Rapid and efficient diagnosis of leptospirosis in an aborted foal by PCR of gastric juice.
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 been employed for
diagnosis leptospirosis as a reproductive disease in
horses. Leptospiral DNA has been detected in tissues
(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 been 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 live 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
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
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 been 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 tissues
are not in good condition.
The aa. are thankful for the help of Dr. E. Kraus. This
study was supported by CNPq and FAPERJ, Brazil. WL is a
Veterinary Microbiology 160 (2012) 274–275
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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.
Laboratory of Veterinary Bacteriology,
Universidade Federal Fluminense, Rua Hernani Mello 101,
Sala 309, Niteroi, RJ 24210-130, Brazil
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: email@example.com (W. Lilenbaum).
9 May 2012
Letter to the Editor / Veterinary Microbiology 160 (2012) 274–275