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

Abstract

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods. ß

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

“…Therefore these results point toward the use of highly conserved within eubacterial kingdom REP and ERIC sequences for genomic analysis of haemoprotozoa as has been done for long time with bacteria (Versalovic et al 1991). The greater discriminatory capability of ERIC-PCR for intra-species genomic differentiation and complexity of bands of the REP-PCR is in agreement with that found in the differentiation of Actinobacillus seminis (Appuhamy et al 1998), Bartonella henselae strains (Sander et al 1998) and in different Brazilian isolates of the Rickettsia Anaplasma marginale (Ferreira et al 2001).…”

“…For REP-PCR, the following program was used: 95ºC for 5 minutes, 1 cycle; 94ºC for 1 minute, 45ºC for 1 minute, 65ºC for 8 minutes, 40 cycles, and 65ºC for 16 minutes, 1 cycle. The thermocycling of ERIC-PCR was 95ºC for 5 minutes, 1 cycle; 94ºC for 1 minute, 52ºC for 1 minute and 30 seconds, 72ºC for 8 minutes for 40 cycles and 72ºC for 16 minutes, 1 cycle (Ferreira et al 2001).…”

“…Random amplification of polymorphic DNA (RAPD) (Williams et al 1990), conventional restriction fragment length polymorphism (RFLP) (Conrad et al 1987) and its variation such as riboprinting, which involves polymerase chain reaction (PCR) amplification of the small-subunit ribosomal RNA (SSU-rRNA) (Clark et al 1995), pulsed field gel electrophoresis (PFGE) (Morzaria et al 1990) and amplified restriction fragment length polymorphism (Masiga et al 2000) are some of these techniques. Repetitive extragenic palindromic (REP) elements (Stern et al 1984) and enterobacterial repetitive intergenic consensus (ERIC) sequences (Hulton et al 1991) have been used to determine fingerprinting in the genome of various prokaryotic organisms (Versalovic et al 1991, Ferreira et al 2001) and in to a lesser extend in eukaryotic organisms (Gillings & Holley 1997) are also techniques that amplify fragments without the need of DNA sequencing. Among all, RAPD, REP-PCR and ERIC-PCR are the simplest reactions and use far less DNA than other methods.…”

Characterisation and genetic diversity via REP-PCR of Escherichia coli isolates from polluted waters in southern Brazil

“…The study of the microbial genetic diversity may be of use when planning bacterial control strategies and this study of intra‐specific diversity of E. coli isolates was the first step in the implementation, continuance, and evaluation of projects that address the recovery of the waters of Arroio Feijó. The use of REP sequences has been validated for an increasing number of bacterial genera, particularly those that raise health concerns [24–26], isolates of relevance in industrial activities [27,28], and phytopathogenic isolates [29]. The use of REP to assess isolates from the natural environment is restricted, but with the advances in local micro‐environment studies for the maintenance and recovery of the macro‐environment, the technique may be more widely used.…”

Identificación y caracterización molecular de plásmidos transmisibles por conjugación de aislamientos locales en Sinorhizobium meliloti

“…De acuerdo a lo que hemos presentado en la sección 2 precedente y en acuerdo con resultados publicados por otros autores con distintas bacterias, es común poner en evidencia una abundante diversidad genotípica en aislamientos de una misma especie cuando se emplean como herramientas de comparación genómica técnicas de "huella digital" por PCR (Aguilar et al, 1998;Ferreira et al, 2001). Como puede verse en la Tabla IILL, en S. meliloü hemos obtenido índices de diversidad del mismo orden cuando la generación de las unidades taxonómicas (OTUs) fueron realizadas ya sea a partir de "huellas digitales" por PCR, o mediante el empleo de perfiles plasmídicos.…”