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Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

ABSTRACT: The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium.We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene (gltA).Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp. BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (T m ~55.8 °C); E. chaffeensis and E. ewingii (T m ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (T m ~62.0 °C); and the Panola Mountain Ehrlichia (T m ~65.5 °C). The detection limit of the FRET-qPCR was?~?5 gene copies in a reaction and the sequences of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found 12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E. canis or very closely related organisms.In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E. canis or very closely related organisms.

SUBMITTER: Zhang J

PROVIDER: S-EPMC4595018 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

Zhang Jilei J Kelly Patrick P Guo Weina W Xu Chuanling C Wei Lanjing L Jongejan Frans F Loftis Amanda A Wang Chengming C

Parasites & vectors 20151006

<h4>Background</h4>The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium.<h4>Methods</h4>We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a sing ...[more]

PMID: 26438311

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Project description:BackgroundInformation on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST).ResultsA total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but some of the MLVA genotypes are described here for the first time.ConclusionsGenotyping revealed a substantial genetic diversity among domestic ruminants from Northern Spain.

Dataset Information

Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

Publications

Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

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