VECTOR-BORNE AND ZOONOTIC DISEASES

Volume 14, Number 1, 2014

ORIGINAL ARTICLE
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2013.1324

Serological and PCR Detection of Feline

Leptospira in Southern Taiwan

Kun-Wei Chan,1 Yun-Hsiu Hsu,1 Wei-Ling Hu,1 Ming-Jeng Pan,2 Jyh-Mirn Lai,1
Kwo-Ching Huang,3 and Shih-Jen Chou1

Abstract

Taiwan is in the subtropical zone and has typhoons every year. Leptospirosis is an endemic disease in Taiwan,
and feline leptospirosis in Taiwan remains unknown so far. From January, 2010, to September, 2011, 233 cats in
south Taiwan (159 stray cats and 74 household cats) were sampled in this research. Leptospira antibody titer was
detected by the serology gold standard, the microscopic agglutination test (MAT). Both serum and urine were
examined for Leptospira DNA by polymerase chain reaction (PCR) with two sets of primers. In this study, the
serological survey showed 21 (9.3%) examined sera contained antibodies specific for pathogenic Leptospira
serogroups. The results of PCR revealed that 25 (19.1%) serum and 80 (67.8%) urine samples were found positive
for leptospiral DNA sequences. All products amplified from PCR reactions were sequenced by an automated
method for further confirmation. This is the first study concerning the epidemiology of pathogenic Leptospira in
stray and household cats’ urine, and the results demonstrate that some of the cats are susceptible to pathogenic
Leptospira and have the potential to shed pathogenic Leptospira into the environment. This could be an issue of
public health.

Key Words: Cat serum and urine—Leptospirosis—Taiwan—Microscopic agglutination test—Polymerase chain

reaction.

Introduction of one serovar and serve as an accidental host for another

serovar (Levett 2001, Felt et al. 2011). Rodents are generally

L eptospirosis is a worldwide zoonotic disease caused

by pathogenic Leptospira species. Humans can be infected
by pathogenic Leptospira when in contact with contaminated
urine or water directly or indirectly (Levett 2001). Taiwan is
located in the subtropical climate zone with typhoons and
suffers from serious rainfall and floods every year. The inci-
dence rate of leptospirosis of humans in Taiwan, from 2001 to
2010, was about 0.11 to 0.88 per 100,000 population according
to data from the Centers for Disease Control of Taiwan
(Centers for Disease Control of Taiwan 2010).
Both domestic and wild animals are natural reservoirs and
carriers of Leptospira, acting as maintenance or accidental
hosts (Felt et al. 2011). In Taiwan, the seroprevalence of lep-
tospirosis was 11.49% (64/557) in small mammals and ro-
dents, 33.6% (395/1175) in cattle, 58.8% (83/141) in swine,
and 44.2% (102/231) in stray dogs (Lin 2001, Lai 2004, Lin
2004, Pan et al. 2006). An animal can be the maintenance host
the maintenance host of serovars belonging to serogroups
Icterohaemorrhagiae and Ballum, and dogs may serve as the
reservoir host of serovar Canicola (Levett 2001; Millán et al.
2009). In Taiwan, the most prevalent serogroup by the highest
titers in the microscopic agglutination test (MAT) were Po-
mona and Bataviae in small mammals and rodents, Pomona
and Shermani in cattle, Shermani in swine, Shermani and
Canicola in stray dogs, and Shermani in humans (Lin 2001,
Lai 2004, Lin 2004, Pan et al. 2006, Centers for Disease Control
of Taiwan 2010).
Limited reports are published on Leptospira in feline. Lep-
tospira seroprevalence of felines was 4.9–33.3% in different
countries (Shophet 1979, Agunloye and Nash 1996, Mylonakis
et al. 2005, Jamshidi et al. 2009, Millán et al. 2009, Mosalla-
nejad et al. 2011). The serologic evidence above suggests that
cats are susceptible to Leptospira. However, attention was
seldom paid to significant zoonotic risk of feline leptospirosis,

1
Department of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan.
2
Central Taiwan University of Science & Technology, Institute of Medical Biotechnology, Taichung, Taiwan.
3
Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taipei, Taiwan.

1
2 CHAN ET AL.
although Larsson et al. proved that cats infected with Leptos-
pira experimentally were able to scatter potential zoonotic
Leptospira through their urine for up to 3 months (Larsson
et al. 1985).
Cats are one of the most common pets in Taiwan. It had
been pointed out that domestic cats may act as a potential
source of leptospiral infection of human or other domestic
animals (Larsson et al. 1985). Yet there is no information about
the incidence of cat leptospirosis in Taiwan. In this study, we
detected the DNA sequence of pathogenic Leptospira in feline
urine and serum by polymerase chain reaction (PCR), and
analyzed the antibodies against L. interrogans sensu lato in
felines using MAT. The aim of this research was to determine
the prevalence of pathogenic Leptospira in household and
stray cats in southern Taiwan. Meanwhile, we detected
pathogenic Leptospira DNA sequences in cats’ urine, which
suggested that cats might disseminate pathogenic Leptospira
via urine.
Materials and Methods
Study area
Situated in the subtropical zone, the average rainfall in
southern Taiwan is about 200 mm per month. During the
typhoon season, June to October, the amount of the rainfall
can reach 400 mm per month, according to the records of the
Central Weather Bureau of Taiwan. Typhoons that bring in
large amounts of rainfall could contribute to the epidemic of
leptospirosis in Taiwan (Su et al. 2011). Southern Taiwan is a
plains area, and the main labor force is employed in agricul-
ture and animal husbandry. Rice is the principal crop growing
along southern Taiwan. Rice needs to be grown in flooded
plains called paddies, which is a decent environment for
Leptospira. We suspect that the muddy environment will in-
crease the incidence of farmers becoming infected with
pathogenic Leptospira.
FIG. 1. Flow chart of laboratory tests for cat pathogenic Leptospira. MAT, microscopic agglutination test; PCR, polymerase
chain reaction.
Sample collection
All cat sample collection and laboratory research for this
study were conducted between January, 2010, and Septem-
ber, 2011. We collected samples from three local animal hos-
pitals and five animal shelters around southern Taiwan. Two
of the animal shelters were in the countryside and the other
shelters and animal hospitals were in urban areas. Cats were
anesthetized with Zoletil (Virbac, France) or Dexdometor
(Pfizer, USA), with the owners’ and the shelters’ permission,
to ensure that all cats were under low stress during the sample
collection process. The sample collection procedure was fol-
lowed the rules of Animal Protection Act of Taiwan, and this
project was approved by Bureau of Animal and Plant Health
Inspection and Quarantine, Council of Agriculture, Executive
Yuan.
A total number of 233 cats were sampled, including 159
stray cats and 74 household cats. The methodology of this
study is illustrated as Figure 1. Due to the various volumes of
collected serum, each sample might not have been sufficient
for both MAT and PCR. To obtain a sterile urine sample, a
22-gauge needle and 10-mL syringe were used for ventral cy-
stocentesis. Urine was obtained only when the urinary bladder
had stored more than 3 mL of urine, which was confirmed by
ultrasonography. There were 225 serum samples (155 stray cats
and 70 household cats) examined for pathogenic Leptospira
antibody titer by MAT. For PCR, 131 serum samples (101 stray
cats and 30 household cats) and 118 urine samples (92 stray cats
and 26 household cats) were examined for pathogenic Leptos-
pira DNA sequences. Serum samples were obtained from ve-
nipunctures of cephalic veins and collected in serum separator
tubes. After clotting at room temperature for 15 min, serum
was separated and frozen at - 20C.
Laboratory procedures
DNA extraction. Total genomic DNA in serum was ex-
tracted using QIAamp DNA Mini Kits (QIAgen, Germany).
FELINE LEPTOSPIRA IN TAIWAN
Table 1. Serovars of Leptospiroa Interrogans Sensu Lato Tested for Microscopic
Agglutination in 225 Cats in Southern Taiwan
Species Serogroup
Leptospira interrogans Canicola
Leptospira interrogans Icterohaemorrhagiae
Leptospira interrogans Pyrogenes
Leptospira interrogans Australis
Leptospira interrogans Pomona
Leptospira interrogans Bataviae
Leptospira interrogans Autumnalis
Leptospira interrogans Panama
Leptospira borgpeter senii Javanica
Leptospira borgpeter senii Tarassovi
Leptospira santarosai Shermani
Urine samples were kept in sterile tubes and centrifuged at
15,000 · g for 30 min at 4C (Branger et al. 2005). Urine sedi-
ment was suspended in 200 lL of phosphate-buffered saline
(PBS) solution (Harkin et al. 2003). Genomic DNA in urine
was extracted by QIAamp RNA Mini Kits (QIAgen, Ger-
many) because this commercial kit is able to inactivate the
PCR inhibitors in urine. All the extraction procedures were
performed according to the manufacturer’s protocols.
Microscopic agglutination test. Serum was examined for
antibodies against 11 antigens, shown as Table 1. These an-
tigens were 6- to 10-day-old live cultures of L. interrogans
(serovars Canicola, Icterohaemorrhagiae, Pyrogenes, Aus-
tralis, Pomona, Bataviae, Autumnalis, and Panama), L.
borgpetersenii (serovars Javanica and Tarassovi), and L. san-
tarosai (serovar Shermani), as shown in Table 1. The test was
performed as described by Cole et al. (1973). Briefly, the se-
rum was diluted separately (1:50–1:6400) and then mixed
with the individual Leptospira cultures mentioned above. We
performed the MAT test starting from the serum dilution of
1:50. Antibody titers ‡ 1:100 were determined as a cutoff
value for positive ( Jamshidi et al. 2009, Mosallaneijad et al.
2011).
Polymerase chain reaction. Serum and urine samples
were tested using two pairs of primers. One primer set G1/G2
amplified a 285-bp sequence by PCR from strains of all
pathogenic Leptospira spp. except L. kirschneri. The reactions
were carried out as described previously (Gravekamp et al.
1993). The other pair of primers amplified a 331-bp sequence
of the Leptospira rrs (16S) gene. The PCR assay was performed
according to Mérien et al. (1992). After the amplifications, 5 lL
of PCR products were analyzed using 1% agarose gel that was
subsequently stained with HealthView Nucleic Acid Stain
(Genomics BioSci & Tech Corp, Taiwan) for detection of the
expected product under ultraviolet (UV) light. All of the 285-
bp and 331-bp products amplified from PCR reactions were
sequenced by an automated method. Because primers G1 and
G2 amplified only pathogenic Leptospira, as mentioned pre-
viously, samples were considered positive when the diag-
nostic 285-bp band was observed, whether the amplified
products were sequenced successfully or not. The primer set–
amplified Leptospira rrs (16S) was not able to distinguish be-
tween nonpathogenic L. biflexa and pathogenic Leptospira spp.
Therefore, the products of 331 bp must sequence as patho-
3
Serovar Strain
Canicola Hond Utecht IV
Icterohaemorrhagiae CF1
Pyrogenes Salinem
Australis Ballico
Pomona Pomona
Bataviae Van Tienen
Autumnalis Akiyami A
Panama CZ 214 K
Javanica Valdrat Bataviae 46
Tarassovi Perepeltsin
Shermani ACTT 43286
genic Leptospira or the products would be considered as
negative.
Results
Of 225 cats tested for pathogenic Leptospira antibody titer
by MAT, 21 cats reacted positively for one or two leptospiral
antigens, including 17 stray cats and 4 household cats (Table
2). The titers were ranging from 1:100 to 1:400 are shown in
Table 3. For stray cats, 10 were serologically positive against
Shermani, five against Javanica, two against Icterohaemor-
rhagiae, two against Australis, and one against Pyrogenes.
Antibodies against more than one leptospiral antigen were
found in three cats. One cat had antibodies reacting with Ic-
terohaemorrhagiae (1:100) and Shermani (1:100), one with
Australis (1:200) and Javanica (1:100), and the other one with
Shermani (1:400) and Australis (1:200). These multiple reac-
tions were not considered to be cross-reactions due to the high
dilution titers. No positive titers were obtained with Pomona,
Panama, Autumnalis, Canicola, Bataviae, and Tarassovi.
Among 131 serum samples tested for pathogenic Leptospira
DNA by PCR with two sets of primers, 19.1% (25/131) were
positive for pathogenic Leptospira DNA in serum (16 stray cats
and 9 household cats). As for urine samples, 67.8% (80/118)
were positive for pathogenic Leptospira DNA (71 stray cats
and 9 household cats) (Table 2). All of the 285-bp (n = 100) and
331-bp (n = 88) bands were confirmed by DNA sequencing, as
shown in Table 4. Of n the 100 products amplified with G1/
G2 primers, 82 products (12 serum samples and 70 urine
samples) were sequenced as L. interrogans. The other 18
samples failed to be sequenced due to inadequate PCR
product concentration, although these 18 samples showed the
expected 285-bp products generated by the G1/G2 primer set.
We still considered these 18 samples as positive for patho-
genic Leptospira DNA, because the primer G1/G2 is specific
for pathogenic Leptospira. Of the 88 331-bp sequences of the
Leptospira rrs (16S) gene, 53 samples were sequenced with the
following results: L. interrogans (n = 12), L. kirschneri (n = 2),
Brevundimonas intermedia (n = 3), Pasturella pneumotrpica
(n = 1), and uncultured bacterium (n = 35) (Table 4). The re-
maining 35 samples of 16S PCR failed to be sequenced be-
cause of the low copy numbers, and these samples were
considered as negative because of the inability to differentiate
between pathogenic and nonpathogenic Leptospira by ampli-
fication of the Leptospira rrs gene.
4 CHAN ET AL.

Table 2. Prevalance of Leptospiral Antibodies and DNA in the Cats of South Taiwan

Group Serology (MAT) Leptospira DNA sequence Leptospira DNA sequence

in serum (PCR) in urine (PCR)

Stray cats (n = 159) 11.0% (17/155) 15.8% (16/101) 77.2% (71/92)

Household cats (n = 74) 5.7% (4/70) 30.0% (9/30) 34.6% (9/26)
Total 9.3% (21/225) 19.1% (25/131) 67.8% (80/118)

2010–2011 (total 233 cats). 225 cats were tested using MAT (microscopic agglutination test) and 9.3% (21/225) were found to be positive for
the leptospiral antibody. 131 serum samples were tested for Leptospira using PCR with two sets of primers. Twenty-five cats were tested
positive for pathogenic Leptospira in serum and 118 cats were examined for leptosiral DNA in their urine, and 80 cats were tested positive.

Discussion Taiwan in this study. However, these differences may be a

consequence of different cutoff values and serovars panels.
Pathogenic Leptospira thrives in warm and humid weather,
The sources of the cats’ infection could be wildlife, contami-
therefore leptospirosis is an endemic disease in Taiwan, es-
nated water, cohabitating dogs, and rodents (Green et al.
pecially after the typhoon and flood seasons (Su et al. 2009).
2011). Mosallanejad et al. suggested that different environ-
This is the first study of feline leptospirosis in southern Tai-
mental factors related to the duration of survival of leptospires
wan using serological and PCR detection. Southern Taiwan
may affect the prevalence of leptospirosis (Mosallanejad et al.
was chosen for the study area because this area has suffered
2011).
more floods than other areas of Taiwan. Rice farms in
Ten stray cats in the present study had antibodies against
southern Taiwan may also increase the incidence of lepto-
Shermani, and this serogroup was also found in rodents,
spirosis.
cattle, swine, stray dogs, and humans in Taiwan (Lin 2001, Lai
The results of serological survey showed that 21 cats
2004, Lin 2004, Pan et al. 2006, Centers for Disease Control of
had antibodies against L. interrogans (serovars Icterrohae-
Taiwan 2010); hence Shermani could be one of the most
morrhagiae, Pyrogenes, and Australis), L. santarosai (serovar
widespread serogroups in Taiwan. The serogroup Javanica
Shermani), and L. borgpetersenii (serovar Javanica) in
was diagnosed in five stray cats in this study, and this ser-
dilutions ‡ 1:100. The lowest serum dilution used in Shophet’s
ogroup was also diagnosed previously in rodents and stray
study was 1:24. The author indicated that for some serovars
dogs in Taiwan (Lai 2004, Pan et al. 2006). Three sera of
animals may shed Leptospira through urine with a low or
household cats in this survey had titers to Icterohaemor-
undetectable antibody titer. Therefore, low serum dilutions in
rhagiae. Out of 231 stray dogs in Taiwan, which had never
the MAT test should be included to estimate the ser-
been vaccinated against Leptospira, 17 also had antibodies
oprevalence of cats (Shophet 1979). The antibody titer of 1:100
reacting with Icterohaemorrhagiae (Lai 2004). In view of these
was considered as a cutoff value for positive reaction in this
finding, cats might infect Leptospira directly by ingesting
study to rule out the cross-reaction at low titers. The positive
contaminated prey (Shophet and Marshall 1980) or indirectly
MAT results of cats in this study may indicate their previous
by the water contaminated by domestic animals, rodents, and
exposure to Leptospira, because cats in Taiwan were not rou-
stray dogs (Green et al. 2011). However, more studies need to
tinely vaccinated against Leptospira. The seroprevalence of
be done to clear out the pathogen transmission between do-
leptospirosis in cats is different from place to place. Leptos-
mestic animals, rodents, dogs, and cats in Taiwan.
piral prevalence was reported to be 4.9% (5/102) in stray cats
As shown in Table 2, there were 25 serum samples found
in Ahvaz (Mosallanejad et al. 2011), 27% (30/111) in stray and
positive for Leptospira DNA. The duration of leptospiremia
household cats in Tehran ( Jamshidi et al. 2009), 20.0% (5/25)
in cats is very short because of the rapid rise of leptospiral
in feral cats in Spain (Millán et al. 2009), 33.3% (33/99) in
antibodies (for review, see Larsson et al. 1985). The short oc-
household cats in a feline hospital population in Greece
currence of leptospiremia makes detection of Leptospira
(Mylonakis et al. 2005), and 9.3% (21/225) in cats in southern
DNA in serum a last-resort method to diagnose leptospirosis.
Leptospiral culture of feline urine was used to monitor the
shedding of Leptospira in experimentally and naturally in-
Table 3. Results of MAT Titer (Total 238 Cats) fected cats. None of Leptospira was isolated in Agunloye’s and
Serogroups 100 · 200 · 400 · Prevalencea Nash’s investigation in cats in Scotland (Agunloye and Nash
1996). In Larsson’s experiment, two cats inoculated with L.
Shermani 6 1 3 4.2% interrogans canicola had the presence of L. interrogans detected
Javanica 5 2.1% by urine bacterial cultures between 2nd and 10th week after
Icterohaemorrhagiae 3 2 2.1% inoculation (Larsson et al. 1985). In our study, Leptospira DNA
Australis 3 1.3% sequences were detected using PCR in 80 urine specimens.
Pyrogenes 1 0.4% The detection rate of Leptospira DNA in cats’ urine was con-
21 cats tested positive for the leptopsiral antibody. 18 cats were siderable, whereas urine culture for leptospires was not done
positive for only 1 serogroup. 3 cats were positive for 2 different in this study and the viability of leptospires in cats’ urine was
serogroups. (One was positive for Australis 200 · and Javanica unknown. More stray cats (n = 71) were detected Leptospira
100 · ; one was positive for Shermani 100 · and Icterohaemorrhagiae DNA in urine than household cats (n = 8). This could be due to
100 · ; the other one was positive for Shermani 400 · and Australis
200 · ). the fact that stray cats are at a higher risk of exposure to the
a
positive rate within 238 cats. environmental infections. The detection rate of urine using
MAT, microscopic agglutination test. PCR (67.8%) was obviously higher than the results of serum
FELINE LEPTOSPIRA IN TAIWAN 5

Table 4. DNA Sequence Analysis (Total 188 Samples) of PCR Products

Primers L. intrrogans L. kirchneri Pasturella Brevundimonas Unculture Fail to Total

(products) pneumotropica intermedia bacterium sequence

rrs (16S) 12 2 1 3 35 35 88
(331-bp)
G1/G2 82 0 0 0 0 18 100
(285-bp)

135 samples were sequenced successfully and 53 were sequenced unsuccessfully. The sequenced results of 285-bp products were 82 L.
interrogans. Only 14 of 331-bp products were found as L. interrogans (n = 12) or L. kirchneri (n = 2).

using PCR and MAT, 19.1% and 9.3%, respectively. In view of
these data, the prevalence of Leptospira DNA in cat urine
may be underestimated if only MAT was used for detecting
leptospirosis.
Our sequence results presented here confirm the previous
description of Gravekamp et al. that the G1/G2 primers are
specific for pathogenic Leptospira, but DNA from L. kirschneri is
unable to be amplified using this set of primers (Gravekamp
et al. 1993). Palaniapan et al. claimed that L. biflexa can be am-
plified using G1/G2 primers (Palaniappan et al. 2005), but
L. biflex was not amplified in our study. To detect L. kirschner, the
other primer set was used to amplify sequences from the Lep-
tospira rrs (16S) gene. Fourteen 331-bp products were sequenced
as L. interrogans (n = 12) and L. kirchneri (n = 2), but 39 331-bp
bands were sequenced as Pasturella pneumotropica (n = 1), Bre-
vundimonas intermedia (n = 3), and uncultured bacterium (n = 35).
Therefore, we conclude that the primer-amplified sequence
from the Leptospira rrs (16S) gene was not specific for Leptospira
and sequencing was necessary for further confirmation.
As shown in Table 5, 85 cats were examined with all three
methods used in this study, including MAT and PCR of serum
and urine. Some animals infected with Leptospira may shed
the spirochete with an undetectable or low serological titer, as
reported by Shophet in 1979; this was again confirmed in this
investigation. In the experiment of feline leptospirosis, no
positive result was obtained for the blood cultures of Leptos-
pira due to the early rise of antibody after infection, and the
urine culture was positive 14 days after inoculation. In our
data, 13 cats had Leptospira DNA detected both in serum and
urine at the same time. Reinfection might be one of the reasons
that Leptospira presented in serum and urine simultaneously.
Table 5. Different Results of Cats Tested
with 3 Methods Used in this Study
(Total 85 Samples)
Serology Leptospires Leptospires No. of
(MAT) in serum (PCR) in urine (PCR) samples
+ + + 1
+ + - 0
+ - + 7
- + + 12
+ - - 2
- + - 1
- - + 43
- - - 19
One household cat not only had antibodies against Icter-
ohaemorrhagiae but also was positive for Leptospira DNA in
both serum and urine. It is possible that the low titer of anti-
body (1:100) was not enough to eliminate the antigens in se-
rum. In Larsson’s experiment, no urine culture was positive in
the cats inoculated with Icterohaemorrhagiae (Larsson et al.
1985). In our study, this household cat with a titer of 1:100
antibodies against Icterohaemorrhagiae had DNA of L. in-
terrogans detected in its urine.
Seldom were clinical signs noticed in cats infected with
Leptospira despite the presence of leptospiremia and leptos-
piruria (Green et al. 2011). However, three cats naturally in-
fected with Leptospira were reported by Arbour et al. (2012) to
have polyuria, polydipsia, uveitis, and lameness. These in-
vestigators said that clinical signs might develop eventually
after a long period of infection (Arbour et al. 2012). No clinical
signs were observed in all the cats involved in this study. It
was a pity that we did not persistently monitor the cats with
evidence of leptopsiral infection.
This study demonstrates that cats in southern Taiwan were
exposed to pathogenic Leptospira and the DNA sequences of
Leptospira were found both in serum and urine. Clinical signs
might not be noticed in cats with leptospirosis. This evidence
indicated that cats can be naturally infected by Leptospira, and
cats could be the potential source of human and animal Lep-
tospira infection. However, the viability of Leptospira in cat
urine was unknown because urine culture was not done in
this study.
Acknowledgments
This study was approved and sponsored by the Bureau of
Animal and Plant Health Inspection and Quarantine,
Council of Agriculture, Executive Yuan. We followed the
rules of the Animal Protection Act of Taiwan for sample
collection procedures. We thank Dr. Min-Liang Wong (De-
partment of Veterinary Medicine, College of Veterinary
Medicine, National Chung-Hsing University, Taichung, and
Taiwan) and microbiologist John P. Thibideau for his critical
appraisal.
Author Disclosure Statement
All authors declare that no competing financial interests
exist.
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Address correspondence to:
Shih-Jen Chou
Department of Veterinary Medicine
National Chiayi University
300 Syuefu Road
Chiayi City 60004
Taiwan
E-mail: sjchou@mail.ncyu.edu.tw