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Trop Anim Health Prod (2014) 46:427–432

DOI 10.1007/s11250-013-0508-y

REGULAR ARTICLES

Leptospirosis in pigs, dogs, rodents, humans, and water

in an area of the Colombian tropics
Alfonso Calderón & Virginia Rodríguez & Salim Máttar &
Germán Arrieta

Accepted: 5 November 2013 / Published online: 21 November 2013

# Springer Science+Business Media Dordrecht 2013

Abstract Leptospirosis is a reemerging zoonosis of global Introduction

distribution and is one of the causes of hemorrhagic fevers in
the tropics. We sought to determine seroprevalence in humans Leptospirosis is a zoonosis of global distribution; it is consid-
and animals and isolate Leptospira interrogans sensu lato in ered a reemerging disease, endemic in tropical countries,
domestic animals, rodents, and water sources. The study was which, because of their geoclimatic and social conditions,
conducted in a tropical area of the middle Sinú in Cordoba, favor its transmission (Guerra 2013). In domestic and wild
Colombia. In a prospective descriptive study, we collected blood animals, chronic renal carriers maintain viable leptospires
and urine from pigs and dogs, sera from rural human workers, which may be excreted via urine becoming a source of infec-
sera and kidney macerates of rodents, and water samples from tion for humans and other animals (Monahan et al. 2009).
environmental sources. We used microagglutination to screen Humans can be infected with any serovar of Leptospira from
for antibodies to 13 serovars. Strains were cultured on the any animal or environment, basically, through contact with
Ellinghausen–McCullough–Johnson–Harris medium and con- fluids from infected animals (Lim 2009; Marga et al. 2013).
firmed by PCR amplifying lipL32 gene. Seroprevalence was The organisms may enter the body via lacerations in the
55.9 % in pigs, 35.2 % in dogs, and 75.8 % in humans; no skin, contact with mucosa or conjunctiva, inhalation of aero-
antibody was detected, and no Leptospira were isolated from sols, or ingestion of contaminated food or beverages
kidney macerates of rodents. Seven L. interrogans sensu lato (Monahan et al. 2009; Musso and La Scola 2013). Leptospi-
strains were isolated: three from pigs, two from dogs, and two rosis is considered an occupational disease, primarily affecting
from water. High seroprevalence in pigs, dogs, and humans, farmers, fishermen, veterinarians, and workers in sewers and
concomitant to isolation of strains, demonstrates that in slaughterhouses (Brown et al. 2011). A risk of infection for
Cordoba, transmission exists among animals, the environment, humans is the exposure during water-related recreational ac-
and humans, which warrants the implementation of public tivities (Monahan et al. 2009; Marga et al. 2013).
health intervention measures to reduce the epidemiological The tropical environment favors survival of Leptospira
impact of leptospirosis in the region. outside reservoirs in stagnant or slowly flowing warm water.
Water and soil contaminated by excreta of infected animals are
Keywords Epidemiological surveillance . Transmission . also infection sources (Ganoza et al. 2006; Adler and Peña
Zoonoses . Public health . Disease reservoirs . Social 2010). In recent years, this ecological aspect has gained im-
environment portance, because of the increased incidence of exposure to
contaminated environments (Vijayachary et al. 2008). Our
aim was to measure the seroprevalence of Leptospira infec-
tion in humans and animals and isolate Leptospira
A. Calderón (*) : V. Rodríguez : S. Máttar : G. Arrieta
interrogans sensu lato from animals and water sources.
Department of Animal Sciences, Institute of Investigations Biologics
of Tropic, University of Cordoba (Colombia), Cordoba, Colombia
e-mail: [email protected] Materials and methods
G. Arrieta
Public Health and Healthcare, Corporación Universitaria del Caribe, Type of study and geographic location We conducted a pro-
Sincelejo, Colombia spective, descriptive study between 2009 and 2011. The farms
428 Trop Anim Health Prod (2014) 46:427–432

studied were dedicated to pig farming and located in Monteria There were no rodents captured in five farms. The study
(8°46′15″N, 75°51′31″O), Cerete (8°52′22″N, 75°46′47″O), included 62 workers who inhabited and worked on the farms
and Cienaga de Oro (8°52′44″N, 75°42′8″O). The three mu- and volunteered to participate.
nicipalities (Fig. 1) are located according to the departmental
territorial geographic plan in a middle region of the Sinú River Study of water We analyzed 54 water sources that were
in the department of Cordoba, Colombia. proportionally distributed among the farms selected.
These sources included animal drinking water (n =18),
Study of pigs, dogs, rodents, and humans The total population water from wells (n = 18), and wastewater from pig
of pigs in reproductive age in the three municipalities studied stables (n =18).
was 19.328. Considering a seroprevalence maximum (50 %),
95 % reliability, and 5 % standard error, the sample size for the Serology There were 383 sera of pigs, 54 of dogs, 39 of
microagglutination test (MAT) was 377, but blood samples rodents, and 62 of humans analyzed by MAT. We screened
were taken from 383 pigs in 18 farms. Bacterial cultures were for antibodies against L. interrogans sensu lato using the
performed according to the MAT results, and we took at each MAT according to specifications of the International Office
farm ten urine samples, such as four pigs with titres from of Epizootics; 11 serovars were used: serogroup Australis
1:100 to 1:400, four with titres ≥1:400, and two with negative (serovar Bratislava), serogroup Autumnalis (serovar
serologic results. This study included 54 dogs living on the Autumnalis), serogroup Batavie (serovar Batavie), serogroup
study farms, and 39 rodents were captured with Sherman traps Serjoe (serovar Hardjo), serogroup Grippotyphosa (serovar
randomly distributed in different areas of the farms such as Grippotyphosa), serogroup Icterohaemorrhagiae, (serovar
domestic areas, stables, and package store; the distribution of Icterohaemorrhagiae), serogroup Mini (serovar Mini),
rodent species was Mus musculus , 49 % (n =19); Rattus serogroup Pomona (serovar Pomona), serogroup Pyrogenes
norvegicus, 31 % (n =12); and Rattus rattus, 21 % (n =8). (serovar Zanoni), serogroup Tarassovi (serovar Tarassovi),

Fig. 1 Geographical distribution

of L. interrogans sensu lato in
Cordoba, Colombia
Trop Anim Health Prod (2014) 46:427–432 429

and serogroup Canicola (serovar Canicola). Titres ≥1:100 Results

were considered positive.
Serological results The seroprevalence found were as follows:
L. interrogans culture in urine and water Following MAT, pigs, 55.9 % (214/383); dogs, 35.2 % (19/54); humans, 75.8 %
183 pig samples were selected for bacteriological cultures, but (47/62); and rodents, 0 % (0/39). The percentage distribution by
12 urine samples were lost, and only 171 were cultured: 20 serogroups in the species studied is shown in Table 1.
with negative serology, 75 with titres between 1:100 and
1:400, and 76 with titres >1:400. The urine samples of the Pig urine culture Of 171 pig urine samples cultured, three
54 dogs and 54 water samples were also cultured. Samples Leptospira strains were isolated in females in Montería and
were cultured in Ellinghausen–McCullough–Johnson–Harris two of them were obtained in the same farm. Two females
(EMJH) medium (Difco, Detroit, MI, USA), enriched with showed titres ≥1:800 for serovars Pomona, Tarassovi,
1 % rabbit serum with and without supplementing nalidixic Canicola, Grippotyphosa, and Icterohaemorrhagiae, while in
acid and 5-fluorouracil. Approximately 0.5 ml of the water the third female, the higher reactivity was 1.400 for serovar
and urine samples was seeded directly onto the EMJH medi- Pomona.
um; 2 ml was filtered through a 0.45-μm nitrocellulose mem-
brane, and 0.5 ml was inoculated onto the EMJH medium. Dog urine culture Two isolates were obtained. One was from
The cultures were incubated at 29–30 °C and examined week- a seronegative dog; the other dog had titres of 1:100 for Serjoe
ly under dark-field microscopy for 4 months. and Batavie serogroups (1:200 for Grippotyphosa, Canicola,
Tarassovi, and Mini serogroups; and 1:400 for Australis,
L. interrogans from cultures of kidney macerates of Icterohaemorrhagiae, Pyrogenes, and Autumnalis serogroups).
rodents Upon necropsies, one kidney was collected from each
rodent, sectioned in half, and macerated in 2 ml PBS at pH Rodent culture From 39 cultures of kidney macerates, no
7.2; 0.5 ml of the suspension was inoculated directly in the Leptospira strains were isolated.
EMJH medium. The rest was deposited in 5 ml of PBS at pH
7.2 and allowed to stand at 30 °C for 30 min. After this Water culture From 54 water samples, nine strains were iso-
incubation, 0.5 ml of the supernatant medium was inoculated lated, but only two were identified as L. interrogans sensu lato
in EMJH medium. in Monteria. The first strain was obtained from animal drink-
ing water and the second from wastewater.
Molecular identification The DNA was extracted from field
isolates, meaning Leptospira isolates (strains) obtained in the Rodents No Leptospira were isolated from the kidney of
field study and maintained in the laboratory. Cultures were rodents.
used between 6 and 10 days of growth; 5 ml was taken and
centrifuged at 13,000 rpm. The pellet was resuspended in 1 ml Molecular identification The PCR identified seven isolates
of TE. The DNA extraction was done with the Qiagen kit (three in pigs, two in dogs, and two water samples) like
(QIAamp® DNA Mini Kit). Confirmation of pathogenic pathogenic strains, which amplified a product of about
strains was conducted by using 270F and 692R-specific 423 bp of the gene lipL32 of pathogenic Leptospira.
primers that amplify a 423-bp fragment, which encodes for
the Lip32 protein (Levett et al. 2005). The conditions of the Table 1 Percentage distribution of serogroups of L. interrogans sensu
PCR reaction mixture were as follows: 1× PCR buffer, lato among different species in the middle Sinú area
1.25 nM dNTPs, 2.5 mM MgCl2, 1 mM 270F initiator,
Species/serogroup Pigs % Dogs % Humans %
1 mM 692 R initiator, and Taq DNA pol 0.4 U. Each PCR
cycle was 94 °C×1 min, 55 °C×1 min, and 72 °C×2 min; Mini 28.7 7.4 32.2
34 cycles were performed. Batavie 34.7 9.2 37.1
Serjoe 40.2 9.2 43.5
Ethical aspects The Ethics Committee at the Institute of In- Tarassovi 40.4 14.8 35.4
vestigations Biologics of Tropic (IIBT) at the University of Autumnalis 42.8 11.1 50.0
Cordoba reviewed and approved the study protocol, consid- Australis 43.6 14.8 38.7
ering articles 5 and 87 from decree 309 of 25 February 2000 Grippotyphosa 47.7 18.5 53.2
from the Colombian Ministry of the Environment (2000). For Pyrogenes 50.3 11.1 38.7
the human population, farm workers were invited to partici- Icterohaemorrhagiae 56.4 14.1 54.8
pate on a volunteer basis through signed consent; the work Pomona 61.6 18.5 56.4
was classified as minimum risk according to the Colombian Canicola 62.4 14.1 64.5
Ministry of Health (008430/1993).
430 Trop Anim Health Prod (2014) 46:427–432

Discussion Sejroe (Table 1). The Pomona serogroup has been classified
as emergent in dog’s leptospirosis, and these titres may be due
Isolates of Leptospira from the urine of pigs and dogs and to contact with other dogs and with reservoir skunks or rac-
from water sources demonstrate high endemicity and a poten- coons for these serogroups (Ribotta et al. 2000), which are
tial public health problem in this area of the Colombian common animals in these tropical areas of Colombia. In
Caribbean. The infection was confirmed with high seroprev- Brazil, only 7.1 % seroprevalence was found in dogs, and it
alence in pigs and dogs, which helps to explain the very high was concluded that the presence of Leptospira in the environ-
prevalence of antibody in the human population at risk. ment was the source of infection (Table 2).
In contrast with our results from the Caribbean, lower The very high seroprevalence in humans may be due to the
seroprevalences have been reported in the Colombian Andes population evaluated (occupational risk), to the endemicity of
(Table 2). The higher seroprevalence in pigs in the Caribbean leptospirosis in humid tropical regions where environmental
region may be due to geoclimatic conditions such as high conditions favor transmission of Leptospira, and to perma-
precipitation and relative humidity, sanitary deficiencies, and nent contact with pigs and dogs. The highest seroreactivity of
a higher number of nontechnical pig farms in the Caribbean. the exposed human population was to serogroups Canicola,
Our negative findings for Leptospira infection in domestic Pomona, and Icterohaemorrhagiae (Table 1). Close contact of
rodents could indicate the existence of an unknown wild or these individuals with dogs was noted in the farms. Ours is the
domestic reservoir that is important for the maintenance of first study on a human population in Colombia that reports on
infection in the pig population in our study area. The highest antibodies against the Mini, Tarassovi, and Batavie
seroprevalence in pigs in our study was for serogroups serogroups (Table 1). Antibody to these serovars was also
Canicola, Pomona, and Icterohaemorrhagiae (Table 1), sug- found in dogs and pigs from these same farms. Forty percent
gesting transfer of specific serovars between pigs and dogs. (25/62) of the seropositive humans had no compatible symp-
Given the lack of vaccination records against leptospirosis tomatology, and 35 % (22/62) manifested compatible symp-
in dogs, the 35.2 %, seroprevalence in dogs probably repre- toms during the previous 9 months. The most frequent symp-
sents past infections. In our study, the Pomona and toms were cephalalgia, myalgia, fever, and malaise. The lack
Grippotyphosa serogroups presented the highest frequency, of symptomatology compatible with a clinical picture of lep-
followed by Canicola, Icterohaemorrhagiae, Australis, and tospirosis (jaundice, kidney failure, liver failure) confirms the

Table 2 Seroprevalence of
Leptospira interrogans sensu lato Country Area of influence Seroprevalence (%) Serovars References
in different countries
Pigs
Colombia Caribbean 55.9 13 Present study
Dogs
Brazil Paraná 7.1 24 Oliveira et al. 2012
Brazil Paraná 12.2 30 Fonzar and Langoni 2012
USA Michigan 24.9 6 Stokes et al. 2007
USA Washington 17.1 6 Davis et al. 2008
Thailand Bangkok 83.5 ND Jittapalapong et al. 2009
Mozambique Mayotte 93.1 8 Desvars et al. 2012
87.5
Colombia Tolima 20.2 5 Romero et al. 2010
Colombia Tolima 21.4 5 Romero et al. 2010
Colombia Caribbean 35.2 13 Present study
Humans
Brazil Paraná 8.0 30 Fonzar and Langoni 2012
Peru Lima 1.2 22 Platts et al. 2011
USA 2.5 6 Whitney et al. 2009
Portugal Azores Islands 22.0 26 Vieira et al. 2006
Mexico Veracruz 4.0 13 Navarrete et al. 2006.
Italy Bari 0.0 19 Monno et al. 2009
New Zealand 9.5 2 Benschop et al. 2009
Colombia Tolima 6.0 5 Romero et al. 2010
Colombia Caribbean 75.8 13 Present study
ND not determined
Trop Anim Health Prod (2014) 46:427–432 431

high endemicity in the study area associated with asymptom- Furthermore, from the environmental view, it allows inves-
atic infections or anicteric forms characterized by general tigating whether or not the water sources (rivers, ponds,
nonspecific signs and symptoms; however, those patients also troughs) are contaminated with pathogenic leptospires. In
did not remember clinical manifestations, suggesting that France, qPCR was performed using the detection and quanti-
leptospirosis reveals the possibility of the disease being fication of Lip32 gene in water samples. The method repre-
asymptomatic or underreported, or associated with other hem- sents a tool that could be integrated into future programs of
orrhagic diseases such as dengue, malaria, hepatitis A and E, public health surveillance in recreational waters (Vein et al.
fever, and other hemorrhagic fevers (Musso and La Scola 2012). The detection and monitoring of pathogenic leptospires
2013; Chaudhry et al. 2013). in water allow intervention measures to help reduce the oc-
The serological and bacteriological negative results in ro- currence of leptospirosis in humans and animals.
dents may be due to serial rodent control on the farms. Domestic
cats were also observed; cats could have prevented a permanent
population of rodents on the farms. In contrast to our results, Conclusion
Bunnell et al. (2000) found 20 % seroprevalence in wild rodents
in the Peruvian Amazon. Agudelo et al. (2010) found 25.2 % The high seroprevalence that we found in pigs, dogs, and
seroprevalence in R. norvegicus in a food market in Medellin, humans and the isolation of seven strains of L. interrogans
Colombia, and concluded that serovar Icterohaemorrhagiae has sensu lato in pigs, dogs, and water demonstrate that transmis-
been established in rodents, and these act as carriers of sion exists among animals, the environment, and humans,
pathogenic leptospires for susceptible humans. which originates from endemic zones demarcated for this
Presence of two pathogenic Leptospira was demonstrated in zoonoses in Cordoba. The high prevalence of L. interrogans
water sources, which constitutes a risk to human and animal sensu lato in Cordoba warrants the implementation of public
public health. Studies on the role of water in transmitting health intervention measures to reduce the epidemiological
leptospirosis such as those of Lesem et al. (2010) in Israel impact of leptospirosis in the region.
reported that most human cases were acquired from activities
related to water sources. Viau and Boehm (2011) in Hawaii
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