01_02_0086.
QXD 10/12/2005 2:15 PM Page 636
Evaluation of antibiotics for treatment of cattle
infected with Leptospira borgpetersenii
serovar hardjo
David P. Alt, DVM, PhD; Richard L. Zuerner, PhD; Carole A. Bolin, DVM, PhD
RUMINANTS
Objective—To evaluate antibiotics for treatment of
cattle with leptospirosis caused by Leptospira borg-
petersenii serovar hardjo.
Design—Randomized controlled trial.
Animals—42 healthy mixed-breed cattle.
Procedure—Cattle were inoculated via conjunctival
instillation with L borgpetersenii serovar hardjo. After
infection and urinary shedding of L borgpetersenii
were confirmed, cattle were treated with various
antibiotics. To determine effectiveness of antibiotic
treatment, urinary shedding of L borgpetersenii was
monitored for 4 to 6 weeks after administration of
antibiotics, using darkfield microscopic examination,
microbial culture, immunofluorescence testing, and a
polymerase chain reaction assay.
Results—All inoculated cattle developed leptospiro-
sis and shed leptospires in their urine. The following
antibiotic treatments resulted in elimination of urinary
shedding of leptospires: a single injection of oxytetra-
cycline (20 mg/kg [9 mg/lb] of body weight, IM), tilmi-
cosin (10 mg/kg [4.5 mg/lb], SC), or a combination
product that contained dihydrostreptomycin-penicillin
G (25 mg/kg [11.4 mg/lb], IM) or multiple injections of
ceftiofur sodium (2.2 or 5 mg/kg [1 or 2.3 mg/lb], IM,
once daily for 5 days, or 20 mg/kg, IM, once daily for
3 days).
Conclusions and Clinical Relevance—Successful
resolution of leptospirosis in cattle by administration
of dihydrostreptomycin-penicillin G confirms results
obtained by other investigators. Three other antibi-
otics (oxytetracycline, tilmicosin, and ceftiofur) also
were effective for resolving leptospirosis and may be
useful substitutes for dihydrostreptomycin, an antibi-
otic that is no longer available for use in food-produc-
ing animals in the United States. Cost, safety, and
withdrawal times of these various treatment options
need to be considered. (J Am Vet Med Assoc
2001;219:636–639)
L eptospirosis is an important zoonotic disease of
food-producing animals caused by infection with
pathogenic serovars of Leptospira spp. Leptospirosis in
cattle is most commonly caused by infection with L
From the Bacterial Diseases of Livestock Research Unit, National
Animal Diseases Center, USDA, Agricultural Research Service,
2300 N Dayton Rd, Ames, IA 50010 (Alt, Zuerner); and the
Animal Health Diagnostic Laboratory, College of Veterinary
Medicine, Michigan State University, East Lansing, MI 48824
(Bolin).
Presented in part at the 75th Annual Meeting of the Conference of
Research Workers in Animal Diseases, Chicago, Nov 14 and 15,
1994.
The authors thank John Foley, Annette Olson, Rebecca Rheinhart,
and Sara K. Harris for technical assistance.
636 Scientific Reports: Original Study
borgpetersenii serovar hardjo.1-3 Leptospirosis in cattle
attributable to serovar hardjo can cause mastitis,
decreased milk production, abortion, birth of weak
calves, and infertility.4-7
Serovar hardjo often is directly transmitted among
cattle. Transmission involves contact with infected urine,
placental fluids, or milk. Serovar hardjo also may persist
in the reproductive tract2 of cattle, which can lead to
venereal5 or transplacental transmission.4 Leptospires
invade the body after being deposited on mucous mem-
branes or damaged skin. After a variable incubation peri-
od (3 to 20 days), leptospires circulate in the blood.
During this period, serovar hardjo enters and replicates
in many tissues, including the liver, spleen, kidneys,
reproductive tract, eyes, and CNS. Agglutinating anti-
bodies can be detected in serum soon after the lep-
tospires are in the bloodstream. Appearance of circulat-
ing antibodies coincides with clearance of leptospires
from blood and most organs. However, serovar hardjo
remains in the kidneys, and infected cattle may shed lep-
tospires in their urine for months or years after infection.8
Chronic, persistent, subclinical infections are com-
mon in herds infected with L borgpetersenii serovar hard-
jo. Infected cattle are difficult to identify, because they
often do not have clinical signs and have low antibody
titers against serovar hardjo.9 Therefore, movement of
cattle within and among herds represents an important
risk factor for transmission of leptospirosis attibutable to
serovar hardjo. Cattle infected with serovar hardjo also
present a major health risk for humans.
Vaccination and antibiotic treatment have been
used to reduce the risk of transmission of leptospirosis
in cattle, and results have been variable. Current vac-
cines for prevention of infection attributable to L borg-
petersenii serovar hardjo provide limited protection.
Although several investigators have reported that these
vaccines can decrease the frequency of infection and
duration of leptospiral shedding, reproducibility of
those results are dependent on an artificial route of
exposure.10,11 More recent studies, using an exposure
route that more closely resembles natural infection,
revealed that vaccination has little effect on infection
with L borgpetersenii or persistence of urinary shed-
ding.12-14 Vaccination of an animal that is already infect-
ed with serovar hardjo does not have an impact on uri-
nary shedding. For these reasons, antibiotic treatment
is routinely used to decrease the risk of transmission of
leptospirosis among cattle being prepared for import or
export and during quarantine of cattle before introduc-
tion to a herd. Clearing endemically infected herds of L
borgpetersenii serovar hardjo may be possible by com-
bining use of an aggressive vaccination program with
antibiotic treatment.
JAVMA, Vol 219, No. 5, September 1, 2001
01_02_0086.QXD 10/12/2005 2:15 PM Page 637
Dihydrostreptomycin can be an effective treat-
ment for cattle with leptospirosis.15-18 Use of a single
injection of dihydrostreptomycin (25 mg/kg [11.4
mg/lb] of body weight, IM) has traditionally been rec-
ommended to decrease the risk of transmission of lep-
tospirosis and is specified for use in animals for import
or export.19 However, dihydrostreptomycin is not
always effective, and persistent infection of the kid-
neys and genital tract of treated cattle has been report-
ed.8 Furthermore, dihydrostreptomycin currently is
not available in the United States. Therefore, alterna-
tive antibiotics for treatment of cattle with leptospiro-
sis are needed.
Leptospira spp are susceptible to many antibiotics
in vitro, but in vitro efficacy does not correlate well
with in vivo efficacy.19 In addition, the predictive value
of models that have involved the use of small animals
to assess treatment of chronic infections, similar to
those seen in cattle infected with serovar hardjo, may
be of questionable value.20
The purpose of the study reported here was to
evaluate several antibiotics for efficacy in treatment of
cattle with persistent leptospirosis. Several classes of
antibiotics were chosen for evaluation on the basis that
they had meat withdrawal times of ≤ 30 days, had ther-
apeutic effects, as described in another study,20 and
were available for use in cattle in the United States.
Materials and Methods
Cattle—Forty-two mixed-breed cattle weighing 300 to
450 kg [660 to 990 lb] and that did not have serum antibod-
ies against L borgpetersenii serovar hardjo as determined by
results of the microscopic agglutination test21 were used in
the study. Cattle were housed in separate pens in Association
for Assessment and Accreditation of Laboratory Animal Care
International-approved BL2 biocontainment barns.
Exposure to L borgpetersenii serovar hardjo—
Leptospires were grown in commercial liquid mediuma at 29
C. Cattle were inoculated with 106 L borgpetersenii serovar
hardjo (type hardjo-bovis strain 203) via conjunctival instil-
lation on 3 successive days, as described elsewhere.14 Urine
samples were collected weekly from each of the cattle.
Samples were collected during natural voiding that took
place approximately 10 minutes after administration of an
injection of furosemide (500 mg, IV). Cattle were shedding
serovar hardjo in their urine, as documented by results of
immunofluorescence testing and darkfield microscopy. The
interval from inoculation until confirmed shedding typically
was 3 to 4 weeks. After shedding was confirmed, cattle were
assigned to various treatment groups.
Experimental design and antibiotic treatment—
Initially, oxytetracycline,b ceftiofur,c tylosin,d tilmicosin,e and a
combination productf that contained dihydrostreptomycin-
penicillin G were evaluated. There were 10 treatment groups
with 3 cattle/group. Antibiotic regimens included single injec-
tions of a combination product containing dihydrostrepto-
mycin-penicillin G (25 mg/kg [11.4 mg/lb], IM), oxytetracy-
cline (20 mg/kg [9.1 mg/lb], IM), or tilmicosin (10 mg/kg
[4.5mg/lb], SC) or multiple injections of oxytetracycline (11
mg/kg [5 mg/lb], IM, once daily for 3 days), ceftiofur (20
mg/kg, IM, once daily for 3 days; 2.2 mg/kg [1 mg/lb], IM, once
daily for 3 or 5 days; or 5 mg/kg [2.3 mg/lb], IM, once daily for
3 or 5 days), or tylosin (18 mg/kg [8.2 mg/lb], IM, once daily
for 5 days). Two cattle served as untreated control animals.
Antibiotics were obtained as commercial preparations
JAVMA, Vol 219, No. 5, September 1, 2001
formulated for administration to large animals. All antibiotics
were administered once daily IM in the cranial aspect of the
gluteal muscles, except tilmicosin, which was administered
SC in the cervical region.
Cattle were monitored for 4 to 6 weeks to detect urinary
shedding of L borgpetersenii serovar hardjo. Urine samples
were collected weekly as described previously and examined
by use of darkfield microscopy, immunofluorescence testing,
polymerase chain reaction (PCR) assay, and microbial culture.
A second group of 10 infected cattle was treated with a sin-
gle dose of oxytetracycline (20 mg/kg, IM). These cattle were
monitored for 6 weeks after treatment to detect urinary shedding.
Microbial culture of leptospires—An aliquot (10 ml) of
RUMINANTS
urine was filtered through a 0.45-µm filter.g Filtered urine
was processed for microbial culture, as described elsewhere.14
Semisolid culture medium was prepared by addition of puri-
fied agarh (1.5 g/L) and 5-fluorouracili (100 µg/ml). Aliquots
(200 to 250 µl) of urine at dilutions of 1:10 and 1:100 were
inoculated into culture medium, incubated, and examined,
as described elsewhere.14
Immunofluorescence testing—Unfiltered urine samples
were processed, stained with fluorescein-labeled anti-hardjo
conjugate, and counterstained with flazo orange.22
Leptospires were identified on the basis of typical shape and
specific fluorescence when viewed during incident light fluo-
rescent microscopy.
PCR assay—Urine samples were prepared for PCR
analysis, as described elsewhere.23,24 The PCR was prepared in
accordance with the manufacturer’s directionsj and was used
to amplify a 522-base pair IS1533-derived product25 with the
following primersk: IPL-1, 5'-AACCAACAGAAGGAC-
GAATG-3' and IPR-1, 5'-CCGTCCAATACTTCGTTGTG-3'.
The DNA was amplified in a thermocycler,l using a program
of 30 cycles of 30 seconds at 94 C, 30 seconds at 60 C, and 2
minutes at 72 C, with the extension time increased automat-
ically by 10 s/cycle after the fifth cycle. The PCR-amplified
products were separated by use of agarose gel electrophore-
sis, stained, and examined, as described elsewhere.26
Sensitivity of this PCR assay, on the basis of analysis of seri-
ally diluted cells, was determined to be between 1 and 10
cells (approx 40 to 400 copies of the target sequence).
Results
All cattle inoculated with L borgpetersenii serovar
hardjo became infected, as determined on the basis of
detection of leptospires in their urine. Before initiating
antibiotic treatment, infection was confirmed in each
of the cattle by use of darkfield microscopy and
immunofluorescence testing.
In the initial group, administration of dihy-
drostreptomycin-penicillin G, tilmicosin, oxytetracy-
cline (20 mg/kg, IM, once or 20 mg/kg, IM, once daily
for 3 days), or ceftiofur (2.2 or 5 mg/kg, IM, once daily
for 5 days) eliminated urinary shedding of serovar
hardjo (Table 1). All cattle in each of these treatment
groups did not have detectable urinary shedding of lep-
tospires, as determined by results of darkfield
microscopy, immunofluorescence testing, PCR assay,
and microbial culture 4 to 6 weeks after treatment. In
contrast, cattle treated with oxytetracycline (11 mg/kg,
IM, once daily for 3 days), ceftiofur (2.2 or 5 mg/kg,
IM, once daily for 3 days), or tylosin (18 mg/kg, IM,
once daily for 5 days) continued to shed leptospires in
their urine. The 2 untreated control cattle shed lep-
tospires in their urine for the duration of the study.
Scientific Reports: Original Study 637
01_02_0086.QXD 10/12/2005 2:15 PM Page 638
Table 1—Results of antibiotic treatment of cattle infected with
Leptospira borgpetersenii serovar hardjo on urinary shedding of
leptospires during the period 4 to 6 weeks after treatment
Dosage Duration of
Antibiotic (mg/kg [mg/lb]) treatment (d) Results*
Oxytetracycline
20 [9] 1 0/13
11 [5] 3 1/3
Ceftiofur
20 [9] 3 0/3
5 [2.3] 3 1/3
5 [2.3] 5 0/3
2.2 [1] 3 2/3
2.2 [1] 5 0/3
RUMINANTS
Tylosin 18 [8.2] 5 1/3
Tilmicosin 10 [4.5] 1 0/3
Dihydrostreptomycin
-penicillin G 25 [11.4] 1 0/3
Nontreated infected
control cattle — —
*No of cattle detected shedding leptospires/No. of cattle tested.
— = Not applicable.
In the second group, the use of a single dose of
oxytetracycline (20 mg/kg, IM) was retested in a new
group of cattle to assure its effectiveness. Use of this
antibiotic in accordance with this regimen has several
advantages over the other successful antibiotic regi-
mens tested, including the fact that it involves only a
single injection, is inexpensive, and is safe for use in
humans and animals. For these reasons, administration
of oxytetracycline in accordance with this regimen was
chosen for additional study. Before antibiotic treat-
ment, we again confirmed that all inoculated cattle
were infected and shedding serovar hardjo in their
urine. Cattle were monitored weekly for 6 weeks
beginning 1 week after administration of oxytetracy-
cline, but none of the treated cattle shed detectable
amounts of leptospires in their urine after treatment.
Discussion
In the study reported here, we tested and identified
several antibiotics suitable for treating cattle with lep-
tospirosis. Although dihydrostreptomycin is an effective
treatment, it is no longer available for use in the United
States; therefore, alternative treatments need to be identi-
fied. The focus of this study was to test several families of
antibiotics with differing target specificities. We selected
antibiotics with short withdrawal times (≤ 30 days) that
appeared to be effective on the basis of a preliminary study.20
In the study reported here, we attempted to con-
trol critical aspects of the infection model to reduce
potential variables. This study used cattle infected with
L borgpetersenii serovar hardjo via conjunctival instilla-
tion. This path of inoculation is believed to mimic nat-
ural exposure and has been used in several studies of
vaccine efficacy. Results of those studies12-14 suggest that
the infection that develops after conjunctival instilla-
tion closely resembles the course of natural infection.
Cattle were infected with serovar hardjo strain 203, a
strain that is used routinely and produces a reliable and
reproducible model of bovine leptospiral infection. We
achieved an infection rate of 100%, and as expected,
untreated cattle shed detectable amounts of leptospires
in their urine for the duration of the study.
638 Scientific Reports: Original Study
Antibiotic treatment is expected to reduce prolifer-
ation and urinary shedding of Leptospira organisms.
Thus, the ability to detect a small number of leptospires
is essential to be able to assess the effectiveness of antibi-
otic treatment accurately and to determine with confi-
dence the elimination of productive shedding. Four
methods were used to detect urinary shedding of lep-
tospires (ie, darkfield microscopy, immunofluorescence
testing, PCR analysis, and microbial culture). Each of
these detection methods has advantages and disadvan-
tages. Darkfield microscopy can be used to detect organ-
isms in unprocessed urine, but it is subjective and has a
detection limit of 104 leptospires/ml of urine.
Immunofluorescence testing is more sensitive than
darkfield microscopy, but it cannot differentiate viable
and nonviable organisms. Similar to immunofluo-
2/2 rescence testing, PCR cannot differentiate viable and
nonviable organisms, but it does provide excellent sen-
sitivity and, therefore, can detect an extremely low num-
ber of organisms. Microbial culture is time consuming,
difficult to accomplish, and, similar to darkfield
microscopy, requires a sufficient number of organisms
for detection; however, it is useful in determining
whether leptospires found in the urine of infected cattle
are viable. By combining results of these methods, we
increased the reliability for detection of leptospires.
Dihydrostreptomycin-penicillin G was used as a pos-
itive-control antibiotic and was given in a single injection
(25 mg/kg, IM), as recommended for treatment of lep-
tospirosis.18 Analysis of the data reported here confirmed
that treatment with dihydrostreptomycin-penicillin G is
effective for treatment of cattle infected with L borg-
petersenii serovar hardjo. It should be mentioned that
dihydrostreptomycin is not uniformly effective for elimi-
nation of urinary shedding of serovar hardjo.19 Strains of
at least 2 Leptospira spp are classified as serovar hardjo
and infect cattle (L borgpetersenii [also known as type
hardjo-bovis] and L interrogans [known as type hard-
joprajitno]).27 Infection of cattle with a mixture of these 2
species and subsequent treatment with dihydrostrepto-
mycin has been unsuccessful.1 Although dihydrostrepto-
mycin effectively clears infections caused by L borg-
petersenii serovar hardjo,m it may not be effective for treat-
ment of cattle infected with L interrogans serovar hardjo.
This is of little concern in the United States, because L
interrogans serovar hardjo (hardjoprajitno) has not been
isolated from cattle with leptospirosis in this country.
An important finding in this study was the effective-
ness of a single injection of oxytetracycline (20 mg/kg,
IM) to cause cessation of urinary shedding of leptospires.
This antibiotic may be a suitable replacement for dihy-
drostreptomycin in the treatment of cattle with lep-
tospirosis. The oxytetracycline treatment reported here is
not intended for use in lactating dairy cattle, but it could
be used during the nonlactating period in dairy cattle or
for treatment of beef cattle, providing producers comply
with the 28-day withdrawal period before slaughter.
Other antibiotics tested in this study were effective
for treatment of cattle with serovar hardjo infections,
but they sometimes required a higher dosage than rec-
ommended by the manufacturer to be effective.
Tilmicosin (10 mg/kg, SC, once) and ceftiofur (2.2 or
5 mg/kg, IM, once daily for 5 days) eliminated
JAVMA, Vol 219, No. 5, September 1, 2001
01_02_0086.QXD 10/12/2005 2:15 PM Page 639
detectable urinary shedding of leptospires. These drugs
may be useful for various situations. Tilmicosin has the
advantage of a 28-day withdrawal time before slaugh-
ter and can be given in a single dose. However, tilmi-
cosin is not suitable for use in dairy cows ≥ 20 months
old, veal calves, calves < 1 month old, or calves fed an
all-milk diet. Similarly, tilmicosin has not been evalu-
ated for safety in treatment of breeding-age or pregnant
cattle. Tilmicosin may be most useful in feedlots to
treat cattle with clinically apparent leptospirosis. When
used in accordance with label directions, ceftiofur does
not have milk or meat withdrawal concerns, which
would allow continued processing of milk or meat
from treated cattle. This factor may outweigh the
expense of ceftiofur and the need to administer the
drug for 5 days to achieve successful treatment. We
also found that ceftiofur was effective when we used a
higher dosage than recommended by the manufacturer
(20 mg/kg, IM, once daily) for a shorter duration (3
days). However, use of antibiotics at extralabel dosages
raises concerns regarding residues in food-producing
animals or their products. This would not be a concern
for use in treating breeding stock or cattle that would
be used for import or export markets.
Leptospira borgpetersenii serovar hardjo persists in
the male and female genital tracts2,19 and in the kidneys.
In the study reported here, the reproductive tracts of
cattle were not examined for persistence of L borg-
petersenii following treatment with antibiotics.
Anecdotal evidence suggests that a single dose of
oxytetracycline (20 mg/kg, IM) decreases infertility in
cows persistently infected with serovar hardjo. This
evidence suggests but does not prove that this antibi-
otic may help in clearing L borgpetersenii serovar hard-
jo from the reproductive tract. Definitive studies are
needed to examine this issue.
a
PLM5, Intergen Co, Purchase, NY.
b
Liquamycin LA-200, Pfizer Animal Health, New York, NY.
c
Naxcel, Pharmacia Co, Kalamazoo, Mich.
d
Tylan 200 injection, Eli Lilly Co, Indianapolis, Ind.
e
Micotil 300 injection, Eli Lilly Co, Indianapolis, Ind.
f
Combiotic, Pfizer Animal Health, New York, NY.
g
Millex-HA 0.45-µm filter, Millipore Products Division, Bedford,
Mass.
h
BBL Microbiology Systems, Cockeysville, Md.
i
5-Fluorouracil, Sigma Chemical Co, St Louis, Mo.
j
AmpliTaq, Perkin Elmer Co, Norwalk, Conn.
k
Nucleic acid facility, Iowa State University, Ames, Iowa.
l
PCR system 9600, Perkin Elmer Co, Norwalk, Conn.
m
Ellis WA, Department of Agriculture for Northern Ireland,
Veterinary Sciences Division, Stormont, Northern Ireland: Personal
communication, 1994.
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