Diagnostic and epidemiological nucleic acid probe for bovine leptospirosis

A distinctive repetitive genomic element of Leptospira interrogans serovar hardjo-type hardjo-bovis permits distinguishing this pathogen type from other commonly found leptospires in North American cattle using single-stranded RNA probes. Plasmids carrying DNA templates for useful probes have been deposited as NRRL Accession Nos. B-18462, B-18463, and B-18464. The probes are sufficiently sensitive to detect hardjo-bovis in as few as 1.times.10.sup.2 cells/ml. The diagnostic capabilities of the probes render them useful not only as herd management tools, but also in epidemiology studies designed to determine the origin and migration of L. interrogans isolates.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
Leptospirosis, caused by Leptospira interrogans, is a disease of animals 
and humans which has a worldwide distribution. L. interrogans is an 
immunologically diverse species and contains several distinct genetic 
groups. At least six serologically distinct types (serovars) have been 
identified in North America and about 190 serovars throughout the world. 
In North America, the most common cause of bovine leptospirosis is L. 
interrogans serovar hardjo-type hardjo-bovis. Hardjo-bovis and the 
reference strain for serovar hardjo, hardjoprajitno, are both associated 
in cattle with the causation of abortions, stillbirths, production of weak 
offspring, and infertility. In addition, cattle infected with serovar 
hardjo develop persistent renal infections and shed leptospires in their 
urine. Exposure to urine containing hardjo-bovis is considered to be the 
primary source of infections within herds. 
The two hardjo types can be differentiated by restriction endonuclease 
analysis of genomic DNA. However, the existence of similar antigens shared 
by hardjo-bovis and hardjoprajitno prevents these two bacteria from being 
differentiated by classical serological techniques. 
This invention relates to a sensitive diagnostic probe for distinguishing 
hardjo-bovis from other pathogenic leptospires, particularly those which 
commonly infect domestic animals in North America. 
2. Description of the Prior Art 
Diagnosis of leptospirosis usually depends upon demonstration of serum 
antibodies. The serologic method of choice is the microscopic 
agglutination test reported by Cole et al. [Appl. Microbiol. 25: 976-980 
(1973)]. However, interpretation of microscopic agglutination test results 
is often subjective and is complicated by numerous factors, including 
previous vaccination or infection and antigenic heterogeneity among L. 
interrogans. Since cattle infected with hardjo-bovis may fail to produce 
detectable antibodies, an accurate diagnosis of infection with 
hardjo-bovis requires direct demonstration of L. interrogans in tissues, 
blood, or urine. This is achieved either by bacteriological culture or by 
immunological techniques. Isolation of serovar hardjo from clinical 
specimens is labor intensive and inconsistent and requires weeks or months 
before results are obtained. Similarly, antigens may be degraded or 
blocked in some clinical specimens and thus prevent immunological 
detection of bacteria. 
Several investigators have utilized DNA-DNA hybridization methods for rapid 
and reliable detection of L. interrogans in biological samples (blood, 
urine, and tissue homogenates) [B. D. Millar et al., Vet. Microbiol. 15: 
71-78 (1987); W. J. Terpstra et al. I, J. Gen. Microbiol. 133: 911-914 
(1987); and W. J. Terpstra et al. II, J. Med. Microbiol. 22: 23-28 
(1986)]. The probes for these hybridizations consist of genomic DNA 
labeled by nick translation with radiolabeled or biotinylated nucleotides. 
Although these probes are specific for L. interrogans, they demonstrate 
extensive cross-hybridization among pathogenic serovars (Terpstra et al. 
II, supra). LeFebvre [J. Clin. Microbiol. 25: 2236-2238 (1987)] and Van 
Eys et al. [J. Gen. Microbiol. 134: 567-574 (1988)] disclose cloned DNA 
probes which differentiate hardjo-bovis from hardjoprajitno in DNA blot 
hybridization studies. These probes have not been well characterized, nor 
used to detect hardjo-bovis in biological material. Whereas the probe 
described by Lefebvre recognizes a discrete fragment of the hardjo-bovis 
genome which apparently exists as a single copy, probes described by Van 
Eys et al. may exist as several copies in the hardjo-bovis genome. The 
restriction enzyme maps of the probes described by Van Eys et al. are 
distinct from the probes described herein and are likely to detect 
different sequences than the probes of the invention. 
SUMMARY OF THE INVENTION 
I have now discovered a repetitive sequence element from hardjo-bovis and 
have cloned this fragment to develop a sensitive diagnostic 
single-stranded RNA probe for the detection of hardjo-bovis shed in the 
urine of infected cattle. This probe not only distinguishes hardjo-bovine 
from other pathogenic leptospires known to infect domestic animal species 
in North America, but it is also useful for distinguishing L. interrogans 
isolates of wildlife species as well. The probe is designed to bind 
specifically to the repetitive sequences contained within the 
hardjo-bovine genome while not cross-hybridizing with the genomic 
sequences of many other commonly encountered pathogenic leptospires. 
In accordance with this discovery, it is an object of the invention to 
provide a probe for diagnosis of bovine leptospirosis, and particularly a 
probe which is specific for L. interrogans serovar hardjo-type 
hardjo-bovis. The probe is envisioned for use primarily as a replacement 
for bacteriological culture or fluorescent antibody screening techniques 
for the diagnosis of bovine leptospirosis. 
It is also an object of the invention to provide plasmids carrying template 
DNA for transcribing the novel probes. 
It is also an object of the invention to provide a sensitive, reliable, and 
rapid assay for hardjo-bovis suitable for large-scale herd screening. 
More specifically, it is an object of the invention to identify infected 
cattle shedding hardjo-bovis in their urine. 
Another object of the invention is to provide a probe for use in 
conjunction with restriction fragment length polymorphism (RFLP) 
technology as an epidemiological tool for distinguishing among different 
hardjo-bovis isolates. 
A further object of the invention is to provide a diagnostic basis for 
designing an effective control program for hardjo-bovis in cattle herds. 
Other objects and advantages of this invention will become readily apparent 
from the ensuing description.

Glossary 
For purposes of this invention, the following standard abbreviations and 
terms used herein have been defined below. Also included are a listing of 
biological materials and reagents mentioned in the specification. 
ABBREVIATIONS 
bp=basepairs 
CAP=calf-alkaline phosphatase 
DEAE=diethylaminoethanol 
DNA=deoxyribonucleic acid 
dH.sub.2 O=distilled water 
dpm=disintegrations per minute 
EDTA=ethylenediaminetetraacetic acid 
FA=fluorescent antibody 
kb=kilobases (1000 basepairs) 
kd=kilodalton 
nt=nucleotide 
PAGE =preparative agarose gel electrophoresis 
REA=restriction enzyme analysis 
RFLP=restriction fragment length polymorphism 
RNA=ribonucleic acid 
SDS=sodium dodecyl sulfate 
SSC=0.15M sodium chloride and 0.015M sodium citrate 
ssRNA=single stranded RNA 
TE=10 mM Tris-HCl (pH 8.0), 1 mM EDTA 
TERMS 
clone/cloning: in reference to DNA, the product or process of isolating a 
segment of DNA, linking it to a vector, and introducing it into a host for 
propagation. 
cloning vector: a plasmid or other nucleic acid sequence which is able to 
replicate in a host cell characterized by one or a small number of 
restriction endonuclease recognition sites at which the sequence may be 
cut in a predetermined fashion, and which contains a marker suitable for 
use in the identification of transformed cells, e.g., tetracycline 
resistance or ampicillin resistance. 
downstream: refers to the direction toward the 3' end of the DNA template. 
DNA sequence: a linear series of nucleotides connected one to the other by 
phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. 
transcription vector: a plasmid or other nucleic acid sequence comprising a 
polymerase promoter which is able to induce transcription of an inserted 
gene or sequence of heterologous DNA to single-stranded RNA. 
gene: a segment of DNA which encodes a specific protein or polypeptide. 
genome/genomic: referring to the complete set of genetic instructions for 
an organism as defined by the chromosomal nucleic acid. 
heterologous DNA: a DNA sequence inserted within or connected to another 
DNA sequence which codes for polypeptides not coded for in nature by the 
DNA sequence to which it is joined. 
hybridization: the pairing together or annealing of complementary 
single-stranded regions of nucleic acids to form double-stranded 
molecules. 
infection: the introduction of bacteria or virus into cells or into a 
living organism wherein the bacteria or virus can replicate. 
linker: synthetic oligonucleotide containing a site for a restriction 
enzyme. 
multiple cloning site: a DNA sequence containing a multitude of different 
restriction enzyme sites. 
nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety 
(pentose), a phosphate, and a nitrogenous heterocyclic base. The base is 
linked to the sugar moiety via the glycosidic carbon (1' carbon of the 
pentose) and that combination of base and sugar is a nucleoside. The base 
characterizes the nucleotide. The four DNA bases are adenine ("A"), 
guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, 
G, C and uracil ("U"). "N" is commonly used to represent any of these five 
bases. 
phage: a bacteriophage; a virus which infects bacteria. 
plasmid: a non-chromosomal, double-stranded DNA sequence capable of 
autonomous replication within a host cell. 
polylinker: synthetic oligonucleotide containing multiple restriction 
enzyme sites. 
probe: a nucleic acid sequence (DNA or RNA) that can be used to detect, by 
hybridization or complementary base-pairing, another nucleic acid sequence 
which is homologous or complementary. 
promoter: a recognition sequence defining a site for binding of RNA 
polymerase and initiating transcription. 
recombinant DNA molecule: a hybrid DNA sequence comprising at least two DNA 
sequences, the first sequence not normally being found together in nature 
with the second. 
restriction site: A nucleotide sequence, usually 4 to 6 basepairs long, 
which is recognizes and susceptible to cleavage in a specific fashion by a 
restriction enzyme. 
sequence: two or more DNA or RNA nucleotides in a given order. 
serogroup: serological classification of Leptospira in which all strains in 
the same serogroup share a common "serogroup" antigen which is not present 
in strains outside of this serogroup. 
serovar: serological classification of Leptospira in which strains within 
the same serogroup are serologically distinct from each other. 
subclone: in reference to DNA, the product or process of cloning a portion 
of an already cloned DNA segment. 
transcription: the process of producing messenger RNA (mRNA) from a 
structural gene. 
transform: to change in a heritable manner the characteristics of a host 
cell in response to DNA foreign to that cell. 
transformant/transformation system: a host cell such as E. coli which has 
been transformed by intoduction of a vector containing DNA foreign to the 
cell. 
type: classification term for Leptospira in which two strains serologically 
classified as belonging to the same serovar may be differentiated on the 
basis of restriction endonuclease analysis. 
vector: a derivative of a virus or plasmid constructed by recombinant DNA 
techniques and having a cloning site or sites for inserting new DNA 
sequences. 
BIOLOGICAL MATERIALS AND REAGENTS 
______________________________________ 
Source 
______________________________________ 
Enzymes: 
T4 DNA ligase New England BioLabs, Inc. 
Genes: 
Ap.sup.r = ampicillin resistance gene 
on pBSM13- 
galactosidase gene 
on pBSM13- 
Plasmids: 
Accession No. 
pBSM13- Stratagene Corp. 
pLI16 NRRL B-18461 
pLI17 NRRL B-18462 
pLI18 NRRL B-18463 
pLI19 
pLI20 NRRL B-18464 
pUC19 Bethesda Research Lab. Inc. 
Polymerases: 
T3: bacteriophage T3 
Bethesda Research Lab. Inc. 
T4: bacteriophage T4 
Bethesda Research Lab. Inc. 
T7: bacteriophage T7 
New England BioLabs, Inc. 
______________________________________ 
______________________________________ 
Restriction Enzymes: 
Cleavage Site 
AccI 
##STR1## 
ClaI 5'. .AT.sup. OGAT. .3' 
DraII 
##STR2## 
EcoRI 5'. .G.sup. AATTC. .3' 
EcoRV 5'. .GAT.sup. ATC. .3' 
HhaI 5'. .GOG.sup. C. .3' 
HindIII 5'. .A.sup. AGCTT. .3' 
HinPI 5'. .G.sup. OGC. .3' 
NarI 5'. .GG.sup. CGCC. .3' 
PstI 5'. .CTGCA.sup. G. .3' 
SacI 5'. .GAGCT.sup. C. .3' 
XmnI 5'. .GAANN.sup. NNTTC. .3' 
Transformation Systems: 
Source 
E. coli strain JM107 
J. Neill, NADC 
______________________________________ 
The L. interrogans bacterial strains disclosed herein are summarized below 
in Table I. 
TABLE I 
__________________________________________________________________________ 
Bacterial Strains 
Organism and serogroup 
Serovar 
Type and/or strain 
Source.sup.a 
__________________________________________________________________________ 
L. interrogans 
Sejroe hardjo Hardjo-bovis 93U 
NADC 
hardjo Hardjoprajitno 
CDC/NADC 
Canicola portland-vere 
Lt63-69 CDC/NADC 
Grippotyphosa 
grippotyphosa 
RM-52 NADC 
Icterohaemorrhagiae 
copenhageni 
M20 CDC/NADC 
Pomona pomona Kennewicki RM211 
NADC 
Shermani babudieri 
CI40 CDC 
Icterohaemorrhagiae 
birkini 
Birkin CDC 
Canicola broomi patane CDC 
Pyrogenes camlo LT64-47 CDC 
Gryppotyphosa 
canalzonae 
cz188 CDC 
Ballum castellonis 
castelloni 3 
CDC 
Celledoni celledoni 
celledoni CDC 
Cynopteri cynopteri 
3522C CDC 
Javanica flumininse 
Aa3 CDC 
Autumnalis fortbragg 
fort bragg 
CDC 
Djasiman gurungi 
gurung CDC 
Louisiana lanka R740 CDC 
Australis meunchen 
Munchen c-90 
CDC 
Bataviae paidjan 
paidjan CDC 
Sarmin rio Rr5 CDC 
Sejroe romanica 
LM294 CDC 
Pomona tropica 
Ca299U CDC 
Sejroe wolffi 3705 CDC 
L. biflexa 
Semaranga Patoc Patoc I N. Charon 
__________________________________________________________________________ 
.sup.a NADC strains are from the National Leptospirosis Reference 
Laboratory, National Animal Disease Center, Ames, IA. N. Charon is from 
West Virginia University, Morgantown. CDC strains are from K. Sulzer at 
the Center for Disease control, Atlanta, GA. CDC/NADC strains are 
originally from the Center for Disease Control. 
DETAILED DESCRIPTION OF THE INVENTION 
The original single-stranded RNA (ssRNA) probes of the invention were 
isolated as a result of discovering a repetitive sequence element which 
occurs collectively about 40-50 times within the hardjo-bovis genome and 
putative plasmid DNA. The 35-40 copies in the genome constitute about 2% 
of the genomic DNA. This element was identified by restriction 
endonuclease analysis (REA) of purified genomic DNA revealing a 1.4-kb 
NarI fragment which is present at a high copy number within the 
hardjo-bovis genome. This fragment was isolated by preparative agarose gel 
electrophoresis (PAGE) and thereafter cloned into the plasmid vector pUC19 
to yield plasmid pLI16. Upon digestion of pLI16 with the restriction 
endonuclease HindIII, a 1,100 basepair (bp) fragment was recovered, and 
this fragment was subcloned into the RNA transcription vector, pBSM13-. 
Hybridization probes were synthesized from SacI-digested template DNA in 
the resulting plasmid, pLI17, by runoff transcription using phage T3 RNA 
polymerase and [.alpha..sup.-32 P]uridine triphosphate. 
Contemplated within the scope of this invention are ssRNA probes which are 
complementary to, and will hybridize with, the repetitive element 
described above; that is, with the element comprising the 1.4-kb NarI 
fragment. Generally, the complementary portion of these probes should be 
at least about 250 bp in length so that the hybridization products can be 
visualized by autoradiograph or other assay. Probes having a length of 
about 250-350 are preferred for the reason that they tend to be more 
specific than longer probes in hybridizing to target DNA. For the purpose 
of differentiating hardjo-bovis from hardjoprajitno genomic DNA, at least 
about 80% homology is required between the probe and the target DNA. 
Noncomplementary sequences may flank the complementary portion of the 
probe provided that such sequences do not interfere with the hybridization 
of the DNA in the repetitive element to the extent that the hybridization 
products are not identifiable. Such extraneous sequences may arise at the 
5' end of the probe as a result of transcription initiation by RNA 
polymerase within vector sequences prior to the heterologous DNA located 
3' to the initiation site. It is likely that the terminal ends of the 
repetitive element are clipped by the NarI enzyme. Other endonucleases 
which would preserve these ends or a portion thereof could be substituted 
for the NarI in providing suitable template DNA for synthesis of the ssRNA 
probes. 
For visualization by autoradiography, the probes are of course suitably 
labeled as with [.alpha.-.sup.32 P]uridine triphosphate. This is readily 
achieved by labeling about 50% of the uridine triphosphate provided in the 
transcription medium during synthesis of the RNA probe. 
Also contemplated by this invention are the templates and other progenitor 
DNA sequences for these probes, including plasmids useful in cloning and 
transcribing these sequences. An exemplary cloning plasmid is the 
above-mentioned pLI16 (FIG. 1) derived by insertion of the 1.4-kb NarI 
fragment into the pUC19 cloning vector which has been digested with NarI 
and AccI. This plasmid can be used to transform E. coli for expansion of 
the DNA insert. The above-described plasmid pLI17 (FIG. 2) illustrates a 
suitable plasmid for the synthesis of the ssRNA probes. As mentioned, it 
is constructed by excising the 1,100 bp HindIII fragment from the NarI 
insert of pLI16 and inserting this fragment into the transcription vector 
pBSM13-. In addition to the probe template, pLI17 comprises the promoter 
and other regulatory sequences for T3 and T7 RNA polymerases. Other 
transcription plasmids similarly derived from pLI16 include pLI18 (FIG. 
3), pLI19 described in Example 5 and pLI20 (FIG. 4) described in Example 
9 below. Plasmids pLI16, pLI18, and pLI20 have been deposited under the 
Budapest Treaty with the Agricultural Research Service Culture Collection 
in Peoria, IL, and have been assigned Accession Nos. NRRL B-18461, NRRL 
B-18462, NRRL B-18463 and NRRL B-18464, respectively. 
It is envisioned that plasmids substantially equivalent to those which have 
been deposited could be readily derived by the skilled artisan by 
following the procedures described herein. Functional derivatives of these 
plasmids may also be prepared by making minor deletions, substitutions, or 
insertions in the nucleotides of the probe template DNA and/or by making 
deletions, substitions, or insertions in the vector sequences. 
The ssRNA probes are useful in DNA blot experiments utilizing restriction 
endonuclease digested DNA (Southern blots). Briefly, the L. interrogans 
DNA is immobilized on a membrane, and the membrane is washed in a solution 
of a radiolabeled probe. The probe binds to the appropriate segment of the 
repetitive element by complementary basepairing and can be detected by 
autoradiography. The probes can also be used in slot blot analysis of 
urine or other body fluid from infected livestock. Insofar as L. 
interrogans is also known to infect humans, it is understood that 
reference herein to body fluids and tissues of animals is meant to include 
the same from humans as well. 
The strategy of repetitive sequence DNA used in this invention is 
attractive for the development of diagnostic probes, since the target 
sequences for probes are amplified naturally within the genome. This 
attribute enables detection of fewer organisms with the probes directed to 
repetitive sequence elements compared with probes directed to single-copy 
sequences. It is imperative for diagnostic purposes that the minimal 
number of cells detectable by a probe be relevant to the levels of 
bacteria shed by infected animals. As illustrated in Example 2 below, the 
probes contemplated herein detect as few as 1.times.10.sup.2 cells/ml. 
These probes therefore detect typical levels of hardjo-bovis shed in the 
urine of cattle and offer an effective method to rapidly identify 
potential sources of hardjo-bovis infections within herds. The sensitivity 
and consistency of the repetitive element-based probe compares favorably 
with existing techniques but does not require prior cultivation of the 
organism. 
The other significant attribute of diagnostic probes manifested by those of 
the invention is selectivity. As illustrated in Example 4, the subject 
probes demonstrates little detectable hybridization with any of the 
serovars commonly isolated from domestic animals in North America 
(serovars grippotyphosa, hardjo, copenhageni, pomona, and portland-vere) 
except for hardjo-bovis. Likewise, little cross-hybridization is observed 
with hardjoprajitno, the reference strain for serovar hardjo. 
On the other hand, the ssRNA probes cross-hybridize with the DNA from 
several L. interrogans serovars isolated from wildlife. The presence of 
these serovars probably represent commensal infections. The selectivity 
against isolates of domestic animals and cross-reactivity with wildlife 
isolates render the ssRNA probes suitable for epidemiological studies in 
determining the origin and migration of a given isolate. Furthermore, the 
ability to type L. interrogans isolates, particularly those obtained from 
wildlife, using repetitive element-based probes, provides a more rapid 
typing system than using serological methods. 
For distinguishing differences among isolates in epidemiology studies of 
hardjo-bovis infections, the enzymes EcoRI, HhaI, and XmnI were found to 
be most suitable for digesting the genomic DNA prior to probing. For RFLP 
analysis for identifying serovars, the enzymes EcoRI, ClaI, BamHI, 
HindIII, and XmnI were found to be most useful. 
The following examples are intended only to further illustrate the 
invention and are not intended to limit the scope of the invention which 
is defined by the claims. 
EXAMPLE 1 
Isolation of the Repetitive Element 
The repetitive element was cloned by digesting 10 .mu.g L. interrogans 
serovar hardjo-type hardjo-bovis strain 93U DNA (prepared by the method of 
Thiermann et al., [J. Clin. Microbiol. 21: 585-587 (1985)] with 
approximately 10 units of restriction endonuclease NarI at 37.degree. C. 
for 6 hr in a solution containing 10 mM Tris-Hcl (pH 7.4), 10 mM 
MgCl.sub.2, 10 mM 2-mercaptoethanol. The resulting restriction fragments 
were resolved in a 1% agarose gel buffered with 89 mM Tris, 89 mM boric 
acid, 2 mM EDTA at 50 V for 20 hr. The gel was stained with 1 .mu.g 
ethidium bromide/ml for 1 hr and the DNA visualized by illumination with 
ultraviolet light. The 1.4 kb NarI fragment was excised from the gel and 
transferred to an NA-45 DEAE membrane (Schleicher and Schuell, Inc.) by 
electrophoresis at 100 V for 2 hr. The membrane was rinsed with 
electrophoresis buffer and the DNA eluted in 200 .mu.l, 1.0M NaCl, 0.1 mM 
EDTA, 20 mM Tris-Hcl (pH 8.0), at 60.degree. C. for 2 hr. The membrane was 
discarded and the solution volume brought to 500 .mu.l with distilled 
water (dH.sub.2 O). This DNA was precipitated with 50 .mu.l 3M sodium 
acetate (pH 5.2), and 1 ml 95% ethanol, and harvested by centrifugation at 
11,000.times.g for 15 min. The precipitate was suspended in 500 .mu.l TE 
[10 mM Tris-HCl (pH 8.0), 1 mM EDTA] and extracted twice with 500 .mu.l 
phenol-chloroform-isoamyl alcohol (50:50:1). DNA was recovered from the 
aqueous phase by precipitation with sodium acetate and ethanol, and the 
precipitate washed with 500 .mu.l 70% ethanol. The precipitate was 
suspended in 20 .mu.l dH.sub.2 O and 10 .mu.l (ca. 100 ng) was mixed with 
approximately 100 ng pUC19 which had previously been digested with NarI 
and AccI, and treated with calf-alkaline phosphatase (CAP). This mixture 
was treated with 400 units of phage T4 DNA ligase in a 20 .mu.l reaction 
volume containing 50 mM Tris-Hcl (pH 7.8), 10 mM MgCl.sub.2, 20 mM 
dithiothreitol, 1 mM adenosine triphosphate, and 50 .mu.g bovine serum 
albumin/ml at 16.degree. C. for 6 hr. The mixture was heated at 65.degree. 
C. for 15 min then used to transform Escherichia coli JM107 to ampicillin 
resistance by standard technique. Transformants harboring recombinant 
plasmids were identified by inactivation of the vector-encoded lacZ gene 
by using the chromogenic lactose analog 
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside [J. Messing, Methods 
Enzymol. 101: 20-78 (1983)]. Plasmid DNA was prepared from randomly picked 
transformants using an alkaline lysis technique [D. Ish-Horowitcz et al., 
Nucl. Acids Res. 9: 2989-2996 (1981)] and examined by REA. One of these 
clones, pLI16 was used for subsequent analysis and subcloning. A schematic 
restriction enzyme map of pLI16 is shown in FIG. 1, wherein the ampicillin 
resistance gene is indicated as "Ap.sup.r." Vector DNA sequences are shown 
as double lines, whereas cloned hardjo-bovis DNA is shown as single lines. 
Construction of Probe DNA Template by Subcloning Hardjo-Bovis DNA into 
pBSM13-. 
pLI16 (ca. 10 .mu.g) as prepared above was digested with 10 units of 
HindIII in 100 .mu.l of a solution containing 50 mM NaCl, 50 mM Tris-HCl 
(pH 8.0), 10 mM MgCl.sub.2 at 37.degree. C. for 6 hr. The digestion 
products were fractionated by electrophoresis in a 1% agarose gel and the 
1100 bp HindIII fragment isolated onto NA-45 membrane as described above. 
The DNA was eluted from the NA-45 membrane, concentrated, and ligated to 
HindIII-digested, CAP-treated RNA transcription vector pBSM13- by the 
method described above for ligating the bacterial DNA to the digested 
pUC19. Plasmid pBSM13- contains the RNA polymerase promoters for both 
bacteriophage T3 and T7. E. coli JM107 was transformed to ampicillin 
resistance with the ligation mixture and plasmid DNA isolated from 
randomly picked transformants and examined by REA. One plasmid, pLI17, was 
used for subsequent probe synthesis. The restriction map of pLI17 is shown 
in FIG. 2, wherein T7 and T3 represent the T7 and T3 promoters, 
respectively. 
Preparation of ssRNA Probe 
Five micrograms of purified pLI17 DNA was digested with 5 units of 
restriction endonuclease SacI at 37.degree. C. for 2 hr in a solution 
containing 10 mM Tris-HCl (pH 7.4), 10 mM MgCl.sub.2, 10 mM 
2-mercaptoethanol, and a final volume of 50 .mu.l. The plasmid DNA was 
precipitated by addition of 5 .mu.l 3 M sodium acetate (pH 5.2) and 200 
.mu.l 95% ethanol, harvested by centrifugation for 15 min at 
11,000.times.g, and then washed with 500 .mu.l 70% ethanol. The 
precipitated DNA was dissolved in solution by sequential addition of 2 
.mu.l 0.1M dithiothreitol, 2.4 .mu.l 0.1 mM uridine triphosphate, 4 
.mu.l/2.5 mM each ATP, GTP, CTP, 1 .mu.l dH.sub.2 O, 5 .mu.l 
[.alpha.-.sup.32 P]uridine triphosphate [ca. 650 Curies (Ci)/mmol, 10 
mCi/ml)], 4 .mu.l buffer [200 mM Tris-HCl (pH 8.0), 40 mM MgCl.sub.2, 10 
mM spermidine, 125 mM NaCl] and the reactions initiated with 10 units of 
phage T3 RNA polymerase. After incubating the reaction at 37.degree. C. 
for 1 hr, it was terminated by addition of 1 .mu.l 20% sodium lauryl 
sulfate, 75 .mu.l dH.sub.2 O, 10 .mu.l 3M sodium acetate (pH 5.2), and 500 
.mu.l 95% ethanol. The DNA template was harvested by centrifugation and 
washed as before. The resultant 1,100 bp radiolabeled single-stranded RNA 
(ssRNA) probe was suspended in 100 .mu.l TE, heated at 85.degree. C. for 5 
min, and then quickly chilled on ice in preparation for use in the 
hybridization analyses described below in Example 2. 
EXAMPLE 2 
Diagnostic Screening 
Positive urine samples were collected from four experimentally infected 
animals demonstrating no clinical signs of disease 4, 8, and 12 wk after 
exposure to hardjo-bovis and from one naturally infected cow. In this 
experiment, the negative-control sample was a composite from nine animals 
collected prior to experimental infection. 
The slot blot analyses were performed by using a modification of the 
procedure described by Millar et al. [Vet. Microbiol. 15: 71-78 (1987)]. 
Bacteria were concentrated from 1-10 ml of the collected urine by 
centrifugation at 11,000.times.g for 5-15 min. The cells were suspended in 
100 .mu.l phosphate buffered saline, mixed with an equal volume of 0.5M 
NaOH, 1.5M NaCl, and incubated at room temperature for 1 hr. Solutions 
were neutralized with 200 .mu.l 1M Tris-HCl (pH 8.0), 1.5M NaCl and 
filtered through nylon membrane filters (Hybond-N, Amersham Corporation) 
using a commercially available filtering apparatus (Minifold II, 
Schleicher and Schuell Corporation). The filters were rinsed with 
2.times.SSC (1.times.SSC: 0.15M NaCl, 0.015M sodium citrate), and air 
dried. Membranes were incubated for a minimum of 2 hr at 61.degree. C. in 
hybridization solution [6.times.SSC, 5.times.Denhardts solution 
(1.times.Denhardts: 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% 
bovine serum albumin), 1% sodium lauryl sulfate, 100 .mu.g denatured 
salmon sperm DNA/ml, and 100 .mu.g yeast RNA/ml]. This prehybridization 
solution was discarded, replaced by fresh hybridization solution 
containing the radiolabeled ssRNA probe prepared in Example 1 and 
incubated overnight at 61.degree. C. These filters were then washed twice 
in 2.times.SSC at room temperature for 15 min each, twice in 2.times.SSC 
at 61.degree. C. for 15 min each, and twice in 2.times.SSC, 0.1% SDS at 
61.degree. C. for 30 min each. Blots were used to expose autoradiographic 
film (Eastman Kodak, Inc.) at -80.degree. C. for 1-3 da before developing. 
Autoradiographic images were quantitated by scanning the autoradiographs 
with a laser densitometer (Ultrascan XL, LKB Instruments, Inc., Rockville, 
MD) and subsequent analysis with Gelscan XL software (LKB Instruments). 
To quantitate the amount of hardjo-bovis shed in the urine of infected 
cattle, autoradiographic signals obtained in the urine samples were 
compared to signals obtained with specific cell numbers. The cell 
concentration of cultured bacteria was determined with a Petroff-Hauser 
counting chamber by using dark-field microscopy. Cell concentrations were 
adjusted to 5.times.10.sup.7 cells per ml with phosphate-buffered saline 
and serially diluted, by using twofold dilutions, to 2.5.times.10.sup.4 
cells per ml. Samples (100 .mu.l) of these diluted cell suspensions were 
mixed with 100 .mu.l of 0.5M NaOH-1.5M NaCl and incubated for 1 hr at room 
temperature. These suspensions were neutralized with 200 .mu.l of 1M Tris 
hydrochloride-1.5M NaCl, pH 8.0, and 40 .mu.l of the suspension was 
applied to Hybond-N by using a slot blot apparatus (Schleicher & Schuell). 
The results of this experiment are summarized in Table II below and 
demonstrate that the pLI17-derived probe detects hardjo-bovis shed in 
urine from infected animals. The number of leptospires detected in the 
urine of infected animals ranged from &lt;1.times.10.sup.2 cells per ml to 
approximately 3.times.10.sup.4 cells per ml. Most but not all of the urine 
samples tested were found to contain hardjo-bovis either by culture or by 
fluorescent antibody (FA). Additionally, culture and FA results of urine 
samples from cow 86 taken at other times during the infection were 
positive, thus confirming that this cow was infected and shedding L. 
interrogans in its urine. 
TABLE II 
______________________________________ 
Test of pLI17 Probe with Cattle Urine Samples 
Time 
postinfection 
Concn 
Sample (mo).sup.a (cells/ml).sup.b 
Culture.sup.c 
FA.sup.d 
______________________________________ 
Negative control &lt;1 .times. 10.sup.2 
- - 
Cow 103 1 3 .times. 10.sup.2 
+ + 
2 4 .times. 10.sup.3 
- + 
3 1 .times. 10.sup.2 
+ + 
Cow 79 2 7 .times. 10.sup.3 
+ + 
3 2 .times. 10.sup.3 
- + 
Cow 86 2 4 .times. 10.sup.2 
- - 
3 1 .times. 10.sup.2 
- - 
Cow 80 2 3 .times. 10.sup.4 
+ + 
40U 4 .times. 10.sup.3 
+ + 
______________________________________ 
.sup.a Time following experimental infection of cattle with hardjobovis. 
40U was naturally infected, and time of exposure could not be determined. 
.sup.b Bacterial cell concentrations in urine normalized to cells per 
milliliter as determined with pLI17. 
.sup.c Results of attempts to culture urine samples. 
.sup.d Results of fluorescent antibody with antihardjo-bovis conjugate. 
EXAMPLE 3 
Sensitivity and Selectivity Comparison of ssRNa Probe and DNA-Probe 
The sensitivity of the radiolabeled ssRNA probe synthesized from pLI17 in 
Example 1 was compared with that of a radiolabeled genomic DNA probe. 
Various serovars and types of L. interrogans and L. biflexa were cultured 
and then were serially diluted from 5.times.10.sup.7 cells per ml to 
2.5.times.10.sup.4 cells per ml and lysed. The DNA was denatured, and a 
portion of these suspensions was filtered through a nylon membrane. The 
immobilized DNA was used to hybridize either the pLI17-derived 
radiolabeled ssRNA probe or serovar hardjo-type hardjo-bovis genomic DNA 
radiolabeled by nick translation. Both of these probes were radiolabeled 
to specific activities of approximately 10.sup.9 dpm/.mu.g of nucleic 
acid. The resulting autoradiographs demonstrated that the pLI17-generated 
probe can detect as few as 1.times.10.sup.3 hardjo-bovis cells, while the 
detection limit for the radiolabeled genomic DNA probe was approximately 
4.times.10.sup.3 cells. The level of specificity of these two probes for 
hardjo-bovis was assessed by quantitating autoradiographic signals by 
scanning laser densitometry and then comparing the values obtained in 
heterologous reactions with those obtained in homologous reactions. The 
results of this analysis (Table III) indicate that the pLI17-derived probe 
is more specific for hardjo-bovis than the genomic hardjo-bovis DNA probe. 
Both probes were species specific, as where was no detectable 
cross-hybridization between either probe and the saprophyte. L. biflexa 
serovar patoc. 
TABLE III 
______________________________________ 
Quantitative Comparison of pLI17 and Genomic 
DNA Probe Specificities 
% Hybridization with 
nucleic acid probe from: 
Organism and serovar.sup.a 
pLI17.sup.b 
Hardjo-bovis.sup.c 
______________________________________ 
L. interrogans 
Hardjo-type hardjo-bovis 
100 100 
Hardjo-type hardjoprajitno 
8 11 
Pomona-type kennewicki 
2 5 
Grippotyphosa 5 16 
Portland-vere 1 27 
Copenhageni 1 11 
L. biflexa 
Patoc &lt;1 &lt;1 
______________________________________ 
.sup.a Samples containing 2.5 .times. 10.sup.5 cells. 
.sup.b Comparison of autoradiographic signals with signals obtained with 
hardjobovis using pL117 probe. 
.sup.c Comparison of autoradiographic signals with signals obtained with 
hardjobovis using hardjobovis genomic probe. 
EXAMPLE 4 
Selectivity of pLI17-Derived ssRNA Probe Against Domestic Animal Isolates 
The selectivity of the ssRNA probes synthesized from pLI17 as described in 
Example 1 for the 1.4 kb NarI repetitive fragment characteristic of 
hardjo-bovis was demonstrated by Southern blot analysis. Genomic DNA (2.5 
.mu.g) isolated from each of L. interrogans serovars hardjo-type 
hardjo-bovis, hardjo-type hardjoprajitno, pomona-type kennewicki, 
grippotyphosa, copenhageni, and portland-vere was digested with NarI. 
Digestion products were fractionated by electrophoresis at 50 V overnight 
in 0.7% agarose gels as described previously. The gels were treated with 
0.5M NaOH, 1.5M NaCl for 1-2 hr, and then neutralized with 1M Tris-HCl (pH 
8.0), 1.5M NaCl for 1-2 hr. DNA was blotted to nylon membranes by 
capillary action overnight with 20.times.SSC. The nylon membranes were 
washed with 2.times.SSC, air dried, prehybridized, and hybridized with the 
.sup.32 P-labeled ssRNA probe as described in Example 2. The membranes 
were washed as in Example 2 with an additional wash of 0.2.times.SSC at 
61.degree. C. The membranes were used to expose AR film at -80.degree. C. 
for 1 hr before developing. The autoradiographs indicated at least 12 
distinct bands for the hardjo-bovis DNA and no bands for any of the other 
serovar or type DNA assayed. 
EXAMPLE 5 
Preparation of pLI18- and pLI19-Derived Probes 
Following the procedure of Example 1 for construction of a probe DNA 
template and preparation of ssRNA probe, pLI16 was digested with HinPI, 
and a recovered 1,000 bp fragment was inserted into the AccI site of 
pBSM13-. The two selected plasmids, pLI18 and pLI19 carry this fragment in 
opposite orientations. pLI18 is shown schematically in FIG. 3. 
Probes generated by runoff transcription of Eco-RI-digested pLI18 detect 
some fragments which are not detectable with pLI17-derived probes since 
these fragments are homologous to the small EcoRI-NarI fragment of the 
repetitive element missing in pLI17. 
EXAMPLE 6 
Selectivity of the pLI18-Derived ssRNA Probe Against Wildlife Isolates 
The ability of the ssRNA probe from pLI18 prepared in Example 1 to 
distinguish two L. interrogans wildlife isolates, serovars wolffi and 
romanica, from one another was determined. These serovars are genetically 
similar and cannot be differentiated by restriction endonuclease analysis 
of genomic DNA. Genomic DNA from cultured cells of these serovars was 
prepared by the method of Thiermann as referenced in Example 1 and 
digested with EcoRI, HindIII, or XmnI. The resulting fragments were 
fractionated by agarose gel electrophoresis, blotted to a nylon membrane, 
and probed with the radiolabeled probe as in Example 2, except the 
hybridization and washing were conducted at 58.degree. C. The subtle 
differences between these two serovars could be detected as illustrated by 
the autoradiograph of FIG. 5. The odd-numbered lanes represent wolffi, and 
the even-numbered lanes represent romanica. The DNA of lanes 1 and 2 was 
digested with EcoRI, that of lanes 3 and 4 was digested with HindIII, and 
that of lanes 5 and 6 was digested with XmnI. The arrows depict bands 
representing fragments not characteristic of the related serovar. 
EXAMPLE 7 
Differentiation of Hardjo-Bovis Isolates with pLI17-Derived Probe 
Genomic DNA (2.5 .mu.g) from six hardjo-bovis isolates originally obtained 
from cattle in Iowa, Colorado, Florida, Alberta (Canada), Switzerland, and 
Chile were digested with HhaI or EcoRI. The digestion products were 
electrophoresed in agarose, blotted to a nylon membrane, and probed with 
the pLI17-derived probe, all as described in Example 4. Autoradiographs 
showed distinct patterns among the isolates for each of the enzyme 
digests. This experiment demonstrates the usefulness of the ssRNA probes 
in differentiating isolates from different geographical locations. 
EXAMPLE 8 
Differentiation of L. interrogans Serovars 
Genomic DNA (2.5 .mu.g) from L. interrogans serovars meunchen, fortbragg, 
castellonis, paidjan, lanka, broomi, celledoni, cynopteri, rio, gurungi, 
canalzonae, birkini, flumininse, tropica, camlo, babudieri were digested 
with EcoRI, electrophoresed in a 0.7% agarose gel, and blotted to a nylon 
membrane, essentially as described in Example 6. Radiolabeled probes were 
synthesized by the procedure of Example 1 except the pLI17 plasmid was 
digested with EcoRV and the transcription was initiated with phage T7 RNA 
polymerase. The autoradiograph hybridization pattern representing each of 
the 16 serovars was distinct, thereby providing a basis for distinguishing 
one from the other. 
EXAMPLE 9 
Preparation of pLI20 Derived Probe 
Following the procedure of Example 1 for construction of a probe DNA 
template and preparation of ssRNA probe, pLI18 was digested with EcoRI and 
a recovered 3,450 bp fragment was self-ligated using T4 DNA ligase. The 
resulting plasmid, pLI20, differs from pLI18 by the deletion of a 750 bp 
EcoRI fragment. pLI20 contains approximately 250 bp of hardjo-bovis DNA 
inserted between the EcoRI and AccI sites of pBSM13- and is shown 
schematically in FIG. 4. Radiolabeled ssRNA probes are synthesized from 
HinPI-digested pLI20 by run-off transcription with T7 phage RNA 
polymerase. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention.