish not only between viable and nonviable cells but also between typical and atypical strains of A. salmonicida. Using RT-MPCR, two DNA fragments, of 542 and 1,258 bp, were amplified from RNA of typical A. salmonicida, whereas only one DNA fragment, of 542 bp, was amplified from the RNA of the atypical ones. The proposed assay was also used successfully to detect A. salmonicida in artificially infected rainbow trout (Oncorhyncus mykiss).

Development of an antibody ELISA for potency testing of furunculosis (Aeromonas salmonicida subsp salmonicida) vaccines in Atlantic salmon (Salmo salar L).

The study was conducted in Atlantic salmon to establish the initial and basic scientific documentation for an alternative batch potency test for salmon furuculosis vaccines. We assessed the antibody response development for Aeromonas salmonicida vaccines at different immunisation temperatures (3, 12 and 18 °C), by an enzyme-linked-immunosorbent assay (ELISA) 3, 6, 9 and 12 weeks post vaccination, and the correlation between antibody response and protection in cohabitation challenge experiments performed 6 and 12 weeks post vaccination. Fish immunised with a vaccine containing full antigen dose had a significant increase in antibody response after 252 day degrees and the measured values correlated well with protection after 500 day degrees. Fish vaccinated with a reduced antigen dose showed a significant lower antibody response than fish vaccinated with the full dose vaccine at all samplings, and showed a similar low relative percent survival (RPS) in the challenges. The results from this study indicate that an antibody ELISA can discriminate between vaccines of different antigen content and the method may replace challenge tests in batch potency testing of furunculosis vaccines in Atlantic salmon. An immunisation temperature of 12 °C and sampling after 6-9 weeks, seemed to be the most appropriate time for using antibody responses to confirm batch potency.

QTL detection for Aeromonas salmonicida resistance related traits in turbot (Scophthalmus maximus).

BACKGROUND: Interactions between fish and pathogens, that may be harmless under natural conditions, often result in serious diseases in aquaculture systems. This is especially important due to the fact that the strains used in aquaculture are derived from wild strains that may not have had enough time to adapt to new disease pressures. The turbot is one of the most promising European aquaculture species. Furunculosis, caused by the bacterium Aeromonas salmonicida, produces important losses to turbot industry. An appealing solution is to achieve more robust broodstock, which can prevent or diminish the devastating effects of epizooties. Genomics strategies have been developed in turbot to look for candidate genes for resistance to furunculosis and a genetic map with appropriate density to screen for genomic associations has been also constructed. In the present study, a genome scan for QTL affecting resistance and survival to A. salmonicida in four turbot families was carried out. The objectives were to identify consistent QTL using different statistical approaches (linear regression and maximum likelihood) and to locate the tightest associated markers for their application in genetic breeding strategies. RESULTS: Significant QTL for resistance were identified by the linear regression method in three linkage groups (LGs 4, 6 and 9) and for survival in two LGs (6 and 9). The maximum likelihood methodology identified QTL in three LGs (5, 6 and 9) for both traits. Significant association between disease traits and genotypes was detected for several markers, some of them explaining up to 17% of the phenotypic variance. We also identified candidate genes located in the detected QTL using data from previously mapped markers. CONCLUSIONS: Several regions controlling resistance to A. salmonicida in turbot have been detected. The observed concordance between different statistical methods at particular linkage groups gives consistency to our results. The detected associated markers could be useful for genetic breeding strategies. A finer mapping will be necessary at the detected QTL intervals to narrow associations and around the closely associated markers to look for candidate genes through comparative genomics or positional cloning strategies. The identification of associated variants at specific genes will be essential, together with the QTL associations detected in this study, for future marker assisted selection programs.

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues.

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non-salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold-conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13-nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody-coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10(4) CFU mL(-1) , and results were obtained within 45 min. The antibody-coated gold nanoparticles were stable for at least 2 months at 4 ° C. The immunoassay using antibody-coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.

Quantitative genetics of disease resistance in vaccinated and unvaccinated Atlantic salmon (Salmo salar L.).

Furunculosis (Aeromonoas salmonicida) is an important disease in Atlantic salmon (Salmo salar) farming. Vaccination and selective breeding for increased resistance to the disease on the basis of challenge tests of unvaccinated fish are used as complementary prophylactic methods. An important issue is whether genetic predisposition to infection is consistent across vaccinated and unvaccinated fish. Hence, the main objective of this study was to determine the magnitude of the genetic associations (correlations) between resistance to furunculosis in vaccinated and unvaccinated fish, and to estimate the magnitude of the correlation of resistance to furunculosis with resistance to the viral diseases infectious pancreatic necrosis (IPN) and infectious salmon anaemia (ISA). Sub-samples of unvaccinated and vaccinated salmon from 150 full-sib families were subjected to separate cohabitation challenge tests. Substantial genetic variation was found in resistance to furunculosis in both the unvaccinated (heritabilities of 0.51 ± 0.05) and vaccinated (0.39 ± 0.06) fish. However, the genetic correlation between resistance to furunculosis in the two groups was low (0.32 ± 0.13), indicating a weak genetic association between resistance in the two groups. Hence, the current selection strategy on the basis of challenge tests of unvaccinated fish is likely to produce low genetic improvement in resistance to furunculosis under field conditions, where fish are vaccinated with an effective vaccine. Evidence was found of significantly favourable genetic associations of resistance to furunculosis in unvaccinated (but less so for vaccinated) fish with resistance to both IPN and ISA (unvaccinated fish), indicating that vaccination 'mask' genetic associations between resistance to different diseases.

In vitro adherence of two candidate probiotics from Atlantic cod and their interference with the adhesion of two pathogenic bacteria.

The potential of two candidate probiotic bacteria (GP21 and GP12), isolated from the gut of Atlantic cod, to adhere to primary cultures of the epithelial cells from the different regions of the intestine and to interfere with the adhesion of two pathogens, Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida were investigated. The intestinal isolates showed clear preference in adhering to the cells from the different intestine segments. GP12 adhered strongly to the fore- and mid intestine cells. The adherence of GP21 was most to the cells from the hind intestine followed by those from the mid-segment. The adhesion of V. anguillarum was affected by both GP21 and GP12; GP12 interfered through competition, but a specific mode of action was not observed for GP21. In the case of A. salmonicida, competition was the principal mechanism by which GP21 interfered with their adhesion, while exclusion mechanism was favoured by GP12. In addition, GP21 was more auto-aggregative than GP12, but the latter was more co-aggregative with both the pathogens. The isolates were also capable of lowering lactate dehydrogenase activity compared to that by the pathogen and they reduced the caspase-3 activity in the epithelial cells from the hind intestine, to which the pathogens adhered the most. Thus it could be concluded that the adhesion of the candidate probiotics is segment-specific and their interference with the adhesion of pathogens is dependent on both source of the epithelial cells and the mechanism adopted by the isolates. This information is novel in the case of fish and the manner in which potential probiotic organisms interfere with the pathogen adhesion provides supportive information for disease control.

Furuncular myiasis caused by the human bot-fly Dermatobia hominis in a domestic cat from Brazil.

This paper reports a case of furuncular myiasis caused by the human bot-fly Dermatobia hominis in a domestic cat from Brazil. A crossbred shorthaired female cat of approximately 3 years old, presented with three boil-like cutaneous lesions at the left cranioventral region of the neck. These were diagnosed as furuncular myiasis. The animal was sedated, and after shaving the fur, bot-fly larvae were removed from the lesion by digital compression. Afterwards, the wounds were treated with 10% iodine solution and also with wound-healing cream containing sulfanilamide, urea and beeswax. Maggots were identified as third-stage larvae of D hominis. Clinical case reports of human bot-fly myiasis in cats are relevant due to its scarce occurrence in feline veterinary practice in some countries.

First description of atypical furunculosis in freshwater farmed Atlantic salmon, Salmo salar L., in Chile.

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.

Mutations in the Aeromonas salmonicida subsp. salmonicida type III secretion system affect Atlantic salmon leucocyte activation and downstream immune responses.

Deletion mutants of Aeromonas salmonicida subsp. salmonicida were used to determine the effect of the type three secretion system (TTSS) on Atlantic salmon anterior head kidney leucocytes (AHKL). One strain had a deletion in the outer membrane pore gene, ascC; and the other in three effector genes: aopO, aopH and aexT (we call this strain Deltaaop3). Host cell invasion success and 24h survival were depressed in DeltaascC, as was 24h survival of Deltaaop3, when compared to the wild type strain. Challenge of AHKLs with A449 or TTSS mutants stimulated expression of the inflammatory mediators IL-8, IL-1 and TNFalpha at two bacterial concentrations (A(600) 0.1, 0.01). Expression of IL-12 was not stimulated in DeltaascC challenged cells, whereas A449 and Deltaaop3 challenge resulted in an up-regulation of IL-12 in AHKLs, 2- and 4-fold higher than PBS, respectively. Only the wild type strain elicited a significant increase in IL-10 expression (5.5x at A(600) 0.1). Inducible nitric oxide synthetase (iNOS) and arginase (I+II) genes were also significantly up-regulated upon exposure to all strains. However, iNOS:arginase ratio was elevated in the effector mutant challenge. These results suggest that A. salmonicida subsp. salmonicida may enhance survival within the host cell through polarization of macrophages/leucocytes to an alternative, rather than classical, activation state. Furthermore, the short-term survival and lack of T-cell signalling cytokine stimulation in DeltaascC, may help explain its inefficiency at providing protection to subsequent wild type challenge.

Atypical furunculosis vaccines for Atlantic cod Gadhus morhua: impact of reattached Aeromonas salmonicida A-layer protein on vaccine efficacy.

Atypical furunculosis caused by atypical Aeromonas salmonicida bacteria is reported as an increasing problem in farmed Atlantic cod Gadus morhua in Norway. At present, furunculosis vaccines adapted for cod or other marine fish species are not available. To identify bacterial components important for inducing protection in cod, we compared oil-adjuvanted vaccines based on A. salmonicida isolates phenotypically differing in their major cell surface constituents, such as the A-layer protein and lipopolysaccharide O-chains. Also included was an A-layer-deficient isolate with physically reattached A-layer protein. Vaccines containing A. salmonicida A-layer-producing cells elicited significantly better protection than vaccines with A-layer-deficient cells or with a supernatant with secreted A-layer protein. The A. salmonicida cells with reattached A-layer-protein resulted in significant and equal protection to the A-layer-producing cells and protected significantly better than the A-layer-deficient isolate. These results indicate that the A-layer protein when attached to the cell surface plays a role in inducing protective immunity in cod.

Tri-trophic linkages in disease: pathogen transmission to rainbow trout through stonefly prey.

Relationships between macroinvertebrates and microorganisms in aquatic environments are only poorly understood despite the fact that many aquatic macroinvertebrates feed on microbial biofilms during some life stage. Better understanding of trophic interactions between microbial biofilms, macroinvertebrates, and fish may also help control fish diseases and loss of natural resources. It has also been suggested that pollution, habitat fragmentation, and poor water quality may contribute to increased pathogenesis and mortality in fish. Increased disease incidence is difficult to assess, however, in part because of the complexity of pathogen transmission dynamics. Several environmental pathogens exist whose reservoir(s) and means of transmission remain poorly understood, highlighting the need to study pathogen ecology and interactions with organisms other than susceptible hosts. Aeromonas salmonicida is rarely isolated from freshwater sediments. However, stonefly nymphs were found to frequently harbor A. salmonicida and were shown to preferentially feed on the bacterium. Rainbow trout juveniles were presented with different feeding regimes to determine the transmission capacity of nymphs, and all fish fed stoneflies harboring A. salmonicida expressed symptoms of disease. Although current rates of furunculosis in freshwater ecosystems are unknown, trout primarily feed on stoneflies when water oxygen levels are high and temperatures are low (winter months), which is presumed to correspond to high resistance to the pathogen. Given that furunculosis is associated with physiological stress and higher water temperatures, its natural incidence may change in response to global or regional climatological effects.