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Background   Brucella melitensis  bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of  Brucella  to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection.    Results  Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize  B. melitensis  RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered  Brucella  RNA from supramammary lymph nodes of  B. melitensis -infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of  Brucella  gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at  B. melitensis  gene expression patterns in vivo, the subset of  Brucella  genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction.    Conclusions  Overall, this study provides the first example of recovery plus characterization of  B. melitensis  RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in  Brucella  species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in  Brucella  persistence in natural host systems.    Electronic supplementary material  The online version of this article (10.1186/s12867-018-0111-x) contains supplementary material, which is available to authorized users.

Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis