Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli

Peleg, Ofer; Baneth, Gad; Eyal, Osnat; Inbar, Jacob; Harrus, Shimon

doi:10.1016/j.vetpar.2010.06.039

Cited by 64 publications

(61 citation statements)

References 22 publications

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“…Several PCR platforms, targeting a variety of determinants i.e. , 16S rRNA, p28 , p30 , dsb , Vir B9, were developed and demonstrated [7–12]. Although these PCR based techniques were useful for experimental investigations, the limitation of PCR in term of the lack of assay simplicity, its time consumption and its dependence on laboratory facilities both for a thermal cycler device and technical hands-on experience, have created a barrier to field site application of the technique as a point-of-care test approach.…”

Section: Introductionmentioning

confidence: 99%

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

Muangchuen

Chaumpluk

Suriyasomboon

et al. 2014

Sensors

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Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.

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Section: Introductionmentioning

confidence: 99%

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

Muangchuen

Chaumpluk

Suriyasomboon

et al. 2014

Sensors

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“…They identify antibodies specific for E. canis which are bound to specific, purified, recombined antigen of E. canis. The most sensitive diagnostic method of erlichiosis is, however, the identification of the microorganisms in cell cultures of histiocytes or monocytes, together with the isolation of the Ehrlichia's DNA with PCR technique (Brouqui et al 1994, McBride et al 1996, Pusterla et al 2000, Peleg et al 2010.…”

mentioning

confidence: 99%

Monocytic Ehrlichiosis in dogs

Procajło¹,

Em²,

Bladowski³

et al. 2011

Polish Journal of Veterinary Sciences

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Ehrlichiosis is the multiorgan infectious disease caused by small, intracellular rickettsias from the genus Ehrlichia. These microorganisms are known as an etiologic factor of infections world wide in humans and in different species of animals. Dog ehrlichiosis can be caused by several species of Ehrlichia attacking different groups of blood cells, but most often an infection by Ehrlichia canis is diagnosed with special relation to monocytes. A vector for E. canis are Rhipicephalus sanguineus and Ixodes ricinus, commonly occurring in Poland. Disease caused by E. canis is known as Canine Monocytic Ehrlichiosis (CME). The disease most often has an asymptomatic course which can, in favourable circumstances, run into acute or chronic forms. The acute form of CME proceeds usually with fever, apathy, weakness and accompanying respiratory symptoms, lameness and disturbances in blood coagulation. In laboratory examinations thrombocytopenia, anemia and leucopenia are ascertained. The chronic form of CME proceeds among gentle, unspecific symptoms which may last even 5 years. The CME diagnosis is difficult and often demands parallel different diagnostic methods. A medicines of choice in the ehrlichiosis treatment are antibiotics from the group of tetracyclines, given at least for 28 days. They are largely efficient during treatment of the acute CME, causing the quick improvement. Instead, in the case of chronic form, answer for treatment can be weak, and cases of resistance to antibiotics ave known.

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“…Özellikle uluslararası seyahat aktivitelerinin artması, insanların seyahatlerinde köpeklerini yanlarında götürmeleri, iklim ve ekolojik değişiklikler enfeksiyonların global yayılmasına imkan sağlayarak risk potansiyelini artırmaktadır. Bu çerçevede köpeklerde klinik ve subklinik seyirli babesiosis, hepatozoonosis, anaplasmosis ve ehrlichiosis gibi kenelerlerle nakledilen enfeksiyonların takipleri gereklidir (12,24,36,38).…”

Section: Tartışma Ve Sonuçunclassified

“…Son yıllarda köpeklerde bu hastalıkların moleküler epizootiyolojileri ile ilgili önemli bilgilerin elde edildiği ve birçok yeni etkenin tanımlandığı görülmektedir (12,24,36,38 E. canis TrKysEcan1-3 izolatlarının 16S rRNA gen bölgesine göre kendi aralarında %100 identiklik gösterdiği, dünyadaki izolatlarla aralarında %0,1 farklılık bulunduğu saptanmıştır. A. phagocytophilum TrKysAp1-3 izolatları, ankA gen bölgesine göre kendi aralarında %99,8±0,2 identik bulunmuş, dünyadan filogenetik analizlere dahil edilen diğer izolatlarla ise aralarında %0,9±0,3 genetik farklılık saptanmıştır.…”

Section: Introductionunclassified

Köpeklerde kene kaynaklı bazı protozoon ve rickettsial enfeksiyonların Real Time PCR ile araştırılması ve saptanan izolatların moleküler karakterizasyonları

Önder; İNCİ

2014

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Özet: Bu canis vogeli ve B. gibsoni izolatlarının 18S rRNA gen bölgesine ait sekanslarının dünyadaki aynı türden diğer izolatlarla ikili kıyaslamalarında %1,4±0,2, %0,3±0,2 ve %0,9±0,3 genetik farklılık saptanmıştır. E. canis için 16S rRNA gen bölgesi yönünden üç izolatın kendi aralarında %100 identik olduğu, dünyadaki izolatlarla ise %0,1 farklılık gösterdiği; A. phagocytophilum için ankA gen bölgesi yönünden üç izolatın kendi aralarında %99,8±0,2 identik olduğu, dünyadaki izolatlarla ise %0,9±0,3 farklılık gösterdiği; H. canis için 18S rRNA geni yönünden iki izolatın kendi aralarında %100 identik olduğu, dünyadaki izolatlarla ise %0,2±0,1 farklılık gösterdikleri tespit edilmiştir. Sonuç olarak bu çalışmayla Kayseri yöresinde köpeklerde kene kaynaklı protozoon ve rickettsial enfeksiyonların moleküler prevalansları saptanmış ve enfeksiyonlara yol açan türlerin çeşitli gen bölgeleri analiz edilerek moleküler karakterizasyonları yapılmıştır. Tüm izolatların Genbank kayıtları gerçekleştirilmiştir.Anahtar sözcükler: Anaplasmosis, babesiosis, ehrlichiosis, hepatozoonosis, köpek, moleküler karakterizasyon, Real Time PCR. B. gibsoni, A. phagocytophilum, H. canis, and B. canis vogeli was detected as 14.5%, 12.0%, 9.0%, 7.8%, 5.3%, and 2.3%, respectively while B.canis rossi was not detected in the examined samples. 182 (89.7%) out of the 400 samples were found to be infected with a single parasite species, and 21 (10.3%) were found to be infected with two species. According to the pairwise comparisons of 18S rRNA gene region of the isolates under B. canis canis, B.canis vogeli and B. gibsoni, 1.4±0.2%, 0.3±0.2%, and 0.9±0.3% genetic distance were detected with the other similar isolates from the world, respectively. Based on the 16S rRNA gene sequence alignments, 100% identity was found among the 3 isolates of E. canis while 0.1% genetic difference was determined with the isolates from the world. With respect to ankA gene region of A. phagocytophilum, 99.8±0.2% identity and 0.9±0.3% genetic difference were found among the 3 isolates obtained from Kayseri region and the isolates from the world, respectively. The 2 H. canis isolates were showed 100% identity to each other and 0.2±0.1% genetic difference were determined with the other isolates from the world with respect to 18S rRNA gene region. In conclusion, the molecular prevalence of tick-borne protozoon and rickettsial infections in dogs in Kayseri region was determined and the molecular characterizations of the obtained isolates were performed by analyzing the various gene regions in this study. All isolates were recorded to GenBank.

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Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli

Cited by 64 publications

References 22 publications

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

Monocytic Ehrlichiosis in dogs

Köpeklerde kene kaynaklı bazı protozoon ve rickettsial enfeksiyonların Real Time PCR ile araştırılması ve saptanan izolatların moleküler karakterizasyonları

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