Abstract Book of EAVLD2012 - eavld congress 2012

S4 - P - 02
DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR ASSAY FOR THE DETECTION OF
LEPTOSPIRA BORGPETERSENII SEROVAR HARDJO IN URINE AND KIDNEY
Zbigniew Arent 1 , Caroline Frizzell 2 , Colm Gilmore 1 , William Ellis 1
1
OIE Leptospirosis Reference Laboratory, Veterinary Sciences Division, AFBI, Belfast, Northern Ireland
2
Department of Veterinary Science, School of Agriculture and Food Science, The Queen’s University of Belfast
Leptospira, leptospirosis, real-time PCR
Introduction
Leptospira borgpetersenii serovar Hardjo is the most common
cause of leptospirosis in cattle throughout the world. Infection of
cattle with Hardjo can cause abortion, stillbirth, birth of weak
calves and decreased milk production (1, 2). Infected animals
can act as lifelong renal carriers without clinical signs of the
disease (3) and shed live organisms over a long period of time
(4).
There is no satisfactory method available for the rapid
identification of carrier animals. Culturing of leptospires is the
most definitive test. However this is possible only on fresh
samples where viable leptospires are present. . PCR assays
have the potential to detect both viable and non-viable
leptospires and in recent years, several real-time PCR assays
have been described. Levett et al. (5) developed a real-time
assay using SYBR green chemistry, in which the target was the
gene for the outer-membrane protein LipL32. Expression of the
LipL32 gene is a unique virulence factor for pathogenic
leptospires. LipL32 is an outer membrane lipoprotein that is
conserved genetically and immunologically in pathogenic
leptospires. It is one of the most abundant proteins in Leptospira.
In this study we developed and validated a TaqMan PCR using
novel primers which target a 200 bp fragment of the LipL32 gene.
Materials & methods
Eight animals that had no evidence of exposure to serovar Hardjo
were experimentally infected with L. borgpetersenii serovar
Hardjo type Bovis via conjunctival instillation of 2.5 x 10 6
organisms on 3 consecutive days. A total of 8 urine samples
were collected from each animal prior to and at intervals up to 40
days after infection. In addition , 24 culture negative urine
samples obtained from herds with no evidence of leptospirosis
and 72 kidney samples collected post mortem from animals with
serological evidence of Leptospira serovar Hardjo exposure were
used in the study. All samples were cultured using Leptospira
semisolid Tween 80/40 medium and biphasic culture medium
with different variants of inhibitory substance addition.
DNA was extracted from bacterial cultures and kidneys using the
QIAamp DNA Mini kits (Qiagen) and from urines using the
QIAamp Viral RNA Mini Kit (Qiagen) according to the
manufacturer’s recommendations.
The TaqMan assay to amplify a 200 bp fragment of the LipL32
gene was optimized (primer/probe concentration, annealing
temperature and incubation time with the QuantiTec Probe PCR
Kit (Qiagen) for urine samples and kidney according to the
manufacturer’s instructions. As a control for PCR inhibitors,
specific primers and probe were designed to a housekeeping
gene, β-actin, in cattle for kidney samples but in case of urine
samples TaqMan Exogenous Internal Positive Control reagent
(Applied Biosystems) was added to the amplification reaction.
The limit of detection was ten template copies. Thirty two urine
samples were used to determined diagnostic specificity (DSp). All
of them were culture negative. Also thirty two urine samples and
20 kidney samples that contained serovar Hardjo, as determined
by culture were used to determine diagnostic sensitivity (DSe).
The table below gives the results.
Table 1: Diagnostic sensitivity and specificity of the LipL32 realtime
PCR.
Sensitivity % Specificity %
Urine 84.4 90.6
Kidney 95.0 Not tested
Discussion & conclusions
This study evaluated the real-time PCR assay targeting LipL32
gene for detection of Leptospira spp in bovine kidney and urine.
The results indicate that the sensitivity of the diagnostic assay
varies for kidney and urines. Presumably, some false negative
results were caused by substances in the urine that inhibited
extraction of the DNA, as the exogenous internal positive control
did not show inhibition of the amplification reaction.
In view of the difficulties associated with isolation of leptospires
and the fact that serology does not imitate the carrier or shedding
status, the LipL32 real-time PCR assay may be used as method
for the detection of pathogenic leptospires in bovine urine and
kidney samples
References
1. Ellis, W.A., (1984) Bovine Leptospirosis in the tropics: Prevalence,
Pathogenesis and Control. Pre Vet Med 2: 411-421.
2. Ellis, W.A., O’Brien, J.J., Bryson, D.G., Mackie, D.P., (1985) Bovine
leptospirosis: some clinical features of serovar Hardjo infection. Vet Rec
117: 101-104.
3. Ellis, W.A., Songer, J.G., Montgomery, J., Cassells, J.A., (1986)
Prevalence of Leptospira interrogans serovar Hardjo in the genital and
urinary tracts of non-pregnant cattle. Vet Rec 118: 11-13.
4. Ellis, W.A., (1994) Leptospirosis as a cause of reproductive failure. Vet
Clin North Am Food Anim Pract 10: 463-478.
5. Levett, P.N., Morey R.E., Galloway R.L., Turner D.E., Steigerwalt A.G.,
Mayer L.W. (2005) Detection of pathogenic leptospires by real-time
quantitative PCR. J Med Microbiol 54: 45-49.
The real time PCR was tested against a panel of bacterial
species known to be ruminant pathogens to determine the
analytical specificity of the assay. The analytical sensitivity was
calculated using a dilution series of target DNA. A genome size of
3.9 Mb was used to determine the genomic equivalent (GE) per
microlitre of the purified DNA. The diagnostic sensitivity and
specificity of the assay was determined by testing samples with
only known culture status.
Results
The LipL32 target was amplified from 35 strains tested which
belonged to 6 pathogenic leptospiral species (L.interrogans,
L.borgpetersenil, L.kirschneri, L. santarosai, L.noguchii, L. weilii).
The non-pathogenic Leptospira biflexa, intermediate L. fainei and
other 20 bovine pathogens included were not amplified.