Skip directly to local search Skip directly to A to Z list Skip directly to navigation Skip directly to site content Skip directly to page options
CDC Home

Volume 6, Number 5—October 2000

Research

Naturally Occurring Ehrlichia chaffeensis Infection in Coyotes from Oklahoma

Alan KocanComments to Author , Gena Crowder Levesque, Lisa C. Whitworth, George L. Murphy, Sidney A. Ewing, and Robert W. Barker

Author affiliations: Oklahoma State University, Stillwater, Oklahoma, USA

Article in Chinese

Main Article

Figure

Agarose gel electrophoresis of results of PCR amplification of Ehrlichia chaffeensis nss rRNA gene from whole blood samples of coyotes numbers 9-11.* Lane 1= negative control (no DNA); Lane 2= coyote 9 (+); Lane 3= coyote 10 (+); Lane 4= coyote 11 (-); Lane 5 = positive control (E. chaffeensis-infected DH82 cells). M = 100-bp DNA ladder (Life Technologies, Rockville, MD).* * Ehrlichia forward primer ECC (5'-AGAACGAACGCTGGCGGCAAGC-3') and Ehrlichia reverse primer ECB (5'-CGTATTACCGCGGCTGCTGGCA-3'

Figure. Agarose gel electrophoresis of results of PCR amplification of Ehrlichia chaffeensis nss rRNA gene from whole blood samples of coyotes numbers 9-11.* Lane 1= negative control (no DNA); Lane 2= coyote 9 (+); Lane 3= coyote 10 (+); Lane 4= coyote 11 (-); Lane 5 = positive control (E. chaffeensis-infected DH82 cells). M = 100-bp DNA ladder (Life Technologies, Rockville, MD).* * Ehrlichia forward primer ECC (5'-AGAACGAACGCTGGCGGCAAGC-3') and Ehrlichia reverse primer ECB (5'-CGTATTACCGCGGCTGCTGGCA-3') amplified all Ehrlichia spp (12,18). These reactions (50 l) contained 10 l of template DNA in 10 mM Tris-Cl (pH 8.3), 0.2 mM each deoxynucleoside triphosphate (dNTP), 2 mM MgCl2, 50 mM KCl, 0.5 m each primer, and 1.25 U of Taq DNA polymerase (Promega Corporation, Madison, WI). A hot-start PCR was used in which each enzyme was added to reactions after an initial 3-min denaturation step at 94°C. Reactions consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 2 min, and extension at 72°C for 2 min. Products of this reaction were used as template with species-specific primer sets for three nested reactions. Primers HE1 (5'-CAATTGCTTATAACCTTTTGGTTATAAAT-3') and HE3 (5'-TATAGGTACCGTCATTATCTTCCCTAT-3') (17) were used for E. chaffeensis-specific amplifications. Primers ECAN5 (5'-CAATTATTTATAGCCTCTGGCTATAGGA-3') (12,13) and HE3 were used for E. canis-specific amplifications, and primers EE5 (5'-(CAATTCCTAAATAGTCTCTGACTATTTAG-3') (this study) and HE3 were used for E. ewingii-specific amplifications. Reactions (50 l) contained 10 l of the reaction product with ECC and ECB primers as template, and the remaining reaction components as above. A hot-start PCR was used in which the enzyme was added to reactions after an initial 3-min denaturation step at 94°C. Reactions with species-specific primers were in two stages. The first consisted of three cycles of denaturation at 94°C for 1 min, annealing at 55oC for 2 min, and extension at 72°C for 1.5 min. The second consisted of 37 cycles of denaturation at 92°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 1.5 min. Distilled, deionized water served as a negative control. Positive control DNA samples were purified from E. chaffeensis-infected DH82 cells, blood from a dog experimentally infected with E. canis, and diluted general primer PCR reactions of synovial fluid from a dog experimentally infected with E. ewingii. To prevent contamination of samples, DNA purification, PCR master mix assembly, and amplifications were performed in separate rooms. Positive displacement pipetters and aerosol-free pipette tips were also used as further precautions.

Main Article

Top of Page

 

Past Issues

Select a Past Issue:

World Malaria Day - April 25, 2014 - Invest in the future, defeat malaria

20th Anniversary - National Infant Immunization Week - Immunization. Power to Protect.

Art in Science - Selections from Emerging Infectious Diseases
Now available for order



CDC 24/7 – Saving Lives, Protecting People, Saving Money. Learn More About How CDC Works For You…

USA.gov: The U.S. Government's Official Web PortalDepartment of Health and Human Services
Centers for Disease Control and Prevention   1600 Clifton Rd. Atlanta, GA 30333, USA
800-CDC-INFO (800-232-4636) TTY: (888) 232-6348 - Contact CDC–INFO