Development of a novel molecular detection method for clustered regularly interspaced short palindromic repeats (CRISPRs) in Taylorella organisms.
Contagious equine metritis is a bacterial infectious disease of horses caused by Taylorella equigenitalis, a Gram-negative eubacterium. The disease has been described in several continents, including Europe, North America and Asia. A novel molecular method was developed to detect clustered regularly interspaced short palindromic repeats (CRISPRs), which were separated by non-repetitive unique spacer regions (NRUSRs) of similar length, in the Taylorella equigenitalis EQ59 strain using a primer pair, f-/r-TeCRISPR-ladder, by PCR amplification. In total, 31 Taylorella isolates (17 T. equigenitalis and 14 Taylorella asinigenitalis) were examined. The T. equigenitalis isolates came from thoroughbred and cold-blooded horses from nine countries during 1980-1996, whilst the T. asinigenitalis isolates all originated from donkey jacks in France and the USA during 1997-2006. PAGE fractionated all of the 13 CRISPRs separated by 12 NRUSRs in T. equigenitalis EQ59. Permutation examples of CRISPRs, which were separated by NRUSRs for small-sized ladders, consisting of two doublet bands were shown. Putative CRISPRs separated by NRUSRs were amplified with 14/17 (82.4 %) geographically disparate T. equigenitalis isolates using the newly designed primer pair. Approximately 82.4 % of the T. equigenitalis isolates had CRISPRs separated by NRUSRs. The CRISPR locus was also found in the French T. asinigenitalis strain MCE3. Putative CRISPRs separated by NRUSRs were detected similarly in 4/14 (28.6 %) T. asinigenitalis isolates. Overall, a more detailed understanding of the molecular biology of CRISPRs within Taylorella organisms may help elucidate the pathogenic virulence and transmission mechanisms associated with this important equine pathogen.
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Yasushi
Y
Hara
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Takuya
T
Nakajima
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Marie
M
Akamatsu
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Motoki
M
Yahiro
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Shizuko
S
Kagawa
AFSSA, Laboratoire d'Etudes et de Recherches en Pathologie Equine, IPC, Goustranville 14430, France.
Sandrine
S
Petry
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Motoo
M
Matsuda
Centre for Infection and Immunity, Queen's University, Belfast BT9 7AB, UK.|Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast BT9 7AD, UK.
John E
JE
Moore
D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D021901:DNA, Intergenic / Q000235:genetics; D005260:Female; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D006734:Horse Diseases / Q000175:diagnosis* / Q000382:microbiology; D006736:Horses; D008297:Male; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020625:Taylorella equigenitalis / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D021901:DNA, Intergenic