PLOS ONE
RESEARCH ARTICLE
Cats shedding pathogenic Leptospira spp.—An
underestimated zoonotic risk?
Roswitha Dorsch ID1*, Javier Ojeda2, Miguel Salgado3, Gustavo Monti ID3,
Bernadita Collado3, Camillo Tomckowiack4, Carlos Tejeda3, Ananda Müller ID2¤,
Theo Eberhard5, Henricus L. B. M. Klaasen6, Katrin Hartmann1
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1 Medizinische Kleintierklinik, Ludwig-Maximilians-Universität Munich, Munich, Germany, 2 Instituto de
Ciencias Clı́nicas, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile, 3 Instituto
de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia,
Chile, 4 Escuela de Graduados, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia,
Chile, 5 University of Applied Sciences Munich, München, Germany, 6 Department of Global Companion
Animals R&D, MSD Animal Health, Boxmeer, The Netherlands
¤ Current address: Department of Biomedical Sciences, Ross University School of Veterinary Medicine,
Saint Kitts and Nevis, West Indies
* roswitha.dorsch@lmu.de
Abstract
OPEN ACCESS
Citation: Dorsch R, Ojeda J, Salgado M, Monti G,
Collado B, Tomckowiack C, et al. (2020) Cats
shedding pathogenic Leptospira spp.—An
underestimated zoonotic risk? PLoS ONE 15(10):
e0239991. https://doi.org/10.1371/journal.
pone.0239991
Editor: Kalimuthusamy Natarajaseenivasan,
Bharathidasan University, INDIA
Received: May 27, 2020
Accepted: September 16, 2020
Published: October 22, 2020
Peer Review History: PLOS recognizes the
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https://doi.org/10.1371/journal.pone.0239991
Copyright: © 2020 Dorsch et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Shedding of DNA of pathogenic Leptospira spp. has been documented in naturally infected
cats in several countries, but urinary shedding of infectious Leptospira spp. has only recently
been proven. The climate in Southern Chile is temperate rainy with high annual precipitations which represents ideal preconditions for survival of Leptospira spp., especially during
spring and summer. The aims of this study were to investigate shedding of pathogenic Leptospira spp. in outdoor cats in Southern Chile, to perform molecular characterization of isolates growing in culture, and to assess potential risk factors associated with shedding. Urine
samples of 231 outdoor cats from rural and urban areas in southern Chile were collected.
Urine samples were investigated for pathogenic Leptospira spp. by 4 techniques: qPCR targeting the lipL32 gene, immunomagnetic separation (IMS)-coupled qPCR (IMS-qPCR),
direct culture and IMS-coupled culture. Positive urine cultures were additionally confirmed
by PCR. Multilocus sequence typing (MLST) was used to molecularly characterize isolates
obtained from positive cultures. Overall, 36 urine samples (15.6%, 95% confidence interval
(CI) 11.4–20.9) showed positive results. Eighteen (7.8%, 95% CI 4.9–12.1), 30 (13%, 95%
CI 9.2–18), 3 (1.3%, 0.3–3.9) and 4 cats (1.7%; 95% CI 0.5–4.5) were positive in qPCR,
IMS-qPCR, conventional culture, and IMS-coupled culture, respectively. MLST results of 7
culture-positive cats revealed sequences that could be assigned to sequence type 17 (6
cats) and sequence type 27 (1 cat) corresponding to L. interrogans (Pathogenic Leptospira
Subgroup 1). Shedding of pathogenic Leptospira spp. by cats might be an underestimated
source of infection for other species including humans. The present study is the first one
reporting growth of leptospires from feline urine in culture in naturally infected cats in SouthAmerica and characterisation of culture-derived isolates. So far, very few cases of successful attempts to culture leptospires from naturally infected cats are described worldwide.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
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Funding: The Bavarian Academic Center for Latin
America supported the study by paying for travel
expenses (between Valdivia and Munich on two
occasions) for planning the study. The Dirección de
Investigación y Desarollo of the Universidad
Austral de Chile supported the study by covering
part of the costs for the material necessary for
sampling and part of additional costs, e.g.
transport and compensation of owners for study
participation. For compensation, all included cats
were vaccinated and dewormed. MSD Animal
health provided the resources for sampling, clinical
investigation of cats and laboratory investigations.
Part of the laboratory work was funded by
Proyecto FIC 15-8 (código BIP 30421496) del
Gobierno Regional de Los Rı́os, Chile.
Competing interests: H.L.B.M. Klaasen is an
employee of a company (MSD Animal Health)
developing and marketing animal vaccines and
pharmaceuticals. Otherwise the authors declare
that they have no conflict of interest with respect to
the research, authorship and/or publication of this
article. This does not alter our adherence to PLOS
ONE policies on sharing data and materials.
Leptospira shedding by cats in Southern Chile
Introduction
Leptospirosis is a worldwide zoonotic disease affecting most mammalian species. Prevalence
of Leptospira species (spp.) infection is associated with certain environmental conditions and
shows a positive association with increased rainfall and temperatures [1–4]. Leptospires survive for months in water and moist soil. Contamination of the environment with pathogenic
Leptospira spp. mainly occurs via urinary shedding of reservoir hosts, such as small rodents
that are considered the most important reservoir species. These species usually do not show
clinical signs after infection but harbor leptospires in their renal tubules and shed them for
prolonged periods of time [5]. In livestock animals (cows and pigs), leptospirosis causes
chronic infections affecting fertility and reproduction [6, 7].
Only few reports of clinical cases of leptospirosis in cats are published [8–10], and Leptospira spp. infection appears to be underinvestigated in cats compared to other animal species. It
has, however, been demonstrated that cats can be infected and develop specific antibodies [11,
12]. Reported antibody prevalence in naturally infected cats ranges from 0.8% to 33.3%,
depending on the geographic area and the cats´ lifestyle [13–16]. The highest prevalence of
antibody-positive cats have been found in Greece (33.3%) [17] and in rural areas of Southern
Chile (25.2%) [18]. In addition, there are reports that infection with pathogenic Leptospira
spp. in cats might be, although rarely, associated with acute kidney injury or liver disease but
also with the development of chronic kidney disease. A study from Canada identified a significantly higher antibody prevalence in cats with chronic kidney disease (14.9%) when compared
to healthy cats (7.2%) [12].
Still there are uncertainties concerning the role of cats as carrier of Leptospira spp. and their
potential zoonotic risk. Few studies demonstrated shedding of Leptospira spp. in cats, first by
identification of leptospiral agglutinins in the urine [19] and later by PCR [11, 12, 14–16, 20].
Experimentally infected cats can shed leptospires via urine intermittently up to 10 weeks after
infection with Leptospira interrogans serovar Canicola [19]. Prevalence of cats shedding pathogenic Leptospira spp. in naturally infected cats has also been documented using urine PCR,
ranging from 0.8% in Thailand [15], 1.7% in Spain [16], 3.3% in Canada and Germany [12,
14], 4.9% in Malaysia [20], up to 67.8% in Taiwan [11].
The climate in Southern Chile is temperate rainy with an average temperature of 16.7˚C. In
the Los Rios region, the reported annual precipitations exceed 2400 mm (www.wetterkontor.
de). This represents ideal environmental conditions for survival of pathogenic Leptospira spp.
in the environment, particularly during spring and summer. A study performed in rural communities in Southern Chile demonstrated the ubiquity of leptospires in household environments [21]. Water samples from puddles, containers, animal troughs, rivers, canals, and
drinking water from 236 households were investigated for the presence of pathogenic Leptospira spp. using PCR. Pathogenic Leptospira spp. were identified in 13.5% of all samples; e.g. in
11.3% of open containers, 14.5% of animal drinking sources, 15.8% of human drinking
sources, and 19.3% of puddles. This indicates that there is a considerable risk of infection for
humans and mammalian animals living in this environment. Leptospirosis has emerged to
become a major public health threat in Latin America. Nevertheless, little is currently known
regarding its incidence [22]. In countries, such as Peru and Chile, where leptospirosis has been
studied systematically in rural areas, a large proportion of the human population has antibodies indicating previous exposure to pathogenic leptospires (36,6% and 12,0% respectively) [23,
24].
In Chile, a variable human to cat ratio ranging from 9.3:1 to 1.4:1 has been reported [25]
suggesting that there is a relatively large feline population that could be a potential source of
Leptospira spp. infection. So far, it is not known how many cats in this geographic area actively
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Leptospira shedding by cats in Southern Chile
shed pathogenic Leptospira spp., while the reported antibody prevalence in cats in rural areas
(25.2%) [18] is among the highest reported worldwide. Therefore, the role of cats as a reservoir
for pathogenic Leptospira spp. could be very important.
Thus, to provide scientific knowledge for a better understanding of the role and relative
importance of cats as a source of pathogenic Leptospira spp. infection, the aims of the study
were: (1) to determine the proportion of cats shedding pathogenic Leptospira spp. in Southern
Chile, (2) to determine the molecular profile of the cultured isolates, and (3) to identify possible risk factors regarding Leptospira spp. shedding.
Material and methods
Ethical note
Procedures were approved by the Universidad Austral de Chile Animal Care Committee (No.
269/2016). Informed written owner consent was obtained for all cats included in the study.
Study design
The sampling size was statistically estimated by assuming an expected prevalence of urinary
shedding of pathogenic Leptospira of 4% with a 95% confidence interval (CI) of the estimation
with a 5% precision, indicating that at least 236 cats had to be included. Between October 2016
and March 2017, cats of rural and urban origin from the Los Rı́os and Los Lagos region in
Southern Chile were included, trying to cover a geographic area in which a high antibody prevalence in cats had been reported previously [18].
Cats
Included cats were patients of the small animal hospital of the Universidad Austral de Chile in
Valdivia presented for health issues or for elective procedures (e.g. neutering, vaccination), client-owned cats participating in castration projects offered by the city of Valdivia or other communities, and cats presented by their owners specifically for study enrolement. Cats were
offered one free vaccination and deworming as compensation for participating, if this was clinically justifiable. Cats were excluded from the study if they had received antimicrobial treatment within the last four weeks before sampling due to a decreased likelihood for detection of
Leptospira spp. even if they were infected, or if they were living exclusively indoors or had only
access to a balcony because of a low risk for infection.
Sampling
Urine samples were collected via cystocentesis using a 21 G needle attached to 5 ml syringe.
Cystocentesis was performed with palpation of the urinary bladder or ultrasound-guided. At
the time of sampling, a physical examination was performed in each cat. A questionnaire with
information about the cat and potential risk factors was answered by the owner. It included
questions about signalment, living conditions, consumption of raw meat, drinking out of puddles, contact with rodents or eating rodents, and contact with other cats, dogs, or livestock and
vaccination status.
Detection of pathogenic Leptospira spp. by qPCR and culture
To increase the overall diagnostic sensitivity for the detection of pathogenic Leptospira spp. in
urine, four diagnostic tools were used in each sample: lipL32 quantitative qPCR, immunomagnetic-separation (IMS)-coupled with lipL32 qPCR (IMS-qPCR), culture for Leptospira spp.,
and IMS-coupled with culture (Fig 1).
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Leptospira shedding by cats in Southern Chile
Quantitative real-time lipL32 polymerase chain reaction. For each sample, at least 1 mL
was centrifuged at 6,500 rpm for 15 minutes. The pellet was resuspended in 1 mL of 1X phosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4
(pH 7)] and then transferred to a 1.5 mL microcentrifuge tube and re-centrifuged at 11,000
rpm for 5 minutes. Finally, the supernatant was discarded and the pellet was resuspended in
1mL of 1X PBS [26]. After this first cleaning and purification step, 100 μL were separated for a
pretreatment step of immune-separation (see below) before proceeding with DNA extraction
and direct qPCR. The rest of the urine was subjected to a DNA extraction-purification protocol using the High Pure PCR Template Preparation kit (Roche), following the manufacturer
instructions.
The DNA templates obtained were analyzed in a qPCR system (Roche LightCycler 2.0),
using a TaqMan probe and targeting the lipL32 gene which is specific only for pathogenic Leptospira spp. [26]. The amplification mixture for each sample included 0.7 μM primers, 0.15 μM
probe, 10 μL Master Mix TaqMan universal (Roche) and 5 μL DNA template, in a total volume
of 20 μL. Samples were amplified with the following program: Initial denaturation at 95˚C for
2 min, followed by 40 cycles of denaturation for 5 s at 95˚C for 5 and annealing/elongation for
30 s at 58˚C [26, 27]. The PCR system included a negative and positive control in order to survey the proficiency of the reaction as well as DNA extraction-negative and -positive controls.
The positive control for both, DNA extraction and PCR protocols, corresponded to a pure culture of pathogenic Leptospira spp. in two dilutions with a known concentration of leptospires
(104/mL and 102/mL), where the two dilutions were used. Amplification efficiency (E) was
calculated from the slope of the standard curve in each run using the following formula
(E = 10−1/slope).
Immunomagnetic-separation (IMS)-coupled lipL32 quantitative polymerase chain reaction. The urine sample aliquot was pre-treated with an IMS method published previously
[28], followed by a DNA extraction purification and molecular confirmation qPCR. The IMSqPCR consisted of 4 steps (in silico peptide production; polyclonal antibody production; coating magnetic beads with polyclonal antibodies, and IMS–qPCR Leptospira spp. detection). The
magnetic separation of pathogenic Leptospira spp. from urine samples and the subsequent
washing steps were carried out using the automated BeadRetrieverTM System (Invitrogen Life
Technologies, Grand Island, NY). Pathogenic Leptospira spp. were selectively concentrated
from 0.5 mL processed samples obtained from the coated magnetic beads, as described above.
The final product was suspended in 0.5 mL PBS for DNA extraction purification followed by
qPCR for pathogenic Leptospira spp. as described above.
Direct culture. Two dilutions of urine (1:10 and 1:100) with 5 ml of liquid EllinghausenMcCullough-Johnson-Harris (EMJH) medium supplemeted with 5-fluorouracil (0.2 mg/ml)
were prepared immediately after urine sampling. Samples were cultured at 29˚C for 13 weeks
according to previous recommendations [29]. In this liquid medium, growth was defined as
turbidity that becomes apparent at an estimated bacterial density of 1x108 bacteria/mL. Each
sample was observed for evidence of growth using dark-field microscopy for observation of
viable, motile Leptospira spp. bacteria. DNA of positive isolations was used for molecular characterization using the above described extraction protocol.
Immunomagnetic separation coupled to culture. After immunomagnetic separation as
described above, 100 μL of the final product were transferred into 5 ml EMJH medium and
cultured at 29˚C for 13 weeks. Each sample was observed for evidence of colony growth, as
defined above. DNA of positive cultures was used for molecular characterization.
Estimation of pathogenic Leptospira spp. shedding load. Pathogenic Leptospira spp. cell
numbers (genome equivalents) were estimated from either of the detection methods. Estimation was based on the concentration of Leptospira spp. DNA, obtained from positive samples,
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Leptospira shedding by cats in Southern Chile
Fig 1. Methods applied for identification of urinary shedding of pathogenic Leptospira spp.
https://doi.org/10.1371/journal.pone.0239991.g001
that was measured in a Nanoquant spectrophotometer (TECAN group, Männedorf, Schweiz)
adjusted for a 106 dilution. With the reference of the molecular weight of the genome of Leptospira interrogans serovar Hardjo-prajitno (GenBank accession number EU357983.1) a standard curve for estimation of pathogenic Leptospira spp. numbers in urine using a Roche 2.0
real-time PCR, according to the following equation was established [30, 28]:
Genome equivalent ¼
DNA concentration ðng=mlÞ � ð6:022 � 1023 mol 1 Þ
ð4:659 � 106 base pairsÞ � ð1 � 109 ng=g � 660g=molÞ
ðaccesion number EU357983:1Þ ðbase massÞ
Molecular characterization by multilocus sequence typing (MLST). The "Multi Locus
Sequence Typing" (MLST) method was used to molecularly characterize strains obtained from
positive cultures. MLST was performed according to a protocol described by Thaipadungpanit
et al. (2007) with the following housekeeping genes: glmU, pntA, sucA, tpiA, pfkB, mreA, and
caiB [31]. Briefly, PCR amplification was carried out using Leptospira spp. DNA obtained
from positive urine cultures, where the initial denaturation step was 94˚C for 5 min, followed
by 35 cycles of 94˚C for 30 sec, annealing/elongation was at 50˚C for 60 sec for all genes, extension was at 72˚C for 50 sec, and then the final extension was at 72˚C for 7 min. The PCR products were purified using the Geneaid™ "Gene/DNA Genetic Extraction" kit, and sequencing
was done in both directions with primers initially used for PCR amplification. Sequencing was
performed using the sequencer kit "BigDye Terminator v. 3.1 cycle sequencing kit (ABI)" and
the automated DNA sequencer "ABI Prism 3130xl Genetic". The sequences were analyzed
using the Chrommas and Bioedits programs, and these were derived from the international
database for free use (https://pubmlst.org/leptospira/) to obtain the allelic profile and to assign
the sequence type (ST).
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Leptospira shedding by cats in Southern Chile
Data analysis
The overall prevalence of urinary shedding (as detected by any of the 4 techniques) of pathogenic Leptospira spp. with their 95% confidence was calculated. Mann-Whitney test was used
for comparison of Leptospira spp. loads between direct qPCR and IMS-qPCR. Statistical analysis to assess risk factors was performed by ordinary and mixed-logistic regression models
using the location of the cat as a random slope effect to account for potential environmental
heterogeneity.
The strategy for building the model consisted in obtaining unconditional models for initial
screening of variables. Variables associated with the outcome (P<0.25) were eligible for inclusion in the conditional model that was built using a forward approach and a P value of 0.05
was used for interpreting the outcomes. Potential interactions were tested based on their biological significance. The best model was assessed by using the Bayesian information criterion
(BIC) index as a measure of goodness-of-fit. Two analysis were performed: a) one for cats positive with any of the 4 techniques, combined as “Leptospira-shedding” cats, and b) one for “viable Leptospira-shedding” cats (culture- and IMS-coupled culture-positive cats). Analysis was
done with lme4 package [32] of R (R Development Core Team (2008)) [33]. A power post hoc
analysis was performed using WebPower package [34] for a evaluation of variables that were
statistically not significant in the model.
Results
Shedding of pathogenic Leptospira spp.
In total, 231 cats from 11 locations at 7 municipal districts were included into the study (Fig
2). Overall, 30 cats were PCR-positive (Table 1), and 7 cats were culture-positive.
All 18 cats that were positive on qPCR were also positive on IMS-qPCR (Table 2). IMSqPCR identified 12 additional cats as shedders of pathogenic Leptospira spp. Seven cats were
positive in culture, 3 cats in direct culture and 4 other cats in IMS-coupled culture. None of
them was positive in both culture methods. One of the cats that was positive in IMS-coupled
culture was also positive in conventional qPCR and on IMS-qPCR. The other 6 culture-positive cats were PCR-negative. Individual data on signalment and health status are illustrated in
Table 2. Data of geographic distribution of positive cats are combined in Table 3.
Multi locus sequence typing of isolates obtained of 7 positive cultures revealed that
sequences of 6 cats were identical to available sequences of L. interrogans sequence type (ST)
17, and the sequence of one cat was identical to available sequences of L. interrogans ST 27
(Table 4).
Risk factor analyses
For unconditional model (Table 5), health status (P<0.001), eating rodents (P = 0.08), reproductive status (P = 0.11), previous vaccination (P = 0.11), and age >1 year (P = 0.12) were variables primary associated with leptospiruria, and used for building a conditional model for cats
that were PCR- and/or culture positive.
Conditional logistic regression after evaluating potential interactions and confounders is
illustrated in Table 6 and included four risk factors. Two of them (being sick and being vaccinated) were significantly associated with shedding of pathogenic Leptospira spp. while the
other two (age >1year and eating rodents) were not. The random intercept was statistically
significant and accounted for 22.9% of the extra variability due to the place where the cat lived.
The power estimation for this model was 90% which is considered high enough.
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Leptospira shedding by cats in Southern Chile
Fig 2. Map of Latin America and of the region Los Rios in Southern Chile. Sampled cats originated from the 7 delineated municipal
districts.
https://doi.org/10.1371/journal.pone.0239991.g002
Table 1. Percentage and 95% Confidence Intervals (CI) of positive results from 231 urine samples using quantitative real-time PCR (qPCR), immunomagnetic-separation-coupled qPCR (IMS-qPCR), direct culture and IMS-coupled culture for identification of cats shedding pathogenic Leptospira spp. in urine.
Assay
Percentage (number) of positive samples
Estimated bacterial load/mL
% (n)
95% CI
qPCR
7.8 (18)
4.9–12.1
32.7
17–49
IMS-qPCR
13.0 (30)
9.2–18.0
1,683,123
578,206–2,788,040
Direct culture
1.3 (3)
0.3–3.9
n. d.
n. d.
IMS-coupled culture
1.7 (4)
0.5–4.5
n. d.
n. d.
15.6 (36)
11.4–20.9
n. d.
n. d.
Total
Mean
95% CI
CI = confidence interval; n. d. = not determined
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Leptospira shedding by cats in Southern Chile
Table 2. Signalment, origin and health status of cats with positive results on qPCR, immunomagnetic-separation (IMS)-coupled PCR (IMS-qPCR), direct culture
and/or IMS-coupled culture and respective bacterial concentrations in number of genomic equivalents/mL (GE/ml).
Cat Sex
Age
(years)
Location
Status
Health status
qPCR GE/ml estimated by
qPCR
IMSqPCR
GE/mL estimated by
IMS-qPCR
Culture
IMS-coupled
culture
2
fs
1,5
Valdivia
priv.
ear mites, chronically ill�
neg
0
neg
0
neg
pos
8
fs
5
Valdivia
priv.
healthy
neg
0
pos
119
neg
neg
10
f
1,75
Valdivia
priv.
healthy
pos
35
pos
7,160,000
neg
neg
11
f
1
Valdivia
priv.
healthy
pos
26
pos
330,000
neg
neg
12
f
0,75
Valdivia
priv.
healthy
pos
96
pos
3,380,000
neg
neg
13
f
4
Valdivia
priv.
healthy
pos
42
pos
8,380,000
neg
neg
18
f
1
Paillaco
priv.
chronically ill�
pos
19
pos
124,800
neg
neg
22
f
NA
Lago
Ranco
stray
cat
healthy
pos
3
pos
2,680
neg
neg
25
fs
2
Valdivia
priv.
unilateral nasal discharge
pos
22
pos
9,440,000
neg
neg
26
mc
3
Valdivia
priv.
hematuria
pos
16
pos
632,000
neg
pos
29
mc
2
Valdivia
priv.
acute renal failure
pos
12
pos
6,840,000
neg
neg
30
m
2
Valdivia
priv.
ear mites chronically ill�
neg
0
pos
1,178
neg
neg
34
f
1
Valdivia
priv.
healthy
pos
124
pos
562,000
neg
neg
38
f
1
Lanco
priv.
healthy
neg
0
pos
630
neg
neg
40
mc
8
Lanco
priv.
healthy
neg
0
pos
3,020
neg
neg
43
f
6
Lanco
priv.
healthy
neg
0
pos
1,966
neg
neg
45
f
1
Valdivia
priv.
healthy
pos
62
pos
6,800,000
neg
neg
47
f
0,6
Valdivia
priv.
stomatitis
neg
0
pos
14,720
neg
neg
48
m
1
Valdivia
priv.
stomatitis
neg
0
pos
16,600
neg
neg
49
f
1,5
Valdivia
priv.
healthy
neg
0
pos
566
neg
neg
50
mc
4
Valdivia
priv.
idiopathic cystitis,
urethral stricture
neg
0
pos
1,804
neg
neg
56
m
8
Valdivia
priv.
healthy
pos
14
pos
4,720,000
neg
neg
65
fs
4
Valdivia
priv.
healthy
neg
0
neg
0
neg
pos
66
f
1
Valdivia
priv.
healthy
neg
0
neg
0
pos
neg
67
f
3
Valdivia
priv.
vomiting
neg
0
neg
0
pos
neg
68
mc
10
Valdivia
priv.
oral tumor
pos
8
pos
352,000
neg
neg
69
m
6
Valdivia
priv.
mammary tumors
pos
16
pos
314,000
neg
neg
71
m
NA
Mafil
priv.
healthy
pos
14
pos
402,000
neg
neg
76
m
1
Corral
priv.
healthy
pos
45
pos
354,000
neg
neg
90
mc
5
Valdivia
priv.
HCM
pos
19
pos
394,000
neg
neg
91
m
4
Valdivia
priv.
healthy
neg
0
neg
0
neg
pos
121
f
0,4
Valdivia
priv.
healthy
neg
0
pos
5
neg
neg
130
fs
6
Valdivia
priv.
healthy
pos
14
pos
262,000
neg
neg
181
f
1,5
Valdivia
priv.
healthy
neg
0
pos
3,480
neg
neg
182
f
1,5
Valdivia
priv.
healthy
neg
0
pos
132
neg
neg
209
m
1,5
Valdivia
priv.
healthy
neg
0
neg
0
pos
neg
f = female, fs = female spayed, HCM = hypertrophic cardiomyopathy, m = male, mc = male castrated, NA = information not available, neg = negative, pos = positive,
priv. = privately owned; bold = positive in the respective test;
Cats had the habitus of a chronically ill animal presenting with a reduced body condition score, dull fur and lethargy;
�
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Table 3. Description of sampling locations, number of cats sampled and number of cats positive in quantitative real-time PCR (qPCR) and/or immunomagneticseparation (IMS)-coupled qPCR, as well as in direct culture and/or IMS-coupled culture.
Sampling location (Municipality)
All sampled cats n
PCR-positive cats n (%)
Culture-positive cats n (%)
All positive cats n (%)
Valdivia
186
23 (12.4)
7 (3.8)
29 (15.6)
Corral
12
1 (8.3)
0
1 (8.3)
Paillaco
10
1 (10.0)
0
0
Lanco
7
3 (42.9)
0
3 (42.9)
La Unión
6
0
0
0
Mafil
5
1 (20.0)
0
1 (20.0)
Lago Ranco
Total
5
1 (20.0)
0
1 (20.0)
231
30 (13.0)
7 (3.0)
36
https://doi.org/10.1371/journal.pone.0239991.t003
In addition, another model was used to evaluate potential risk factors associated with shedding of viable pathogenic Leptospira spp. (only cats positive on direct culture or IMS-coupled
culture). For the unconditional models, only 3 variables were potential candidates: age >1
year, the reproductive status of the cat, and eating rodents were significantly associated with
Leptospira spp.-positive urine cultures.
Although a mixed model was also assessed, comparison of the goodness-of-the-fit between
both indicated that the mixed-model did not fit better. Therefore, ordinary logistic regression
model results are shown. The conditional logistic regression model (Table 7) included 2 variables (age >1 year and eating rodents) that were significantly associated with shedding of viable pathogenic Leptospira spp.
Discussion
Overall, 15.6% of cats included in the study were identified as shedders of pathogenic Leptospira spp‥ This is considerably higher than the shedding rates from 0.8% to 4.9% in Thailand,
Spain, Germany, Canada, and Malaysia [12, 14–16, 20]. One possible explanation might be the
diagnostic techniques used in the present study as the application of an additional Leptospira
spp. concentration technique (IMS-qPCR) increased the proportion of Leptospira spp.-shedding cats identified by PCR from 7.8% to 13.0% (12 additional cats). With direct culture and
IMS-coupled culture another 6 shedding cats were identified. Other possible explanations
could be related to the humid climatic conditions in the area where the study was performed
with very high annual precipitations and the types of pathogenic Leptospira spp. circulating in
the study area. In the present study, serovar identification was not performed, but the culturegrown leptospires could be characterized as L. interrogans ST 17 and ST 27 (pathogenic Subgroup 1) based on MLST. Other studies using whole-genome sequencing provided evidence
that strains of pathogenic leptospires differ in genetic features and virulence factors for
Table 4. Multi locus sequence typing of PCR products, allelic profile, and sequence types (ST) from 7 Leptospira spp.-positive urine cultures.
Cat #
caiB
glmU
mreA
pfkB
pntA
sucA
tpiA
ST
2
8
11
4
10
1
21
2
17
Product
L. interrogans Icterohaemorrhagiae Copenhageni
26
8
11
4
10
2
21
65
17
L. interrogans Icterohaemorrhagiae Copenhageni
65
8
4
4
10
2
21
2
17
L. interrogans Icterohaemorrhagiae Copenhageni
66
19
11
4
10
1
21
65
17
L. interrogans Icterohaemorrhagiae Copenhageni
67
8
4
4
10
2
21
2
17
L. interrogans Icterohaemorrhagiae Copenhageni
91
8
4
4
10
1
21
2
17
L. interrogans Icterohaemorrhagiae Copenhageni
209
19
11
13
10
12
3
3
27
L. interrogans Autumnalis Autumnalis
https://doi.org/10.1371/journal.pone.0239991.t004
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Leptospira shedding by cats in Southern Chile
Table 5. Unconditional logistic regression model results for pathogenic Leptospira spp. shedding cats (PCR- and/or culture-positive cats).
Variable
Age
Gender
Reproductive status
Origin
Ownership
Category
n
Leptospira spp. shedding
Positive
Negative
Odds ratio
n (%)
n (%)
�1 year
98
11 (11.22)
87 (88.78)
Ref.
>1 year
112
23 (20.54)
89 (79.46)
1.8�
Female
138
22 (15.94)
116 (84.06)
Ref.
Male
89
14 (15.73)
75 (84.27)
0.98
Neutered
52
12 (23.07)
40 (76.92)
Ref.
Intact
175
24 (13.71)
151(86.29)
0.53�
Rural
81
15 (18.51)
66 (81.48)
Ref.
Urban
144
20 (13.89)
124 (86.11)
0.70
Feral
11
2 (18.18)
9 (81.82)
Ref.
Privatly owned
213
33 (15.49)
180 (84.51)
0.83
Weight
>3kg
99
14 (14.14)
85 (85.86)
Ref.
�3 kg
96
11 (11.46)
85 (88.54)
1.22
Health status
Healthy
208
23(11.06)
185 (88.94)
Ref.
Sick
23
13 (56.52)
10 (43.48)
4.12�
No
159
25 (15.72)
134 (84.28)
Ref.
Yes
66
10 (15.15)
56 (84.85)
0.97
No
103
14 (13.59)
89 (84.41)
Ref.
Drinking out of puddles
Contact with rodents
Eating rodents
Consumption of raw meat
Contact with other cats
Contact with dogs
Yes
122
21 (17.21)
101 (82.79)
1.35
No
170
22 (12.94)
148 (87.06)
Ref.
Yes
43
10 (23.26)
33 (76.74)
2.08�
No
163
28 (17.18)
135 (82.82)
Ref.
Yes
53
6 (11.32)
47 (88.68)
0.64
No
12
2 (16.67)
10 (83.33)
Ref.
Yes
211
33 (15.64)
178 (84.36)
1.07
No
49
8 (16.33)
41 (83.67)
Ref.
Yes
173
27 (15.61)
146 (84.39)
0.96
Contact with livestock
No
172
29 (16.86)
143 (83.12)
Ref.
Yes
52
6 (11.54)
46 (88.46)
0.643
Previous vaccinations#
No
121
13 (10.7)
108 (89.3)
Ref.
Yes
44
9 (20.4)
35 (79.6)
2.13�
�
#
95% CI
0.86–3.90
0.46–2.02
0.25–1.15
0.34–1.47
0.20–5.63
0.79–1.86
1.81–9.23
0.42–2.10
0.65–2.86
0.87–4.75
0.24–1.58
0.38–2.67
0.14–3.78
0.25–1.65
0.82–5.39
P<0.25; CI = Confidence interval, Ref. = Reference category
vaccinations against panleukopenia, feline herpesvirus and calicivirus and rabies in variable combinations and intervals
https://doi.org/10.1371/journal.pone.0239991.t005
persistence outside the host, during entry into the host, as well as for colonization and persistence within the host organism [35, 36], suggesting that not all pathogenic Leptospira spp. are
equally virulent [37]. Therefore, it cannot be excluded that the identified strains in the present
study might have a higher virulence or be better adapted to invade the species cat than strains
in other geographic regions.
There is only one study with a much higher proportion (68%) of shedding cats in Taiwan
[11]. However, this study was performed after a typhoon with an associated outbreak of leptospirosis in humans [38] and included a high proportion of stray cats (68%), whereas in the
present study most of the cats were privately owned well-fed cats, and only 31% of them had
been observed by their owners eating rodents. In addition, in the study from Taiwan, a
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Leptospira shedding by cats in Southern Chile
Table 6. Conditional logistic regression model results for pathogenic Leptospira spp. shedding cats (PCR- and/or
culture-positive cats).
Variable
Category
Odds ratio
90% CI
Age
>1 year
1.36
0.54–3.42
�1 year
Ref.
Health status
Eating rodents
Previous vaccinations#
Healthy
Ref.
Sick
3.04�
No
Ref.
Yes
1.20
No
Ref.
Yes
2.93�
1.10–8.39
0.40–3.55
1.18–7.24
�
P<0.05; Bayesian information criterion index = 144.9
CI = confidence interval, Ref. = Reference category
#
vaccinations against panleukopenia, feline herpesvirus and calicivirus and rabies in variable combinations and
intervals
https://doi.org/10.1371/journal.pone.0239991.t006
different PCR technique (not a quantitative PCR) was applied and not all primers used in that
study were specific for pathogenic Leptospira spp.
While PCR can detect DNA of viable and killed bacteria, culture is only positive when a sufficient number of viable Leptospira spp. is present. Recently, a study from Malaysia demonstrated cultivation of pathogenic Leptospira spp. from renal tissue in 3/82 clinically healthy
shelter cats and from both, renal tissue and urine, in 1/82 of these cats [20]. In addition to that
study, there is one case report of a cat with a positive Leptospira spp. culture originating from
the same geographic area as the present study [10]. The present study also yielded positive Leptospira spp. culture results from feline urine. Feline urine with its higher osmolality compared
to dogs and humans [39] is a hostile environment for bacterial growth and therefore could
prohibit the shedding of viable Leptospira spp. in many cats. This might explain the low proportion of culture-positive cats in the present study. Low pH is another physicochemical property of the urine contributing to the defense system against bacteria [40] and Leptospira spp.
do not survive well in acidic urine but remain viable in alkaline urine [41]. However, in the
present study, the mean urine pH was identical in culture-positive and in culture-negative
cats.
Usage of magnetic separation enhances the analytical specificity and sensitivity of the subsequent detection method, given that it helps to remove the presence of many inhibitory substances or other organisms [42]. Still, only 7 cats (3.0%) were culture-positive (3 direct culture,
4 IMS-coupled culture) compared to 30 (13.0%) PCR-positive cats. However, detection of
positive cultures nevertheless demonstrates that feline urine can be a source of possible
Table 7. Conditional logistic regression model results for cats with positive urine cultures (direct culture and/or
IMS-coupled culture) for Leptospira spp.
Variable
Category
Odds ratio
90% CI
Age
>1 year
6.61
1.08–40.40
�1 year
Ref.
No
Ref.
Yes
3.87
Eating rodents
1.03–14.49
CI = confidence interval, Ref. = Reference category
https://doi.org/10.1371/journal.pone.0239991.t007
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Leptospira shedding by cats in Southern Chile
contamination of the environment with viable Leptospira spp. One possible explanation for
the discrepancy in the results of direct and IMS-coupled cultures is the hypothesis that antibodies used in IMS could have a neutralizing effect for the growth of Leptospira spp. in culture.
In the present study, only 1 of the 7 culture-positive cats was also PCR-positive (qPCR and
IMS-qPCR). One possible reason for the negative PCR in the culture-positive cats could be the
presence of PCR inhibitors. In biologic fluids, such as urine, presence of PCR inhibitors has
been reported to cause false negative PCR results [43]. However, positive controls in the present study showed hardly or no inhibition. Interestingly, a similar discrepancy was seen in the
only other study in cats reporting the isolation of Leptospira spp. from feline kidneys and urine
[20]. In that study, all 4 culture-positive cats had negative urine PCR results. This indicates
that there might be substances/chemicals present in the PCR processing but not in the samples
that are cultured that could be inhibitory for certain steps in the PCR. Other reasons might
also be the level of detection on qPCR and possibly the selection of primers that do not bind to
all DNA targets of Leptospira spp. However, molecular characterization of isolates later on
revealed sequence types that should have been recognized by PCR.
In the present study, serovar identification was not performed. Nevertheless, it was possible
to molecularly characterize isolates via MLST, and identified sequences were assigned to ST 17
and ST 27, which corresponds to L. interrogans (Pathogenic Leptospira Subgroup 1) [44].
Although the Taxonomic Subcommittee does not yet accept MLST for serovar identification,
the information reported from the MLST database used in the analysis of the isolated strain in
this study, reports the ST 17 as Leptospira interrogans serogroup Icterohaemorrhagiae serovar
Copenhageni, and the ST 27 as L. interrogans serogroup Autumnalis serovar Autumnalis. Previously characterized Leptospira spp. isolates from cats´ kidney or urine had high similarity to
Leptospira interrogans serovar Pomona (1 cat from this area of Chile) [10] or to Leptospira
interrogans serovar Bataviae (4 cats from Malaysia) [20]. Whereas, Leptospira interrogans Bataviae is reported to be the most common isolated serovar in humans, dogs and rats in Malaysia
[45–47], L. interrogans serovar Autumnalis is among the most commonly reported serovars in
dogs and cats in Chile [18, 48, 49], and antibodies against L. interrogans Icterohaemorrhagiae
have previously been reported in rodents in this area [50].
Four factors (age (�1 year/>1year), health status, eating rodents, and vaccination status)
were present in the final conditional model for all Leptospira spp. shedding cats (cats positive
in any of the 4 techniques), and 2 factors (age (�1 year/>1year), eating rodents) for the “viable
Leptospira spp. shedding” cats (culture- or IMS-coupled culture-positive cats).
Adult stage (>1 year) was significantly associated with leptospiruria (odds ratio 6.8, CI 90%
1.08–40,4). Previous studies already reported older age as a risk factor for Leptospira spp. infection [15, 51]. A study in Thailand identified age >4 years as the only risk factor for infection
(urine qPCR-positive status and/or serum antibodies in MAT) in a multivariate analysis [15].
A possible explanation for this is longer exposure to infectious environmental sources and
therefore a higher risk of being infected. It is not known how long cats remain carriers after
infection or if and under which circumstances they can eliminate the infection. Risk factors for
Leptospira spp. infection have been investigated in several studies. Contact of cats with livestock animals was significantly associated with presence of Leptospira spp. antibodies in a
study performed in Chile [18] but such an association between shedding and contact with livestock animals was not identified in the present study.
The proportion of sick cats was significantly higher in Leptospira spp. shedding cats
(36,1%) than in non-shedding cats (5,1%). Clinical cases of leptospirosis in cats are rarely
described. In one case series with 3 cats and 2 further reports of single cases, cats presented
with acute onset of polydipsia and polyuria, lethargy, anorexia, and hematuria, and had various
degrees of kidney disease or increased liver enzymes [8–10]. In a more recent case report, a cat
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presented with a 2-months history of hematuria, had no abnormalities in serum chemistry but
a marked leukocytosis [10]. One leptospiruric cat (positive in qPCR and IMS-qPCR) of the
present study also suffered from acute renal failure (history of acute lethargy and anorexia,
severe renal azotemia), and 2 presented with hematuria (1 cat positive in qPCR, IMS-qPCR
and IMS-coupled culture, 1 cat positive in IMS-qPCR). These clinical signs could indeed be
associated with leptospirosis and represent acute infection. In the other 10 sick cats with different organs affected, a causal relationship of infection with Leptospira spp. is not likely. However, the higher proportion of sick cats shedding pathogenic Leptospira spp. in the present
study suggests that Leptospira spp. could be one of the etiological factors in the development of
these clinical problems, and that diagnostic testing Leptospira spp. infection should also be
considered in cats with relevant clinical signs. Alternatively, it could be discussed that sick cats
might be immunosuppressed and that an impaired immune status increases the risk of leptospiral infection and subsequent shedding. This, however, has not been reported so far. On the
other hand, sick cats shedding pathogenic Leptospira spp. could represent chronically infected
individuals. It would have been interesting to further monitor these cats to see if they remained
chronic shedders.
Eating rodents was found to be associated with an increased risk of shedding viable pathogenic leptospires. In general, rodents are a natural reservoir pathogenic Leptospira spp., and
rats are reservoir hosts of the Icterohaemorrhagiae and Copenhageni serovars [52]. Therefore,
prey–predator transmission between cats and rodents is possible by hunting and is believed to
be the main source of infection in cats [12, 53]. In the contrary, a negative association between
cat ownership and the presence of Leptospira antibodies in people in an urban population in
North America was demonstrated, and cats appear to have a protective role by decreasing contact of people with rodents that act as reservoir hosts [54].
An unforeseen result was the statistically significant association of an increased risk of shedding pathogenic Leptospira spp. and previous vaccination against feline panleukopenia, feline
herpes- and calicivirus infection and/or rabies. The reason for this association is unknown.
Immunological host-pathogen processes that could affect shedding of leptospires by cats in
any direction are conceivable but specific information is missing. Weather there is an interference in susceptibility of vaccinated cats to act as shedders of Leptospira spp. is an interesting
aspect that should be further investigated.
Results of the present study show that cats can shed viable pathogenic Leptospira spp. and,
therefore, could act as transmitters to humans and livestock. Results of PCR and culture with
and without separation and concentration, taken together, showed that: i) the bacterial load in
feline urine is relatively low; ii) in 90% of feline shedders it was not possible to culture the
organism suggesting that viability of the bacteria in feline urine is negatively affected by the
hostile environment, e.g. the relatively high osmolality of feline urine; and iii) separation and
concentration followed by qPCR significantly increases the sensitivity of detection of leptospiral DNA in feline urine.
Apart from the possible negative effect on bacterial viability of the hostile environment that
is provided by feline urine and the stage of disease, there is another potential cause of negative
culture result. There is a known difficulty to isolate Leptospira spp. from infected hosts and get
them growing in vitro. Despite the presence of viable leptospires in clinical samples of an
infected host, it is in many cases very difficult to isolate the bacteria in media due to the in vivo
phenotype of the host-derived bacteria and their lack of capacity to adapt to in vitro conditions.
Based on this general problem with cultural isolation of Leptospira spp. from infected humans
and animals, it can be concluded that, despite this difficulty, viable Leptospira spp. were
detected in the urine of 7 cats, which demonstrates the zoonotic potential of this infection in
cats.
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Leptospira shedding by cats in Southern Chile
Conclusion
In this region of Southern Chile, 15.6% of cats were leptospiruric and 3.0% of cats were Leptospira spp. culture-positive. The present study is one of the first reporting growth of Leptospira
spp. from feline urine in culture and describing genetic characteristics of culture-derived isolates. Even though feline urine appears to be a hostile environment for leptospires, shedding
cats present a potential risk for zoonotic transmission.
Supporting information
S1 Questionnaire.
(DOCX)
S2 Questionnaire.
(DOCX)
Acknowledgments
We thank all cat owners and cats for participation in the study.
Author Contributions
Conceptualization: Roswitha Dorsch, Miguel Salgado, Ananda Müller, Katrin Hartmann.
Data curation: Roswitha Dorsch, Javier Ojeda, Theo Eberhard.
Formal analysis: Roswitha Dorsch, Javier Ojeda, Gustavo Monti, Theo Eberhard.
Funding acquisition: Roswitha Dorsch, Gustavo Monti, Ananda Müller, Katrin Hartmann.
Investigation: Roswitha Dorsch, Javier Ojeda, Miguel Salgado, Bernadita Collado, Camillo
Tomckowiack, Carlos Tejeda, Theo Eberhard.
Methodology: Miguel Salgado, Bernadita Collado, Camillo Tomckowiack, Carlos Tejeda,
Ananda Müller.
Project administration: Roswitha Dorsch, Gustavo Monti.
Resources: Roswitha Dorsch, Javier Ojeda, Miguel Salgado.
Software: Roswitha Dorsch, Gustavo Monti.
Supervision: Roswitha Dorsch, Miguel Salgado.
Validation: Miguel Salgado, Gustavo Monti, Henricus L. B. M. Klaasen.
Visualization: Roswitha Dorsch, Theo Eberhard.
Writing – original draft: Roswitha Dorsch, Miguel Salgado, Gustavo Monti, Katrin
Hartmann.
Writing – review & editing: Roswitha Dorsch, Javier Ojeda, Miguel Salgado, Gustavo Monti,
Ananda Müller, Theo Eberhard, Henricus L. B. M. Klaasen, Katrin Hartmann.
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