M_bovis_Ravenel
Efficacy and immunogenicity of Mycobacterium bovis delta RD1 against aerosol M.bovis infection in neonatal calves
Genomic DNA fromthe M. bovis Ravenel strainwas prepared for  sequencing using standard protocols from the manufacturer (Illumina,  San Diego, CA). In brief, DNA was nebulized, end-repaired,  ligated to adapter oligos and 130–150 bp fragments were isolated  by size selection.DNA fragmentswere amplified by single molecule  PCR on the Illumina cluster station and then sequencing by synthesis  was performed through 36 cycles. The sequencing images  were analyzed using the Solexa pipeline v. 0.2.2.6 software and the  resulting 9.1 million reads were aligned to the M. bovis AF2122/97  [27] or the M. bovis BCG Pasteur 1173P2 sequence using SOAP software  [28]. Ninety-nine percent of the genome could be aligned to  reads to a depth of ten reads or greater where readswere aligned to  the genome with less than or equal to five mismatches. Nucleotide  changes from the reference sequence were called where at least  six separate reads called the alternate letter, and where the sum  of the Solexa quality scores for the alternate letter was fivefold  greater than the sum of the Solexa quality score for the reference  letter. A newreference sequencewas generated incorporating  these changes and the reads were then aligned with SOAP against  the edited genomic sequence. This process of alignment, detection  of polymorphisms, and sequence correction was iterated nine  times.  Solexa reads were aligned to reference sequences consisting of  transcripts plus 100 bp of flanking genomic sequence upstream and  downstream. SNPs were called by the criteria above, and new reference  sequence generated to reflect the changes. Nucleotides that  did not have a Solexa quality score sum of at least 120were deemed  not confirmable, andwere recorded as “N”. The resulting transcript  sequence with M. bovis Ravenel SNPs was aligned by BLASTX [36]  to M. bovis BCG Pasteur proteins (collected from the bovis Pasteur  page of TubercuList (http://genolist.pasteur.fr/BCGList/). 3414  (87%) of the M. bovis Ravenel translated sequences were perfect  matches to the BCG reference. 370 of the M. bovis Ravenel translated  sequences have amino acid differences compared to corresponding M. bovis BCG Pasteur proteins. All translated protein sequences  that contain non-determinable amino acids (secondary to nondeterminable  nucleotides) are listed in the supplementary Tables  2 and 3 and contain an “X” at the position of the unknown amino  acid.  In order to gain insight into potential evolutionary relationships  between the strains studied and the possible role of SNPs  in observed phenotypes, the SNPs were analyzed that resulted in  amino acid substitutions, deletions and gene truncations between  the previously sequenced M. bovis AF2122/97 and M. bovis Ravenel.  As a representative set, 115 of the 233 SNPs that resulted in differences  in the proteins coded for by annotated genes were analyzed.  Of the 115 SNPs characterized, nearly half of the M. bovis Ravenel  sequences had the consensus residue for the M. tuberculosis (MTB)  complex.
An attenuated Mycobacterium bovis RD1 deletion ( RD1) mutant of the Ravenel strain was constructed,  characterized, and sequenced. This M. bovis RD1 vaccine strain administered to calves at 2 weeks of age  provided similar efficacy as M. bovis bacillus Calmette Guerin (BCG) against low dose, aerosol challenge  with virulent M. bovis at 3.5 months of age. Approximately 4.5 months after challenge, both  RD1- and  BCG-vaccinates had reduced tuberculosis (TB)-associated pathology in lungs and lung-associated lymph  nodes and M. bovis colonization of tracheobronchial lymph nodes as compared to non-vaccinates. Mean  central memory responses elicited by either  RD1 or BCG prior to challenge correlated with reduced  pathology and bacterial colonization. Neither  RD1 or BCG elicited IFN-  responses to rESAT-6:CFP-10  prior to challenge, an emerging tool for modern TB surveillance programs. The  RD1 strain may prove  useful for bovine TB vaccine programs, particularly if additional mutations are included to improve safety  and immunogenicity.