Review

The Worm Turns: Trematodes Steering the Course of Co-infections

Veterinary Pathology 2014, Vol. 51(2) 385-392 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985813519655 vet.sagepub.com

L. Garza-Cuartero1, A. Garcia-Campos1, A. Zintl, A. Chryssafidis1, J. O’Sullivan1, M. Sekiya1, and G. Mulcahy1

Abstract A reductionist approach to the study of infection does not lend itself to an appraisal of the interactions that occur between 2 or more organisms that infect a host simultaneously. In reality, hosts are subject to multiple simultaneous influences from multiple pathogens along the spectrum from symbiotic microflora to virulent pathogen. In this review, we draw from our own work on Fasciola hepatica and that of others studying helminth co-infection to give examples of how such interactions can influence not only the outcome of infection but also its diagnosis and control. The new tools of systems biology, including both the ‘‘omics’’ approaches and mathematical biology, have significant promise in unraveling the as yet largely unexplored complexities of co-infection. Keywords co-infection, cattle, immunoregulation, helminth, trematodes

The advent of infection biology can be traced to Pasteur, Koch, and others who formulated the ‘‘germ’’ theory of disease. The idea of a pathogen, having entered into a host and causing a disease, took root, and ‘‘Koch’s postulates’’ were coined to determine when a particular organism could be held responsible for a particular disease state. With further development of understanding that the outcome of infection with a given organism does not always equate to disease and that pathogens are not always culturable, Koch’s postulates have been modified accordingly to fit 21st-century understanding.24 The major effect of the genetic background of the host is also recognized as an important factor in the outcome of any infectious disease process. An additional factor that must be considered is the potential intersecting influence of coinfecting organisms within the same host. Because of their wellrecognized effects as regulators of the host immune response, parasitic helminths have received significant attention with respect to their potential to affect the outcome of infection with other organisms present in the same host at the same time—that is, co-infections. In this review, we discuss the potential of helminths to influence the response to and outcome of co-infections, drawing on fundamental concepts of parasitism and symbiosis and providing examples from our own work and that of others on the influence of trematodes (blood and liver flukes) in this regard. Finally, we argue that a systems biology approach is beneficial in studying co-infections where a reductionist approach has, to date, provided an incomplete picture of the interactions between host and infecting organisms.

Parasitism and Symbiosis The interactions between infecting organisms and their hosts range from those in which there is mutual benefit (mutualism) to one in which one organism is exploited and harmed at the expense of the other (parasitism). Rather than understanding parasitism as the diametrical opposite of mutualism, today’s interpretation sees these 2 types of interspecific relationships as the extremes of an exploitation continuum.5 This continuum extends from optimal mutual benefit for both partners (symbiosis) at one end to maximum exploitation of one partner (the host) by the other (the parasite) at the opposite end.15 Numerous hypotheses have been proposed to predict the location of any given symbiotic/parasitic relationship on this continuum. The oldest of these is the ‘‘association time hypothesis,’’ which holds that parasites that have a long-standing evolutionary association with their host are less pathogenic than other less-well-adapted ‘‘upstarts’’ that can cause considerable disease to their more recently acquired hosts. The most frequently cited examples are the African trypanosomes, which are much better tolerated in the taurine cattle breeds (Bos 1 School of Veterinary Medicine and Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Veterinary Sciences Centre, Belfield, Dublin, Ireland

Corresponding Author: G. Mulcahy, School of Veterinary Medicine and Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Veterinary Sciences Centre, Belfield, Dublin 4, Ireland. Email: [email protected]

386 taurus), brought into Africa in 2 large waves around 7000 and 4600 years ago, than in the more recently introduced humpback zebu (Bos indicus), which did not become numerous in Africa until after the Arab invasion in 699 AD. Murray et al,36 however, 70 years ago, warned that while there is circumstantial evidence that certain long-standing host-parasite associations may have resulted in reduced pathogenicity, this phenomenon did not apply universally. Specifically, these authors cautioned against the assertion that relative harmlessness was prima facie evidence for long parasitism or that severe pathogenicity was equal evidence for its relative newness. Among the numerous examples provided are 2 of the oldest and yet most severe human parasitic diseases on record: leishmaniasis and schistosomiasis. The first is thought to have been present in the New World before Columbus and is pictured on Inca drinking vessels, while the latter is mentioned in papyri of ancient Egypt. Furthermore, Schistosoma egg stages were discovered in mummies dating from 1250 BC, testifying to a very longstanding association with their human host. Nevertheless, there cannot be any doubt that parasites and hosts coevolve, exerting mutual selective pressures. If it is assumed that high parasite density not only increases the probability of transmission but also reduces host survival, the trade-off between these factors should result in an intermediate level of virulence.3 However, while intuitively these assumptions seem justified, there is little practical evidence to support them. The rate of transmission is subject to a myriad of environmental, host, and parasite factors, and a direct link with parasitemia is difficult to establish. Similarly, the relationship between parasitemia and morbidity is often not linear. For instance, even relatively small numbers of tissue-migrating helminthic larvae can cause severe disease because much of the injury is caused by the host immune response rather than a direct result of larval activity.47 At the same time, even relatively large numbers of adult worms in the intestine are generally well tolerated. Host health and fitness relate to the degree to which that host resists or tolerates an infection. Raberg and colleagues40 argued that host resistance aimed at reducing or eliminating invaders would lead to an open-ended antagonistic coevolution between parasite and host. This ‘‘escalated arms race’’ would be driven by parasites developing ever-new ways to overcome host defenses, on one hand, and by hosts mounting evermore aggressive resistance mechanisms, on the other. As considerable resources are required for this type of response, it would only be of selective advantage for the host if parasites were rare but highly pathogenic.51 Conversely, common but relatively avirulent helminths would be expected to select for host tolerance. In contrast to resistance, this response to infection (resilience) is a cost-reduction strategy aimed at limiting any harm caused by the parasite while imposing little or no selective pressure on the invader.40 Furthermore, tolerance/resilience mechanisms differ from resistance in that they do not eliminate the selection pressures that favor them and, as a result, are likely to go to fixation. Intriguingly, these authors hypothesized that numerous tolerance mechanisms have become fixed in host populations and that that is why helminth infections are

Veterinary Pathology 51(2) generally quite well tolerated. Instead of producing sterile immunity, host immune responses often serve chiefly to limit helminth multiplication and to localize infections by ‘‘walling off’’ invaders.48 In part, this is no doubt due to ingenious immune evasion mechanisms that have been developed by parasitic helminths. However, direct immunosuppression is also likely to play an important role. This immunosuppression may be general (bystander effect) or specific to the helminth itself or, indeed, to particular life cycle stages (concomitant immunity). In some cases, it is due to a direct inhibition of immune effector mechanisms, while in others it involves stimulation host immune regulatory mechanisms that help to dampen inflammatory responses.57 The bystander effect of these sophisticated host-parasite interactions are intrinsically linked to helminth-mediated alteration of co-infections. We argue that alteration of the effect on the host of co-infections may be a significant factor in the coevolution of host and helminth—one that has received relatively little attention. By extension, the hygiene hypothesis proposes that alteration of the ecosystem of common helminth and other pathogens in modern industrialized societies results in loss of beneficial immunoregulatory effects, effectively providing a loophole through which deleterious responses to both infectious organisms and self-antigens can be generated.

Microbiota, Ecosystems, Health, and Disease In some cases, helminth-induced bystander immunoregulation may confer an advantage on the host in terms of limiting potentially pathogenic inflammatory and immune responses, including those directed at self-antigens. In others, it may increase the impact of infectious disease or interfere with diagnostic tests. Much interest has been focused of late on seeking applications for the potential of helminth infection to downregulate excessive and pathogenic inflammatory responses. The increased prevalence of autoimmune and inflammatory diseases in industrialized societies and in northern hemispheres may well be related to lower exposure of populations to helminths and other organisms along the mutualism-parasitism scale. The most high-profile studies of the potential clinical use of helminthmediated immunoregulation have been in the use of gutdwelling helminths to moderate inflammation in inflammatory bowel disease.57 However, there is now evidence of beneficial effects on autoimmune diseases beyond the gut, including multiple sclerosis.17 Efforts to produce defined pharmaceutical treatments using helminth-derived products have yet, however, to deliver clinical benefits. These well-recognized helminth-mediated effects, however, may just be the tip of the iceberg. If we accept that a certain level of helminth infection is a feature of the ‘‘evolutionary ecosystem’’ of higher organisms, the context in which these effects are manifested becomes clear. High-throughput sequencing techniques and bioinformatics analysis have enabled a step change in studying microbial ecosystems within higher organisms (reviewed in Robles Alonso and Guarner43). The influences of gut flora on health and disease is profound, with

Garza-Cuartero et al the diversity and balance of bacterial species present having an effect on phenomena as diverse as obesity, inflammatory bowel disease, and neurologic function. Helminths, friend or foe, depending on the circumstances, can in many respects be seen as just one component of the normal ecosystem of a host. The dramatic increase in prevalence of autoimmune and inflammatory conditions in developed human societies and the more subtle immunoregulatory effects seen in specific co-infection in both animals and humans can be viewed as disturbances in this larger ecosystem. This opens pathways to restoring health and increasing productivity through manipulation of the microbiota—for example, through use of probiotics or prebiotics—or maintaining helminth infections at levels below which they interfere with health and productivity but above that required for beneficial immunoregulation. Mechanistic explanations of how gastrointestinal helminths might influence microbial flora and downregulate inflammatory responses may involve IL-22, a cytokine of the IL-10 family, involved in the production of antimicrobial peptides and in repair functions in the gut, including maintaining integrity of the mucosal barrier.32

Effects of Helminths as Coinfecting Organisms There is a growing body of evidence describing the modulation of pathogenicity, disease course, and disease outcome in the presence of co-infection with helminths. A recent review54 provides a wide overview of the breadth of hosts and disease systems where this is relevant. At its most simplistic, the basic model hypothesis used to explain helminth co-infection outcomes proposes that helminth-induced stimulation of the type 2 / T-regulatory arm of the immune system results in bystander immunosuppression of type 1 / proinflammatory innate and adaptive responses (reviewed in Ezenwa and Jolles16 and Moreau and Chauvin35). This can be linked directly to the de novo induction of Foxp3expressing Treg cells by helminth-derived molecules.26 A schematic diagram showing the major mechanisms proposed as mediating helminth immunoregulation is shown in Figure 1. In spite of demonstrated evidence for the interaction of helminths and their products with host immune and inflammatory pathways, however, the result of helminth co-infection in terms of altered disease course, disease resistance, and other effects is highly variable, depending on the host species and coinfecting pathogens. Here we discuss examples arising from our own work and that of others, and we identify avenues for further research. The trematodes include some of the most socioeconomically devastating human parasites, as well as some important veterinary pathogens. Some of the best-studied immunoregulatory effects in human helminth infection involve the blood flukes or schistosomes, while the work of our laboratory has involved a related trematode, the liver fluke (Fasciola hepatica). Through contact with contaminated water, the blood flukes (Schistosoma spp) infect about 200 million people and 165 million of animals annually.53 The liver flukes (F. hepatica and F. gigantica) affect a range of mammals, including humans, with approximately 170 million of people at risk of infection. In the

387

Figure 1. Schematic showing the principal immunologic cell types typically activated following helminth infection, with the downstream effects that may affect the outcome of infection with other pathogens in helminth-infected hosts. Typical helminth infections affect the behavior of most cell types involved in orchestrating immune responses in specific ways. The outcome for the helminth itself is often chronic infection, with downregulation of inflammatory cytokines and modulation of chronic tissue damage. Innate lymphoid cells are recently recognized players in the initiation of the typical type 2 responses associated with helminth infection.

livestock industry worldwide, Fasciola spp are estimated to cause annual losses of about €2.5 billion (US$3.5 billion). It has been recognized for some time that trematodes can alter the host immune response to concurrent infection with other pathogens. This immune modulation is thought to be due to the stimulation of the anti-inflammatory cytokines IL-10 and TGF-b as well as the promotion of alternatively activated macrophages. IL-10 and TGF-b suppress Th1 and enhance T-regulatory activity, while alternatively activated macrophages have anti-inflammatory and tissue repair functions, with reduced antibacterial and antiprotozoal activity.25 A simplistic analysis of the effects of helminth co-infection would lead to the conclusion that the severity of disease induced by pathogens that elicit Th1 responses would be increased. However, in many cases, the host immune and inflammatory response is, in itself, a factor in the outcome of an infection process. Under these circumstances, helminth-induced immune suppression or regulation might even cause a reduction in pathogenicity. Finally, the site and timing of infection, as well as the compartmentalization of the immune response and/or immune suppression, may be of significance. The balance of these effects depends on the pathogens involved, with the background of the host and tissues affected.

Co-infection With Schistosomes and Other Trematodes There are numerous well-documented examples of a modulating effect in schistosome co-infection in laboratory animals.

388 Mice coinfected with Schistosoma mansoni and the ascarid nematode Toxocara canis had significantly lower mortality rates than animals infected with T. canis alone.31 Interestingly, levels of anti-Toxocara antibodies were also reduced in the coinfected group. In co-infections with Plasmodium berghei, schistosome infection prevented severe Plasmodium-associated brain lesions, although it did not protect from parasitemia and weight loss.1,6 This was linked to the release of T-regulatory molecules, including IL-10, which downregulated the highly polarized Th1 response and potentially lethal inflammatory response otherwise seen. In contrast, co-infection with schistosomes increased the severity of other protozoal diseases, including toxoplasmosis, trypanosomiasis, and leishmaniasis. In the case of Leishmania co-infection in a mouse model, Schistosoma-induced hepatic egg granulomas favored the growth of Leishmania in those niches, promoting the replication of amastigotes.28 The potentially significant effect of schistosomes on vaccination was demonstrated by Chen et al,7 who showed that mice with chronic Schistosoma japonicum infection had reduced responses to hepatitis B vaccine. This was manifested by reduced specific antibody responses, IFN-g production, and IL-2 production. The effects were reversible following deworming. Apart from its immunoregulatory effect, the cytokine IL-10 also plays an important role in the shift from Th1- to Th2dominated immune responses. This has been observed in cases of bacterial diseases where, for instance, Th1 cytokines (IFN-g) normally produced by mice infected with Mycobacterium avium were replaced by Th2 cytokines such as IL-4 in mice infected with both M. avium and S. mansoni. Moreover, granulomas in concurrently infected mice also included eosinophils, a characteristic that is never seen in granulomas from animals infected with M. avium only.45 In this case, S. mansoni was found to enhance susceptibility to tuberculosis and increase the bacterial burden, by stimulating the release of Th2 and T-regulatory molecules.22 Additionally, a suppressed Th1 response has been shown to be responsible for reduced efficacy of BCG vaccination against tuberculosis in mice with schistosomiasis. This was observed by the higher bacterial loads, more tissue damage in lungs, and lower Th1 cytokine– mediated protection in coinfected mice.22 An overview of experimental Schistosome co-infection in mice therefore indicates that immunoregulatory features can potentially downregulate inflammatory responses and Th1biased responses induced by coinfecting pathogens. This may in some circumstances modulate pathogenic effects, as in cerebral malaria, and it may in others enhance pathogenicity through reducing immune control of pathogen growth. The greatest effects tend to be seen in the immediate vicinity, or ‘‘immunological niches,’’ occupied by the helminths. Demonstration of the extent of the effect of schistosome co-infection in target species, rather than experimental animals, especially at an epidemiologic level, has proven to be less clear-cut. A positive association between the prevalence of Plasmodium infection and that of Ascaris lumbricoides, Trichura trichiura, and S. mansoni has been recorded in a population in southern Ethiopia.10 However, the cause-effect

Veterinary Pathology 51(2) relationship in this situation is not clear. The same constraint applies to many other reports on schistosome co-infection. A study involving a small population of children in Mali demonstrated an apparently enhancing effect on the production on memory B cells against Plasmodium falciparum when Schistosoma haematobium co-infection was present,33 thus indicating a potentially beneficial effect of co-infection between these 2 parasites in this particular population. A potential beneficial effect for populations in which schistosomes and other helminths are endemic can also be seen in reduction of allergic/atopic responses. Strong inverse correlation, for example, between intensity of S. haematobium infection and atopic responses has been demonstrated in a population in Zimbabwe.44 The inverse of this phenomenon—in other words, rampant allergic and autoimmune diseases in the developed world where helminth infections have been largely eliminated—is a key element of the hygiene hypothesis. In summary, in spite of extensive studies and improved mechanistic understanding of immune responses against schistosomes in mice and demonstration of immunoregulation in experimental co-infection, examples and quantification of effects in humans or domestic animals remain rare. What studies there are involve relatively small populations, and the true epidemiologic relevance of schistosomes to co-infection dynamics remains elusive. In our laboratory, we are attempting to unravel the nature, mechanisms, and epidemiologic importance of co-infection of a related trematode: the liver fluke, F. hepatica, in cattle. We believe that these studies contribute to the overall understanding of the ‘‘real-world’’ impact of helminth co-infection. Our model system provides us with ready access to infected populations, as fasciolosis is highly prevalent in cattle in Ireland and Great Britain and experimental infections are also straightforward. We have concentrated to date on studying the interaction between F. hepatica infection and mycobacterial infections in cattle. This presents the opportunity to study co-infection in a context where both coinfecting pathogens are of high importance to animal health and to the agri-food economy, considering that both bovine tuberculosis and paratuberculosis remain problematic disease issues. There is abundant evidence that F. hepatica follows the typical pattern of helminth infection in causing a switch from a Th1- to a Th2-driven immune response in cattle.9 In the first approach to studying co-infection in our laboratory, cattle were immunized with M. bovis BCG and subsequently tested for mycobacterial immune responses via 2 tests used in diagnosis of bovine tuberculosis: the single comparative intradermal tuberculin test and the IFN-g assay.19 Experimental co-infection with F. hepatica modulated the inflammatory response to the intradermal tuberculin test, thereby pushing weak positives into a negative diagnosis. Fewer than half the animals reacted to the test when they were inoculated with M. bovis BCG first and subsequently infected with F. hepatica. Even more significant, no animals infected with F. hepatica first, followed by BCG inoculation second, showed a positive reaction to the tests.

Garza-Cuartero et al This experiment was followed with a similar study but this time using a low-dose respiratory challenge with virulent M. bovis. A reduction in INF-g production and an increase in TGF-b1 and IL-10 in vitro after restimulation of peripheral blood mononuclear cells from coinfected animals were observed.21 As with the previous study, a significant decrease in responsiveness in the single intradermal comparative cervical tuberculin was also observed in the coinfected group.8 The potential significance of the effect of F. hepatica infection on the diagnosis of bovine tuberculosis infection on a large scale was also examined.8 In this work, dairy herds in England and Wales served as a target population. A significant negative correlation was found between F. hepatica infection and bovine tuberculosis reactor status. One of the implications of this study is that in areas where F. hepatica is endemic, the sensitivity of the single comparative intradermal tuberculin test may be reduced by a margin sufficient to impede the eradication campaign for bovine tuberculosis. Simple measures could resolve this issue, such as treating herds with a flukicide in advance of testing. Further work on the effect of F. hepatica infection on the course of mycobacterial disease remains to be done. By extension, the influence of F. hepatica on related diseases in cattle, such as paratuberculosis, also warrant examination. In terms of mechanistic explanations for the effects seen in coinfected hosts, increased production of TGF-b during the early stages of F. hepatica infection and IL-10 during the chronic phase of infection has been recorded.20 However, the cellular sources of these remain to be identified. Some of the unanswered questions include an assessment of F. hepatica co-infection on the course of infection and on mycobacterial burdens and lesion distribution. We are currently attempting to address these questions through in vitro studies of granuloma formation and by gene expression studies at the individual cell population level. A summary of the known immunoregulatory effects, and the mechanisms involved where known, is presented in Table 1. These studies show that by acting as immune modulators, trematodes not only have the potential to affect the severity of concurrent infections but can also alter the efficacy of certain diagnostic tests. In addition, there are indications that responses to BCG vaccination are influenced by infection with Schistosoma spp and Fasciola spp infections. Considering that infections with these and other trematodes are endemic in many parts of the world, both in the human population and in livestock, this poses serious questions in relation to disease management and control programs.

Helminth Co-infection: Practical Implications in Human and Veterinary Medicine While significant gaps remain in our understanding of co-infection, which a systems biology approach may help to fill, emerging evidence about co-infection phenomena on a population scale points in some cases to the possibility of new interventions to aid in disease control. Claridge et al,8 for example, have shown that F. hepatica infection in dairy herds in England and Wales can decrease the sensitivity of the skin test for the diagnosis of bovine

389 Table 1. Known Immunologic Parameters Altered by Fasciola hepatica Infection.a Effect

Host System

Studies in ruminants Epidemiological evidence and Downregulation of host experimental infection in cattle8 responses to skin testing for bovine tuberculosis Reduction in IFN-g production Experimental infection, cattle21 in cattle co-infected with Mycobacterium bovis Experimental infection, cattle19 Alteration of macrophage function, reduced IFN-g production in animals immunized with M. bovis BCG Studies in experimental animal models Suppression of Th1 responses, Experimental infection, mice4 exacerbation of co-infection with Bordetella pertussis No effect on co-infection with Experimental infection, mice34 Toxoplasma gondii (but T. gondii did reduce Th2 responses to F. hepatica) Action of F. hepatica derived Impairment of macrophage helminth-defense molecules in antigen processing and mouse model41 presentation Action of F. hepatica derived Reduced release of helminth-defense molecules in inflammatory mediators mouse model42 from macrophages Action of F. hepatica derived Enhancement of Th2 peroxiredoxin in mouse responses and alternative model11 activation of macrophages Suppression of dendritic cell Action of F. hepatica derived maturation and function tegumental antigen in mouse model27 Suppression of cytokine Action of F. hepatica derived secretion by mast cells tegumental antigen in mouse model55 Attenuation of autoimmunity Experimental infection in a mouse model56 and suppression of Th17/ Th1 responses a Parameters that interact with the host response to bacterial infections and/or to stimulation with bacterial antigens in natural and experimental situations, in vivo and in vitro.

tuberculosis sufficiently to reduce the number of herds where reactors are detected. Routine flukicidal treatment in advance of skin testing, therefore, is a strategy worth examining as a simple cost-effective method of improving the sensitivity of this test. A secondary test for diagnosis of bovine tuberculosis, the g-INF test, which relies measuring the release of this cytokine following restimulation of peripheral blood mononuclear cells in vitro with M. bovis purified protein derivative,19 is also affected by F. hepatica infection. We have shown, however, that this change is accompanied by a shift to the production of IL-4 and IL-10 in response to purified protein derivative stimulation in coinfected cattle, pointing the way toward the development of alternative or multiplex ELISA cytokine assays that may overcome challenges of diagnosis in coinfected animals.

390 The potential benefits of eliminating or controlling helminth infections to minimize the direct effect on other infectious diseases are less clear. Increased susceptibility to tuberculosis13,39 and Bordetella pertussis4 has been shown in mouse models. However, other authors have demonstrated no effect of a preexisting helminth infection on mycobacterial infection in mice,23 and there is some evidence that the acute phase of a helminth infection can be associated with slower mycobacterial growth.12 These conflicting results in mouse models reflect the gaps of our mechanistic knowledge of helminth co-infection and highlight again the need for a systems, rather than reductionist, approach to understanding the myriad of interactions involved.

Systems Biology as an Approach to Studying Co-infection Since Descartes, scientific approaches to biology had been chiefly reductionist, studying the sciences from the smallest elements. This approach began to broaden in the middle of the 19th century when the scientific community required an understanding of not only each building block separately but also the relation and mutual effects among them. This requirement with the acquisition of a large body of new data from different organisms triggered the origin of an idea now known as systems biology. Claude Bernand is viewed as the first ‘‘systems biologist,’’37 and eminent researchers such as William Harvey and Gregor Mendel followed in his footsteps. Thanks to the emergence of new ‘‘omics’’ technologies in the last decades, the potential of increasing biological knowledge through a systems approach has been increased exponentially. For example, as far as proteomics are concerned, very few publications existed on parasites in 2002, whereas in 2011 alone more than 70 articles were published. In the beginning, much of the parasite proteomics research focused on important protozoan parasites of humans, such as P. falciparum18,30 Cryptosporidium parvum,46 and Toxoplasma gondii.58 The next necessary step was to find ways to integrate the acquired data across all the different organisms and scales with the purpose of understanding complex interactions among biological entities as well as functional and large-scale organizations and their underlying pathways.38 Systems biology, with its twin discipline of computational biology, offers a way of tackling this issue. The domain of systems biology relies on the integration of ‘‘wet’’ approaches—such as proteomics, glycomics, genomics, transcriptomics, and clinical studies—with ‘‘dry’’ approaches, which involve bioinformatics, mathematics, and computer modeling. In other words, data generation, interpretation, hypothesis formation, and validation must be considered together. Systems biology can be carried out from different biological starting points: bottom-up, top-down, middle-out, and the landscape concept.29 Of these, the landscape concept is the most complex approach. It applies mathematical theoretical modeling to interpret multidimensional interactions among different parameters in a biological system and to predict the changes in one parameter caused by manipulation of another.

Veterinary Pathology 51(2) It is a useful tool for formulating hypotheses and for determining the feasibility and practical starting point of an experiment. In spite of the complexity and the set of open problems that the landscape concept may cover,2 it is most commonly used metaphorically and quantitatively in biological research to understand different biological processes, such as the cell cycle or the combined activity of several drugs and their pathways within the same host and/or against the same pathogen or disease. A useful application of systems biology would be to compare the effects of a single pathogen on a host with the consequences of multiple concurrent infections, based on both static and temporal data. Specifically in respect of helminths that may have complex life cycles—sometimes involving multiple hosts and long-lived environmental transmission stages—life cycle–specific gene expression and translational profiles can be included in the model to characterize host-parasite interactions at each stage of the life cycle. A number of computational approaches to the study of infectious diseases that allow for the interactions among coinfecting pathogens as well as between host and pathogen have already been developed.49,52 Systems biology approaches have been proposed for studying the dynamic interactions of complex pathogens (eg, fungi) with the host as well as for pinpointing evolutionary transitions between commensalism and pathogenicity.50 Game theory, not normally thought of as a technique to be employed by infection biologists, has been beneficially used to analyze persistent/ chronic bacterial infections.14 It seems to us that approaches to studying co-infection dynamics will require such approaches to understand the complexity involved.

Conclusions The study of infection biology has moved from centering on the pathogen as a cause of disease to one in which the outcome of infection is understood to involve the interplay between pathogen and host response, with the latter contributing not only to control of infection but also in many cases to the disease itself. We argue that a paradigm change should now take place in which the entire host ecosystem—including resident microbiota and incidental infections—is taken into account in the study of infectious disease. Helminth infections, because of their welldescribed effects in orchestrating host immunoregulation and chronic infections, have provided examples of the potential of co-infections to alter the diagnosis, course, and outcome of disease processes. Data from experimental studies and epidemiologic investigations of trematode infections, as highlighted in this article, have shown that these effects should be considered as factors relevant to disease control programs. In an era where systems biology provides tools for studying the interaction of multiple pathways and complex interactions, the potential of such studies to influence a wide area of biomedicine is clear. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Garza-Cuartero et al Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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