The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.     

Phage-Driven Loss of Virulence in a Fish Pathogenic Bacterium

Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio) as a model system, the ancestral rhizoid morphotypes were associated with a 25–100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types), did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.     

Emergence of Novel Streptococcus iniae Exopolysaccharide-Producing Strains following Vaccination with Nonproducing Strains

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.     

Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444–2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ∼40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.     

Invasion Ability and Disease Dynamics of Environmentally Growing Opportunistic Pathogens under Outside-Host Competition

Most theories of the evolution of virulence concentrate on obligatory host-pathogen relationship. Yet, many pathogens replicate in the environment outside-host where they compete with non-pathogenic forms. Thus, replication and competition in the outside-host environment may have profound influence on the evolution of virulence and disease dynamics. These environmentally growing opportunistic pathogens are also a logical step towards obligatory pathogenicity. Efficient treatment methods against these diseases, such as columnaris disease in fishes, are lacking because of their opportunist nature. We present a novel epidemiological model in which replication and competition in the outside-host environment influences the invasion ability of a novel pathogen. We also analyze the long-term host-pathogen dynamics. Model parameterization is based on the columnaris disease, a bacterial fresh water fish disease that causes major losses in fish farms worldwide. Our model demonstrates that strong competition in the outside-host environment can prevent the invasion of a new environmentally growing opportunist pathogen and long-term disease outbreaks.     

Intensive Farming: Evolutionary Implications for Parasites and Pathogens

An increasing number of scientists have recently raised concerns about the threat posed by human intervention on the evolution of parasites and disease agents. New parasites (including pathogens) keep emerging and parasites which previously were considered to be 'under control' are re-emerging, sometimes in highly virulent forms. This re-emergence may be parasite evolution, driven by human activity, including ecological changes related to modern agricultural practices. Intensive farming creates conditions for parasite growth and transmission drastically different from what parasites experience in wild host populations and may therefore alter selection on various traits, such as life-history traits and virulence. Although recent epidemic outbreaks highlight the risks associated with intensive farming practices, most work has focused on reducing the short-term economic losses imposed by parasites, such as application of chemotherapy. Most of the research on parasite evolution has been conducted using laboratory model systems, often unrelated to economically important systems. Here, we review the possible evolutionary consequences of intensive farming by relating current knowledge of the evolution of parasite life-history and virulence with specific conditions experienced by parasites on farms. We show that intensive farming practices are likely to select for fast-growing, early-transmitted, and hence probably more virulent parasites. As an illustration, we consider the case of the fish farming industry, a branch of intensive farming which has dramatically expanded recently and present evidence that supports the idea that intensive farming conditions increase parasite virulence. We suggest that more studies should focus on the impact of intensive farming on parasite evolution in order to build currently lacking, but necessary bridges between academia and decision-makers.     

How sea lice from salmon farms may cause wild salmonid declines in Europe and North America and be a threat to fishes elsewhere

Fishes farmed in sea pens may become infested by parasites from wild fishes and in turn become point sources for parasites. Sea lice, copepods of the family Caligidae, are the best-studied example of this risk. Sea lice are the most significant parasitic pathogen in salmon farming in Europe and the Americas, are estimated to cost the world industry €300 million a year and may also be pathogenic to wild fishes under natural conditions.Epizootics, characteristically dominated by juvenile (copepodite and chalimus) stages, have repeatedly occurred on juvenile wild salmonids in areas where farms have sea lice infestations, but have not been recorded elsewhere. This paper synthesizes the literature, including modelling studies, to provide an understanding of how one species, the salmon louse, Lepeophtheirus salmonis, can infest wild salmonids from farm sources. Three-dimensional hydrographic models predicted the distribution of the planktonic salmon lice larvae best when they accounted for wind-driven surface currents and larval behaviour. Caligus species can also cause problems on farms and transfer from farms to wild fishes, and this genus is cosmopolitan. Sea lice thus threaten finfish farming worldwide, but with the possible exception of L. salmonis, their host relationships and transmission adaptations are unknown. The increasing evidence that lice from farms can be a significant cause of mortality on nearby wild fish populations provides an additional challenge to controlling lice on the farms and also raises conservation, economic and political issues about how to balance aquaculture and fisheries resource management.     

Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G₄ and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.     

Biofilm Formation by the Fish Pathogen Flavobacterium columnare: Development and Parameters Affecting Surface Attachment

Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems.     

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.     

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Aims: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Methods and Results: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. Conclusions: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. Significance and Impact of the Study: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.     

Proteomic analysis of the fish pathogen <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>

Background  

Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

Deposition of pathogenic Mycoplasma gallisepticum onto bird feeders: host pathology is more important than temperature-driven increases in food intake

Although ambient temperature has diverse effects on disease dynamics, few studies have examined how temperature alters pathogen transmission by changing host physiology or behaviour. Here, we test whether reducing ambient temperature alters host foraging, pathology and the potential for fomite transmission of the bacterial pathogen Mycoplasma gallisepticum (MG), which causes seasonal outbreaks of severe conjunctivitis in house finches (Haemorhous mexicanus). We housed finches at temperatures within or below the thermoneutral zone to manipulate food intake by altering energetic requirements of thermoregulation. We predicted that pathogen deposition on bird feeders would increase with temperature-driven increases in food intake and with conjunctival pathology. As expected, housing birds below the thermoneutral zone increased food consumption. Despite this difference, pathogen deposition on feeders did not vary across temperature treatments. However, pathogen deposition increased with conjunctival pathology, independently of temperature and pathogen load, suggesting that MG could enhance its transmission by increasing virulence. Our results suggest that in this system, host physiological responses are more important for transmission potential than temperature-dependent alterations in feeding. Understanding such behavioural and physiological contributions to disease transmission is critical to linking individual responses to climate with population-level disease dynamics.     

Adaptation to enemy shifts: rapid resistance evolution to local Vibrio spp. in invasive Pacific oysters

One hypothesis for the success of invasive species is reduced pathogen burden, resulting from a release from infections or high immunological fitness of invaders. Despite strong selection exerted on the host, the evolutionary response of invaders to newly acquired pathogens has rarely been considered. The two independent and genetically distinct invasions of the Pacific oyster Crassostrea gigas into the North Sea represent an ideal model system to study fast evolutionary responses of invasive populations. By exposing both invasion sources to ubiquitous and phylogenetically diverse pathogens (Vibrio spp.), we demonstrate that within a few generations hosts adapted to newly encountered pathogen communities. However, local adaptation only became apparent in selective environments, i.e. at elevated temperatures reflecting patterns of disease outbreaks in natural populations. Resistance against sympatric and allopatric Vibrio spp. strains was dominantly inherited in crosses between both invasion sources, resulting in an overall higher resistance of admixed individuals than pure lines. Therefore, we suggest that a simple genetic resistance mechanism of the host is matched to a common virulence mechanism shared by local Vibrio strains. This combination might have facilitated a fast evolutionary response that can explain another dimension of why invasive species can be so successful in newly invaded ranges.     

A Brucella Virulence Factor Targets Macrophages to Trigger B-cell Proliferation

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.     

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires'' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae''s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.     

Increasing water temperature and disease risks in aquatic systems: Climate change increases the risk of some, but not all, diseases

Global warming may impose severe risks for aquatic animal health if increasing water temperature leads to an increase in the incidence of parasitic diseases. Essentially, this could take place through a temperature-driven effect on the epidemiology of the disease. For example, higher temperature may boost the rate of disease spread through positive effects on parasite fitness in a weakened host. Increased temperature may also lengthen the transmission season leading to higher total prevalence of infection and more widespread epidemics. However, to date, general understanding of these relationships is limited due to scarcity of long-term empirical data. Here, we present one of the first long-term multi-pathogen data sets on the occurrence of pathogenic bacterial and parasitic infections in relation to increasing temperatures in aquatic systems. We analyse a time-series of disease dynamics on two fish farms in northern Finland from 1986 to 2006. We first demonstrate that the annual mean water temperature increased significantly on both farms over the study period and that the increase was most pronounced in the late summer (July-September). Second, we show that the prevalence of infection (i.e. proportion of fish tanks infected each year) increased with temperature. Interestingly, this pattern was observed in some of the diseases (Ichthyophthirius multifiliis, Flavobacterium columnare), whereas in the other diseases, the pattern was the opposite (Ichthyobodo necator) or absent (Chilodonella spp.). These results demonstrate the effect of increasing water temperature on aquatic disease dynamics, but also emphasise the importance of the biology of each disease, as well as the role of local conditions, in determining the direction and magnitude of these effects.     

Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen

Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens.     

Food provisioning alters infection dynamics in populations of a wild rodent

While pathogens are often assumed to limit the growth of wildlife populations, experimental evidence for their effects is rare. A lack of food resources has been suggested to enhance the negative effects of pathogen infection on host populations, but this theory has received little investigation. We conducted a replicated two-factor enclosure experiment, with introduction of the bacterium Bordetella bronchiseptica and food supplementation, to evaluate the individual and interactive effects of pathogen infection and food availability on vole populations during a boreal winter. We show that prior to bacteria introduction, vole populations were limited by food availability. Bordetella bronchiseptica introduction then reduced population growth and abundance, but contrary to predictions, primarily in food supplemented populations. Infection prevalence and pathological changes in vole lungs were most common in food supplemented populations, and are likely to have resulted from increased congregation and bacteria transmission around feeding stations. Bordetella bronchiseptica-infected lungs often showed protozoan co-infection (consistent with Hepatozoon erhardovae), together with more severe inflammatory changes. Using a multidisciplinary approach, this study demonstrates a complex picture of interactions and underlying mechanisms, leading to population-level effects. Our results highlight the potential for food provisioning to markedly influence disease processes in wildlife mammal populations.