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Usefulness of PCR-RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic Eimeria spp. (Apicomplexa: Eimeriidae) of European bison, Bison bonasus L. with histopathological correlation.

ABSTRACT: Eimeria spp. infection was investigated in 10 free-roaming European bison aged three months to 26 years by anatomopathological, histopathological, coproscopic and PCR-RFLP examination. The coproscopic study identified Eimeria oocysts in the faeces of five bison. The most prevalent morphotypes were E. bovis, present in all positive samples, and E. zuernii, in all but one. Additionally, mixed infections consisting of E. bovis, E. zuernii, E. alabamensis, E. auburnensis, E. canadensis, E. cylindrica, E. ellipsoidalis and E. subspherica were diagnosed in two bison calves. Besides being the most prevalent form, E. bovis also demonstrated the highest OPG (2,750). The presence of oocysts in the faeces was associated with those of macrogamonts, microgamonts and oocysts in the epithelium of the large intestine. Intestinal coccidiosis associated with lymphoplasmacytic enteritis was observed in many bison, not only those with positive OPG. Four animals with negative coproscopy results demonstrated early-stage gametogony in the large intestine; one case presented no endogenous stages of coccidians in the histopathological sections of the intestine, nor oocysts in the faecal samples. A 530 bp product of E. bovis 18S rDNA (GenBank: MK951685) was obtained from both the colon wall and oocysts; this was subjected to PCR-RFLP analysis based on AluI and Hin1II (NlaIII) restriction enzymes. Both samples yielded a consistent seven-band pattern, four of which (270 bp, 40 bp, 180 bp and 84 bp) were expected, and the other three represented undigested fragments. The obtained digestion pattern is indicative of Eimeria spp. infection, and can serve as a first-step diagnostic approach in detection of infection. The result of computer-based virtual digestion of the PCR product suggests that double digestion with Mval (BstNI) and KpnI restriction enzymes may be used as a second-step tool to distinguish between E. bovis, E. zuernii and E. alabamensis, all of which are highly-pathogenic species.

SUBMITTER: Pyziel AM

PROVIDER: S-EPMC7190760 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Usefulness of PCR-RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic Eimeria spp. (Apicomplexa: Eimeriidae) of European bison, Bison bonasus L. with histopathological correlation.

Pyziel Anna M AM Demiaszkiewicz Aleksander W AW Osińska Barbara B Dolka Izabella I Anusz Krzysztof K Laskowski Zdzisław Z

International journal for parasitology. Parasites and wildlife 20200403

Eimeria spp. infection was investigated in 10 free-roaming European bison aged three months to 26 years by anatomopathological, histopathological, coproscopic and PCR-RFLP examination. The coproscopic study identified Eimeria oocysts in the faeces of five bison. The most prevalent morphotypes were E. bovis, present in all positive samples, and E. zuernii, in all but one. Additionally, mixed infections consisting of E. bovis, E. zuernii, E. alabamensis ...[more]

PMID: 32368487

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Project description:BackgroundClinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo).MethodsSix single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome.ResultsComplete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens.ConclusionsMitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

Project description:BackgroundOur recent macro- and scanning electron microscopic study of tongue conducted on domesticated cattle, wild living European bison, and Bison bonasus hybrid revealed various spatial arrangement and number of gustatory and mechanical papillae between parental species and their hybrid. Furthermore, scanning electron microscopy analysis of gustatory papillae indicated the variable distribution of fungiform papillae (Fu) over the surface of the tongue, which could be significant in differentiated taste perception during feeding in studied wild living and domesticated husbandry ruminants. To specify the detailed microstructure of Fu papillae with connective tissue cores (CTC) and intraepithelial taste buds system, the first time the three-dimensional computer-aided analysis of serial histoslides resulted in the rendering of 3D reconstructions of Fu papillae.ResultsThe comparative analysis of 3D models Fu papillae conducted in six areas of lingual mucosa of each tongue revealed information about, microstructural diversity of Fu papillae in studied ruminants. The estimation of number and density of Fu papillae on tongues, rate of protrusion of papillae over mucosa, and a number of taste buds per papilla allowed to state the ventral surface of the lingual apex and posterolateral surfaces of the lingual torus as regions important in taste perception, as in the preselection of taken food, as well in the analysis of food during rumination, respectively. On the 3D models were observed three structural types of CTC of different distribution on the tongue in studied species. The quantitative data of the number of taste buds on Fu papillae have regional functional differences in the taste system important in feeding and veterinary practice. Moreover, our analysis determined specific features in examined hybrid and showed similarities of some studied features with cattle, i.e., maternal species.ConclusionsThe 3D reconstruction method used for the first time in the field of study of the lingual papillae and taste buds system can be considered as an innovative and effective tool in assessing of the microstructures of Fu papillae, and it could be suitable for further studies of taste system structures in normal and pathological condition.

Dataset Information

Usefulness of PCR-RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic Eimeria spp. (Apicomplexa: Eimeriidae) of European bison, Bison bonasus L. with histopathological correlation.

Publications

Usefulness of PCR-RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic <i>Eimeria</i> spp. (Apicomplexa: Eimeriidae) of European bison, <i>Bison bonasus</i> L. with histopathological correlation.

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