Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals. Parasite species – specific prevalences are especially important when studying evolutionary questions in wild populations. We tested whether increased host population density increases prevalence of individual Eimeria species at the farm level, as predicted by epidemiological theory. We studied free-living commensal populations of the house mouse ( Mus musculus ) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred niche of each parasite species along the distal-proximal axis of the intestine. Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.7%; 95% CI 13.2–20.7), E. falciformis (4.2%; 95% CI 2.6–6.8) and E. vermiformis (1.9%; 95% CI 0.9–3.8). We also find that mice in dense populations are more likely to be infected with E. falciformis and E. ferrisi . We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We show and discuss how such data can help to test hypotheses in ecology, evolution and epidemiology on a species level.

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.

Species of Eimeria (Apicomplexa:Coccidia) differ in the timing of lifecycle progression and resulting infections vary in host immune reactions and pathology they induce. Eimeria infections in house mice are used as models for basic immunology and the most commonly used isolates have been passaged in laboratory mice for over 50 years. We questioned in how far such isolates are still representative for infections in natural systems.In the current study, we address this question by comparing the "laboratory isolate" E. falciformis BayerHaberkorn1970 with a novel, wild derived isolate E. falciformis Brandenburg88, and contrast this with another novel wild derived isolate, E. ferrisi Brandenburg64. We compare parasite lifecycle progression. We relate this to immune cell infiltration at the site of infection (in the caecum) and cytokine gene expression in the spleen as a measure of host immune response. We assess host weight loss as a measure of pathogenicity.A species-specific slower parasite lifecyle progression and higher pathogenicity are observed for E. falciformis vs. E. ferrisi. Host cytokines, in contrast, are expressed at significantly higher level in the 1 spleen of mice infected with the E. falciformis laboratory isolate than in both wild derived isolates, irrespective of the species. Differences in histopathology are observable between all three isolates: The E. falciformis BayerHaberkorn1970 laboratory isolate induces the strongest inflammation and cellular infiltration (with lymphocytes, plasma cells and eosinophilic granulocytes) followed by the wild derived E. falciformis Brandenburg88 isolate. E. ferrisi Brandenburg64 is inducing milder histological changes than both E. falciformis isolates.It can be speculated that the serial passaging of E. falciformis BayerHaberkorn1970 has resulted in evolutionary divergence rendering this isolate more virulent in NMRI mice. Caution is needed when findings from experimental infection with laboratory strains should be integrated with observations in natural systems.Highlights  E. ferrisi has a shorter pre-patency than wild-derived and laboratory isolates of E. falciformis. E. ferrisi is less virulent than both E. falciformis isolates and the timing of maximal oocyst shedding relative to host weight loss differs. The laboratory strain of E. falciformis induces stronger cytokine expression in the spleen than both wild derived strains of E. falciformis and E. ferrisi. The laboratory strain of E. falciformis induces stronger tissue infiltration of immune cells than the wild-derived strain. E. ferrisi infections are associated with the lowest infiltration.

Host defense mechanisms evolve to alleviate the detrimental effect of parasites. They can be categorized into two components: resistance and tolerance (Råberg et al., 2009). Resistance is the ability of a host to reduce parasite burden, resulting from defense against parasite infection or proliferation early after infection (Schmid-Hempel, 2013). The negative effect of resistance on parasite fitness can lead to antagonistic coevolution. According to theoretical models, fluctuating host and parasite genotypes arise, and

Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals.Parasite species -specific prevalences are especially important when studying evolutionary questions in wild populations. We might expect, for example, highly prevalent parasite species to have a lower virulence than lowly prevalent species.We studied free-living commensal populations of the house mouse (Mus musculus) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred parasite occurrence along the distal-proximal axis of the intestine.Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.1%; 95% CI 12.7 -20.2), E. falciformis (4.2%; 95% CI 2.6 -6.8) and E. vermiformis (1.1%; 95% CI 0.4 -2.7).We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We discuss the need for broader species level assessment in Coccidia. Prevalence negatively correlates with virulence for Eimeria species of house mice, as the more prevalent E. ferrisi has been shown to be less virulent than E. falciformis. It is an open question whether house mouse Eimeria are host specialist and whether prevalence in our system correlates with host range.

Intracellular parasites of the genus Eimeria are described as tissue/host‐specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species. With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non‐Mus isolates, even with this high‐resolution data. In conclusion, we show that species‐level resolution should not be assumed for COI and 18S markers in coccidia. Host–parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

Background Counting parasite transmission stages in faeces is the classical measurement to quantify “parasite load”. DNA-based quantifications of parasite intensities from faecal samples are relatively novel and often validated against such counts. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts. Here, we investigate this issue using the example of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus). Methods We performed an infection experiment of house mice with E. ferrisi, in which the intensity of infection correlates with increased health impact on the host, measured as temporary weight loss during infection. We recorded the number of parasite transmissive stages (oocysts) per gram of faeces (OPG) and, as a DNA-based measurement, the number of Eimeria genome copies per gram of faeces for 10 days post-infection (dpi). We assessed weight loss relative to the day of experimental infection as a proxy of host health and evaluated whether DNA or oocyst counts are better predictors of host health. Results Absolute quantification of Eimeria DNA and oocyst counts showed similar but slightly diverging temporal patterns during 10 dpi. We detected Eimeria DNA earlier than the first appearance of oocysts in faeces. Additionally, Eimeria OPGs within each dpi did not explain parasite DNA intensity. Early dpi were characterized by high DNA intensity with low oocyst counts, while late infections showed the opposite pattern. The intensity of Eimeria DNA was consistently a stronger predictor of either maximal weight loss (1 value per animal during the infection course) or weight loss on each day during the experiment when controlling for between-dpi and between-individual variance. Conclusions Eimeria ferrisi oocyst counts correlate weakly with parasite intensity assessed through DNA quantification. DNA is likely partially derived from life-cycle stages other than transmissive oocysts. DNA-based intensities predict health outcomes of infection for the host more robustly than counts of transmissive stages. We conclude that DNA-based quantifications should not necessarily require validation against counts of transmissive stages. Instead, DNA-based load estimates should be evaluated as complementary sources of information with potential specific biological relevance for each host-parasite system. Graphical Abstract