BRIEF NOTE

A glass bead protocol for recovery of host cell free Ehrlichia canis and quantification by Sybr-green real-time PCR

G. P. Cardozo¹, E. V. Santos¹, A. L. Fachin¹, S. C.  França¹, and M. Marins^1,2,* 

^1.  Unidade de Biotecnologia, UNAERP, Ribeirão Preto, Brazil.
^2.  Heranza - Biotecnologia, Ribeirão Preto, Brazil.

*Address correspondence to:

Mozart Marins.
Unidade de Biotecnologia, UNAERP, Ribeirão Preto, Brazil. Tel.: (+55-16) 3603 6892. Fax: (+55 16) 3603 7030. E-mail: marins@heranza.com.br

Received: May 27, 2010.
Acepted: March 30, 2011.

Abstract: E. canis infection of the canine cell line DH82 is a routine in studies with this bacteria. A protocol for isolation of host cell free bacteria was developed based on the use of glass beads. Improvement of infection with E. canis isolated by this method was detected by real-time PCR.

Key words: Ehrlichia canis; Glass bead; Real-time PCR.

The Anaplasmataceae family of Gram-negative endobacteria are tick-borne pathogens of human and veterinary interest. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, is one of the most important species affecting dogs worldwide. The in vitro cultivation of these bacteria in the canine macrophage cell line DH82 is a powerful tool for studies of host-pathogen interaction and validation of susceptibility to antibiotics (Branger et al., 2004; Cheng and Ganta, 2008). Moreover, studies of gene expression of in vitro cultured E. canis have the potential of identifying targets for the development of new drugs to combat endobacteria. Methods for purifying host cell-free E. canis have been described and are an important pre-requisite for such studies (Zhang et al., 2007; Cheng and Ganta, 2008). In this communication we present our protocol, modified from Cheng and Ganta (2008) for isolation and storage by freezing of host cell free E. canis. We also present a Sybr-Green real-time PCR, based on the major surface protein 4 gene sequence (msp4), for detection of the bacteria.
The E. canis São Paulo strain (Aguiar et al., 2008) was used for infection of DH82 cells cultured in 25 cm² flasks, in cubated at 37ºC, 5% CO₂. After reaching 90 to 100% infection, the cells were detached from the flask bottom with the help of a disposable scraper and homogenized. A 5 ml aliquot was transferred to a sterile centrifuge conical tube containing about 200 glass beads. After homogenization, tubes were centrifuged for 1 minute at 150 g. The supernatant was divided into 1 ml aliquots and transferred to 1,5 ml microtubes. After centrifugation for 15 minutes, at 15.500 g and 4ºC, the supernatant was discarded and the cell pellet ressuspended in 500 µl of  freezing medium consisting of 70% culture media, 20% fetal bovine serum and 10% DMSO. Stocks were frozen at -80ºC.
The defrosted content of a microtube was used for infecting healthy DH82 cells and the presence of morulae was confirmed by Diff-Quick staining (LaborClin®, Brasil), after five days infection. These results demonstrate that our isolation and freezing method generates viable E. canis cells for post infection of DH82 cells.
A Sybr-Green real time PCR assay was also employed for quantification of E. canis after isolation by the glass beads method. The DNEasy Blood & Tissue kit (Qiagen) was used for the extraction of genomic DNA of the isolated bacteria. Two newly designed primers p28fwd3 5'-CAAGCATGTCCTCCGCAAG-3' and p28rev3 5'-ATCAGTACCAACACCTGCAC-3' were used for amplification and were based in the msp4 gene sequence of the E. canis Jake strain (GenBank: CP000107). The amplicon genereated was 146 pbs long and encompassing positions 1279114 to 1279259 of the reference sequence. PCR reactions and fluorescence detection were performed using a Stratagene Mx3000P (Stratagene, La Jolla, CA) and a Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA). The final volume of reactions were 25 µl and contained 150 nM of each primer. The thermal cycler program was 95ºC for 10 min, followed by 40 cycles of 95ºC for 15 s, 60ºC for 20 s, 72ºC for 20 s, followed by a dissociation curve.
The comparison of Ct values in the samples with known amounts of DNA indicates that the concentration of isolated bacteria are 1,63x10⁵ bacterial cells/µL, when glass beads were used for isolation, and 1,3x10³ bacterial cells/µL when glass beads were not used. The protocols described in the literature for isolation of Ehrlichia sp. use sonication for homogenization and release of the bacteria from host cells ^{(3, 6)}. Alternativelly, density gradient centrifugation is also used ⁽⁵⁾. Ganta et al. (2007) used glass beads for dispersing E. chaffeensis, a related E. canis species, and immediate use without freezing in the infection of mice. Zhang et al. (2004) also used sonication and storage by freezing of E. chaffeensis for infection of the human monocyte cell line THP1. In the case of E. canis, only the methods using sonication were described until now and without freezing the isolated bacterial cells before infection.
Our method uses glass beads in the step of homogenization to increase the release and concentration of isolated bacterial cells. Further optimization of this protocol will allow sinchronization of infection of DH82 cells. This will facilitate, for example, in vitro experiments for screening of drugs which can block adherence of the bacteria to the host cell or the process of lysosome and phagosome fusion during the life cycle of Ehrlichia spp (Zhang et al., 2007).

Acknowledgments

This study was supported by funds from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant. 07/07234-6). We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for schoolarships granted to G.P.C. and E.V.S., the staff of the Biotechnology Unit-UNAERP for general support. We thank Prof. Dr. M.B. Labruna and D.M.Aguiar for kindly supplying the E. canis São Paulo strain.

References

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