Diagnostic tests for a new spirochete, Borrelia lonestari sp. nov.

Abstract

Bites from Amblyomma americanum , a hard tick, have been associated with a Lyme disease-like illness in the southeastern and south-central United States. Present in 2% of ticks collected in four states were uncultivable spirochetes. Through use of the polymerase chain reaction, partial sequences of the flagellin and 16s rRNA genes of microorganisms from Texas and New Jersey were obtained. The sequences showed that the spirochete was a Borrelia sp. but distinct from other known members of this genus, including B. burgdorferi , the agent of Lyme disease. Species-specific differences in the sequences of the flagellin protein, the flagellin gene and the 16s rRNA gene between the new Borrelia species and previously known species provide compositions and methods for assay for determining the presence of this new spirochete, or for providing evidence of past or present infection by this spirochete in animal reservoirs and humans.

1 - 36 . (canceled)  
     
     
         37 . A purified nucleic acid segment comprising a nucleotide sequence of at least 14 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         38 . The purified nucleic acid segment of  claim 37 , wherein the purified nucleic acid segment comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         39 . The purified nucleic acid segment of  claim 38 , wherein the purified nucleic acid segment comprises at least 20 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         40 . The purified nucleic acid segment of  claim 39 , wherein the purified nucleic acid segment comprises at least 30 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         41 . The purified nucleic acid segment of  claim 40 , wherein the purified nucleic acid segment comprises at least 50 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         42 . The purified nucleic acid segment of  claim 41 , wherein the purified nucleic acid segment comprises at least 100 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         43 . The purified nucleic acid segment of  claim 42 , wherein the purified nucleic acid segment comprises at least 200 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         44 . The purified nucleic acid segment of  claim 43 , wherein the purified nucleic acid segment comprises at least 400 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         45 . The purified nucleic acid segment of  claim 44 , wherein the purified nucleic acid segment comprises the nucleotide sequence of SEQ ID NO: 1, or its complement.  
     
     
         46 . The purified nucleic acid segment of  claim 37 , wherein the purified nucleic acid segment comprises at least one  B. lonestari  sp. nov.-specific nucleotide selected from the group consisting of G70, G96, T141, A193, G199, G228, A231, T269, C270, T271, A273, A300, T308, G315, A376, G380, A406, G418, G423, G505, A510, G546, T572, and C603, or its complement.  
     
     
         47 . The purified nucleic acid segment of  claim 37 , wherein the purified nucleic acid segment comprises the nucleotide sequence of SEQ ID NO: 7, or its complement.  
     
     
         48 . The purified nucleic acid segment of  claim 37 , wherein the purified nucleic acid segment comprises the nucleotide sequence of SEQ ID NO: 8, or its complement.  
     
     
         49 . The purified nucleic acid segment of  claim 37 , wherein the purified nucleic acid segment encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or its complement.  
     
     
         50 . An expression vector comprising a nucleic acid segment operably attached to a promoter, wherein the nucleic acid segment comprises at least 14 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         51 . The expression vector of  claim 50 , wherein the purified nucleic acid segment comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         52 . The expression vector of  claim 51 , wherein the purified nucleic acid segment comprises at least 20 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         53 . The expression vector of  claim 52 , wherein the purified nucleic acid segment comprises at least 30 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         54 . The expression vector of  claim 53 , wherein the purified nucleic acid segment comprises at least 50 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         55 . The expression vector of  claim 54 , wherein the purified nucleic acid segment comprises at least 100 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         56 . The expression vector of  claim 55 , wherein the purified nucleic acid segment comprises at least 200 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         57 . The expression vector of  claim 56 , wherein the purified nucleic acid segment comprises at least 400 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         58 . The expression vector of  claim 57 , wherein the purified nucleic acid segment comprises the nucleotide sequence of SEQ ID NO: 1, or its complement.  
     
     
         59 . The expression vector of  claim 50 , further defined as a viral vector.  
     
     
         60 . The viral vector of  claim 59 , further defined as an adenoviral vector.  
     
     
         61 . The expression vector of  claim 50 , further defined as a plasmid vector.  
     
     
         62 . A host cell comprising an expression vector comprising a nucleic acid segment operably attached to a promoter, wherein the nucleic acid segment comprises at least 14 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         63 . The host cell of  claim 62 , wherein the host cell is  E. coli  LE392  
     
     
         64 . A kit for the detection of  B. lonestari  sp. nov. in a sample, the kit comprising: 
 (a) a carrier adapted to receive a plurality of containers in close confinement therewith;    (b) a first container comprising a nucleic acid sequence of at least 14 contiguous nucleotides of SEQ ID NO: 1, or its complement.    
     
     
         65 . The nucleic acid sequence of  claim 64 , wherein the purified nucleic acid segment comprises at least one  B. lonestari  sp. nov.-specific nucleotide selected from the group consisting of G70, G96, T141, A193, G199, G228, A231, T269, C270, T271, A273, A300, T308, G315, A376, G380, A406, G418, G423, G505, A510, G546, T572, and C603.  
     
     
         66 . The nucleic acid sequence of  claim 65 , wherein the purified nucleic acid segment comprises at least 20 contiguous nucleotides of SEQ ID NO: 1, or its complement.  
     
     
         67 . The nucleic acid sequence of  claim 66 , wherein the purified nucleic acid segment comprises at least 100 contiguous nucleotides of SEQ ID NO: 1, or its complement.