Tropical Animal Health and Production

https://doi.org/10.1007/s11250-020-02203-y

REGULAR ARTICLES

High frequency of seropositive and carriers of Leptospira spp. in pigs

in the semiarid region of northeastern Brazil
Juciê Jales Fernandes 1 & João Pessoa Araújo Júnior 2 & Camila Dantas Malossi 2 & Leila Sabrina Ullmann 2 &
Diego Figueiredo da Costa 1 & Maria Luana Cristiny Rodrigues Silva 1 & Clebert José Alves 1 & Sergio Santos de Azevedo 1 &
Severino Silvano dos Santos Higino 1

Received: 16 April 2019 / Accepted: 11 January 2020

# Springer Nature B.V. 2020

Abstract
Carrier animals are considered key in the transmission cycle of leptospirosis. Although investigations have been carried out on
several species, the role of pigs in the epidemiology of the disease is still poorly studied in the semi-arid region. Thus, the
objective of this study was to determine the presence of Leptospira spp. in the genitourinary tract of pigs intended for slaughter.
Fifty pigs were used: adults and unvaccinated. Samples of the kidney, urine, and vaginal fluid were collected for the molecular
detection of Leptospira spp. and blood samples for the serological test. The molecular test was performed using the polymerase
chain reaction (PCR), and the serological test was performed with the microscopic agglutination test (MAT). Samples with DNA
amplification were submitted to genetic sequencing. Twenty (40%) animals were found with anti-Leptospira spp. antibodies, and
the majority of the reactions (50%) occurred for the serogroup Tarassovi. Leptospiral DNA was found in the tissue of 11 (22%)
pigs. The gene from a urine sample was sequenced and showed similarity to L. borgpetersenii. The results evidenced a high rate
of porcine carriers; therefore, they appear to be important sources of agent infection, being potential transmitters of the disease to
other animal species and man.

Keywords Leptospirosis . Genital carriers . Slaughterhouses . Pigs

Introduction genitals during the chronic phase of the disease (Figueiredo

Leptospirosis is one of the most widespread zoonoses in the
world (Loffler et al. 2014), being considered endemic in Latin
America and responsible for broad losses in livestock (Adler
2015). Several domestic and wild animals are reservoirs of
leptospirosis, which can also infect humans (Millán et al.
2009). Its occurrence is directly related to environmental fac-
tors and the presence of reservoirs, such as rodents and wild
animals (Adler and Moctezuma 2010).
In pigs the infection is most often subclinical, leading to
large losses due to reproductive failures, which may be due to
fetal infection during the acute phase and also to lesions in the
* Severino Silvano dos Santos Higino
higinosss@gmail.com
1
Federal University of Campina Grande (UFCG), Av. Universitária,
no number, Santa Cecília, Patos, Paraíba, Brazil
2
Paulista State University (Unesp), Botucatu, Brazil
et al. 2013b). Pigs are considered maintenance hosts for the
Pomona, Bratislava, and Tarassovi serovars, as well as acci-
dental hosts of the serovars Icterohaemorrhagiae, Canicola,
Autumnalis, Hardjo, and Grippotyphosa (Gonçalves and
Costa 2011).
The microscopic agglutination test (MAT) is the technique
most used for the diagnosis (Soto et al. 2007), as it is recom-
mended by the World Organization for Animal Health (OIE
2014). Surveys in pigs in Brazil showed frequencies ranging
from 4.7% in Maranhão (Gonçalves et al. 2011), 16.5% in São
Paulo (Azevedo et al. 2006) to 78.6% in Rio Grande do Norte
(Leite et al. 2018). In Paraíba state, northeastern Brazil, there
are epidemiological surveys in sheep (Costa et al. 2017;
Higino et al. 2010; Silva and Alves 2015), goats (Higino
et al. 2012), and cattle (Pimenta et al. 2014). In swine, the
disease is still poorly studied in the region, especially the role
of carrier in these animals, but serological surveys have al-
ready been carried out, in which Azevedo et al. (2008) and
Figueiredo et al. (2013a) found frequencies of 33.6% and
14.6%, respectively.

Indirect tests with MAT may become less sensitive due to
the absence of detectable levels of serum antibodies, so that
direct diagnostic techniques like PCR that detect the DNA of
the microorganism are being increasingly used because of the
sensitivity and practicality that other techniques not present
(Hamond et al. 2014). PCR can identify the leptospiral DNA
even if these microorganisms are already lysed (Lilenbaum
et al. 2008a, b), so this is an important tool for the diagnosis
of the disease and epidemiological investigations (Soto et al.
2007; Lilenbaum et al. 2008a, b). Most of the investigations of
swine carriers using the PCR technique were carried out in
other regions of Brazil, such as Shimabukuro et al. (2003) in
São Paulo, Oliveira et al. (2007) in Rio Grande do Sul,
Miraglia et al. (2008) in São Paulo, and Gonçalves et al.
(2014) in Rio de Janeiro; therefore, the carrier role of this
species in semiarid conditions is still little studied, which in
fact limits the understanding of the epidemiology of the
disease.
Carrier animals are considered key in the transmission cy-
cle of leptospirosis (Lilenbaum et al. 2008a, b). Some studies
have pointed out other species that carry the agent in the semi-
arid region, such as cattle (Oliveira et al. 2016) and sheep
(Director et al. 2014; Silva et al. 2019), but data on the disease
in pigs are still scarce. Thus, the objective of the present study
was to detect the presence of antibodies against Leptospira
spp. and the DNA of the agent in slaughtered pigs in the
Brazilian semiarid region.
Material and methods
Study area and sample collection
The study was carried out in the public slaughterhouse of the
municipality of Brejo do Cruz, state of Paraíba, an area inserted
in the Brazilian semiarid. According to the climatic classifica-
tion of Köppen and Geiger (1928), the region is characterized
by the climate of the type (BSh). The collections were carried
out between November 2016 and March 2017. The period is
corresponding to the end of the dry season and beginning of the
rainy season, but in this occasion the region was undergoing
extensive drought. The production system in the region is char-
acterized by family subsistence creations where there is no
effective sanitary control. Samples were collected from 50 un-
vaccinated adult pigs, 22 males and 28 females. Blood, urine,
kidney, and vaginal fluid samples were collected. Blood was
collected at the time of bleeding in 10-mL tubes without anti-
coagulant, and after centrifugation the serum was stored at −
20 °C until the serological test was performed. The urine was
collected by direct puncture of the bladder using sterile syrin-
ges, then deposited in microtubes and stored at − 20 °C.
Samples of renal tissue, approximately 1 g, were collected
using surgical scissors and sterile anatomical tweezers.
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Vaginal fluid was collected by sterile swabs and both samples
were frozen at − 20 °C until processing.
Serological test
The presence of anti-Leptospira spp. antibodies was determined
by the microscopic agglutination test (MAT), as recommended
by the World Organization for Animal Health (OIE 2014). A
collection of live antigens containing 20 serovars: Castellonis,
Tarassovi, Australis, Bataviae, Bratislava, Guaricura, Panama,
Hebdomadis, Canicola, Grippotyphosa, Copenhageni, Hardjo,
Pomona, Icterohaemorrhagiae, Pomona From, Djasiman,
Wolffi, Shermani, Sejroe, and Autumnalis. All samples with
binder activity at the 1:100 dilution were considered positive
and then serially titrated for a ratio of two. The highest titer
obtained was used to identify the infecting serogroup.
Molecular detection and phylogenetic analysis
DNA in urine, vaginal fluid, and renal tissue was extracted
using the Dneasy Blood and Tissue Kit (Qiagen, Hilden,
Germany), following the manufacturer’s recommendations.
The polymerase chain reaction (PCR) was performed as previ-
ously described by Hamond et al. (2014). The LipL 32-45F (5′-
AAG CAT TAC CGC TGG TG-3′) and Lip L 32-286R (5′-
GAA CTC CCA TTT CAG CGA TT-3 ′) primers, designed by
Stoddard et al. (2009), were used to amplify the gene LipL32,
which is specific for pathogenic leptospires. The L. interrogans
serogroup Pomona and serovar Kennewicki was used as posi-
tive control, and ultrapure water as negative control.
A phylogenetic tree was generated using the o software
Seaview4 (Gouy et al. 2010). Phylogenetic trees were con-
structed based on the maximum-likelihood (ML) method with
1000 bootstraps, model General Time Reversal GTR, using
PhyML. Trees were visualized in FigTree v1.4.3 (http://tree.
bio.ed.ac.uk/). The phylogenetic reconstruction program
included sequences from Leptospira spp. for comparison.
Results
MAT has detected anti-Leptospira spp. antibodies in the se-
rum of 20 (40%) pigs, and PCR identified the leptospiral DNA
in 11 (22%) animals. Only three (6%) animals were positive
for the two techniques, 17 (34%) positive animals in MAT and
negative PCR, and 8 (16%) positive in PCR and negative in
MAT (Table 1).
The most frequent serogroup was Tarassovi (50%), follow-
ed by Australis (25%), Pomona (15%), Bataviae (5%), and
Icterohaemorrhagiae (5%), with titers varying between 100
and 400, as shown in Chart 1.
The DNA of Leptospira spp. was found in eight (29.6%)
urine samples, two (5.1%) kidneys, and three (12%) vaginal
Trop Anim Health Prod

Table 1 Relationship of positive animals according to the diagnostic 2011), 16.1% (Valença et al. 2013), and 4.7% (Gonçalves
technique, for swine slaughtered in the semi-arid region, Brazil
et al. 2011). In the state of Paraíba, 33.6% (Azevedo et al.
MAT PCR Total 2008) and 14.6% (Figueiredo et al. 2013a) of pigs tested pos-
itive. Thus, it is evidenced that even with several years of rain
Positive Negative scarcity in the State, which prevented the formation of ade-
Positive 3 (6%) 17 (34%) 20 (40%)
quate environments for the presence of bacteria, it remains
being maintained among the animals of the region. Other fac-
Negative 8 (16%) 22 (44%) 30 (60%)
tors may be contributing to this reality, such as the fact that
Total 11 (22%) 29 (58%) 50 (100%)
swine breeding in the region is characterized mainly by clan-
destine creations, absent from adequate management and san-
fluid samples (Table 2). PCR positive samples of kidney were itary conditions, where the animals are fed with all type of
also positive in urine, so of the eight positive animals in the organic material and have greater possibility of contact with
urine, five were negative in the kidney PCR and one of them other reservoirs. The conditions of the extensive creations lead
could not be verified. Positive animals in vaginal fluid PCR to greater predisposition (Campos et al. 2011), because man-
did not present positive reactions in the kidney or urine. agement failure and contact with other animals are important
A positive sample from urine was sequenced, and a 260- risk factors (Leite et al. 2018).
nucleotide fragment in phylogenetic analysis showed identity The pigs are adapted to the main serogroups found
with Leptospira borgpetersenii sequences (Fig. 1). (Tarassovi, Australis, and Pomona) (Gonçalves and Costa
2011; Strutzberg-Minder and Kreienbrock 2011; Leite et al.
2018), and thus, even if they do not develop clinical signs,
they are disseminating the agent, making it possible to infect
Discussion the pigs themselves and other animal species. Favero et al.
(2002) already indicated an evolution in the change of the
The frequency of anti-Leptospira spp. antibodies of 40% was serological profile in swine, in which the reactions by the
elevated in comparison with other studies, 25.6% (Cavalcanti serogroup Icterohaemorrhagiae would exceed the Pomona,

Chart 1 The serogroups found

and their respective titers of anti-
Leptospira sp. antibodies in swine
slaughtered in semi-arid region,
Brazil

Table 2 Frequency of positive samples by PCR according to the organ used, for swine slaughtered in the semi-arid region, Brazil
Organ used for PCR Total number of samples
Urine 27
Vaginal fluid 25
Kidney 39
which has been verified in recent studies (Hashimoto et al.
2008; Osava et al. 2010; Gonçalves et al. 2011; Leite et al.
2018), corroborating with Pinto et al. (2017) who in a system-
atic review found this serogroup as the most frequently report-
ed in swine, thus allowing the possible participation of
synanthropic rodents, which fosters the importance of control
of these hosts, since representatives of this serogroup are as-
sociated with large reproductive losses in pigs in Brazil
(Ramos et al. 2006), possibly because the creations in the
semi-arid are not technified, which would make intraspecific
transmission difficult. There is great environmental depen-
dence for the presence of the agent in the pigs (Figueiredo
et al. 2013b), but in the present work a higher frequency of
serogroups adapted to the species was observed, possibly be-
ing facilitated by means of direct contact, such as venereal
transmission and close contact with urine (Neibergs and
Zanella 2010).
The detection of (22%) positive animals by PCR is similar to
the study of Santos et al. (2011) who diagnosed 19.44% using
the same technique. Some studies such as the ones from More
et al. (2017) and Oliveira et al. (2007) verified higher positivity
by molecular diagnosis and confirmed the higher sensitivity of
this technique compared with the MAT; however, in the current
study the highest positivity was through MAT, a fact that is
justified because just contact with the agent is enough for the
production of antibodies to be induced, not specifically the
characterization of the carrier state or the infection itself. The
difference of reactive animals in serology and PCR has been
followed in different studies independent of the animal species,
as in Costa et al. (2017), Silva et al. (2019) and Sant’ana et al.
Fig. 1 The phylogenetic tree was
constructed by the Phy test, using
the GTR method. The analysis
included 260 nucleotides
sequences with the 1000
replicates in the bootstrap test.
(Black up-pointing triangle)
Reacons
Sequenced sample
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Number of positive reactions Frequency (%) CI (95%) Sequencing
8 29.6 12.4–46.9 1
3 12 4.2–30.0 –
2 5.1 1.4–16.9 –
(2017), emphasizing the importance of associating the two
techniques for reliable diagnosis (Otaka et al. 2013).
The presence of PCR-pos/MAT-neg can be explained by
the pathogenesis of the disease itself, where a gradual reduc-
tion of antibodies occurs until the levels are low or undetect-
able (Latosinski et al. 2018). This fact can be well observed
for serogroups adapted to the host, where the titers remain low
but with the agent present in the organism (Sakoda et al.
2012). The cut-off point used (1:100) may have been an im-
portant factor, in which animals with a lower concentration of
antibodies could not be detected; in addition the leptospiric
DNA detected in the PCR of some samples could belong to a
serogroup not included in the MAT. The most notable were
MAT-pos/PCR-neg animals (17 animals) corroborating with
other studies (Fornazari et al. 2012; Koizumi et al. 2013;
Hamond et al. 2014).
For Benacer et al. (2017) the renal tissue can be limited to
conventional PCR if this material has high concentration of
DNA from the host, which may explain the fact that, in this
paper, there were positive animals in urine PCR and negative
in the kidney test. Another thing that can be considered is the
fact that only a small fragment of the organ is collected; thus,
there is the possibility that the bacterium is not present in
sufficient concentration in this sample for its molecular
detection, even though it is colonizing the kidney of the
animal. Vitale et al. (2005) found a greater number of positives
through the urine, as well as in the present study, where eight
(29.6%) urine samples were reagent.
The little positivity reaction in the kidney 2/39 (5.1%) was
also seen by Gonçalves et al. (2014), unlike studies in which
Percentage and anbody ter
7
6
5 Tarasssovi
4 Australis
Pomona
3
Bataviae
2 Icterohaemorrhagiae
1
0
100 (20%) 200 (55%) 400 (25%)
Trop Anim Health Prod
this organ was the major representative of the positive reac-
tions (Oliveira et al. 2007; Miraglia et al. 2008). Even though
the kidney is an organ of predilection of the bacterium, it may
not always be present there, and it is necessary to use other
organs for molecular detection, considering that there were
three (12%) reactions from the vaginal fluid. As in the present
study, studies on other animal species have demonstrated the
presence of Leptospira in vaginal fluid in an increasingly fre-
quent way (Lilenbaum et al. 2008a, b; Hamond et al. 2014).
Oliveira et al. (2007) detected leptospires in the genital tract of
pigs in Brazil, and studies in other species identified a greater
amount of positives through reproductive organs, as Arent
et al. (2013), Director et al. (2014) and Silva et al. (2019),
evidencing the importance of these organs as extra-renal sites
of infection.
Molecular typing is very important to understand the epi-
demiology of the disease, since it allows to identify the species
of Leptospira through genetic sequencing (Lagadec et al.
2016). Through DNA amplification was detected
L. Borgpetersenii, representatives of this species were se-
quenced in cattle by Allan et al. (2018), and in humans and
Rattus ratus by Guernier et al. (2017), so the pigs represent a
source of infection for other hosts, including humans, since
the sequencing of the same species of Leptospira in humans.
The presence of DNA in vaginal secretion evidences the
possibility of the reproductive tract functioning as an impor-
tant extra-renal site of the disease, and thus the importance of
these animals as carriers of the agent, leading to the risk of
infection to other pigs due to intimate contact that happens
between members of the same species. In addition, because
they are animals of small creations they can easily enter into
contact with other animals and even the human being.
Conclusion
The results evidenced a high rate of positive pigs; thus, con-
sidering that in the semiarid there are many extensive crea-
tions and creations consorted with other animals, there are
great possibilities of contact of these carriers with other animal
species and man, representing a potential risk of transmission
of the disease.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed. This
research was approved by the Research Ethics Committee (CEP) from
the Federal University of Campina Grande (UFCG) under number 031/
2016. This article does not contain any studies with human participants
performed by any of the authors.
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