Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.     

Use of microcosms to determine the survival of the fish pathogen Tenacibaculum maritimum in seawater

The survival of the fish pathogen Tenacibaculum maritimum in different seawater microcosms was investigated during 160 days. The persistence of culturable cells was greater in sterile than in natural seawater. Standard plate counts showed that T. maritimum survived in sterile seawater for more than 5 months at concentration around 10(3) cfu ml(-1). However, T. maritimum proved to be very labile in non-sterile seawater, rendering culturable cells no longer than 5 days. These results were confirmed when DNA-based methods were applied. Regardless of the microcosms used, epifluorescence microscopy counts remained at about 10(6) cells ml(-1) throughout the experiment, even though we can not distinguish T. maritimum in the case of non-sterile microcosms. Resuscitation assays with addition of fresh medium to non-sterile microcosms did not favour the recovery of T. maritimum on solid media. Although morphological changes from filamentous to spheres were observed after 3 days in the non-sterile microcosms, in the case of the sterile microcosms this change was observed at the sixth day. The biochemical, physiological, serological and genetic characteristics were unaffected in the sterile microcosms. The overall results contribute to a better understanding of the behaviour of T. maritimum in natural seawater and suggest that the aquatic bacterial population play an important role in the survival of this fish pathogen.     

Response of antioxidative enzymes to nickel and cadmium stress in hyperaccumulator plants of the genus Alyssum

The term 'phytoremediation' is used to describe the clean-up of heavy metals from contaminated soils by plants. In this study, we examined Alyssum argenteum and Alyssum maritimum for their ability to accumulate Cd²⁺. We also exemined Ni²⁺ accumulation by A. maritimum with comparison with the known Ni-hyperaccumulator A. argenteum , in a hydroponic system. Both species were tolerant to low levels of Cd²⁺, and accumulated high quantities under the experimental conditions. Only very low levels of Ni²⁺ were found in the shoot of A. maritimum , defining it as a non-hyperaccumulator. The role of the antioxidative enzyme system was investigated in relation to Ni²⁺ and Cd²⁺ stress. In both species, superoxide dismutase (SOD) activity was elevated at high Cd²⁺ concentrations, while ascorbate peroxidase (APX) activity remained unchanged and glutathione reductase (GR) activity was reduced. In the presence of Ni²⁺, A. maritimum exhibited a typical antioxidative defense mechanism, as evidenced by the elevated activities of all three enzymes tested. A. argenteum exhibited a different enzyme response pattern, with a significant reduction in SOD activity, and elevated APX and GR activities only at the highest Ni²⁺ concentration.     

Iron uptake mechanisms in the fish pathogen Tenacibaculum maritimum

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. Proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

Mechanisms underlying host persistence following amphibian disease emergence determine appropriate management strategies

Emerging infectious diseases have caused many species declines, changes in communities and even extinctions. There are also many species that persist following devastating declines due to disease. The broad mechanisms that enable host persistence following declines include evolution of resistance or tolerance, changes in immunity and behaviour, compensatory recruitment, pathogen attenuation, environmental refugia, density‐dependent transmission and changes in community composition. Here we examine the case of chytridiomycosis, the most important wildlife disease of the past century. We review the full breadth of mechanisms allowing host persistence, and synthesise research on host, pathogen, environmental and community factors driving persistence following chytridiomycosis‐related declines and overview the current evidence and the information required to support each mechanism. We found that for most species the mechanisms facilitating persistence have not been identified. We illustrate how the mechanisms that drive long‐term host population dynamics determine the most effective conservation management strategies. Therefore, understanding mechanisms of host persistence is important because many species continue to be threatened by disease, some of which will require intervention. The conceptual framework we describe is broadly applicable to other novel disease systems.     

Effects of gill abrasion and experimental infection with Tenacibaculum maritimum on the respiratory physiology of Atlantic salmon Salmo salar affected by amoebic gill disease

The effects of gill abrasion and experimental infection with Tenacibaculum maritimum were assessed in Atlantic salmon Salmo salar with underlying amoebic gill disease. The respiratory and acid-base parameters arterial oxygen tension (P(a)O2), arterial whole blood oxygen content (C(a)O2), arterial pH (pHa), haematocrit and haemoglobin concentrations were measured at intervals over a 48 h recovery period following surgical cannulation of the dorsal aorta. Mortality rates over the recovery period were variable, with gill abrasion and inoculation with T. maritimum causing the highest initial mortality rate and unabraded, uninoculated controls showing the lowest overall mortality rate. Fish with abraded gills tended to show reduced P(a)O2 and lower C(a)O2 compared with unabraded fish. Infection with T. maritimum had no effect on P(a)O2 or C(a)O2. All fish showed an initial alkalosis at 24 h post-surgery/inoculation which was more pronounced in fish inoculated with T. maritimum. There were no significant effects of gill abrasion or infection upon the ratio of oxygen specifically bound to haemoglobin or mean cellular haemoglobin concentration. Histologically, 48 h following surgery, abraded gills showed multifocal hyperplastic lesions with pronounced branchial congestion and telangiectasis, and those inoculated with T. maritimum exhibited focal areas of branchial necrosis and erosion associated with filamentous bacterial mats. All fish examined showed signs of amoebic gill disease with multifocal hyperplastic and spongious lesions with parasome-containing amoeba associated with the gill epithelium. The results suggest that respiratory compromise occurred as a consequence of gill abrasion rather than infection with T. maritimum.     

Eryngium maritimum, biology of a plant at its northernmost localities

The prospects for persistence of Eryngium maritimum in its northern distribution area was evaluated by studying historical data, matrix modelling, dispersal, seed germination and molecular variation. Historical data revealed a fragmented distribution of small populations with decrease in both population numbers and size during the last 150 years. Fruits of E. maritimum were found to have a relative low floating ability, making the prospects of long distance water dispersal more limited than would be expected from the world distribution of the species. Germination was found to be low (< 25 %). Elasticity matrices showed that survival of reproductive plants were more important than reproduction. High juvenile mortality and low germination activity emphasised the importance of rapid growth and survival. The population insurance against natural catastrophes and environmental stochasticity is suggested to be in a "root bank" of established individuals that are able to tolerate disturbances due to clonal reproduction and regrowth from root fragments. Isozyme electrophoresis revealed low molecular variation on a large geographical scale. The chromosome number is 2n = 16. A high degree of fixed heterozygosity suggests that the species is tetraploid with basic chromosome, number x = 4. The persistence prospects of E. maritimum in its northernmost distribution area are considered low considering the small population sizes, the low dispersal ability and low germination.     

Glacial refugia in pathogens: European genetic structure of anther smut pathogens on Silene latifolia and Silene dioica

Climate warming is predicted to increase the frequency of invasions by pathogens and to cause the large-scale redistribution of native host species, with dramatic consequences on the health of domesticated and wild populations of plants and animals. The study of historic range shifts in response to climate change, such as during interglacial cycles, can help in the prediction of the routes and dynamics of infectious diseases during the impending ecosystem changes. Here we studied the population structure in Europe of two Microbotryum species causing anther smut disease on the plants Silene latifolia and Silene dioica. Clustering analyses revealed the existence of genetically distinct groups for the pathogen on S. latifolia, providing a clear-cut example of European phylogeography reflecting recolonization from southern refugia after glaciation. The pathogen genetic structure was congruent with the genetic structure of its host species S. latifolia, suggesting dependence of the migration pathway of the anther smut fungus on its host. The fungus, however, appeared to have persisted in more numerous and smaller refugia than its host and to have experienced fewer events of large-scale dispersal. The anther smut pathogen on S. dioica also showed a strong phylogeographic structure that might be related to more northern glacial refugia. Differences in host ecology probably played a role in these differences in the pathogen population structure. Very high selfing rates were inferred in both fungal species, explaining the low levels of admixture between the genetic clusters. The systems studied here indicate that migration patterns caused by climate change can be expected to include pathogen invasions that follow the redistribution of their host species at continental scales, but also that the recolonization by pathogens is not simply a mirror of their hosts, even for obligate biotrophs, and that the ecology of hosts and pathogen mating systems likely affects recolonization patterns.     

Anopheles gambiae pathogen susceptibility: the intersection of genetics, immunity and ecology

Mosquitoes are the major arthropod vectors of human diseases such as malaria and viral encephalitis. However, each mosquito species does not transmit every pathogen, owing to reasons that include specific evolutionary histories, mosquito immune system structure, and ecology. Even a competent vector species for a pathogen displays a wide range of variation between individuals for pathogen susceptibility, and therefore efficiency of disease transmission. Understanding the molecular and genetic mechanisms that determine heterogeneities in transmission efficiency within a vector species could help elaborate new vector control strategies. This review discusses mechanisms of host-defense in Anopheles gambiae, and sources of genetic and ecological variation in the operation of these protective factors. Comparison is made between functional studies using Plasmodium or fungus, and we call attention to the limitations of generalizing gene phenotypes from experiments done in a single genetically simple colony.     

Evidence of interpopulation variation in the germination of Eryngium maritimum L. (Apiaceae)

Plant Ecology - Germination is considered as one of the most crucial steps in a plant’s lifetime. The germination of Eryngium maritimum, a dune species located across all European coasts, has...     

Experimentally induced marine flexibacteriosis in Atlantic salmon smolts Salmo salar. I. Pathogenicity

Tenacibaculum maritimum causes marine flexibacteriosis in many cultured fish species, including Atlantic salmon Salmo salar in Tasmania, Australia. Several aspects of the pathogenicity of this bacterium were investigated in naive Atlantic salmon smolts using different isolates, growth conditions and doses to produce a model of infection. We found that T. maritimum is pathogenic to Atlantic salmon using either marine Shieh's or marine Ordal's culture medium. The use of aeration in broth culture produced a dose effect in challenge due to a 'clumping' of the bacteria during culture. The virulence of a strain appears to be connected with this 'clumping'; the more adherent the cells, the more pathogenic the strain. Differences in virulence between 3 strains was apparent, with 1 of the strains (89/4747) being non-pathogenic and unable to produce disease in the host. The 2 other strains (89/4762, 00/3280) were highly virulent, resulting in 100% mortalities within 3 d. A reproducible model of infection has been established in the present study using strain 89/4762. Results from the present study provide a better insight into the nature of the disease.     

Linking community and disease ecology: the impact of biodiversity on pathogen transmission

The increasing number of zoonotic diseases spilling over from a range of wild animal species represents a particular concern for public health, especially in light of the current dramatic trend of biodiversity loss. To understand the ecology of these multi-host pathogens and their response to environmental degradation and species extinctions, it is necessary to develop a theoretical framework that takes into account realistic community assemblages. Here, we present a multi-host species epidemiological model that includes empirically determined patterns of diversity and composition derived from community ecology studies. We use this framework to study the interaction between wildlife diversity and directly transmitted pathogen dynamics. First, we demonstrate that variability in community composition does not affect significantly the intensity of pathogen transmission. We also show that the consequences of community diversity can differentially impact the prevalence of pathogens and the number of infectious individuals. Finally, we show that ecological interactions among host species have a weaker influence on pathogen circulation than inter-species transmission rates. We conclude that integration of a community perspective to study wildlife pathogens is crucial, especially in the context of understanding and predicting infectious disease emergence events.     

The interaction between plant competition and disease

It is well documented that pathogens can affect the survival, reproduction, and growth of individual plants. Drawing together insights from diverse studies in ecology and agriculture, we evaluate the evidence for pathogens affecting competitive interactions between plants of both the same and different species. Our objective is to explore the potential ecological and evolutionary consequences of such interactions. First, we address how disease interacts with intraspecific competition and present a simple graphical model suggesting that diverse outcomes should be expected. We conclude that the presence of pathogens may have either large or minimal effects on population dynamics depending on many factors including the density-dependent compensatory ability of healthy plants and spatial patterns of infection. Second, we consider how disease can alter competitive abilities of genotypes, and thus may affect the genetic composition of populations. These genetic processes feed back on population dynamics given trade-offs between disease resistance and other fitness components. Third, we examine how the effect of disease on interspecific plant interactions may have potentially far-reaching effects on community composition. A host-specific pathogen, for example, may alter a competitive hierarchy that exists between host and non-host species. Generalist pathogens can also induce indirect competitive interactions between host species. We conclude by highlighting lacunae in our current understanding and suggest that future studies should (1) examine a broader taxonomic range of pathogens since work to date has largely focused on fungal pathogens; (2) increase the use of field competition studies; (3) follow interactions for multiple generations; (4) characterize density-dependent processes; and (5) quantify pathogen, as well as plant, population and community dynamics.     

Lichen communities and the associated fauna on a rocky sea shore on Bornholm in the Baltic

Thirty-three sampling sites representing 11 lichen dominated plant communities on maritime rocks on Bornholm were analysed with respect to composition of the vegetation, number of animals present and gross taxonomic composition of the fauna. The specific fauna of one moss and seven lichen species was examined.
The lichen communities studied were largely the same as those found in similar studies in NW Europe, except for the recognition of a distinct Ramalina cuspidata community. The highest average number of animals per area was found in the Schistidium maritimum and the Ramalina siliquosa communities, whereas the highest average number of animals g⁻¹ dry plant biomass was recorded in the Parmelia saxatilis community. Among the species examined Anaptychia fusca contained the highest average number of animals g⁻¹ dry thallus and Ramalina cuspidata the lowest number, 114 and 7, respectively. Collemboles appeared to dominate in the moss Schistidium maritimum and in foliose lichens, whereas mites dominated in crustose and fruticose lichens.     

Disease hotspots or hot species? Infection dynamics in multi‐host metacommunities controlled by species identity,not source location

Pathogen persistence in host communities is influenced by processes operating at the individual host to landscape‐level scale, but isolating the relative contributions of these processes is challenging. We developed theory to partition the influence of host species, habitat patches and landscape connectivity on pathogen persistence within metacommunities of hosts and pathogens. We used this framework to quantify the contributions of host species composition and habitat patch identity on the persistence of an amphibian pathogen across the landscape. By sampling over 11 000 hosts of six amphibian species, we found that a single host species could maintain the pathogen in 91% of observed metacommunities. Moreover, this dominant maintenance species contributed, on average, twice as much to landscape‐level pathogen persistence compared to the most influential source patch in a metacommunity. Our analysis demonstrates substantial inequality in how species and patches contribute to pathogen persistence, with important implications for targeted disease management.     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

Inferential biases linked to unobservable states in complex occupancy models

Modeling of species distributions has undergone a shift from relying on equilibrium assumptions to recognizing transient system dynamics explicitly. This shift has necessitated more complex modeling techniques, but the performance of these dynamic models has not yet been assessed for systems where unobservable states exist. Our work is motivated by the impacts of the emerging infectious disease chytridiomycosis, a disease of amphibians that is associated with declines of many species worldwide. Using this host‐pathogen system as a general example, we first illustrate how misleading inferences can result from failing to incorporate pathogen dynamics into the modeling process, especially when the pathogen is difficult or impossible to survey in the absence of a host species. We found that traditional modeling techniques can underestimate the effect of a pathogen on host species occurrence and dynamics when the pathogen can only be detected in the host, and pathogen information is treated as a covariate. We propose a dynamic multistate modeling approach that is flexible enough to account for the detection structures that may be present in complex multistate systems, especially when the sampling design is limited by a species’ natural history or sampling technology. When multistate occupancy models are used and an unobservable state is present, parameter estimation can be influenced by model complexity, data sparseness, and the underlying dynamics of the system. We show that, even with large sample sizes, many models incorporating seasonal variation in vital rates may not generate reasonable estimates, indicating parameter redundancy. We found that certain types of missing data can greatly hinder inference, and we make study design recommendations to avoid these issues. Additionally, we advocate the use of time‐varying covariates to explain temporal trends in the data, and the development of sampling techniques that match the biology of the system to eliminate unobservable states when possible.     

Stimulated growth rate by restriction of P availability at moderate salinity but insensitive to P availability at high salinity in Crithmum maritimum

The halophyte Crithmum maritimum thrives in cracks of calcareous rocks or cliffs at seashores, a situation which associates limited phosphorus availability and high salinity. In order to understand the common patterns of colonization and zonation of this species, seedlings were cultivated for 34 d in inert sandy soil irrigated with a nutrient solution containing or not phosphorus at moderate (50 mM) or high (250 mM) NaCl level. Net assimilation rate and consequently relative growth rate increased in response to P deprivation at moderate saline level, but not at high salinity level. Parallelly, CO2 fixation rate, rubisco capacity, transpiration rate and stomatal conductance were diminished by P deprivation at moderate NaCl level. Intercellular CO2 concentration was therefore not affected. Chlororophyll fluorescence analysis revealed that photosynthetic systems were insensitive to change in P availability at moderate salinity level: neither pigment content, nor effective and maximum quantum yield, photochemical and non photochemical quenching, and electron transport rate were affected by P deprivation. On the contrary, at high salinity level when net photosynthesis, rubisco capacity and the quantum yields of PS2 were severely affected, P deprivation strongly augmented electron transport rate. Stomatal aperture and more modest increase in net photosynthesis, rubisco capacity, photosystem II effective quantum yield and photochemical quenching accompanied this response. This study shows the tolerance of C. maritimum to the phosphorus deprivation combined to moderate or to high saline level which may explain the common patterns of colonization and zonation of this species.     

An Emerging Mycoplasma Associated with Trichomoniasis,Vaginal Infection and Disease

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen.