However, the molecular weight of x-B12 and x-B16 fragments (6 6 a

However, the molecular weight of x-B12 and x-B16 fragments (6.6 and 5.5 kb, respectively) was different from those bearing the extra IS711 copies in 2308 (x-08, 1.9 kb that also includes the 3a copy) and RB51 (x-RB51, 1.5 kb) (Figure 1). Interestingly, whereas AZD1152 Strain B51, which was isolated

from the same sample as B12, displayed the genetic profile typical of B. abortus, strains B16, B49 and B50 showed an identical profile, even though they were from successive outbreaks in the same flock (Figure 1 and Table 1). These results show that it is possible to find B. abortus field isolates with different IS711 distributions. Table 1 Brucella strains used     Genetic profile by:   Strain Relevant features RFLP IS 711 Ava I -Cla I a AMOS enhanced PCR b Reference B. abortus 544 Reference

strain of biovar 1 A A [24] B. abortus 2308 USDA challenge strain; biovar 1 B B [25] B. abortus RB51 Vaccine Compound C rough derivative from 2308 C Trichostatin A B [26] B. abortus B51 c Biovar 1; milk isolate (Río Bueno, Chile; 2004) A A This work B. abortus B12 c Biovar 1; milk isolate (Río Bueno, Chile; 2004) D A [10] B. abortus B16 d Biovar 1; aborted fetus isolate (Osorno, Chile; 2002) E A [10] B. abortus B49 d Biovar 1; aborted fetus isolate (Osorno, Chile; 2000) E A This work B. abortus B50 d Biovar 1; aborted fetus isolate (Osorno, Chile; 2004) E A This work B. ovis 23/290 B. ovis reference strain F C [24] B. ceti NCTC 12891T B. ceti type strain Np e Np [27] B. pinnipedialis Cyclin-dependent kinase 3 NCTC 12890T B. pinnipedialis type strain Np Np [27] B. abortus 2308 NalR Nalidixic acid resistant derivative of 2308 strain Np Np [21] a IS profiles are shown in Figure 1. b A, B. abortus typical pattern; B, B. abortus 2308 pattern; C, B. ovis typical pattern. c B12

and B51 were isolated from the same sample. d B16, B49 and B50 are strains isolated from different outbreaks in the same flock. e Np: Not performed Figure 1 Identification of new IS 711 copies in B. abortus B12, B16, B49 and B50 by Southern blot. The new IS711 copies found in field isolates and the additional IS711 present in 2308 and RB51 are indicated on the left. The IS711-nomenclature proposed by Ocampo-Sosa et al. (2008) and the fragment size are indicated on the right (note that x-08 fragment includes both the additional 2308 strain and 3a copies). The signals marked with an * correspond to IS other than IS711 which show cross-hybridization. Capital letters at the bottom indicate the RFLP IS711 AvaI-ClaI profile (Table 1). We characterized the insertion sites in B12 and B16 (and B49 and B50) to ascertain whether they were new or already present in other brucellae. To this end, we carried out IS-anchored PCR using IS711-bound primers plus a decamer of %GC similar to that of the Brucella genome (Table 2). The resulting amplicons ranged from 0.2-3.

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