Eimeria spp

Jarquín-Díaz, Víctor Hugo, Balard, Alice, Jost, Jenny, Kraft, Julia, Dikmen, Mert Naci, Kvičerová, Jana & Heitlinger, Emanuel, 2019, Detection and quantification of house mouse Eimeria at the species level - Challenges and solutions for the assessment of coccidia in wildlife, International Journal for Parasitology: Parasites and Wildlife 10, pp. 29-40 : 32

3.1. Sampling and Eimeria spp . detection

We used flotation of oocyst from faeces and PCR amplification of a novel diagnostic maker (Ap5) from colon content DNA to detect Eimeria parasites in a total of 378 house. Overall prevalence was 25.9% [95% CI = 21.7–30.7] (98/378) for PCR and 27.0% [95% CI = 22.7–31.7%] (102/378) for flotation. These estimates are not significantly different (Fisher exact test, p> 0.05). However, both techniques considered together estimate a higher prevalence of 37.6% [95% CI = 32.8–42.6] (142/378), meaning that 44 and 40 positive results were detected only by flotation or PCR, respectively ( Fig. 1B ). We further aimed to provide species specific identification and to consolidate results from the two different detection methods.

3.2. Molecular identification of Eimeria isolates - (phylogenetic analysis nu 18S and mt COI)

Eimeria species were identified by phylogenetic analysis of nu 18S and mt COI sequences, the most commonly used molecular markers of apicomplexan parasites. To identify our isolates, sequences were compared with references from the NCBI database. Sequences from three previously described Eimeria species infecting M. musculus showed highest BLAST similarities and phylogenetic clustering. This approach ignores the problem whether isolates from different hosts would be assigned to the same phylogenetic clusters while they are regarded different species by taxonomists.

The nu 18S phylogenetic tree was inferred based on 80 sequences (540–1795 bp), 73 of them from wild house mice generated in our study (3 from ileum tissue, 16 from cecum tissue and 54 from colon content, see below). Eimeria species previously described in house mouse were represented by E. falciformis (AF080614), E. vermiformis (KT184355) and E. ferrisi (KT360995). In addition, one newly generated sequence from E. falciformis strain BayerHaberkorn1970 (MH751998) was also included. Sequence identity of our isolates to this references sequences was above 98% and even 100% in most of the cases for this marker. Isospora sp. sequences identified in Talpa europaea moles were used as outgroup. Both ML and BI rooted trees shared a topology placing our sequences at the same positions in relation to reference sequences with high support (bootstrap values and posterior probabilities are shown in Fig. 2A ). The sequences clustered in three well supported monophyletic groups ( Fig. 2A ).

The phylogenetic tree for mt COI was based on 103 sequences (519–804 bp), 97 of which were obtained from Eimeria infecting wild house mice (3 from ileum, 16 from cecum tissue and 78 from colon content). Reference sequences from house mouse Eimeria ( E. ferrisi, KT 361028; E. falciformis, KX 495129 and MH777539; E. vermiformis JN 205071) identified by BLAST searches showed an identity of above 98% to respective groups of our isolates. We defined Isospora sp. from Talpa europaea as outgroup for rooting. ML and BI rooted trees based on alignments of these COI sequences shared a general topology with respect to the placement of our isolates in relation to reference sequences. Bootstrap values and posterior probabilities for support of these placements are shown in Fig. 2B . The sequences derived from house mice cluster in three monophyletic groups including reference sequences for E. falciformis (n = 17, sequences from our study), E. ferrisi (n = 72) and E. vermiformis (n = 8) ( Fig. 2B ). Phylogenies based on concatenated supermatrices for the two markers show the same topology concerning placement of isolates from the present study (Supplementary data S3 and S4).

Figures

Fig. 1. Geographical localization of house mice (Mus musculus) collected for this study and comparison of diagnostic methods for Eimeria. A) Localization from the 378 mice included in the present study, colors indicate the Eimeria species identified for each. B) Venn diagram showing the overlap between detection methods and successful genotyping identification of the isolates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Phylogenetic trees based on 18S rRNA and COI sequences. Sequences of 18S A) and COI B) were used to infer the molecular identification of wild-derived isolates of Eimeria. In both phylogenies, our isolates clustered in three groups one close to E. falciformis (red), other close to E. ferrisi (green) and finally one to E. vermiformis (yellow). Numbers in the branches represent the Bayesian posterior probability and the non-parametric bootstrap value. In bold are indicate the reference sequences for each species. CE and IL make reference to sequence derived from cecum or ileum tissue DNA, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)