Rapid and efficient diagnosis of leptospirosis in an aborted foal by PCR of gastric juice

Laboratory of Veterinary Bacteriology, Universidade Federal Fluminense, Rua Hernani Mello 101, Sala 309, Niteroi, RJ 24210-130, Brazil.
Veterinary Microbiology (Impact Factor: 2.51). 05/2012; 160(1-2):274-5. DOI: 10.1016/j.vetmic.2012.05.030
Source: PubMed

Full-text

Available from: Gabriel Martins, Apr 12, 2014
Letter
to
the
Editor
Rapid
and
efficient
diagnosis
of
leptospirosis
in
an
aborted
foal
by
PCR
of
gastric
juice
To
the
Editor,
Our
group
has
recently
demonstrated
that
molecular
diagnostics
of
leptospirosis
in
horses
is
becoming
increas-
ingly
important
(Pinna
et
al.,
2011).
At
this
moment,
we
would
like
to
share
our
successful
experience
employing
the
molecular
diagnostic
(PCR)
of
leptospiral
infection
in
the
gastric
juice
of
an
aborted
foal.
Leptospirosis
in
horses
is
an
important
disease
of
the
reproductive
sphere,
since
it
may
lead
to
the
birth
of
weak
foals,
stillbirth
or
neonatal
mortality
and
abortion,
usually
around
the
six
month
of
pregnancy
(Timoney
et
al.,
2011).
Nevertheless,
not
all
infected
animals
present
with
the
acute
disease,
and
subclinical
forms
are
common
in
endemic
regions
(Houwers
et
al.,
2011).
Titers
to
several
serovars
have
been
reported
in
horses.
While
in
North
America
and
other
temperate
countries
serovar
Pomona
seems
to
be
predominant
(Timoney
et
al.,
2011),
in
tropical
countries
serovars
belonging
to
the
Icterohaemorrhagiae
serogroup,
such
as
Icterohaemorrha-
giae
or
Copenhageni,
tend
to
be
most
prevalent
(Hamond
et
al.,
2012).
Although
useful
for
herd
diagnosis,
serology
not
always
is
a
reliable
tool
for
detecting
individual
cases
of
leptospirosis,
since
not
all
animals
produce
detectable
titres
of
specific
agglutinins
(Houwers
et
al.,
2011).
The
isolation
procedures
are
cumbersome,
time
consuming
and
require
fresh
samples
with
a
significant
concentration
of
leptospires
(Hamond
et
al.,
2012).
Therefore,
molecular
tools
such
as
polymerase
chain
reaction
(PCR)
have
been
increasingly
bee n
employed
for
diagnosis
leptospirosis
as
a
reproductive
disease
in
horses.
Leptospiral
DNA
has
been
detected
in
tis sues
(kidney
and
liver)
(Whitwell
et
al.,
2009)
and
in
thoracic
fluid
of
aborted
fetuses
(Pinna
et
al.,
2011).
A
total
of
16
Thoroughbred
mares
of
the
same
flock
were
studied.
The
horses
ranged
from
five
to
seven
years
old.
Despite
it
is
an
endemic
area
for
leptospirosis,
none
of
these
animals
had
bee n
vaccinated
for
leptospirosis.
Six
out
of
the
16
mares
were
struggling
to
get
pregnant
and
miscarried
in
the
eighth
month
of
pregnancy.
In
a
routine
checking
for
reproductive
health
of
the
mares,
serology
(microscopic
agglutination
test
MAT)
was
performed
using
22
liv e
serovars
of
live
leptospires
as
antigens
and
cut-off
point
at
200.
Of
the
16
serum
samples
tested,
14
(87.5%)
were
reactive,
and
Copenha-
geni
was
by
far
the
most
frequent
serovar
(12
out
the
14
reactive
sera).
In
this
meantime,
one
mare
at
the
eight
month
of
pregnancy
aborted.
The
abortion
was
necropsied
and
jaundice
and
petechiae
were
observed.
Since
liver
and
kidneys
were
friable
and
deteriorated,
gastric
juice
was
collected
and
sent
to
the
laboratory
for
bacterial
culturing
and
PCR.
Additionally,
urine
samples
were
collected
from
all
the
mares
for
culturing
and
PCR.
Bacterial
culturing
was
performed
by
inoculation
of
the
samples
into
tubes
with
Fletcher
and
EMJH
media
(BD
Difco,
Franklin
Lakes,
NJ,
USA)
for
20
weeks,
while
PCR
was
performed
by
extraction
of
DNA
with
the
Promega
Wizard
SV
kit
genomic
DNA
Purification
System1
and
amplification
targeting
on
primers
LipL32_45F
(50
AAG
CAT
TAC
TTG
CGC
TGG
TG
30)
and
LipL32_286R
(50
TTT
CAG
CCA
GAA
CTC
CGA
TT
30)
(Pinna
et
al.,
2011).
Serology
of
the
mare
that
aborted
was
also
performed
(MAT).
None
leptospiral
culture
was
obtained.
Nevertheless,
six
(37.5%)
urines
and
the
gastric
juice
were
positive
by
PCR,
what
confirms
the
infection.
Importantly,
although
the
presence
of
leptospiral
DNA
in
aborted
foals
has
already
bee n
demonstrated,
in
various
tissues
and
thoracic
fluid
(Whitwell
et
al.,
2009;
Timoney
et
al.,
2011;
Pinna
et
al.,
2011),
this
is
the
first
report
of
detection
of
leptospiral
DNA
in
gastric
juice
of
an
equine
abortion.
Leptospires
have
already
been
reported
in
the
gastric
juice
of
an
equine
abortion
by
darkfield
microscopy
(Santa
Rosa,
1970),
but,
probably
due
to
the
acid
pH
of
the
gastric
juice,
it
has
never
been
cultured
from
that
sample.
Since
DNA
can
withstand
on
acidic
pH,
PCR
may
be
a
valuable
tool
for
diagnosing
leptospires
in
gastric
juice
of
aborted
foals
when
tis sues
are
not
in
good
condition.
Acknowledgements
The
aa.
are
thankful
for
the
help
of
Dr.
E.
Kraus.
This
study
was
supported
by
CNPq
and
FAPERJ,
Brazil.
WL
is
a
CNPq
fellow.
Veterinary
Microbiology
160
(2012)
274–275
Contents
lists
available
at
SciVerse
ScienceDirect
Veterinary
Microbiology
jou
r
nal
h
o
mep
ag
e:
w
ww
.els
evier
.co
m/lo
c
ate/vetm
ic
0378-1135/$
see
front
matter
ß
2012
Elsevier
B.V.
All
rights
reserved.
http://dx.doi.org/10.1016/j.vetmic.2012.05.030
Page 1
References
Hamond,
C.,
Martins,
G.,
Lawson-Ferreira,
R.,
Medeiros,
M.A.,
Lilenbaum,
W.,
2012.
The
role
of
horses
in
the
transmission
of
leptospirosis
in
an
urban
tropical
area.
Epidemiol.
Infect.
15,
1–3.
Houwers,
D.J.,
Goris,
M.G.,
Abdoel,
T.,
Kas,
J.A.,
Knobbe,
S.S.,
van
Dongen,
A.M.,
Westerduin,
F.E.,
Klein,
W.R.,
Hartskeerl,
R.A.,
2011.
Agglutinat-
ing
antibodies
against
pathogenic
leptospira
in
healthy
dogs
and
horses
indicate
common
exposure
and
regular
occurrence
of
sub-
clinical
infections.
Vet.
Microbiol.
148,
449–451.
Pinna,
A.E.,
Martins,
G.,
Hamond,
C.,
Lilenbaum,
W.,
Medeiros,
M.A.,
2011.
Molecular
diagnostics
of
leptospirosis
in
horses
is
becoming
increas-
ingly
important.
Vet.
Microbiol.
153,
413.
Santa
Rosa,
C.A.,
1970.
Diagno
´
stico
laboratorial
das
leptospiroses.
Rev.
Microbiol.
1,
97–109.
Timoney,
J.F.,
Kalimuthusamy,
N.,
Velineni,
S.,
Donahue,
J.M.,
Artiushin,
S.C.,
Fettinger,
M.,
2011.
A
unique
genotype
of
leptospira
interrogans
serovar
Pomona
type
kennewicki
is
associated
with
equine
abortion.
Vet.
Microbiol.
150,
349–353.
Whitwell,
K.E.,
Blunden,
A.S.,
Miller,
J.,
Errington,
J.,
2009.
Two
cases
of
equine
pregnancy
loss
associated
with
leptospira
infection
in
England.
Vet.
Rec.
165,
377–378.
C.
Hamond
G.
Martins
W.
Lilenbaum
*
Laboratory
of
Veterinary
Bacteriology,
Universidade
Federal
Fluminense,
Rua
Hernani
Mello
101,
Sala
309,
Niteroi,
RJ
24210-130,
Brazil
M.A.
Medeiros
Bio-Manguinhos,
Oswaldo
Cruz
Foundation,
Brazilian,
Ministry
of
Health,
Rio
de
Janeiro,
RJ
21040-360,
Brazil
*Corresponding
author.
Tel.:
+55
21
2629
2435;
fax:
+55
21
2629
2432
E-mail
address:
mipwalt@vm.uff.br
(W.
Lilenbaum).
9
May
2012
Letter
to
the
Editor
/
Veterinary
Microbiology
160
(2012)
274–275
275
Page 2
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Leptospirosis is a zoonotic disease of global importance and has a worldwide distribution. This infection displays clear seasonal nature in some regions of the tropics, where the rainy season is marked by high temperatures. Household and wild animals carry leptospires and contribute to their dissemination in nature. Transmission mainly occurs by contact with water contaminated with the urine of infected animals, and consequently, it is quite widespread especially in times of rain, since many areas are subject to flooding and have poor sanitation. Serological tests demonstrate that Leptospira sp. infection in horses occurs worldwide and that the predominant serovar may vary depending on the region or infection sources. Besides systemic and ocular manifestations, leptospirosis in horses has been recognized as an important disease of the reproductive system, since it leads to the birth of weak foals, stillbirths or neonatal mortality, and mainly to abortion, usually after the sixth month of pregnancy. In this context, this review aims to gather and discuss information about the role of leptospirosis in reproductive disorders in horses.
    Full-text · Article · Aug 2013 · Tropical Animal Health and Production
  • [Show abstract] [Hide abstract] ABSTRACT: Leptospirosis is a global disease of animalsAnimals , which can have a major economic impact on livestock industries and is an important zoonosis. The current knowledge base is heavily biased towards the developed agricultural economies. The disease situation in the developing economies presents a major challenge as humans and animals frequently live in close association. The severity of disease varies with the infecting serovar and the affected species, but there are many common aspects across the species; for example, the acute phase of infection is mostly sub-clinical and the greatest economic losses arise from chronic infection causing reproductive wastage. The principles of, and tests for, diagnosisDiagnosis , treatmentTreatment , controlControl and surveillanceSurveillance are applicable across the species.
    No preview · Article · Jan 2015 · Current topics in microbiology and immunology