We describe here the complete genome sequence of a common clone ofMycobacterium aviumsubspeciesparatuberculosis(Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon.In silicoanalysis identified >3,000 genes with homologs to the human pathogen,M. tuberculosis(Mtb), and 161 unique genomic regions that encode 39 previously unknownMapgenes. Analysis of nucleotide substitution rates withMtbhomologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory duringMtbinfection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence ofMap; andMap-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology inMapand enables the development of new generations of diagnostic tests for bovine Johne's disease.

Leptospirosis is one of the most common zoonotic diseases in the world, resulting in high morbidity and mortality in humans and affecting global livestock production. Most infections are caused by eitherLeptospira borgpeterseniiorLeptospira interrogans, bacteria that vary in their distribution in nature and rely on different modes of transmission. We report the complete genomic sequences of two strains ofL. borgpeterseniiserovar Hardjo that have distinct phenotypes and virulence. These two strains have nearly identical genetic content, with subtle frameshift and point mutations being a common form of genetic variation. Starkly limited regions of synteny are shared between the large chromosomes ofL. borgpeterseniiandL. interrogans, probably the result of frequent recombination events between insertion sequences. TheL. borgpeterseniigenome is ≈700 kb smaller and has a lower coding density thanL. interrogans, indicating it is decaying through a process of insertion sequence-mediated genome reduction. Loss of gene function is not random but is centered on impairment of environmental sensing and metabolite transport and utilization. These features distinguishL. borgpeterseniifromL. interrogans, a species with minimal genetic decay and that survives extended passage in aquatic environments encountering a mammalian host. We conclude thatL. borgpeterseniiis evolving toward dependence on a strict host-to-host transmission cycle.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP^Sc, in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP^Sc was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP^Sc from case 1 showed molecular features similar to typical BSE isolates, whereas PrP^Sc from case 2 revealed an unusual molecular PrP^Sc pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within the range of the prion protein gene sequence diversity previously reported for cattle.

Objective—To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. Animals—24 Hereford heifers that lacked detectable antibodies against serovar hardjo. Procedure—Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. Results—All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urine and had evidence of renal colonization. All 4 control heifers challenged by conjunctival instillation and 2 of 4 control heifers challenged by intraperitoneal inoculation shed leptospires in their urine, and all 8 had evidence of renal colonization. In contrast, leptospires were not detected in the urine or tissues of any of the 8 heifers that received the commercial hardjo vaccine. Heifers that received the commercial hardjo vaccine had significantly higher antibody titers than did heifers that received the reference vaccine. Conclusions and Clinical Relevance—Results suggest that cattle that received 2 doses of the commercial hardjo vaccine were protected against renal colonization and urinary shedding when challenged with L borgpetersenii serovar hardjo strain 203 four months after vaccination. (Am J Vet Res 2001;62:995–1000)

Profiling of 16S rRNA gene sequences is an important tool for testing hypotheses in complex microbial communities, and analysis methods must be updated and validated as sequencing technologies advance. In host-associated bacterial communities, the V1–V3 region of the 16S rRNA gene is a valuable region to profile because it provides a useful level of taxonomic resolution; however, use of Illumina MiSeq data for experiments targeting this region needs validation. Using a MiSeq machine and the version 3 (300 × 2) chemistry, we sequenced the V1–V3 region of the 16S rRNA gene within a mock community. Nineteen bacteria and one archaeon comprised the mock community, and 12 replicate amplifications of the community were performed and sequenced. Sequencing the large fragment (490 bp) that encompasses V1–V3 yielded a higher error rate (3.6 %) than has been reported when using smaller fragment sizes. This higher error rate was due to a large number of sequences that occurred only one or two times among all mock community samples. Removing sequences that occurred one time among all samples (singletons) reduced the error rate to 1.4 %. Diversity estimates of the mock community containing all sequences were inflated, whereas estimates following singleton removal more closely reflected the actual mock community membership. A higher percentage of the sequences could be taxonomically assigned after singleton and doubleton sequences were removed, and the assignments reflected the membership of the input DNA. Sequencing the V1–V3 region of the 16S rRNA gene on the MiSeq platform may require additional sequence curation in silico, and improved error rates and diversity estimates show that removing low-frequency sequences is reasonable. When datasets have a high number of singletons, these singletons can be removed from the analysis without losing statistical power while reducing error and improving microbiota assessment.