Molecular identification of Eimeria species in Spanish bats

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.     

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.     

Acetate,a metabolic product of Heligmosomoides polygyrus,facilitates intestinal epithelial barrier breakdown in a FFAR2-dependent manner

Approximately 2 billion people worldwide and a significant part of the domestic livestock are infected with soil-transmitted helminths, of which many establish chronic infections causing substantial economic and welfare burdens. Beside intensive research on helminth-triggered mucosal and systemic immune responses, the local mechanism that enables infective larvae to cross the intestinal epithelial barrier and invade mucosal tissue remains poorly addressed. Here, we show that Heligmosomoides polygyrus infective L3s secrete acetate and that acetate potentially facilitates paracellular epithelial tissue invasion by changed epithelial tight junction claudin expression. In vitro, impedance-based real-time epithelial cell line barrier measurements together with ex vivo functional permeability assays in intestinal organoid cultures revealed that acetate decreased intercellular barrier function via the G-protein coupled free fatty acid receptor 2 (FFAR2, GPR43). In vivo validation experiments in FFAR2^?/? mice showed lower H. polygyrus burdens, whereas oral acetate-treated C57BL/6 wild type mice showed higher burdens. These data suggest that locally secreted acetate – as a metabolic product of the energy metabolism of H. polygyrus L3s – provides a significant advantage to the parasite in crossing the intestinal epithelial barrier and invading mucosal tissues. This is the first and a rate-limiting step for helminths to establish chronic infections in their hosts and if modulated could have profound consequences for their life cycle.     

Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies

Genetic diversity in animal immune systems is usually beneficial. In hybrid recombinants, this is less clear, as the immune system could also be impacted by genetic conflicts. In the European house mouse hybrid zone, the long‐standing impression that hybrid mice are more highly parasitized and less fit than parentals persists despite the findings of recent studies. Working across a novel transect, we assessed infections by intracellular protozoans, Eimeria spp., and infections by extracellular macroparasites, pinworms. For Eimeria, we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection or increased mortality in the centre of the hybrid zone. This means ecological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focusing on parasite intensity (load in infected hosts), we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.     

Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine

Stockdale P. G. H., Stockdale M. J., Rickard M. D. and Mitchell G. F. 1985. Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine, International Journal for Parasitology15: 447–452. Five inbred strains of mice and three hypothymic (nude) strains were infected orally with different doses of E. falciförmis oocysts. After resolution of primary infection as determined by faecal oocyst output, mice were challenged orally with a second dose of E. falciformis. Amongst the intact mice, BALB/c proved the most resistant to primary infection, while C3H/He mice were most susceptible, in terms of faecal oocyst production. Resistance was far more dramatic in BALB/c mice given high numbers of challenge oocysts. In terms of mortality at high oocyst doses, CBA/H were the most susceptible. All of the strains of mice were highly resistant to reinfection. In the case of nude mice, BALB/c. nu/nu were more susceptible than CBA/H.nu/nu or C57BL/6.nu/nu both in terms of faecal oocyst production and mortality. Thus the most resistant inbred mouse strain (BALB/c) is the least resistant in the absence of T cells. Unlike intact mice, nude mice showed no resistance to reinfection, this result being in line with previous work on this and other Eimeria spp. in nude mice.     

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.     

Supplemented nutrition decreases helminth burden and increases drug efficacy in a natural host–helminth system

Gastrointestinal (GI) helminths are common parasites of humans, wildlife, and livestock, causing chronic infections. In humans and wildlife, poor nutrition or limited resources can compromise an individual''s immune response, predisposing them to higher helminth burdens. This relationship has been tested in laboratory models by investigating infection outcomes following reductions of specific nutrients. However, much less is known about how diet supplementation can impact susceptibility to infection, acquisition of immunity, and drug efficacy in natural host–helminth systems. We experimentally supplemented the diet of wood mice (Apodemus sylvaticus) with high-quality nutrition and measured resistance to the common GI nematode Heligmosomoides polygyrus. To test whether diet can enhance immunity to reinfection, we also administered anthelmintic treatment in both natural and captive populations. Supplemented wood mice were more resistant to H. polygyrus infection, cleared worms more efficiently after treatment, avoided a post-treatment infection rebound, produced stronger general and parasite-specific antibody responses, and maintained better body condition. In addition, when applied in conjunction with anthelmintic treatment, supplemented nutrition significantly reduced H. polygyrus transmission potential. These results show the rapid and extensive benefits of a well-balanced diet and have important implications for both disease control and wildlife health under changing environmental conditions.     

Coupling between tolerance and resistance for two related Eimeria parasite species

Resistance (host capacity to reduce parasite burden) and tolerance (host capacity to reduce impact on its health for a given parasite burden) manifest two different lines of defense. Tolerance can be independent from resistance, traded off against it, or the two can be positively correlated because of redundancy in underlying (immune) processes. We here tested whether this coupling between tolerance and resistance could differ upon infection with closely related parasite species. We tested this in experimental infections with two parasite species of the genus Eimeria. We measured proxies for resistance (the (inverse of) number of parasite transmission stages (oocysts) per gram of feces at the day of maximal shedding) and tolerance (the slope of maximum relative weight loss compared to day of infection on number of oocysts per gram of feces at the day of maximal shedding for each host strain) in four inbred mouse strains and four groups of F1 hybrids belonging to two mouse subspecies, Mus musculus domesticus and Mus musculus musculus. We found a negative correlation between resistance and tolerance against Eimeria falciformis, while the two are uncoupled against Eimeria ferrisi. We conclude that resistance and tolerance against the first parasite species might be traded off, but evolve more independently in different mouse genotypes against the latter. We argue that evolution of the host immune defenses can be studied largely irrespective of parasite isolates if resistance–tolerance coupling is absent or weak (E. ferrisi) but host–parasite coevolution is more likely observable and best studied in a system with negatively correlated tolerance and resistance (E. falciformis).     

The role of host sex in parasite dynamics: field experiments on the yellow-necked mouse Apodemus flavicollis

We investigated the role of host sex in parasite transmission and questioned: ‘Is host sex important in influencing the dynamics of infection in free living animal populations?’ We experimentally reduced the helminth community of either males or females in a yellow‐necked mice (Apodemus flavicollis) population using an anthelmintic, in replicated trapping areas, and subsequently monitored the prevalence and intensity of macroparasites in the untreated sex. We focussed on the dominant parasite Heligmosomoides polygyrus and found that reducing parasites in males caused a consistent reduction of parasitic intensity in females, estimated through faecal egg counts, but the removal of parasites in females had no significant influence on the parasites in males. This finding suggests that males are responsible for driving the parasite infection in the host population and females may play a relatively trivial role. The possible mechanisms promoting such patterns are discussed.     

Expression of parasite genetic variation changes over the course of infection: implications of within-host dynamics for the evolution of virulence

How infectious disease agents interact with their host changes during the course of infection and can alter the expression of disease-related traits. Yet by measuring parasite life-history traits at one or few moments during infection, studies have overlooked the impact of variable parasite growth trajectories on disease evolution. Here we show that infection-age-specific estimates of host and parasite fitness components can reveal new insight into the evolution of parasites. We do so by characterizing the within-host dynamics over an entire infection period for five genotypes of the castrating bacterial parasite Pasteuria ramosa infecting the crustacean Daphnia magna. Our results reveal that genetic variation for parasite-induced gigantism, host castration and parasite spore loads increases with the age of infection. Driving these patterns appears to be variation in how well the parasite maintains control of host reproduction late in the infection process. We discuss the evolutionary consequences of this finding with regard to natural selection acting on different ages of infection and the mechanism underlying the maintenance of castration efficiency. Our results highlight how elucidating within-host dynamics can shed light on the selective forces that shape infection strategies and the evolution of virulence.     

Host age modulates within-host parasite competition

In many host populations, one of the most striking differences among hosts is their age. While parasite prevalence differences in relation to host age are well known, little is known on how host age impacts ecological and evolutionary dynamics of diseases. Using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa, we examined how host age at exposure influences within-host parasite competition and virulence. We found that multiply-exposed hosts were more susceptible to infection and suffered higher mortality than singly-exposed hosts. Hosts oldest at exposure were least often infected and vice versa. Furthermore, we found that in young multiply-exposed hosts competition was weak, allowing coexistence and transmission of both parasite clones, whereas in older multiply-exposed hosts competitive exclusion was observed. Thus, age-dependent parasite exposure and host demography (age structure) could together play an important role in mediating parasite evolution. At the individual level, our results demonstrate a previously unnoticed interaction of the host''s immune system with host age, suggesting that the specificity of immune function changes as hosts mature. Therefore, evolutionary models of parasite virulence might benefit from incorporating age-dependent epidemiological parameters.     

Dissecting the effect of a heterogeneous environment on the interaction between host and parasite fitness traits

Environmental variation can alter the probability of parasitic infection or the fitness consequence of infection, and thus has the potential to dramatically alter the dynamics of host parasite coevolution. Here we investigated the effect of a changing temperature on host-parasite interactions using the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa. By reciprocally varying (1) the temperature at which exposure to parasites occurred and (2) the temperature at which within-host parasite growth occurred, and measuring several fitness-related traits, we show that while there are temperature combinations that favour either host or parasite, there are also conditions that favour neither, that is, negative fitness consequences for the host without fitness benefits for the parasite. This result highlights the importance of considering a heterogeneous rather than static environment in coevolutionary studies, while also showing support for an optimal virulence strategy in castrating parasites.     

CD8+ cells protect mice against reinfection with the intestinal parasite Eimeria falciformis

We investigated cellular immune responses of mice infected with the apicomplexan parasite Eimeria falciformis in order to characterise protective immune mechanisms and effector functions. Adoptive transfer experiments with mesenterial lymph node cells (MLNC) from immune donor mice were performed, and the oocyst output monitored after challenge infection. Phenotypical analysis by fluorescence cytometry and T cell proliferation assay showed that already from day four post infection E. falciformis-specific lymphocytes were present in the MLN. The frequency of parasite-specific, IFN-γ producing CD4+ and CD8+ cells increased in this period by 9.8% and 16.4%, respectively. Infection experiments with IFN-γ deficient mice revealed that IFN-γ is involved in resistance to primary and secondary infection. Transfer of total MLNC from immune donors reduced the oocyst output by 65–74%, as compared to the oocyst output of animals transferred with cells from naïve donors. Transfer of CD8+ cells inhibited parasite development resulting in a reduction of oocyst numbers by 42–64%, whereas CD4+ cells showed no influence on resistance to reinfection.     

Development of Fatal Intestinal Inflammation in MyD88 Deficient Mice Co-infected with Helminth and Bacterial Enteropathogens

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection.     

Partial Protective Effect of Intranasal Immunization with Recombinant Toxoplasma gondii Rhoptry Protein 17 against Toxoplasmosis in Mice

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.     

Within-host dynamics of Trichinella spiralis predict persistent parasite transmission in rat populations

Trichinella spiralis is transmitted and maintained in both a domestic and sylvatic cycle, whereby rats contribute to the spread of T. spiralis from domestic to sylvatic animals and vice versa. As a model for T. spiralis transmission in wildlife, we studied the potential of rats to act as a reservoir species for T. spiralis, by assessing experimentally its within-host infection dynamics, and simulating the between-host dynamics by a Monte Carlo approach. The distribution of parasite burden in individual rats is mathematically defined by roots of the dose response equation intersecting with the diagonal. In simulated between-host dynamics, up to 10⁴ events of uninterrupted parasite transmission were observed. Histograms of parasite burdens per individual rat matched closely with the mixture of two gamma distributions, which were derived from the within-host infection dynamics. In conclusion, T. spiralis transmission persists in a population of rats when they cannibalize their own species. Rats should be included in the minimal set of wildlife species that maintain the life cycle of T. spiralis.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

Microhabitat distributions and species interactions of ectoparasites on the gills of cichlid fish in Lake Victoria,Tanzania

Heterogeneous exposure to parasites may contribute to host species differentiation. Hosts often harbour multiple parasite species which may interact and thus modify each other’s effects on host fitness. Antagonistic or synergistic interactions between parasites may be detectable as niche segregation within hosts. Consequently, the within-host distribution of different parasite taxa may constitute an important axis of infection variation among host populations and species. We investigated the microhabitat distributions and species interactions of gill parasites (four genera) infecting 14 sympatric cichlid species in Lake Victoria, Tanzania. We found that the two most abundant ectoparasite genera (the monogenean Cichlidogyrus spp. and the copepod Lamproglena monodi) were non-randomly distributed across the host gills and their spatial distribution differed between host species. This may indicate microhabitat selection by the parasites and cryptic differences in the host–parasite interaction among host species. Relationships among ectoparasite genera were synergistic: the abundances of Cichlidogyrus spp. and the copepods L. monodi and Ergasilus lamellifer tended to be positively correlated. In contrast, relationships among morphospecies of Cichlidogyrus were antagonistic: the abundances of morphospecies were negatively correlated. Together with niche overlap, this suggests competition among morphospecies of Cichlidogyrus. We also assessed the reproductive activity of the copepod species (the proportion of individuals carrying egg clutches), as it may be affected by the presence of other parasites and provide another indicator of the species specificity of the host–parasite relationship. Copepod reproductive activity did not differ between host species and was not associated with the presence or abundance of other parasites, suggesting that these are generalist parasites, thriving in all cichlid species examined from Lake Victoria.     

Seasonal Patterns of Hormones,Macroparasites, and Microparasites in Wild African Ungulates: The Interplay among Stress,Reproduction, and Disease

Sex hormones, reproductive status, and pathogen load all affect stress. Together with stress, these factors can modulate the immune system and affect disease incidence. Thus, it is important to concurrently measure these factors, along with their seasonal fluctuations, to better understand their complex interactions. Using steroid hormone metabolites from fecal samples, we examined seasonal correlations among zebra and springbok stress, reproduction, gastrointestinal (GI) parasite infections, and anthrax infection signatures in zebra and springbok in Etosha National Park (ENP), Namibia, and found strong seasonal effects. Infection intensities of all three GI macroparasites examined (strongyle helminths, Strongyloides helminths, and Eimeria coccidia) were highest in the wet season, concurrent with the timing of anthrax outbreaks. Parasites also declined with increased acquired immune responses. We found hormonal evidence that both mares and ewes are overwhelmingly seasonal breeders in ENP, and that reproductive hormones are correlated with immunosuppression and higher susceptibility to GI parasite infections. Stress hormones largely peak in the dry season, particularly in zebra, when parasite infection intensities are lowest, and are most strongly correlated with host mid-gestation rather than with parasite infection intensity. Given the evidence that GI parasites can cause host pathology, immunomodulation, and immunosuppression, their persistence in ENP hosts without inducing chronic stress responses supports the hypothesis that hosts are tolerant of their parasites. Such tolerance would help to explain the ubiquity of these organisms in ENP herbivores, even in the face of their potential immunomodulatory trade-offs with anti-anthrax immunity.