Potential Facilitation Between a Commensal and a Pathogenic Microbe in a Wildlife Disease

We assessed the potential for microbial interactions influencing a well-documented host–pathogen system. Mycoplasma agassizii is the known etiological agent of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii), but disease in wild animals is extremely heterogeneous. For example, a much larger proportion of animals harbor M. agassizii than those that develop disease. With the availability of a new quantitative PCR assay for a microbe that had previously been implicated in disease, Pasteurella testudinis, we tested 389 previously collected samples of nasal microbes from tortoise populations across the Mojave desert. We showed that P. testudinis is a common commensal microbe. However, we did find that its presence was associated with higher levels of M. agassizii among the tortoises positive for this pathogen. The best predictor of P. testudinis prevalence in tortoise populations was average size of tortoises, suggesting that older populations have higher levels of P. testudinis. The prevalence of co-infection in populations was associated with the prevalence of URTD, providing additional evidence for an indirect interaction between the two microbes and inflammatory disease. We showed that URTD, like many chronic, polymicrobial diseases involving mucosal surfaces, shows patterns of a polymicrobial etiology.

     

Vector preference for hosts differing in infection status: sharpshooter movement and Xylella fastidiosa transmission

1. Epidemiological theory predicts that vector preference for hosts differing in infection status (i.e. healthy or infected) affects disease dynamics. 2. Numerous studies have documented strong vector preference for or discrimination against infected hosts. However, the significance of these behaviours for pathogen transmission and spread has been poorly described. 3. We conducted a series of choice assays to evaluate orientation preference, feeding preference, and movement rates of an important group of vectors, the sharpshooter leafhoppers, based on host infection status for the generalist plant pathogen, Xylella fastidiosa Wells et al. 4. Sharpshooters did not discriminate between healthy versus infected‐but‐asymptomatic grapevines, but they oriented preferentially to healthy grapevines more frequently than either symptomatic vines or those artificially coloured to mimic disease symptoms. 5. In a field trial three sharpshooter species showed different movement rates and preferences for feeding site, but all species exhibited similar and significant preference for healthy hosts. 6. Although there was no significant difference in acquisition efficiency among vector species, those individuals that spent more time on healthy hosts tended to be less likely to acquire the pathogen. 7. These results suggest that sharpshooters discriminate against infected grapevines, which are likely to be of poorer quality, with visual cues playing a role in host selection. Preference by these vectors may affect pathogen acquisition, which could affect disease spread in the field.     

Mycoplasmal Upper Respiratory Tract Disease Across the Range of the Threatened Mojave Desert Tortoise: Associations with Thermal Regime and Natural Antibodies

Most research of upper respiratory tract disease (mycoplasmal URTD) in the threatened Mojave Desert tortoise (Gopherus agassizii) has worked under the hypothesis that the pathogen, Mycoplasma agassizii, has a relatively consistent and predictable effect on tortoise populations across their natural range. In contrast, we hypothesized that multiple factors influence the prevalence of disease and analyzed biological and environmental variables that vary significantly across the Mojave Desert. We used multiple regression models to analyze associations between mycoplasmal URTD and the genetic structure of 24 tortoise populations, levels of natural antibody (NAb) to M. agassizii in tortoises (one component of the innate immune system), precipitation, and colder thermal regimes. We detected a significant, positive association between mean levels of NAb and seroprevalence to M. agassizii. We hypothesized that NAbs may provide tolerance to mycoplasmal infections and that more tolerant populations may act as host reservoirs of disease. We also detected significant associations between colder winters and mycoplasmal URTD, suggesting that colder winters may depress tortoise immune resistance against M. agassizii or enhance conditions for the growth of M. agassizii.     

Effects of mammal host diversity and density on the infection level of Trypanosoma cruzi in sylvatic kissing bugs

Several reports have described host species diversity and identity as the most important factors influencing disease risk, producing either dilution or amplification of the pathogen in a host community. Triatomine vectors, mammals and the protozoan Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) Chagas are involved in the wild cycle of Chagas disease, in which infection of mammals occurs by contamination of mucous membranes or skin abrasions with insect‐infected faeces. We examined the extent to which host diversity and identity determine the infection level observed in vector populations (i.e. disease risk in humans). We recorded infection in triatomine colonies and on the coexisting host mammalian species in semi‐arid Chile. Host diversity, and total and infected host species densities are used as predictor variables for disease risk. Disease risk did not correlate with host diversity changes. However, the densities of each infected rodent species were positively associated with disease risk. We suggest that the infected host density surrounding the vector colonies is a relevant variable for disease risk and should be considered to understand disease dynamics. It is crucial to pay attention on the spatial scale of analysis, considering the pattern of vector dispersal, when the relationship between host diversity and disease risk is studied.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Disease‐structured N‐mixture models: A practical guide to model disease dynamics using count data

Obtaining inferences on disease dynamics (e.g., host population size, pathogen prevalence, transmission rate, host survival probability) typically requires marking and tracking individuals over time. While multistate mark–recapture models can produce high‐quality inference, these techniques are difficult to employ at large spatial and long temporal scales or in small remnant host populations decimated by virulent pathogens, where low recapture rates may preclude the use of mark–recapture techniques. Recently developed N‐mixture models offer a statistical framework for estimating wildlife disease dynamics from count data. N‐mixture models are a type of state‐space model in which observation error is attributed to failing to detect some individuals when they are present (i.e., false negatives). The analysis approach uses repeated surveys of sites over a period of population closure to estimate detection probability. We review the challenges of modeling disease dynamics and describe how N‐mixture models can be used to estimate common metrics, including pathogen prevalence, transmission, and recovery rates while accounting for imperfect host and pathogen detection. We also offer a perspective on future research directions at the intersection of quantitative and disease ecology, including the estimation of false positives in pathogen presence, spatially explicit disease‐structured N‐mixture models, and the integration of other data types with count data to inform disease dynamics. Managers rely on accurate and precise estimates of disease dynamics to develop strategies to mitigate pathogen impacts on host populations. At a time when pathogens pose one of the greatest threats to biodiversity, statistical methods that lead to robust inferences on host populations are critically needed for rapid, rather than incremental, assessments of the impacts of emerging infectious diseases.     

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml^− 1 to a high of 72,962 pg ml^− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.     

Specificity and seasonal prevalence of anther smut disease Microbotryum on sympatric Himalayan Silene species

Host sympatry provides opportunities for cross‐species disease transmission and compounded disease effects on host population and community structure. Using the Silene–Microbotryum interaction (the castrating anther smut disease), eleven Himalayan Silene species were assessed in regions of high host diversity to ascertain levels of pathogen specificity. We also investigated disease prevalence, seasonal dynamics of infection and flowering patterns in five co‐blooming Silene species. We identified several new Microbotryum lineages with varying degrees of specialization that is likely influenced by degrees of host divergence and ecological similarities (i.e. shared pollinator guilds). Affected species had 15%–40% of plants infected by anther smut. Flowering was seasonally overlapping among host species (except for the species pair S. asclepiadea and S. atrocastanea), but diseased flowering onset was earlier than healthy plants, leading to dramatic seasonal shifts in observed disease prevalence. Overlapping distributions and flowering provides opportunities for floral pathogen movement between host species, but host specialization may be constrained by the plant phylogenetic relatedness, adaptation to micro‐habitats and difference in pollinator/vector guilds.     

Interactive effects between diet and genotypes of host and pathogen define the severity of infection

Host resistance and parasite virulence are influenced by multiple interacting factors in complex natural communities. Yet, these interactive effects are seldom studied concurrently, resulting in poor understanding of host‐pathogen‐environment dynamics. Here, we investigated how the level of opportunist pathogen virulence, strength of host immunity and the host condition manipulated via diet affect the survival of wood tiger moth Parasemia plantaginis (Arctidae). Larvae from “low cuticular melanin” and “high cuticular melanin” (considered as low and high pathogen resistance, respectively) selection lines were infected with moderately and highly virulent bacteria strains of Serratia marcescens, while simultaneously manipulating host diet (with or without antibacterial compounds). We measured host survival and food preference before and after infection to test whether the larvae “self‐medicate” by choosing an anti‐infection diet (Plantago major, i.e., plantain leaf) over lettuce (Lactuca sativa). “High melanin” larvae were more resistant than “low melanin” larvae to the less virulent strain that had slower growth and colonization rate compared with the more virulent strain. Cuticular melanin did not enhance survival when the larvae were infected with the highly virulent strain. Anti‐infection diet enhanced survival of the “high melanin” but not the “low melanin” hosts. Survival was dependent on family origin even within the melanin selection lines. Despite the intrinsic preference for lettuce, no evidence of self‐medication was found. These results demonstrate that the relative benefit of host cuticular melanin depends on both diet and pathogen virulence: plantain diet only boosted the immunity of already resistant “high melanin” hosts, and cuticular melanin increased host survival only when infected with moderately virulent pathogen. Moreover, there was considerable variation in host survival between families within both melanin lines suggesting genetic basis for resistance. These results indicate that although melanin is an important predictor of insect immunity, its effect on disease outcomes greatly depends on other interacting factors.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.     

Initiation of Batrachochytrium dendrobatidis infection in the absence of physical contact with infected hosts – a field study in a high altitude lake

Understanding transmission is a critical prerequisite for predicting disease dynamics and impacts on host populations. It is well established that Batrachochytrium dendrobatidis (Bd), the amphibian fungal pathogen responsible for chytridiomycosis, can be transmitted directly, through physical contact with an infected host. However, indirect pathways of transmission remain poorly investigated. We conducted a five‐week long field infection experiment at a high altitude mountain lake in the French Pyrenees to investigate Bd transmission pathways in larval midwife toads Alytes obstetricans. Uninfected naïve tadpoles were co‐housed either with infected tadpoles (direct and indirect transmission) or with uninfected ones (indirect transmission only). We found that physical contact with an infected host is not necessary for initial infection with Bd and that all tadpoles became infected after only four weeks. However, physical contact with infected tadpoles led to a faster spread within a tadpole group and resulted in higher Bd loads and subsequently higher mortality. Our findings clearly demonstrate that in A. obstetricans, Bd can quickly spread in a population even without physical contact. Our experiment therefore stresses the importance of indirect transmission of Bd zoospores in infected lakes for disease dynamics, especially when a reservoir species such as A. obstetricans is present.     

Sympatry and interference of divergent Microbotryum pathogen species

The impact of infectious diseases in natural ecosystems is strongly influenced by the degree of pathogen specialization and by the local assemblies of potential host species. This study investigated anther‐smut disease, caused by fungi in the genus Microbotryum, among natural populations of plants in the Caryophyllaceae. A broad geographic survey focused on sites of the disease on multiple host species in sympatry. Analysis of molecular identities for the pathogens revealed that sympatric disease was most often due to co‐occurrence of distinct, host‐specific anther‐smut fungi, rather than localized cross‐species disease transmission. Flowers from sympatric populations showed that the Microbotryum spores were frequently moved between host species. Experimental inoculations to simulate cross‐species exposure to the pathogens in these plant communities showed that the anther‐smut pathogen was less able to cause disease on its regular host when following exposure of the plants to incompatible pathogens from another host species. These results indicate that multi‐host/multi‐pathogen communities are common in this system and they involve a previously hidden mechanism of interference between Microbotryum fungi, which likely affects both pathogen and host distributions.     

Estimating distemper virus dynamics among wolves and grizzly bears using serology and Bayesian state‐space models

Many parasites infect multiple hosts, but estimating the transmission across host species remains a key challenge in disease ecology. We investigated the within and across host species dynamics of canine distemper virus (CDV) in grizzly bears (Ursus arctos) and wolves (Canis lupus) of the Greater Yellowstone Ecosystem (GYE). We hypothesized that grizzly bears may be more likely to be exposed to CDV during outbreaks in the wolf population because grizzly bears often displace wolves while scavenging carcasses. We used serological data collected from 1984 to 2014 in conjunction with Bayesian state‐space models to infer the temporal dynamics of CDV. These models accounted for the unknown timing of pathogen exposure, and we assessed how different testing thresholds and the potential for testing errors affected our conclusions. We identified three main CDV outbreaks (1999, 2005, and 2008) in wolves, which were more obvious when we used higher diagnostic thresholds to qualify as seropositive. There was some evidence for increased exposure rates in grizzly bears in 2005, but the magnitude of the wolf effect on bear exposures was poorly estimated and depended upon our prior distributions. Grizzly bears were exposed to CDV prior to wolf reintroduction and during time periods outside of known wolf outbreaks, thus wolves are only one of several potential routes for grizzly bear exposures. Our modeling approach accounts for several of the shortcomings of serological data and is applicable to many wildlife disease systems, but is most informative when testing intervals are short. CDV circulates in a wide range of carnivore species, but it remains unclear whether the disease persists locally within the GYE carnivore community or is periodically reintroduced from distant regions with larger host populations.     

Pathogens manipulate the preference of vectors,slowing disease spread in a multi‐host system

The spread of vector‐borne pathogens depends on a complex set of interactions among pathogen, vector, and host. In single‐host systems, pathogens can induce changes in vector preferences for infected vs. healthy hosts. Yet it is unclear if pathogens also induce changes in vector preference among host species, and how changes in vector behaviour alter the ecological dynamics of disease spread. Here, we couple multi‐host preference experiments with a novel model of vector preference general to both single and multi‐host communities. We show that viruliferous aphids exhibit strong preferences for healthy and long‐lived hosts. Coupling experimental results with modelling to account for preference leads to a strong decrease in overall pathogen spread through multi‐host communities due to non‐random sorting of viruliferous vectors between preferred and non‐preferred host species. Our results demonstrate the importance of the interplay between vector behaviour and host diversity as a key mechanism in the spread of vectored‐diseases.     

PATHOGEN LIFE‐HISTORY TRADE‐OFFS REVEALED IN ALLOPATRY

Trade‐offs in life‐history traits is a central tenet in evolutionary biology, yet their ubiquity and relevance to realized fitness in natural populations remains questioned. Trade‐offs in pathogens are of particular interest because they may constrain the evolution and epidemiology of diseases. Here, we studied life‐history traits determining transmission in the obligate fungal pathogen, Podosphaera plantaginis, infecting Plantago lanceolata. We find that although traits are positively associated on sympatric host genotypes, on allopatric host genotypes relationships between infectivity and subsequent transmission traits change shape, becoming even negative. The epidemiological prediction of this change in life‐history relationships in allopatry is lower disease prevalence in newly established pathogen populations. An analysis of the natural pathogen metapopulation confirms that disease prevalence is lower in newly established pathogen populations and they are more prone to go extinct during winter than older pathogen populations. Hence, life‐history trade‐offs mediated by pathogen local adaptation may influence epidemiological dynamics at both population and metapopulation levels.     

Movement can mediate temporal mismatches between resource availability and biological events in host–pathogen interactions

Global change is shifting the timing of biological events, leading to temporal mismatches between biological events and resource availability. These temporal mismatches can threaten species’ populations. Importantly, temporal mismatches not only exert strong pressures on the population dynamics of the focal species, but can also lead to substantial changes in pairwise species interactions such as host–pathogen systems. We adapted an established individual‐based model of host–pathogen dynamics. The model describes a viral agent in a social host, while accounting for the host''s explicit movement decisions. We aimed to investigate how temporal mismatches between seasonal resource availability and host life‐history events affect host–pathogen coexistence, that is, disease persistence. Seasonal resource fluctuations only increased coexistence probability when in synchrony with the hosts’ biological events. However, a temporal mismatch reduced host–pathogen coexistence, but only marginally. In tandem with an increasing temporal mismatch, our model showed a shift in the spatial distribution of infected hosts. It shifted from an even distribution under synchronous conditions toward the formation of disease hotspots, when host life history and resource availability mismatched completely. The spatial restriction of infected hosts to small hotspots in the landscape initially suggested a lower coexistence probability due to the critical loss of susceptible host individuals within those hotspots. However, the surrounding landscape facilitated demographic rescue through habitat‐dependent movement. Our work demonstrates that the negative effects of temporal mismatches between host resource availability and host life history on host–pathogen coexistence can be reduced through the formation of temporary disease hotspots and host movement decisions, with implications for disease management under disturbances and global change.     

Flow cytometric method for quantifying viable Mycoplasma agassizii,an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii)

Aims: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. Methods and Results: Here, we demonstrate that the nonfluorescent molecule 5‐carboxyfluorescein (5‐CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5‐CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml^?1 can be labelled and counted in less than 1 h. Experiments using temperature‐induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. Conclusions: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. Significance and Impact of the Study: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.     

Two’s company,three’s a crowd: Exploring how host–parasite–microbiota interactions may influence disease susceptibility and conservation of wildlife

A large body of research has demonstrated that host‐associated microbiota—the archaeal, bacterial, fungal and viral communities residing on and inside organisms—are critical to host health (Cho & Blaser, 2012). Although the vast majority of these studies focus on humans or model organisms in laboratory settings (Pascoe, Hauffe, Marchesi, & Perkins, 2017), they nevertheless provide important conceptual evidence that the disruption of host‐associated microbial communities (termed “dysbiosis”) among wild animals may reduce host fitness and survival under natural environmental conditions. Among the myriad of environmental factors capable of inducing dysbiosis among wild animals (Trevelline, Fontaine, Hartup, & Kohl, 2019), parasitic infections represent a potentially potent, yet poorly understood, factor influencing microbial community dynamics and animal health. The study by DeCandia et al. in this issue of Molecular Ecology is a rare example of a host–parasite–microbiota interaction that impacts the health, survival and conservation of a threatened wild animal in its natural habitat. Using culture‐independent techniques, DeCandia et al. found that the presence of an ectoparasitic mite (Otodectes cynotis) in the ear canal of the Santa Catalina Island fox (Urocyon littoralis catalinae) was associated with significantly reduced ear canal microbial diversity, with the opportunistic pathogen Staphylococcus pseudintermedius dominating the community. These findings suggest that parasite‐induced inflammation may contribute to the formation of ceruminous gland tumours in this subspecies of Channel Island fox. As a rare example of a host–parasite–microbiota interaction that may mediate a lethal disease in a population of threatened animals, their study provides an excellent example of how aspects of disease ecology can be integrated into studies of host‐associated microbiota to advance conservation science and practice.     

The community ecology of pathogens: coinfection,coexistence and community composition

Disease and community ecology share conceptual and theoretical lineages, and there has been a resurgence of interest in strengthening links between these fields. Building on recent syntheses focused on the effects of host community composition on single pathogen systems, we examine pathogen (microparasite) communities using a stochastic metacommunity model as a starting point to bridge community and disease ecology perspectives. Such models incorporate the effects of core community processes, such as ecological drift, selection and dispersal, but have not been extended to incorporate host–pathogen interactions, such as immunosuppression or synergistic mortality, that are central to disease ecology. We use a two‐pathogen susceptible‐infected (SI) model to fill these gaps in the metacommunity approach; however, SI models can be intractable for examining species‐diverse, spatially structured systems. By placing disease into a framework developed for community ecology, our synthesis highlights areas ripe for progress, including a theoretical framework that incorporates host dynamics, spatial structuring and evolutionary processes, as well as the data needed to test the predictions of such a model. Our synthesis points the way for this framework and demonstrates that a deeper understanding of pathogen community dynamics will emerge from approaches working at the interface of disease and community ecology.