Detection of Brucella in Tissues and in Formalin-Fixed Paraffin- Embedded (FFPE) Specimens Using PCR

Research Abstract

The present study investigated different diagnostic techniques for the detection of Brucella species in tissue specimens obtained from cattle including culturing technique, universal and Bruce ladder PCR assays based on fresh and formalin fixed paraffin embedding (FFPE) tissues as well as histopathological alterations. Specimens from 102 non-vaccinated brucella seropositive cows were used for isolation of brucellae. Brucella melitensis biovar 3 was isolated from 86 (84.31%) cows. Additionally, universal PCR showed the presence of Brucella DNA in all tissue extracts from which Brucella melitensis has been isolated as well as DNA extracts of their cultures. The assay has amplified the target gene (immunodominant antigen, gene bp26) with fragment size of 450 bp. Bruce ladder multiplex PCR proved the presence of genetic materials of Brucella melitensis in culture DNA extracts as it amplified three fragments (587, 1071 1682 bp in length). On the other hand, Bruce ladder multiplex PCR failed to detect DNA from fresh tissues. The use of FFPE technique on positive culture FFPE specimens, revealed the fingerprints of Brucella spp. DNA in udder, spleen, supramammary and retropharyngeal lymph node targeting 450 bp-fragment using the universal PCR. Moreover, Bruce ladder amplified only the 587 bp-fragment in only one (16.66%) sample out of 6 randomly selected positive universal PCR FFPE specimens. Pathological lesions in tissues of Brucella-infected animals consisted of micro-granulomatous reactions in lymph nodes and spleen. The current study tested brucellosis FFPE specimens and could effectively identify Brucella using specific primers by PCR. The immunodominant antigen, gene bp26, proved a good target for detection of DNA from FFPE tissues

Research Keywords

Brucella Tissue PCR