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Detection of Anaplasma marginale DNA in Larvae of Boophilus microplus Ticks by Polymerase Chain Reaction
Author(s) -
SHIMADA MÁRCIA KIYOE,
YAMAMURA MILTON HISSASHI,
KAWASAKI PAULA MIYUKI,
TAMEKUNI KÁTIA,
IGARASHI MICHELLE,
VIDOTTO ODILON,
VIDOTTO MARILDA CARLOS
Publication year - 2004
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1307.012
Subject(s) - biology , amplicon , polymerase chain reaction , larva , veterinary medicine , dna extraction , tick , anaplasma , dna , nested polymerase chain reaction , microbiology and biotechnology , virology , gene , genetics , botany , medicine
A bstract :Boophilus microplus larvae from two different sources were used for the detection of Anaplasma marginale DNA: larvae A, which were collected from a pasture of an endemic farm, and larvae B, which originated from engorged female ticks fed on calves with no clinical signs of disease and with low rickettsemia (approximately 0.01 to 1.0%). Larvae A were collected monthly, from January to May in 2001. Two hundred engorged female ticks fed on calves that provided larvae B were divided into groups of 10 and kept in a controlled environment at either 18°C or 28°C. Fifty larvae were used from each sample for DNA extraction, and 5 μL of DNA were submitted to amplification of the sequence of msp5 gene of A. marginale by polymerase chain reaction (PCR). Seven out of 50 samples of larvae A (14%) were positive for the presence of DNA of A. marginale showing amplified product of 457 bp. Ten out of 91 samples of larvae B (11%) kept at 18°C were positive, and all larvae B at 28°C were negative. Thus, this study confirmed the presence of A. marginale DNA in B. microplus larvae by PCR. The Eco RI restriction enzyme analysis confirmed the specificity of the amplicon, which resulted in two fragments: 265 bp and 192 bp. The sequencing analysis of the amplicon from larvae demonstrated 98% homology with the msp5 sequence from Florida A. marginale strain.