Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose

Abstract

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations. Anaplasma marginale is an obligately intracellular bacterial pathogen of bovine erythrocytes from the order Rickettsiales (11). A. marginale causes anaplasmosis, a significant, worldwide (20), hemolytic disease of cattle. Many isolates of A. marginale exhibit differences in morphology (9), surface antigens (18), protein structure (1), vector specificity (27), and virulence (10). Knowledge of the relatedness among the various isolates of A. marginale has importance in taxonomic, epidemiological, virulence, and vaccine development

Phylogenetic Analysis of Anaplasma marginale Strains from Paraná State, Brazil, Using the msp1α and msp4 genes

“…Outer membrane proteins of A. marginale have been studied, and variants of the six major surface protein (MSP) genes ( msp 1 α , msp 1, msp 2, msp 3, msp 4 and msp 5) exist (Allred et al., 1990; Barbet and Allred, 1991; Visser et al., 1992; Oberle et al., 1993; Palmer et al., 1994, 2001; Alleman et al., 1997). Some studies have demonstrated genetic variation among different A. marginale strains by means of restriction fragment length polymorphism analysis (Krueger and Buening, 1988), Repetitive Extragenic Palindromic/Enterobacterial Repetitive Intergenic Consensus (REP/ERIC) PCR patterns (Ferreira et al., 2001), PCR assay based on specific sequences of MSPs (de la Fuente et al., 2001; Lew et al., 2002) and protein sequence (de la Fuente et al., 2001, 2004).…”

Use of repetitive DNA elements to define genetic relationships amongAnaplasma marginaleisolates

“…There are few studies about genetic relatedness among A. marginale isolates. The restriction endonuclease patterns produced from five different isolates (Florida, Louisiana, Oklahoma, St. Croix and Virginia) with the restriction enzymes Eco RI, Hin dIII, Bam HI, Sal I and Pst I, were not identical [25]. Restriction endonuclease patterns of Virginia, Florida and South Idaho strains by PFGE were similar, but not identical [6].…”

Use of repetitive DNA elements to define genetic relationships among Anaplasma marginale isolates

“…There are few studies about genetic relatedness among A. marginale isolates. The restriction endonuclease patterns produced from ¢ve di¡erent isolates (Florida, Louisiana, Oklahoma, St. Croix and Virginia) with the restriction enzymes EcoRI, HindIII, BamHI, SalI and PstI, were not identical [25]. Restriction endonuclease patterns of Virginia, Florida and South Idaho strains by PFGE were similar, but not identical [6].…”