Kl. Gage et al., DNA TYPING OF RICKETTSIAE IN NATURALLY INFECTED TICKS USING A POLYMERASE CHAIN REACTION RESTRICTION FRAGMENT LENGTH POLYMORPHISM SYSTEM/, The American journal of tropical medicine and hygiene, 50(2), 1994, pp. 247-260
Citations number
34
Categorie Soggetti
Public, Environmental & Occupation Heath","Tropical Medicine
We used the polymerase chain reaction/restriction fragment length poly
morphism (PCR/RFLP) rickettsial typing system of Regnery and others to
rapidly identify rickettsiae in naturally infected ticks. Unlike prev
iously described methods, our PCR assays type rickettsiae directly fro
m tick tissues without first isolating the organisms. We collected 226
adult Dermacentor andersoni ticks in the Bitterroot Mountains of west
ern Montana and analyzed them for possible rickettsial infection by he
molymph test using the Gimenez stain. Thirteen (5.8%) of these ticks w
ere positive by hemolymph test and selected for further analysis using
the above PCR/RFLP typing system. The PCR assays performed using the
first primer set (RpCS) resulted in amplification of fragments of the
predicted size from nine of the 13 hemolymph test-positive tick sample
s. Only four of these nine tick samples were also positive in similar
PCR assays performed with a second primer set (Rr190) that is presumed
to be spotted fever group specific. The RFLP analyses of material amp
lified from these four ticks indicated they were infected with Rickett
sia rickettsii (one sample) and R. rhipicephali (three samples). The P
CR/RFLP analyses of the five PCR-positive tick samples that were posit
ive only in assays performed with the RpCS primer set indicated that t
hese ticks were infected with R. bellii. The remaining four of 13 hemo
lymph test-positive tick samples gave negative PCR results with both t
he RpCS and Rr190 primer sets. Infected hemocytes from these PCR-negat
ive ticks contained organisms of distinctive bacillary morphology that
appeared similar to those described previously as long forms, and it
is possible that these organisms belong to a genus other than Ricketts
ia. We also examined established laboratory isolates of tick-borne ric
kettsiae from different regions of North America to determine whether
this typing system produces consistent results. Multiple isolates of R
. montana (nine isolates), R. bellii (five isolates), R. rickettsii (H
ip-like) (four isolates), and R. canada (two isolates) were tested and
no significant variations in PCR/RFLP patterns were observed between
members of the same serotypes. However, among the five isolates of R.
rhipicephali tested, two slightly different RFLP patterns were noted.
Our results suggest that this PCR/RFLP typing scheme has wide applicab
ility for identifying rickettsiae directly from D. andersoni or D. var
iabilis tick tissues.