Microbial epidemiology and carriage studies for the evaluation of vaccines.

Respiratory tract infections are responsible for over 2.8 million deaths per year worldwide. Colonization is the first step in the process of microbes occupying the respiratory tract, which may lead to subsequent infection. Carriage, in contrast, is defined as the occupation of microbial species in the respiratory tract. The duration of carriage may be affected by host immunity, the composition and interactions between members of the microbial community, and the characteristics of colonizing bacteria, including physiology associated with being present in a bacterial biofilm. Numerous vaccines have been implemented to control infections caused by bacteria that can colonize and be subsequently carried. Such vaccines are often species-specific and may target a limited number of strains thereby creating a vacant niche in the upper respiratory tract. Epidemiological changes of bacteria found in both carriage and disease have therefore been widely reported, since the vacant niche is filled by other strains or species. In this review, we discuss the use of carriage-prevalence studies in vaccine evaluation and argue that such studies are essential for (1) examining the epidemiology of carriage before and after the introduction of new vaccines, (2) understanding the dynamics of the respiratory tract flora and (3) identifying the disease potential of emerging strains. In an era of increasing antibiotic resistance, bacterial carriage-prevalence studies are essential for monitoring the impact of vaccination programmes.

Ecology and diversity in upper respiratory tract microbial population structures from a cross-sectional community swabbing study.

PURPOSE: Respiratory tract infections (RTIs) are responsible for over 2.8 million deaths per year worldwide with pathobiont carriage a required precursor to infection. We sought to determine carriage epidemiology for both bacterial and viral respiratory pathogens as part of a large population-based cross-sectional carriage study. METHODOLOGY: Nose self-swab samples were collected in two separate time-points, May to August 2012 (late spring/summer) and February to April 2013 (winter/early spring). The presence of six bacterial species: S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, P. aeruginosa and N. meningitidis in addition to respiratory syncytial virus, influenza viruses A and B, rhinovirus/enterovirus, coronavirus, parainfluenza viruses 1-3 and adenovirus was determined using culture and PCR methods.Results/Key findings. Carriage was shown to vary with age, recent RTI and the presence of other species. Spatial structures of microbial communities were more disordered in the 0-4 age group and those with recent RTI. Species frequency distributions were flatter than random expectation in young individuals (X2=20.42, P=0.002), indicating spatial clumping of species consistent with facilitative relationships. Deviations from a neutral model of ecological niches were observed in summer samples and from older individuals but not in the winter or younger individuals (0-4 years), suggesting the presence of seasonal and age-dependent niche processes in respiratory community assembly. CONCLUSION: The application of epidemiological methods and ecological theory to respiratory tract samples has yielded novel insights into the factors that drive microbial community composition.

Pneumococcal conjugate vaccine implementation in middle-income countries.

BACKGROUND: Since 2000, the widespread adoption of pneumococcal conjugate vaccines (PCVs) has had a major impact in the prevention of pneumonia. Limited access to international financial support means some middle-income countries (MICs) are trailing in the widespread use of PCVs. We review the status of PCV implementation, and discuss any needs and gaps related to low levels of PCV implementation in MICs, with analysis of possible solutions to strengthen the PCV implementation process in MICs. MAIN BODY: We searched PubMed, PubMed Central, Ovid MEDLINE, and SCOPUS databases using search terms related to pneumococcal immunization, governmental health policy or programmes, and MICs. Two authors independently reviewed the full text of the references, which were assessed for eligibility using pre-defined inclusion and exclusion criteria. The search terms identified 1,165 articles and the full texts of 21 were assessed for suitability, with eight articles included in the systematic review. MICs are implementing PCVs at a slower rate than donor-funded low-income countries and wealthier developed countries. A significant difference in the uptake of PCV in lower middle-income countries (LMICs) (71%) and upper middle-income countries (UMICs) (48%) is largely due to an unsuccessful process of "graduation" of MICs from GAVI assistance, an issue that arises as countries cross the income eligibility threshold and are no longer eligible to receive the same levels of financial assistance. A lack of country-specific data on disease burden, a lack of local expertise in economic evaluation, and the cost of PCV were identified as the leading causes of the slow uptake of PCVs in MICs. Potential solutions mentioned in the reviewed papers include the use of vaccine cost-effectiveness analysis and the provision of economic evidence to strengthen decision-making, the evaluation of the burden of disease, and post-introduction surveillance to monitor vaccine impact. CONCLUSION: The global community needs to recognise the impediments to vaccine introduction into MICs. Improving PCV access could help decrease the incidence of pneumonia and reduce the selection pressure for pneumococcal antimicrobial resistance.

Can Simpson's paradox explain co-operation in Pseudomonas aeruginosa biofilms?

Co-operative behaviours, such as the production of public goods, are commonly displayed by bacteria in biofilms and can enhance their ability to survive in environmental or clinical settings. Non-co-operative cheats commonly arise and should, theoretically, disrupt co-operative behaviour. Its stability therefore requires explanation, but no mechanisms to suppress cheating within biofilms have yet been demonstrated experimentally. Theoretically, repeated aggregation into groups, interleaved with dispersal and remixing, can increase co-operation via a 'Simpson's paradox'. That is, an increase in the global proportion of co-operators despite a decrease in within-group proportions, via differential growth of groups. We investigate the hypothesis that microcolony formation and dispersal produces a Simpson's paradox that explains bacterial co-operation in biofilms. Using the production of siderophores in Pseudomonas aeruginosa as our model system for co-operation, we use well-documented co-operator and siderophore-deficient cheat strains to measure the frequency of co-operating and cheating individuals, in-situ within-microcolony structures. We detected significant within-type negative density-dependant effects that vary over microcolony development. However, we find no evidence of Simpson's paradox. Instead, we see clear within-microcolony spatial structure (cheats occupying the interior portions of microcolonies) that may violate the assumption required for Simpson's paradox that group members share equally in the public good.

Lack of phenotypic and evolutionary cross-resistance against parasitoids and pathogens in Drosophila melanogaster.

BACKGROUND: When organisms are attacked by multiple natural enemies, the evolution of a resistance mechanism to one natural enemy will be influenced by the degree of cross-resistance to another natural enemy. Cross-resistance can be positive, when a resistance mechanism against one natural enemy also offers resistance to another; or negative, in the form of a trade-off, when an increase in resistance against one natural enemy results in a decrease in resistance against another. Using Drosophila melanogaster, an important model system for the evolution of invertebrate immunity, we test for the existence of cross-resistance against parasites and pathogens, at both a phenotypic and evolutionary level. METHODS: We used a field strain of D. melanogaster to test whether surviving parasitism by the parasitoid Asobara tabida has an effect on the resistance against Beauveria bassiana, an entomopathogenic fungus; and whether infection with the microsporidian Tubulinosema kingi has an effect on the resistance against A. tabida. We used lines selected for increased resistance to A. tabida to test whether increased parasitoid resistance has an effect on resistance against B. bassiana and T. kingi. We used lines selected for increased tolerance against B. bassiana to test whether increased fungal resistance has an effect on resistance against A. tabida. RESULTS/CONCLUSIONS: We found no positive cross-resistance or trade-offs in the resistance to parasites and pathogens. This is an important finding, given the use of D. melanogaster as a model system for the evolution of invertebrate immunity. The lack of any cross-resistance to parasites and pathogens, at both the phenotypic and the evolutionary level, suggests that evolution of resistance against one class of natural enemies is largely independent of evolution of resistance against the other.

Risk of red queen dynamics in pneumococcal vaccine strategy.

Pathogens increasingly evade current vaccines, and new strategies to control them are needed. There is mounting evidence that replacement of vaccine serotypes of Streptococcus pneumoniae with non-vaccine serotypes has taken place following widespread use of limited-serotype conjugate vaccines. New strategies to control vaccine evasion are needed and understanding evolutionary theory is important for the development of such approaches. Hosts are under selection pressure to evolve resistance against pathogens whereas pathogens are under selection pressure to evolve counter-resistance against the resistance mechanism of their host. Evolutionary changes in both host and pathogen lead to a continuous turnover of host and pathogen genotypes; this is known as Red Queen dynamics. We argue that integrating evolutionary thinking into pneumococcal vaccine design will lead to the avoidance of Red Queen dynamics and improved interventions against pneumococci.

L-arginine enhances immunity to parasitoids in Drosophila melanogaster and increases NO production in lamellocytes.

Drosophila melanogaster was used as a model system to explore the link between nutrition and immunity, and to investigate the role of nitric oxide (NO) in enhancing immunity following dietary enhancement with L-arginine. First, we show that adding L-arginine to the food medium increases the ability of D. melanogaster larvae to encapsulate the eggs of the parasitoid Asobara tabida. Secondly, we show that the increase in immunity is specific to L-arginine, and not to an enhanced calorific content, and that immunity decreases when larvae are fed food with added L-NAME, an inhibitor of nitric oxide synthase. Finally, we show that parasitised larvae fed L-arginine have increased haemocyte numbers, and that the lamellocytes (haemocytes which play a key role in encapsulation) show evidence of an increased production of NO. These results suggest that NO plays a key role in immunity and that the effect of NO is mostly targeted via the lamellocytes.

Functional genomics of the evolution of increased resistance to parasitism in Drosophila.

Individual hosts normally respond to parasite attack by launching an acute immune response (a phenotypic plastic response), while host populations can respond in the longer term by evolving higher level of defence against parasites. Little is known about the genetics of the evolved response: the identity and number of genes involved and whether it involves a pre-activation of the regulatory systems governing the plastic response. We explored these questions by surveying transcriptional changes in a Drosophila melanogaster strain artificially selected for resistance against the hymenopteran endoparasitoid Asobara tabida. Using micro-arrays, we profiled gene expression at seven time points during development (from the egg to the second instar larva) and found a large number of genes (almost 900) with altered expression levels. Bioinformatic analysis showed that some were involved in immunity or defence-associated functions but many were not. Previously, we had defined a set of genes whose level of expression changed after parasitoid attack and a comparison with the present set showed a significant though comparatively small overlap. This suggests that the evolutionary response to parasitism is not a simple pre-activation of the plastic, acute response. We also found overlap in the genes involved in the evolutionary response to parasitism and to other biotic and abiotic stressors, perhaps suggesting a 'module' of genes involved in a generalized stress response as has been found in other organisms.

Evolution of host resistance and parasitoid counter-resistance.

By their nature, parasitoids will exert a selection pressure on their hosts to evolve a mechanism through which to resist parasitoid attack. In turn, such a resistance mechanism will lead to parasitoids evolving counter-resistance. In this chapter, we present an overview of the research on the (co)evolutionary interaction between Drosophila and their parasitoids, with the main focus on the cellular immune response of D. melanogaster, and the counter-resistance mechanism of one of its main parasitoids, Asobara tabida. A key aspect of this interaction is the existence of genetic variation: in the field, host resistance and parasitoid counter-resistance vary, both between and within populations. Host resistance and parasitoid counter-resistance are costly, and both these costs turn out to be density dependent. These tradeoffs can explain the existence of genetic variation. We briefly touch upon behavioral aspects of the interaction and the parasites and pathogens that the parasitoids themselves suffer from. We end this chapter by considering the data coming from gene chip experiments: early indications suggest that the genes involved in the actual immune response against parasitoids are mostly different from the genes involved in the evolution of resistance.

Experimental evolution shows Drosophila melanogaster resistance to a microsporidian pathogen has fitness costs.

Most organisms experience strong selection to develop mechanisms to resist or tolerate their pathogens or parasites. Limits to adaptation are set by correlated responses to selection, for example reduced abilities to detect other parasites or trade-offs with other fitness components. For a few model systems it is now becoming possible to compare the evolutionary response to a broad range of natural enemies. In Drosophila, the evolutionary responses to ectoparasitic mites, parasitoids, and fungal and bacterial pathogens have previously been studied. Here replicate lines of D. melanogaster were exposed to the microsporidian parasite Tubulinosema kingi over a period of 61 weeks, with overlapping generations. Compared to controls, exposed lines had higher early-life fecundity and increased longevity when infected suggesting successful selection for resistance or tolerance. In the absence of the pathogen, exposed lines had lower fecundity when reared under harsh environmental conditions, and were poorer larval competitors than controls. They also had relatively higher densities of haemocytes, a component of the cellular immune system. Defense against this pathogen resembles more that against macroparasites than microsparasites, and this is interpreted in the light of what is known about the mechanisms of resistance to microsporidians.

Infection of Drosophila melanogaster by Tubulinosema kingi: stage-specific susceptibility and within-host proliferation.

Despite its importance as a model organism very little is known about the interaction between Drosophila and its microsporidian pathogens. Here we report on the relative susceptibility of Drosophila melanogaster life history stages to infection by Tubulinosema kingi, and on patterns of pathogen proliferation. We find that only larvae can be infected, and that this susceptibility decreases with larval age. Following infection, the pathogen shows little subsequent proliferation in larvae, a limited amount in pupae while it replicates greatly in adults. We present evidence that the host launches a cellular immune response after infection with the pathogen, although its effectiveness remains to be demonstrated.

Wolbachia infection suppresses both host defence and parasitoid counter-defence.

Endosymbiotic bacteria in the genus Wolbachia have been linked to several types of reproductive parasitism, which enhance their own transmission, while their direct effects on the host vary from beneficial to neutral or detrimental. Here, we report negative effects of infection on immunity-related traits of Drosophila simulans and the parasitoid wasp Leptopilina heterotoma. Infected D. simulans showed a reduced ability to encapsulate parasitoid eggs, compared to a tetracycline-treated, bacterium-free line. Challenging the two lines with a fungal pathogen, Beauveria bassiana, on the other hand, revealed no differences in survival. Moreover, elimination of Wolbachia was beneficial for the parasitoid wasp, as eggs laid by uninfected females suffered significantly lower encapsulation rates. We discuss possible origins of these fitness costs and their implications for infection dynamics and the interactions between host species.

An ultrastructural and molecular study of Tubulinosema kingi Kramer (Microsporidia: Tubulinosematidae) from Drosophila melanogaster (Diptera: Drosophilidae) and its parasitoid Asobara tabida (Hymenoptera: Braconidae).

Tubulinosema kingi is a pathogen of Drosophila spp. that was originally described 40 years ago. Although Drosophila melanogaster is widely used as a model organism for biological research, only limited data about microsporidia infecting Drosophila have been published so far and very little is known about the ultrastructure of T. kingi. In this study, we present the results of ultrastructural and molecular examinations of T. kingi. The whole life cycle took place in direct contact with the host cell cytoplasm and all examined life cycle stages contained a diplokaryon. Very few membrane elements were present in early merogonial stages, but their number and order of arrangement increased as the life cycle proceeded. The cell membrane of meronts had a surface coat of tubular elements that encircled the cell. Later, numerous electron-dense strands without any ornamentation accumulated on the plasma membrane, indicating that cells had entered sporogony. The cell membrane of sporonts was covered by electron-dense material. The polar filament in the spores was slightly anisofilar with the last three or four coils being smaller in diameter. The polar filament has 10 to 14 coils which were arranged predominantly in a single row, but in many spores, one winding of the coiled polar filament was located inside the outer coils. In some spores, the polar filament was irregularly arranged in two or even three rows. Molecular analysis showed that all Tubulinosema spp. are closely related and form a clade of their own that is distinct from the Nosema/Vairimorpha clade. All these ultrastructural and molecular features are in concordance with the family Tubulinosematidae and the genus Tubulinosema which reinforces the recent reclassification of this microsporidium.

Genome-wide gene expression in response to parasitoid attack in Drosophila.

BACKGROUND: Parasitoids are insect parasites whose larvae develop in the bodies of other insects. The main immune defense against parasitoids is encapsulation of the foreign body by blood cells, which subsequently often melanize. The capsule sequesters and kills the parasite. The molecular processes involved are still poorly understood, especially compared with insect humoral immunity. RESULTS: We explored the transcriptional response to parasitoid attack in Drosophila larvae at nine time points following parasitism, hybridizing five biologic replicates per time point to whole-genome microarrays for both parasitized and control larvae. We found significantly different expression profiles for 159 probe sets (representing genes), and we classified them into 16 clusters based on patterns of co-expression. A series of functional annotations were nonrandomly associated with different clusters, including several involving immunity and related functions. We also identified nonrandom associations of transcription factor binding sites for three main regulators of innate immune responses (GATA/srp-like, NF-kappaB/Rel-like and Stat), as well as a novel putative binding site for an unknown transcription factor. The appearance or absence of candidate genes previously associated with insect immunity in our differentially expressed gene set was surveyed. CONCLUSION: Most genes that exhibited altered expression following parasitoid attack differed from those induced during antimicrobial immune responses, and had not previously been associated with defense. Applying bioinformatic techniques contributed toward a description of the encapsulation response as an integrated system, identifying putative regulators of co-expressed and functionally related genes. Genome-wide studies such as ours are a powerful first approach to investigating novel genes involved in invertebrate immunity.

Evolutionary change in parasitoid resistance under crowded conditions in Drosophila melanogaster.

Patterns of investment of limiting resources in such processes as competing for food and defense against natural enemies are shaped by trade-offs and constraints. In Drosophila melanogaster artificial selection for increased resistance to parasitoids results in a correlated decrease in larval competitive ability. Here we ask whether selection for competitive ability leads to a correlated reduction in parasitoid resistance. Replicated lines of D. melanogaster were maintained under crowded or uncrowded conditions for eight generations. As expected, the crowded lines evolved higher competitive ability (when tested against a common strain of fly). But instead of parasitoid resistance decreasing, we found a significant increase, and that this was associated with elevated densities of haemocytes in second-instar larvae. To understand these results we measured a variety of life-history traits in the two sets of lines. We find evidence that directly and indirectly selected changes in competitive ability are due to different mechanisms. We also ask why crowded conditions should select for increased resistance to parasitism, and conclude that it is unlikely to be due to correlated selection for resistance to other natural enemies, but might be due to correlated selection for better wound responses.

Ecological and evolutionary implications of immunological priming in invertebrates.

Invertebrates have an immune response that differs considerably from the acquired immune response found in vertebrates. However, new studies indicate that past experience with a pathogen can provide individual invertebrates, or their descendants, with enhanced immunity. This prophylactic effect, termed immunological priming, is functionally similar to the acquired immune response in vertebrates. This newfound complexity of invertebrate immunity begs investigation into the conditions under which immunological priming should evolve, and its consequences for population dynamics.

Selection for parasitoid resistance alters mating success in Drosophila.

Parasite-mediated sexual selection is still a widely discussed hypothesis for the understanding of the evolution of secondary sex traits. Furthermore, it has sparked new fields such as ecological immunology. Despite this, most tests have been restricted to the manipulation of parasite loads. Here, we provide a new experimental approach, where resistance itself was manipulated. Parasitoid-resistant Drosophila melanogaster males achieved a higher mating success compared with non-resistant conspecifics. The underlying mechanism however remains elusive.