Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-24
2003-09-09
Duffy, Patricia A. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S350000
Reexamination Certificate
active
06617441
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of infection and disease. More particularly, it concerns the identification of a new spirochete carried by the hard tick,
Amblyomma americanum
, found by the present inventor to be associated with a Lyme disease-like illness in the southeastern and south-central United States. Most particularly, the invention provides compositions, methods, and kits for the identification of the new spirochete for diagnostic purposes.
DESCRIPTION OF THE RELATED ART
A paradox about Lyme disease is the report of this tick-borne infection from areas in which transmission of the etiologic agent,
B. burgdorferi
, has not been documented (Sigal et al., 1991; Barbour et al., 1993). This phenomenon has been reported from Georgia and Missouri, but may be common in other parts of the southeastern and south-central United States (Centers for Disease Control and Prevention, 1989; 1991). The Lyme disease-like illness is a localized, expanding circular skin rash, sometimes succeeded by persistent, debilitating systemic symptoms (Masters, 1993; Donnell, 1992). Many of the patients with this illness have had negative serologic assays for antibodies to
B. burgdorferi
, a finding that has fueled a controversy about so-called “seronegative Lyme disease” (Sigal et al., 1991; Barbour et al., 1993). Although
Ixodes scapularis
ticks, the usual vector of the Lyme disease agent, has been identified in some of these geographic areas, the more commonly reported exposure for these patients has been to another hard tick,
A. americanum
, known as the “Lone Star tick” (Centers for Disease Control and Prevention 1989; 1991; Masters, 1993; Donnell, 1992). One conclusion from these observations is that the disease is caused by something other than
B. burgdorferi
and that the vector of the putative agent is
A. americanum
(Maupin et al., 1992).
The incompetence of
A. americanum
as a vector of
B. burgdorferi
has been documented (Piesman et al., 1988; Mather et al., 1990; Mukolwe et al., 1992; Ryder et al., 1992). Nevertheless, there have been descriptions in these ticks of spirochetes that cross-react with antibodies to the Lyme disease agents (Maupin et al., 1992; Schulze et al., 1984). Until the discovery of
B. burgdorferi
and related Borrelia species in Ixodes spp. ticks a decade ago, Borrelia spp. had almost exclusively been found in soft or argasid ticks (Barbour et al., 1986).
Reports from several locations in the southeastern and south-central regions of the United States indicate that this Lyme disease-like illness, which is apparently ameliorated by antibiotics, is associated with bites by the Lone Star tick (Centers for Disease Control and Prevention, 1989; 1991; Masters, 1993; Donnell, 1992).
A. americanum
is a common person-biting tick in these areas (Cooney et al., 1974; Koch et al., 1980; Hair et al., 1986; Bloemer et al., 1990). Its usual hosts are white-tailed deer, medium-sized mammals, and ground-feeding birds; rodents are only rarely infested by
A. americanum
. The tick's distribution extends from west-central Texas to Florida and north to Rhode Island (Cooney et al., 1974; Koch et al., 1980; Hair et al., 1986; Bloemer et al., 1990).
Numerous references in the literature relate to aspects of diagnosing and treating Lyme disease. For example: i) U.S. Pat. No. 5,279,938 relates to a nucleotide sequence of a recombinant clone containing a specific segment of
Borrelia burgdorferi
(Bb) DNA, the causative agent of Lyme disease; ii) an abstract by Barthhold (WPI Acc. No.: 92-041321/05) relates to OSPA polypeptides immuno-reactive with antibodies generated by the spirochete
Borrelia burgdorferi
; iii) The Weisburg world patent publication relates to nucleic acid fragments that are used to detect the etiological agent of Lyme disease, Borrelia; iv) The Oliver et al. (1993) abstract relates to a study of the isolation and transmission of the Lyme disease spirochete; v) The Berland et al. (1991) abstract relates to the characterization of a 41 kDa flagellin antigen of
B. burgdorferi
; vi) The Mukolwe et al. (1992) article relates to attempts to transmit the
B. burgdorferi
(Bb) spirochete to three different ticks, one of these being the
Amblyomma americanum
tick. The test results report transfer of the Bb spirochete only to Ixodes scapularis ticks.
Although there is much known about Lyme disease, there are currently no means of identification of the new spirochete associated with the aforedescribed Lyme disease-like pathology and further, no means of diagnosis of infection, compositions for clinical tests, or laboratory assays for diagnosing a patient exhibiting Lyme disease-like symptoms but testing negative for Lyme disease.
SUMMARY OF THE INVENTION
The present invention provides compositions, methods, and kits for the detection of a new spirochete that is associated with a Lyme disease-like illness. The compositions are based on a
Borrelia lonestari
sp. nov.-specific allotype or combination of allotypes of the flagellin protein, or a
Borrelia lonestari
sp. nov.-specific allele or combination of alleles of the flagellin or 16s rRNA genes of the new spirochete. The allotypes and alleles provided by the present invention have been determined by nucleic acid sequencing of portions of the flagellin and rRNA genes from this new spirochete. Detection of a species-specific amino acid or nucleotide as defined herein, or a species-specific combination of amino acids or nucleotides as defined herein, in a subject sample is indicative of infection with
Borrelia lonestari
sp. nov.
“Species-specific allotype” or “species-specific amino acid” or “species-specific epitope” means an amino acid of
B. lonestari
sp. nov. that is different at a particular position of the flagellin protein amino acid sequence than the amino acid at that position of the flagellin protein of other Borrelia species, especially those species needing to be distinguished from
B. lonestari
sp. nov. Table 1 provides a listing of species-specific amino acids of this new spirochete in the context of the amino acid sequence of SEQ ID NO: 2.
“Species-specific combination of allotypes” or “species-specific combination of amino acids” or “species-specific combination of epitopes” is a combination of amino acids of the flagellin protein of
B. lonestari
sp. nov. from Table 1 that is not represented in any of the flagellin proteins of other Borrelia species, especially those species needing to be distinguished from
B. lonestari
sp. nov. Table 1 also provides a listing of amino acids that may be combined with each other to form a combination that is unique to
B. lonestari
sp. nov. in the context of the amino acid sequence of SEQ ID NO: 2.
“Species-specific allele” or “species-specific nucleotide” means a nucleotide of
B. lonestari
sp. nov. that is different at a particular position of the flagellin gene sequence or 16s rRNA gene sequence from the nucleotide at that position of other flagellin gene sequences or 16s rRNA gene sequences of Borrelia species, especially the Borrelia species that need to be particularly distinguished, like
B. burgdorferi
. Tables 2 and 3 provide a listing of species-specific nucleotides of this new spirochete in the context of SEQ ID NO: 1 and 3.
“Species-specific combination of alleles” or “species-specific combination of nucleotides” is a combination of nucleotides of the flagellin gene or 16s rRNA gene of
B. lonestari
sp. nov. from Table 2 or 3 that is not represented in any of the flagellin gene sequences or 16s rRNA gene sequences of other Borrelia species. Tables 2 and 3 provide a listing of nucleotides that may be combined with each other to form a combination that is unique to
B. lonestari
sp. nov. in the context of SEQ ID NO: 1 and 3.
Species-specific flagellin amino acids of
B. lonestari
sp. nov. are listed in Table 1 as the underlined residues in the column Bl and include Val 24, Thr 65, Ala 67, Phe 90, Ser 91, Thr 92, Gly 99, Val 103, Pro 119, Ile 126, Ser 127, Ile 136, Ala 140, Thr 144, Asp 174, and Ile
Barbour Alan G.
Carter Carol
Board of Regents University of Texas
Duffy Patricia A.
Fulbright & Jaworski L.L.P.
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