We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (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 silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs 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 PE͞PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; 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 of Map; and Map-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 in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophilicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.Johne's disease is a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (reviewed in reference 5). The disease occurs in wild and domestic ruminants, including dairy cattle, and has considerable impact on the global agricultural economy. The slow growth of the organism in laboratory culture and its extensive genetic relatedness with Mycobacterium avium subsp. avium (4, 7) have hindered diagnosis of Johne's disease using methods such as bacterial isolation, genomic assays, and serology. Recent investigations showed that the current enzyme-linked immunosorbent assay (ELISA)-based immunoassays have poor sensitivity, detecting fewer than one-third of all infected cattle (6). Furthermore, the use of crude M. avium subsp. paratuberculosis protein mixtures as antigens compromises assay specificity due to conservation of proteins across Mycobacterium avium complex organisms. Hence, identification of suitable M. avium subsp. paratuberculosis antigens that could enable early, sensitive, and specific detection of M. avium subsp. paratuberculosis infection is critically needed to facilitate adequate disease control measures.In order to capitalize on the availability of the complete genome sequence of M. avium subsp. paratuberculosis (10) for novel antigen discovery, we describe here the application of an in vitro transcription and translation system that enables expression of M. avium subsp. paratuberculosis recombinant proteins directly from transcriptionally active PCR (TAP) fragments (11). This approach obviates the need for cloning of individual genes and expression of proteins in a heterologous system. It is also amenable for adaptation to a high-throughput format and enables the expression of hundreds of genes in days versus the months needed for cloning-based expression. Hence, this method is labor-, time-, and cost-effective and is ideal for large-scale antigen discovery.In order to evaluate the utility of this approach for rapid expression and screening of potential antigens for use in M. avium subsp. paratuberculosis immunodiagnostics, we chose two candidate M. avium subsp. paratuberculosis open reading frames (ORFs), MAP1272c and MAP2121c, that have not previously been characterized either functionally or immunologically. Preliminary computational and comparative genomic analyses suggest...

p53 status is an indicator of postsurgical recurrence and overall survival in cats with VAFS. Careful follow-up is important in treating vaccine-site tumors containing allelic deletion of p53, whereas aggressive surgical treatment may be sufficient to control primary vaccination site tumors without the allelic loss.

A case-control study was conducted in order to investigate the association of polymorphisms in the genomic sequence of the feline p53 gene with a predisposition to vaccine-associated feline sarcoma (VAFS). In the study, 50 domestic short hair cats with a confirmed histopathologic diagnosis of VAFS were matched to disease-free controls (1:2) by age, sex, and breed. Cats from both the diseased (case) and control groups were also negative for feline leukemia virus and feline immunodeficiency virus. Germ-line DNA was prepared from blood samples from cats in both groups and analyzed for sequence variation at 8 polymorphic sites in the p53 gene. A strong association was found between VAFS and the presence of specific nucleotides at 2 of the polymorphic sites. The strongest association was observed for a single-base insertion in intron 7 of the gene with an odds ratio of 8.99 (95% confidence interval = 3.42-23.57, P < 0.0001). The results of the study indicate that analysis of the presence or absence of the identified genetic markers in apparently healthy disease-free cats may help in predicting which individual animals are at greater risk of developing the disease.

The establishment of VAFS cell lines provides a tool for the in vitro screening of antitumor drugs. Doxorubicin and mitoxantrone were effective in decreasing the number of viable cells in the 2 cell lines tested. Both of these anthracycline antibiotics have been used to treat various neoplasias in cats, and their efficacy for adjuvant treatment of vaccine-associated sarcomas should be further evaluated.

In an effort to understand the molecular genetic basis of temporal variation in frequency and severity of bacterial disease, genetic relationships among strains of Streptococcus pyogenes that caused scarlet fever epidemics in Canada in the early 1940s and in eastern Germany in the 1960s to 1980s were studied. Application of multilocus enzyme electrophoresis and comparative sequencing of the gene (speA) encoding streptococcal pyrogenic exotoxin A (scarlet fever toxin) revealed that new waves of scarlet fever are associated with an increase in frequency of S. pyogenes clones carrying variant speA alleles. This finding suggests that the occurrence of new scarlet fever epidemics can be predicted by comprehensive monitoring of the frequency of S. pyogenes clones with variant toxin alleles.