Direct Identification of <i>Ehrlichia Canis</i> by a Novel Polymerase Chain Reaction Method and Molecular Analysis of the Citrate Synthase (<i>gltA</i>) Gene from Various Italian Strains

Marsilio, Fulvio; Martino, Barbara Di; Meridiani, I.; Bianciardi, P.

doi:10.1177/104063870601800215

Cited by 9 publications

(9 citation statements)

References 8 publications

(9 reference statements)

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“…The low copy numbers in our samples were also evident from the results of our gltA gene PCRs where we only found positive results after nesting. The gltA gene has also been shown to be highly conserved in E. canis (over 99 %) [ 30 ], but it has greater interspecies variability than the 16S rRNA gene which might make it more useful for differentiating Ehrlichia species [ 9 , 31 ]. The sequences we obtained for our nested gltA , however, were consistent with the 16S rRNA gene findings that the organisms present in the Caribbean domestic ruminants we studied were E. canis or closely related species.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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BackgroundThe 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.MethodsWe 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).ResultsOur 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 (Tm ~55.8 °C); E. chaffeensis and E. ewingii (Tm ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (Tm ~62.0 °C); and the Panola Mountain Ehrlichia (Tm ~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.ConclusionsIn 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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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“…Similarly, the immunoreactive major outer membrane proteins p28 and p30 in U.S. and Venezuelan strains of E. canis appear to be highly conserved (13,29,30,46), an observation that was extended to characterized E. canis strains from six human patients from Venezuela (38). Other genes such as the thio-oxidoreductase gene (dsb) and gltA were also found to be conserved in geographically dispersed strains (23,32).…”

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confidence: 81%

Genetic and Antigenic Diversities of Major Immunoreactive Proteins in Globally DistributedEhrlichia canisStrains

Zhang

Luo

Keysary

et al. 2008

Clin Vaccine Immunol

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The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S3P and P3T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.

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“…In France, Jouret-Gourjault et al (2006) intravenously inoculated four naïve Beagles with the E. canis Borgo 89 strain isolated from sick dog in Corsica, and observed clinical CME comparable to that reported for other strains with one case of unapparent infection. Marsilio et al (2006) used a citrate synthase gene-based PCR assay to detect E. canis in three of 14 blood samples from seropositive dogs in Italy, and amplicon sequence of two of these samples revealed 99.2% identity to the Oklahoma strain. Solano-Gallego et al (2006b) estimated the prevalence of E. canis with real-time PCR from blood of Italian dogs, and reported prevalence in northern, central and southern Italy of 2.9%, 8% and 9.7%, respectively.…”

Section: E Canis Host Surveysmentioning

confidence: 99%

Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis

Stich

Schaefer

Bremer

et al. 2008

Veterinary Parasitology

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The ehrlichioses have been subject to increasing interest from veterinary and public health perspectives, but experimental studies of these diseases and their etiologic agents can be challenging. Ehrlichia canis, the primary etiologic agent of canine monocytic ehrlichiosis, is relatively well characterized and offers unique advantages and opportunities to study interactions between a monocytotropic pathogen and both its vertebrate and invertebrate hosts. Historically, advances in tick-borne disease control strategies have typically followed explication of tick-pathogen-vertebrate interactions, thus it is reasonable to expect novel, more sustainable approaches to control of these diseases as the transmission of their associated infections are investigated at the molecular through ecological levels. Better understanding of the interactions between E. canis and its canine and tick hosts would also elucidate similar interactions for other Ehrlichia species as well as the potential roles of canine sentinels, reservoirs and models of tick-borne zoonoses. This article summarizes natural exposure studies and experimental investigations of E. canis in the context of what is understood about biological vectors of tick-borne Anaplasmataceae.

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Direct Identification of Ehrlichia Canis by a Novel Polymerase Chain Reaction Method and Molecular Analysis of the Citrate Synthase (gltA) Gene from Various Italian Strains

Cited by 9 publications

References 8 publications

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

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

Genetic and Antigenic Diversities of Major Immunoreactive Proteins in Globally DistributedEhrlichia canisStrains

Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis

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