Detection and quantification of house mouse Eimeria at the species level – challenges and solutions for the assessment of Coccidia in wildlife

Abstract

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.

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers

“…Other isolates from house mouse and A. flavicollis and A. sylvaticus clustered in an unresolved internal relationship with house mouse E. vermiformis isolates (Group II). We note that four of five sequences for E. vermiformis from house mice were amplified from ileum tissue, the primary location of infection with this species (in contrast, E. falciformis infects primarily the cecum; Jarquín‐Díaz et al, ). A second clade recovered by nuclear multilocus analysis contained E. falciformis from house mice.…”

“…Multilocus genotyping using apicoplast markers showed some discrepancies with the nuclear analysis. These discrepancies can be attributed to double infections previously discovered in those particular isolates (Jarquín‐Díaz et al, ). Compared to the nuclear genome, the apicoplast genome is present in much higher copy numbers (Heitlinger et al, ).…”

“…Compared to the nuclear genome, the apicoplast genome is present in much higher copy numbers (Heitlinger et al, ). This, combined with more conserved primer binding sites, can lead to amplification of nontarget sequences such as those of the prevalent E. ferrisi (Jarquín‐Díaz et al, ) creating artificial “chimeric” isolates in case of double infections.…”

“…These samples came from rodents captured in farms and private properties in the German federal states of Mecklenburg‐Vorpommern, Bavaria, and Brandenburg (capture permit No. 2347/35/2014) and in Bohemia (Czech Republic) between 2014 and 2017 (Jarquín‐Díaz et al, ). Additionally, DNA from gastrointestinal tract and tissue or feces of Apodemus spp.…”

“…When COI failed to amplify with this protocol, an alternative pair of primers was used: Eim_COI_M_F (ATGTCACTNTCTCCAACCTCAGT) and Eim_COI_M_R (GAGCAACATCAANAGCAGTGT). These primers amplify a ~700 bp fragment of COI and were designed based on the mitochondrial genome of E. falciformis (CM008276.1) (Heitlinger, Spork, Lucius, & Dieterich, ; Jarquín‐Díaz et al, ).…”