Veterinary Microbiology 250 (2020) 108869

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Extra-renal bovine leptospirosis: Molecular characterization of the

Leptospira interrogans Sejroe serogroup on the uterus of non-pregnant cows
Maria Isabel Nogueira Di Azevedo a, Bruno C. Pires a, Hugo Libonati a, Priscila S. Pinto a,
Lucas Figueiredo Cardoso Barbosa a, Filipe Anibal Carvalho-Costa b, Walter Lilenbaum a, *
a
Fluminense Federal University, Laboratory of Veterinary Bacteriology, Biomedical Institute, Niterói, Rio de Janeiro, Brazil
b
Laboratory of Epidemiology and Molecular Systematics, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Bovine genital leptospirosis is a chronic disease that causes reproductive disorders such as abortions, stillbirths,
Uterine infection and estrus repetition, as well as economic losses. Despite clinical signs related to reproductive failure, the ma­
Cattle jority of studies have focused on the detection of Leptospira spp. in the urine, while few have considered the
PCR
reproductive tract. Consequently, the aim of the present study was to investigate the uterus as an important
lipL32
extra-renal site of leptospiral infection in cows. A total of 42 non-pregnant cows were studied at a slaughter­
secY
Bovine genital leptospirosis house. Blood samples and uterine fragments were collected for serology and molecular analysis, respectively.
Concerning serologic results, 20.5 % presented as reactive, all of them against the Sejroe serogroup. Regarding
lipL32 PCR, 26.2 % (11/42) of samples were positive for pathogenic Leptospira sp. Sequencing the secY gene short
region enabled nine strains to be characterized, all of which were L. interrogans, with high identity (98.8 %–99.8
%) with serovar Hardjo. The use of molecular tools substantially improved the sensitivity of Leptospira sp.
detection at species level and demonstrated that the uterus is an important site of bovine leptospiral infection.
The findings of the present study reinforce our understanding that leptospiral uterine infection are associated to
members of the Sejroe serogroup.

1. Introduction that the genital tract appeared to be as important a site of persistence as

Bovine leptospirosis is a chronic disease that causes reproductive
disorders such as abortions, stillbirths, and estrus repetition, as well as
economic losses. Strains of the Sejroe serogroup are the major agents,
mainly genotypes of the serovar Hardjo (bovis and occasionally prajitno
types in Europe and bovis type in the USA) or the Guaricura serovar in
South America (Loureiro et al., 2017). Recently, Loureiro and Lilenbaum
(2020) critically analyzed information about genital leptospirosis in
cattle and considered it as a distinct syndrome, which they denominated
bovine genital leptospirosis (BGL), requiring different approaches to
diagnosis and treatment.
Despite clinical signs related to reproductive failure, most studies
have focused on the detection of viable leptospires or its DNA in urine
(Loureiro et al., 2017). As such, little is known about the presence and
role of leptospires in the reproductive tract of cows. Leptospira sp. was
first recovered from the genital tract of non-pregnant cows more than 35
years ago (Ellis et al., 1986) in Northern Ireland. The authors reported
the urinary tract, findings which were confirmed in a US study (Ellis and
Thiermann, 1986). Nevertheless, despite its importance, little attention
has been subsequently given to uterine infection, and molecular char­
acterization remains practically unexplored. A study using
paraffin-embedded bovine uterine samples stored for 13 years yielded
18 % of PCR-positive results (Pires et al., 2018). More recently, a high
percentage (60.3 %) of uterine carriers of Leptospira sp. was molecularly
detected in sheep in a semi-arid region of northeastern Brazil (Silva
et al., 2019). In this context, the aim of the present study was to
investigate, through an integrative approach using serological and mo­
lecular tools, the role of the uterus as an extra-renal site of leptospiral
infection in cows.

* Corresponding author.
E-mail address: wlilenbaum@id.uff.br (W. Lilenbaum).

https://doi.org/10.1016/j.vetmic.2020.108869
Received 21 May 2020; Accepted 20 September 2020
Available online 29 September 2020
0378-1135/© 2020 Elsevier B.V. All rights reserved.
M.I.N. Di Azevedo et al. Veterinary Microbiology 250 (2020) 108869

2. Material and methods Table 1

Serology and Genetic identification of Leptospira DNA obtained from samples
2.1. Animals and collection of samples collected from asymptomatic cows from a slaughterhouse, Rio de Janeiro, Brazil.
Serum (MAT) Uterine Fragment
This study was approved by the Ethics Committee of Universidade Serogroup (Titre) Sample PCR (lipL32) Sequencing (secY)
Federal Fluminense, Brazil (#863). A total of 42 non-pregnant cows Animal
were selected by convenience from two slaughterhouses located close to 1 NR UF1 NEG NT
Rio de Janeiro, Brazil. Cattle origin, individual history, and herd 2 NR UF2 NEG NT
3 NR UF3 NEG NT
reproductive data were not provided. Blood samples were collected
4 NR UF4 POS L. interrogans
during the bleeding in sterile tubes without anticoagulant and trans­ 5 NR UF5 POS L. interrogans
ported to the laboratory, where they were centrifuged (1000×g for 10 6 NT UF6 NEG NT
min). Serum was stored in 1.5-mL Eppendorf® tubes at − 20 ◦ C for batch 7 NR UF7 POS L. interrogans
testing with serology. For uterine fragment collection, whole uteri were 8 NT UF8 NEG NT
9 NR UF9 POS L. interrogans
recovered from the animals, with no cross-contamination between them. 10 NR UF10 NEG NT
First, urinary bladder was removed from the carcass. After that, whole 11 NT UF11 NEG NT
uteri were removed and put closed on a plastic bag. At the table, it was 12 NR UF12 NEG NT
removed from the bag, opened and then samples were collected. 13 NR UF13 NEG NT
14 NR UF14 NEG NT
Approximately 0.5 cm3 of the uterine horn covering the endometrium
15 NR UF15 NEG NT
and myometrium was cut off, with one sterile scalpel blade used per 16 NR UF16 POS L. interrogans
sample to avoid contamination. The samples were kept frozen until 17 NR UF17 NEG NT
molecular analysis. 18 Sejroe (200) UF18 NEG NT
19 Sejroe (200) UF19 POS L. interrogans
20 Sejroe (100) UF20 POS L. interrogans
2.2. Serology
21 NR UF21 POS L. interrogans
22 Sejroe (100) UF22 NEG NT
A microscopic agglutination test (MAT) was performed according to 23 NR UF23 NEG NT
OIE (OIE, 2018). Sera were tested against a panel of 25 live Leptospira 24 NR UF24 POS L. interrogans
25 NR UF25 POS LQS
cultures as antigens (Table S1). Positive bovine sera were used as posi­
26 NR UF26 NEG NT
tive controls. All strains (except the Guaricura strain) were donated by 27 NR UF27 NEG NT
Institut Pasteur – Paris, France. The Guaricura strain was donated by 28 NR UF28 NEG NT
Universidade de São Paulo (USP) – São Paulo, Brazil. Antigens are 29 NR UF29 NEG NT
monthly tested with polyclonal antisera provided by KIT, Amsterdam. 30 NR UF30 NEG NT
31 NR UF31 POS LQS
Animals were considered serologically positive when titers were ≥100.
32 NR UF32 NEG NT
33 NR UF33 NEG NT
2.3. Molecular analysis 34 NR UF34 NEG NT
35 NR UF35 NEG NT
36 NR UF36 NEG NT
DNA was extracted using DNeasy® Blood & Tissue Kit (Qiagen,
37 Sejroe (100) UF37 NEG NT
California, EUA). Polymerase chain reaction (PCR) targeting a short 38 NR UF38 NEG NT
region of lipL32 gene (242bp), present on pathogenic leptospires 39 Sejroe (100) UF39 NEG NT
(Stoddard et al., 2009), was carried out using primers and under con­ 40 NR UF40 NEG NT
ditions previously described (Hamond et al., 2015). Thereafter, lipL32 41 Sejroe (400) UF41 NEG NT
42 Sejroe (200) UF42 NEG NT
positive samples were submitted to a nested PCR targeting a partial
region of the secY gene (411bp), following conditions described by NR: Non-reactive; NT: Not tested; NEG: Negative; POS: Positive; LQS: Low
Ahmed et al. (2006). The primers used in this study are shown in Quality Sequence.
Table S2. For each set of samples, ultrapure water was used as a negative
control in all reactions, while 10 fg of DNA extracted from Leptospira 3. Results
interrogans serovar Copenhageni (Fiocruz L1− 130) was used as a posi­
tive control. PCR products were analyzed by electrophoresis in 1.5–2% 3.1. Serology
agarose gel and visualized under UV light, after gel red staining.
The secY amplicons were directly sequenced using Big Dye Termi­ It was not possible to obtain serum from three animals. Of the
nator v. 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) remaining 39 sera, eight (20.5 %) presented seroreactivity, all against
in a 3100 automated DNA sequencer according to the manufacturer’s the Sejroe serogroup, with titers of between 100 and 400 (Table 1).
instructions. The nucleotide sequences were deposited in GenBank
under accession numbers MT270420–MT270428. Regarding phyloge­ 3.2. Molecular analysis
netic analysis, genetic distances were calculated using MEGA X (Kumar
et al., 2018), with the Kimura-2-parameter (K2P) model. Pairwise/­ Regarding lipL32 molecular analysis, amplicons with the expected
Blast/NCBI, SeqMan v. 7.0, ClustalW v. 1.35 (Thompson et al., 1994), size were observed in 11/42 (26.2 %) of samples (gel not shown), con­
and BioEdit v. 7.0.1 (Hall, 1999) software were used for editing and firming the presence of leptospiral DNA in the uterine fragment. Of
sequence analysis. A maximum likelihood (ML) tree was constructed these, targeting the secY gene short region, it was possible to amplify (gel
using the K2P model with the gamma distribution in MEGA X software, not shown), and sequencing nine amplicons (UF4, UF5, UF7, UF9, UF16,
as this was determined to be the best-fitting model of DNA substitution UF19, UF20, UF21, UF24). Pairwise/Blast/NCBI comparisons with the
using the Bayesian information criterion. GenBank secY gene dataset identified all of them as L. interrogans (≥98.5
% identity).
Intraspecific genetic distance between sequences from the present
study and others from the Sejroe serogroup serovar Hardjo ranged from
K2P = 0.002 (SE = 0.06) to K2P = 0.012 (SE = 0.05) (Table S3),
reflecting a high identity, varying from 98.8% to 99.8%. The overall

2
M.I.N. Di Azevedo et al.
Fig. 1. Maximum likelihood phylogenetic tree inferred from partial secY gene sequences of L. interrogans serogroup Sejroe from this study (UF) and GenBank se­
quences (accession numbers, strain, serogroup and serovar are shown). Sequences of strains from Brazilian ruminants are indicated. Numbers at nodes are bootstrap
values greater than 50 %. Leptospira biflexa strain ‘Patoc is the outgroup taxa.
mean distance was K2P = 0.01 (SE = 0.00), that is, an identity average
of 99 % between sequences. Phylogenetic analysis based on ML K2P + G
tree confirmed L. interrogans species identification (Fig. 1). Moreover,
sequences from the present study clustered together with strains from
the Sejroe serogroup serovar Hardjo, most of them from Brazilian ru­
minants, reflecting an identity higher than 98.6 % (Fig. 1, Table S3). Not
surprisingly, sequences from the present study are very closely related to
other sequences from the same geographical localization (str. Norma,
str. 2012_OV5, str. 2015_U376, str. 2015_U349) with very low genetic
distances between them (varying from 0.00 to 0.012, i.e., 100 % to 98.8
% of identity) (Table S3).
4. Discussion
Although this was not a prevalence study, it is noteworthy that
infection rates based on serology were not surprising for the region. The
serology indicated around 20 % reactivity against the Sejroe serogroup
with low titers, what is widely described as indicative of chronic infec­
tion in cattle (Correia et al., 2017). Moreover, it has been well estab­
lished that serology has poor resolution for the diagnosis of leptospirosis
on cattle, particularly the chronic infection caused by Sejroe members
and independently of the site of the infection (Otaka et al., 2012; OIE,
2018). In the present study, infected animals (demonstrated by PCR)
present very low titers that are not detected by MAT, as previously re­
ported (Hamond et al., 2014; Libonati et al., 2017; Nally et al., 2018).
It is known that bacterial culturing of leptospires is fastidious, and
difficult to perform. Thus, the use of PCR contributes to improving the
sensitivity of leptospires detection, providing a broader understanding
of the potential significance of infection. Moreover, sequencing pro­
vided useful epidemiological information. Moreover, the uterus is often
neglected as an object of study in bovine leptospirosis, and the molecular
identification of L. interrogans in this site reinforces its association with
the physiopathogenesis of BGL and the need to include it in diagnostic
investigations. Currently, the single report of the detection of leptospiral
DNA in the uterus of cows is limited to a study on paraffin-embedded
samples, which yielded 18 % positivity (Pires et al., 2018).
It is known that Hardjo genotypes are adapted to bovine hosts and
present a preference for the reproductive tract (Ellis et al., 1986; Ellis,
2015), being strictly associated with the chronic reproductive form of
bovine leptospirosis (Loureiro and Lilenbaum, 2020). Although the role
of Leptospira sp. in uterine infection is not well understood, it is known
that the bacterium in the genital tract can lead to reproductive disorders
3
Veterinary Microbiology 250 (2020) 108869
(Ellis, 2015). Abortions, as well as estrus repetition due to early embryo
death, are recognized as a major reproductive disorder worldwide, and
have been associated with leptospirosis (Libonati et al., 2018). Loureiro
and Lilenbaum (2020) highlighted that the effects of BGL can be asso­
ciated with uterine inflammation, resulting in altered uterine conditions
that impair embryonic survival or implantation and leading to early
embryonic loss and consequent estrus repetition. In dogs, the presence of
Leptospira bacteria in the uterus modifies the expression of the extra­
cellular matrix and increases the production of inflammatory uterine
regulators (Wang et al., 2014), potentially impacting on implantation
and viable pregnancies.
A noteworthy highlight and one of the main limitations of the study
is that, since samples were collected at a slaughterhouse, very little in­
formation is available about the reproductive or sanitary history of the
animals. The reason why they were non-pregnant is unknown, but the
possibility that it was due to chronic leptospirosis cannot be neglected.
5. Conclusions
We demonstrated that the uterus is an important extra-renal site of
bovine leptospiral infection and should be included in diagnostic in­
vestigations. The use of molecular tools substantially improved the
sensitivity of Leptospira sp. detection at species level, and genital carriers
of L. interrogans from Hardjo genotypes were demonstrated to be more
common than usually reported. Finally, the findings of the present study
reinforce our understanding that leptospiral uterine infection are asso­
ciated to members of the Sejroe serogroup.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
We thank Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) for partially funding the study – Finance Code
001. We also thank the Bom Pastor and Juiz de Fora slaughterhouses for
allowing access to cows. We are also grateful to the Program for Tech­
nological Development in Tools for Health-PDTIS/FIOCRUZ for allow­
ing us to use its facilities. MINDA and WL are FAPERJ fellows, BCP is a
CAPES fellow, and LFCB and WL are CNPq fellows.
M.I.N. Di Azevedo et al.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.vetmic.2020.108869.
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