https://doi.org/10.52973/rcfcv-e33201
Received: 17/09/2022 Accepted: 24/10/2022 Published: 29/12/2022
1 of 9
Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
ABSTRACT
Cattle raising is the most important livestock activity in Mexico,
highlighting the fact that the Country is eight place worldwide in
the production of bovine meat. However, cattle can be affected by
leptospirosis (a bacterial disease caused by 17 species of spirochetes
of the genus Leptospira), which cause reproductive problems that
translate into great economic losses. Additionally, these zoonotic
agents can cause a rapidly evolving febrile condition in humans, which
can be solved after the rst week of symptoms or could progress to
develop a severe late-phase manifestations. Despite the great impact
of these agents on the economy and Public Health, epidemiological
surveillance against the infectious disease that cause is not present in
municipal slaughterhouses (MS). Therefore, the aim of this study was
to identify, using serological and molecular methods, the circulating
Leptospira species in three MS in Veracruz State. The frequency of
anti-Leptospira antibodies was 67.5%. Additionally, ve sequences
were recovered that were 99% similar to L. interrogans. This work
represents the rst national effort for the evaluation of MS as sentinel
units, that allow establishing the diversity of species of the genus
Leptospira that circulate in cattle and establishing intervention
measures for workers risk mitigation, who come into contact with
the uids and organs of infected animals.
Key words: Leptospirosis; cattle; sentinel units; Leptospira
interrogans
RESUMEN
La ganadería bovina es la actividad pecuaria más importante de
México, destacándose el hecho de que el país ocupa el octavo lugar
a nivel mundial en la producción de carne bovina. Sin embargo, el
ganado puede verse afectado por la leptospirosis (enfermedad
bacteriana causada por 17 especies de espiroquetas del género
Leptospira), que ocasionan problemas reproductivos que se traducen
en grandes pérdidas económicas. Además, estos agentes zoonóticos
pueden causar una condición febril de evolución rápida en humanos, la
cual puede resolverse después de la primera semana de presentación
de síntomas o puede progresar y desarrollar manifestaciones severas
en una fase tardía. A pesar del gran impacto de estos agentes en la
economía y la salud pública, la vigilancia epidemiológica frente a la
enfermedad infecciosa que los provocan no está presente en los
mataderos municipales (MM). Por tanto, el objetivo de este estudio fue
identicar, mediante métodos serológicos y moleculares, las especies
de Leptospira circulantes en tres MM de Veracruz. La frecuencia de
anticuerpos anti-Leptospira fue del 67,5%. Además, se recuperaron
cinco secuencias con un 99% de similitud con L. interrogans. Este
trabajo representa el primer esfuerzo nacional para la evaluación
de los MM como unidades centinela, que permitan establecer la
diversidad de especies del género Leptospira que circulan en los
bovinos y establecer medidas de intervención para mitigar los riesgos
para los trabajadores que entren en contacto con los uidos y órganos
de animales infectados.
Palabras clave: Leptospirosis; ganado; unidades centinelas;
Leptospira interrogans
Serological and Molecular evidence of pathogenic Leptospira species in
cattle from slaughterhouses in Veracruz State, Mexico
Evidencia serológica y molecular de especies patógenas de Leptospira en rastros
de bovinos del estado de Veracruz, México
Jose Luis Ochoa-Valencia
1
, Anabel Cruz-Romero
1
* , Sokani Sánchez-Montes
2,3
, Sandra Cecilia Esparza-González
4
, Dora Romero-Salas
1
,
Belisario Domínguez-Mancera
1
, Jose Rodrigo Ramos-Vázquez
1
, Ingeborg Becker
3
and Marco Torres-Castro
5
1
Universidad Veracruzana, Facultad de Medicina Veterinaria y Zootecnia, región Veracruz. Veracruz, México.
2
Universidad Veracruzana, Facultad de Ciencias
Biológicas y Agropecuarias, región Tuxpan. Veracruz, México.
3
Universidad Nacional Autónoma de México, Facultad de Medicina, Centro de Medicina Tropical,
Unidad de Investigación en Medicina Experimental. Ciudad de México, México.
4
Universidad Autónoma de Coahuila, Facultad de Odontología, Laboratorio de
Cultivo Celular, Unidad Saltillo. Saltillo, México.
5
Universidad Autónoma de Yucatán, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Laboratorio de
Enfermedades Emergentes y Reemergentes.Yucatán, México.
*Email: anabcruz@uv.mx
Leptospira species in cattle from slaughterhouses in Mexico / Ochoa-Valencia et al. _______________________________________________
2 of 9
INTRODUCTION
The impact of leptospirosis in domestic animals and those intended
for consumption, as well as in humans, is not fully understood [9,
24, 39]. The severity of the disease varies according to the serovar
and infecting species of Leptospira, as well as the species of
animal host that is affected [11, 24, 51]. Such is the case with bovine
leptospirosis, which occurs worldwide and is the result of infection
by several species and serovars that affect cattle (Bos taurus). The
acute phase of the disease is mainly subclinical, while the greatest
economic losses are caused as a result of low fertility, abortions
and low milk production, which occur during the chronic phase of
the disease [24, 51]. The genus Leptospira encompasses at least 68
species of spirochetes that are classied into two main groups: 1) the
saprophytic or free-living group, which is divided in two subgroups:
the S1 with 23 species and S2 with ve species; and 2) the pathogenic
group, which is subdivided into two subgroups: P1 (pathogenic) with
19 species and P2 (intermediate) with 21 species (which may or may
not cause disease, depending on the species of host it infects) [28,
55]. It is currently recognized that pathogenic species are the cause
of infections, both in humans and animals, causing lung, kidney and/
or reproductive disorders [2, 3, 11, 24, 55]. It should be noted that the
infection caused by these bacteria is known as leptospirosis, which is
a zoonosis that has a wide distribution worldwide, mainly in Countries
or areas with tropical or subtropical climates. Leptospira pathogenic
species can have direct or indirect transmission from contact with
contaminated water, soil, tissue, or uids [24].
Bovines are the animals most studied with regard to the detection and
manifestations caused by infection with Leptospira spp. [11]. In these
studies, different diagnostic methods (bacteriological, serological,
and molecular) have been used; however, few studies have been
carried out on slaughtered cattle destined for human consumption.
In the period from 1975 to 2020, 44 studies have been recorded in 12
Countries, demonstrating the presence of six pathogenic species and
27 serovars [4, 7, 13, 14, 17, 18, 19, 20, 23, 26, 29] (TABLE I). The prevalence
uctuate between 3.8-85% with an average of 31.1% in 9 Countries
of the Nearctic Region where L. interrogans, L. borgpetersenii and L.
kirschneri were reported; in contrast, in the Neotropical Region the
prevalence uctuate between 2.7-79.8% with an average of 36.4% in 10
Countries with six species detected (L. interrogans, L. borgpetersenii,
L. santarosai, L. kirschneri, L. noguchii, and L. alstonii), Likewise, L.
interrogans and L. borgpetersenii and L. kirschneri are circulate in both
Zoogeographic Regions [30, 31, 33, 34, 41, 42, 43, 44, 45, 46, 47, 48, 50]
(TABLE I). It is necessary to highlight that, during the aforementioned
period, in Mexico, only a single study has been carried out in this type
of animal, in the State of Guerrero, in which 380 animals were sampled,
detecting anti-Leptospira antibodies against two Leptospira species
(L. interrogans and L. borgspetersenii) and four serovars (Australis,
Ballum, Bataviae, and Grippotyphosa) (TABLE I) [8].
Although the municipally slaughterhouses (MS) are sentinel units
in the epidemiological surveillance of other zoonotic pathogens (e.g.,
Brucella and Mycobacterium tuberculosis), in the case of Leptospira,
there is no active surveillance. Therefore, the aim of the present
TABLE I
Studies carried out worldwide in cattle sent to slaughterhouses for the detection of Leptospira spp.
Leptospira species / Serovars Sample Result Country [Ref]
L. interrogans: Hardjo Kidney Isolates 3.7%
Argentina [34]
L. interrogans: Pomona Kidney / Blood Isolates 16%
Australia [3]
L. interrogans: Hebdomadis Blood 44.3% serological evidence
Australia [33]
L. interrogans: Hardjo Foetus AFD en 4.6%
UK [12]
L.interrogans: Hardjo/Grippotyphosa/Pomona Blood / Kidney
MAT 14.5%, ELISA 39.5%,
Isolates 6.4%
USA [52]
L. interrogans: Hardjo Blood/LCR MAT 15.4%, Isolates 66.6%
Canada [26]
L. interrogans: Hardjo Blood Seroreactors 75.3%
Chile [21]
L. interrogans: Hardjo Genital tract Isolates 65%
UK [13]
L. interrogans: Kennewicki | L. borgpetersenii: Hardjo bovis Kidney / Blood Isolates 13.5%, IF 7.6%
Canada [42]
L. interrogans: Hardjo / Pomona Kidney / Blood Isolates 28.6%, MAT 34.4%
Canada [22]
L. interrogans: Hardjo Kidney Isolates 8.3%
Australia [48]
L.borgpetersenii: Hardjobovis Isolates
DNA restriction endonuclease
analysis 91%
Ireland [14]
L. interrogans: Hardjo / Pomona / Grippotyphosa Kidney Isolates 85%
USA [31]
L. interrogans: Grippotyphosa / Pomona | L. borgpetersenii: Hardjo Kidney / Blood Isolates 1.7%, Serology 48.7%
USA [32]
L. interrogans Kidney Isolates 10.4%
Zimbabwe [17]
L. interrogans: Bratislava / Bataviae / Grippotyphosa |
L. borgpetersenii: Hardjo / Ballum
Blood MAT 32.8%
Mexico [8]
L. interrogans: Grippotyphosa / Sejroe / Icterohaemorrhagiae / Hardjo Blood MAT 7.4%
Czech Republic [53]
L. interrogans: Mhou / Marondera Isolates Mhou (3/4); Marondera (1/4)
Zimbabwe [19]
L. santarosai: Gatuni Kidney Two isolates typied by CAAT
Zimbabwe [18]
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
3 of 9
TABLE I (cont...)
Studies carried out worldwide in cattle sent to slaughterhouses for the detection of Leptospira spp.
L. borgpetersenii: Tarassovi / Hardjo Blood / Kidney / Urine MAT 5.7%, Isolates 1.4% Australia [54]
L. interrogans: Pomona / Grippotyphosa / Canicola Blood/Urine MAT 37.7%, Isolates 1%
Iran [45]
L. interrogans: Hardjo / Canicola Blood 10% by MAT
Jamaica [5]
Leptospira sp Kidney / Blood / Urine
PCR: Kidney 79.2, Blood 29.2%,
Urine 55.5%
Iran [4]
L. interrogans: Pomona / Hardjo / Bataviae | L. borgpetersenii: Tarassovi Blood MAT 51%
Tanzania [49]
L. interrogans: Hardjo Blood 3.5% seropositivity
Nigeria [36]
L. interrogans: Bataviae | L. borgpetersenii: Ballum | L. kirschneri: Cynopteri Blood MAT 40%
Egyp [25]
Leptospira sp. Urine Nested PCR 43% Iran [46]
L. borgpeterseni
i, L. kirschneri, L. interrogans Kidney Nested PCR 12.2% Sri Lanka [20]
L. borgpetersenii: Hardjo bovis | L. interrogans: Pomona Urine / Blood / Kidney MAT and PCR
New Zealand [16]
L. borgpetersenii Kidney
DF 59%, DFA 78%, PCR 29.7%,
Isolates 8.1%
USA [44]
L. borgpetersenii: Hardjo bovis | L. interrogans: Pomona Urine / Kidney / Blood qPCR 21.2%, MAT 73%
New Zealand [15]
Leptospira sp. Kidney
PCR 15.4%,
PCR-RFLP 40.8%
Iran [50]
L. santarosai: Sejroe | L. noguchii: Australis VF / Urine
Isolates 4.3%, PCR: Urine 31.0%,
VF 50.4%.
Brazil [30]
L. santarosai, L. alstonii, L. interrogans Urine and/or FV Isolates 2.7%
Brazil [41]
L.borgpetersenii: Hardjo bovis | L. interrogans: Bratislava / Hardjo / Pomona /
Icterohaemorrhagiae | L. kirschneri: Grippotyphosa
Blood / Urine
MAT 20%, Isolates 3.8%,
FAT 5.5%, PCR 8.8%
USA [35]
L. borgpetersenii: Sejroe Blood / Urine / Kidney / FV
MAT 37%, Isolates 5.0%,
PCR 63.9%
Brazil [40]
L. interrogans TU PCR 18%
Brazil [6]
L. borgpetersenii, L. kirschneri Kidney / Urine qPCR: Kidney 7.2%, Urina 5.8%
Uganda [2]
L. borgpetersenii, L. interrogans: Mankarso Blood / Kidney
Seroprevalence 79.8%,
Positive by RT-PCR 18.2%
Saint Kitts [47]
Leptospira sp., L. borgpetersenii, L. kirschneri, L. interrogans, L. santarosai Blood / Urine / Kidney
MAT 46.6%, PCR: Urina 14.9%,
Kidney 5.8%
Brazil [23]
L. kirschneri, L. borgpetersenii: Hardjo bovis / Tarassovi Kidney / Urine Prevalece by real-time PCR 19%
Madagascar [43]
L. noguichii Urine (isolates) PCR and BLAST 98% identity with n=38
Brazil [29]
L. borgpetersenii: Hardjo Blood MAT 27.6%
South Africa [10]
Leptospira sp. Hardjo prajitno / Bratislava Kidney
Isolates 76.4%, MAT 83.3%,
WSs 41.6%, IH 35%
Nigeria [1]
Reference [Ref], Indirect Immunouorescence (AFD), Direct Fluorescent Antibody (DFA), Dark eld (DF), Enzyme-Linked ImmunoSorbent Assay (ELISA), Microscopic Agglutination Test
(MAT), Quantitative Polymerase Chain Reaction (qPCR), Vaginal Fluid (VF), Uterine Tissue (UT), Restriction Fragment Length Polymorphism (RFLP), Basic Local Alignment SearchTool (BLAST),
Fluorescent Antibody Test (FAT), Cross-Agglutinin Absorption Test (CAAT), Reverse Transcription Polymerase Chain Reaction (RT-PCR), Warthin Silver (WSs), immunohistochemical (IH)
study was to identify, using serological and molecular methods, the
circulating Leptospira species in three MS in Veracruz State.
MATERIALS AND METHODS
Sample collection
The study was carried out in three MS in the Central zone of the
State of Veracruz, Mexico. The exact number of livestock production
units (UPP) of origin of the animals slaughtered in the three traces of
study is unknown, however, these cattle destined for consumption
come from UPP that belong to 17 Municipalities of the State of
Veracruz. The MS 1 receives animals from eight Municipalities
(Actopan, Alto Lucero, Emiliano Zapata, Ixhuatán, Paso de Ovejas,
Tlalixcoyan, Teocelo and Úrsulo Galván); MS 2 from nine (Actopan,
Alvarado, Cotaxtla, Cuitláhuac, Ignacio de la Llave, Jamapa, Manlio
Fabio Altamirano, Medellín and Tlalixcoyan); and MS 3 froma single
one (Tierra Blanca). A non-probabilistic convenience sampling was
carried out between April and June 2019, in which samples were
taken from all the cattle that arrived at the MS site on the day of the
visit for sampling, considering only as inclusion criteria cattle that
arrived at the MS.
in which, for the serological diagnosis, 80 blood samples were
collected in vacuum tubes without anticoagulant (BD Vacutainer®
red cap), obtaining a volume of 5 to 7 milliliters (mL) of blood from the
jugular vein during the exsanguination of the animals, at the time of
preparation of the carcass. Likewise, for the molecular identication
of Leptospira spp., 55 samples of kidney lobes were taken after the
evisceration of the animals was performed, which were xed in
containers with 96% ethanol.
Leptospira species in cattle from slaughterhouses in Mexico / Ochoa-Valencia et al. _______________________________________________
4 of 9
Sample processing
The biological samples were transported to the laboratory of Infectious
Diseases in the Diagnostic Unit of the Ranch “Torreón del Molino” of
the Faculty of Veterinary Medicine and Zootechnics of the Universidad
Veracruzana. The tubes with blood were centrifuged in a centrifuge
(Hsiang Tai Model CN-3600, Taiwan, China) at 1,000 G for 10 minutes (min)
to separate the serum from the clot. Once the serum was separated, it
was deposited in 500 microliters (µL) polypropylene microtubes that
were kept frozen at –20 °C in a Freezer (Thermo Scientic, 05LFEETSA,
Massachusetts, United States –USA-) until later use.
Antibodies detection
The detection of anti-Leptospira antibodies was carried out using
the microscopic agglutination technique (MAT) [38]. The analysed sera
were prepared individually, 100 µL of each sample was deposited in
polyethylene tubes with 2,400 µL of Phosphate Buffered Saline (PBS)
to obtain an initial dilution of 1:25, and they were kept refrigerated at
4 °C until later use (LG, LT57BPSX, Busan, South Korea). The MAT was
performed by placing 50 µL of PBS in each of the wells of a 96-well
ELISA plate, adding 50 µL of the initial dilution (1:25); subsequently,
50 µL of the serovar to be evaluated was added to each well. This was
repeated in total with 10 different serovars, which belong to three
different Leptospira species.
The reaction was evaluated using dark eld microscopy (Carl Zeiss,
Axio Lab A1, Jena, Germany). Samples that did not react with 50, 75
or 100% agglutination were discarded. All the samples (n=54) that
presented the aforementioned reaction were titrated until they did
not present a minimum agglutination of 50% of Leptospira, and in this
way, the degree of exposure to the different serovars and species of
Leptospira was assessed, considering as seroreactors all those sera
that reacted to titers ≥ 1:100 [8].
Leptospira genome detection and analysis
Total Deoxyribonucleic acid (DNA) extraction was carried out using
Chelex-100 chelating resin (Bio-Rad®, United States of America (USA))
[37]. The extractions were carried out individually using 3-5 grams (g) of
the central part (kidney lobe) of the kidney samples preserved in ethanol,
which were macerated and placed in polypropylene microtubes. A
total of 500 µL of a 10% Chelex-100 solution and 20 µL of proteinase
K (SIGMA life sciences®, USA) were added to each tube. The samples
were incubated for 24 hours (h) at 56 °C (IVYX Scientic, 0745556232573,
Washington, USA). Subsequently, they were centrifuged at 25,000 G
for 15 min, and the supernatant was collected and deposited in new
polypropylene microtubes that were stored at –20 °C for later use.
For the detection of pathogenic Leptospira DNA, it was
performed a conventional polymerase chain reaction probe (PCR)
in a Veriti 96-Well Fast Thermal Cycler (ThermoFisher Scientic,
4375305, Massachusetts,USA) that amplied a fragment of 430
base pairs (bp) of the LipL32 gene, by using the following pair of
primers: Forward 5’-ATCTCCGTTGCACTCTTTGC-3’ and Reverse
5’-ACCATCATCATCATCGTCCA-3 [56]. PCR amplication was performed
using the following thermal conditions: an initial denaturation cycle
at 95 °C for 5 min, 35 cycles at 95 °C for 1 min, 55 °C for 30 seconds
(s), and 72 °C for 1 min, and a nal extension at 72 °C for 7 min [56].
The PCR products were visualized on 2% agarose gels stained with
uorochrome iQ ™ SYBR® Green Supermix (Bio-Rad®, USA), using 1X TAE
(40 miliMol (mM) Tris-acetate, pH 8.0, 1 mM Ethylenediaminetetraacetic
acid (EDTA)) as a running buffer and a 100 bp Phi X174 DNA molecular
weight marker, for 45 min at 85 volts (V). Subsequently, the gels were
visualized with the aid of the Odyssey CLx Imaging System (LICOR
Biosciences®) [56].
The positive PCR products were puried using the QIAquick kit
(Quiagen®, Hilden, Germany) and sent for sequencing by the Sanger
method (Life Technology® 3500 xl) to Macrogen, Korea. The recovered
sequences were visualized using BioEdit, and once curated, local
alignments were performed using the BLAST-n tool available from
GenBank® (on the NCBI platform). Additionally, reference sequences
of validated Leptospira species deposited in GenBank® were obtained
and aligned with those recovered in the present study, and a
phylogenetic reconstruction was performed using the maximum
likelihood method with the general time reversible substitution model
under 10,000 bootstrap iterations in Mega v.10.
The results were analysed with descriptive epidemiology. Likewise,
the differences between the MS: one (n=30), two (n=30) and three
(n=20), were evaluated calculating the 95% condence intervals (CIs);
the general frequency (of anti-Leptospira antibodies) and specicity
(by serovar) were determined with the statistical program STATA V.14.
RESULTS AND DISCUSSION
Serological assays identied anti-Leptospira antibody titres (from
positive animals) that ranged from 1:100 to 1:3,200, obtaining a general
antibody frequency of 67.5% (54/80; 95% CI 56.1-77.5). The higher
frequency of seroreacting animals was 71.8%, obtained in the rst
slaughterhouse (23/30; 95% CI 53.2-86.2) (TABLE II). The species with
the highest frequency of positive hosts were L. interrogans serovar
Hardjo with 18.7% (15/80; 95% CI 10.9-29.0), followed by L. borgpetersenii
serovar Mini with 25.0% (20/80; 95% CI 15.9-35.9), and L. santarosai
serovar Tarassovi with 37.5% (30/80; 95% CI 26.9-49.0) (TABLES II AND III).
TABLE II
Frequency of anti-Leptospira antibodies by slaughterhouse
Slaughterhouse n Positive F% (
CI
95%
)
1 30 23 71.8 (53.2-86.2)
2 30 15 50.0 (31.2-68.7)
3 20 16 84.2 (60.4-96.6)
Total 80 54 67.5 (56.1-77.5)
F: frequency, Condence Interval at 95%: (CI
95%
)
From the collected kidneys (n=55), 30 of them corresponded to the
rst MS, ve of them to the second MS, and 20 of them to the third
MS, from which six samples amplied positive for the 430 bp of the
LipL32 fragment, which represent a frequency of 10.9% (6/55; 95%
CI 4.1-22.2). The six PCR positive products were sequenced, and ve
full sequences were retrieved for the phylogenetic reconstruction.
Sequences were deposited in GenBank under the following Accession
numbers: OP273650 – OP273655. The ve sequences exhibited a 99%
(429/430 bp) similarity with sequences of L. interrogans deposited in
GenBank. Additionally, the phylogenetic reconstruction grouped our
sequences with those of the L. interrogans group in a monophyletic
subgroup with a support value of 98% (FIG. 1).
FIGURE 1. Phylogenetic tree by the maximum likelihood (ML) method based on the 2-parameter Kimura model. A discrete Gamma distribution (+ G,
parameter = 0.2798) was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The numbers in
parentheses from one to ve correspond to the sequences recovered from the Leptospira hosts
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
5 of 9
TABLE III
Frequency and distribution of positive sera by antibody titles by Leptospira serovars.
Serovar
Antibody titration
F% (CI95%)
1:50 1:100 1:200 1:400 1:800 1:1600 1:3200
L. borgpetersenii
*Mini 34 13 2 2 3 -- -- 25.0 (15.9-35.9)
Sejroe 13 6 2 1 1 -- 1 13.7 (7.0-23.2)
Ballum 4 -- -- -- -- -- -- 0 (0-4.5)
L. interrogans
**Hardjo 41 6 3 4 1 1 -- 18.7 (10.9-29.0)
Icterohaemorrhagiae 18 8 1 -- 1 -- -- 12.5 (6.1-21.7)
Bratislava 37 1 3 1 -- -- -- 6.2 (2.0-13.9)
Pomona 6 2 -- 1 1 -- -- 5.0 (1.3-12.3)
Hardjo 10 1 -- 1 -- 2 -- 5.0 (1.3-12.3)
L. santarosai
*Tarassovi 16 11 9 4 1 4 1 37.5 (26.9-49.0)
Mini 24 10 9 1 3 1 -- 30.0 (20.2-41.2)
Positive seroreactor with titles ≥ 1:100, *serovars with a higher frequency of seroreactors by
Leptospira species, **Hardjo serovar isolated in cattle from the State of Veracruz, Mexico, F:
frequency, Condence Interval at 95%: (CI
95%
)
Leptospira species in cattle from slaughterhouses in Mexico / Ochoa-Valencia et al. _______________________________________________
6 of 9
Due to the fact that the positive animals presented the same
Leptospira species, and the recovered genetic variants have low
polymorphism, it is possible to assume that there is an extensive
dissemination of the pathogen due to contamination of water sources
or due to the movement of animals that are infected in the wild across
these 17 Municipalities. It is important to emphasize that the positive
animals came from MS 1 and 2, with 15 positive animals from the rst
one and ve from the second. Globally, there are few studies focused
on the detection of Leptospira spp. in cattle for human consumption,
particularly in animals from MS, within which, various kind of samples
have been screened (urine, kidney, vaginal uid, whole blood, blood
serum, uterine tissue, and foetal tissue). Also, many diagnostic
techniques have been implemented, such as serology (MAT, ELISA,
AFD and IF), molecular (cPCR, nested PCR, real-time PCR, sequencing,
among other ones) and bacterial isolation (TABLE I).
The prevalence of Leptospira spp. uctuates depending on the
technique used. It is recognized that serological methods tend to
over register cases due to the fact that animals that arrive at MS for
human consumption may present with antibodies as a result of an
immunological memory process generated by previous infections or by
vaccines. However, by means of bacteriological isolation and molecular
methods, it is possible to conrm the circulation of Leptospira spp.
that ranges from 5 to 10%. Since molecular techniques recognize the
presence of bacteria, however, it depends on the amount of tissue,
as well as the extraction method and the number of bacteria in which
they may have a lower sensitivity. In the same way, isolation conceals a
conrmatory technique, but with low yield since the transportation of
the sample, as well as the selection processes of the variants present,
can generate variable results [58, 59].This is important because MS
represent centres of concentrations of animals from different farms in
the same geographic region (facility), which is why multiple pathogenic
species of Leptospira can be present in these in a short period of time
enough for a detectable human chronic infection occur.
In relation to the serovars historically identied by serology of
animals from MS, there are several that have been reported in the
different studies, namely L. interrogans serovar Hardjo (the most
frequently reported) (TABLE I). In the present study, it was identied
the presence of three serovars: L. interrogans serovar Hardjo, being
similar to that reported in bovines slaughtered in the United Kingdom
[12], USA [35, 52], Canada [22, 27], Czech Republic [53], Tanzania
[49], Chile [21], Jamaica [5] and Nigeria [1, 36]; L. borgpetersenii
serovar Mini that, as far it was known, does not coincide with cattle
slaughtered in other Countries (however, there is evidence of other
serovars belonging to this Leptospira species such as Hardjo bovis
[8, 10, 16, 32, 35, 54], Tarassovi [10, 54], Ballum [25] and Sejroe [40];
and L. santarosai serovar Tarassovi.
Compared to the rest of the studies carried out worldwide in cattle
from MS, in the present study, it was possible to identify the species
L. interrogans in kidney samples using molecular essays (PCR), since,
as mentioned above, the recovered sequences presented a 98%
similarity with sequences of L. interrogans deposited in GenBank—
three of them from studies carried out in Brazil (two of them from Bos
taurus and one from Homo sapiens), one from Canis lupus familiaris
reported in Saint Kitts, and nally two from H. sapiens from Malaysia
and Indonesia. This same species of Leptospira has been reported,
by the same diagnostic method, in similar studies carried out in
Countries such as New Zealand [15] and Brazil [6, 40].
It is recognized that L. interrogans is the species most frequently
associated with human leptospirosis, especially with the presence
of Weil’s syndrome, which is the most serious complication of this
disease [57]. For this reason, it is important to consider the risk that
MS workers were exposed to when they come into contact with organs
and uids that may be infected with this bacterium. Likewise, this
study was the rst carried out in the State of Veracruz and the second
in Mexico, although it should be noted that the rst similar study
carried out in Mexico was 25 years ago and exclusively serological. A
previous study showed high antibody titres (24.7%) in one MS workers
in the State of Jalisco, Mexico, for which it is rearmed that it is a
risk group to which a greater number of control studies should be
carried out and provide more protection elements to avoid human
leptospirosis cases [8]. Other studies [2, 23, 27, 32] performed in the
USA, Brazil, and Uganda identied L. borgpetersenii, L. kirschneri, L.
santarosai, L. noguchii, and L. alstonii, in different cattle organs and
uid from animals destinated for consumption (TABLE I).
CONCLUSIONS
With the results obtained and comparing them with what was
reported in other similar studies, it was conclude that L. interrogans is
one of the pathogenic species of this bacterium that is circulating in
cattle slaughtered for consumption in the Coastal State of Veracruz,
Mexico. This study is a pioneer for the detection of pathogenic
species (L. interrogans) that can be found in cattle slaughtered in
MS and in Federal Inspection Type (TIF) traces, which are regulated
by the Mexican government. This study conrmed the presence
of L. interrogans in at least two MS by molecular methods and
demonstrated antibody titters in animals from the three sites, thus
it can be identied that the central region of the state is endemic
for the presence of the microorganism and probably of the disease.
Therefore, it is important to intensify efforts for epidemiological
surveillance against this pathogen in traces so they continue to full
their function as sentinel units to safeguard Public Health, since it
represents a potential risk, not only for the workers of these MS but
also the health risk represented by the management of waste from
these MS, which can affect Public (nal consumers) and Animal Health.
ACKNOWLEDGMENTS
To the Doctorate Program in Agricultural Sciences, Faculty of
Veterinary Medicine and Zootechnics, Universidad Veracruzana, as
well as to CONACYT, for the scholarship granted to one of the authors
of the work with CVU number 781618
Financial support
The project was partially nanced by UNAM-PAPIIT IG201221 and
Laboratory of Infectious Diseases UV-FMVZ.
Conict of interest
The authors certify that they have no aliations with or involvement
in any organization or entity with any nancial interest, nonnancial
interest in the subject matter or materials discussed in this manuscript.
Code or data availability
Sequences generated in the present study are deposited in GenBank
under the following Accession numbers: OM060690, OM108474,
OM108475 and OM108476.
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
7 of 9
Ethical statement
The authors conrm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to. This
project was approved by the Bioethics and Animal Welfare Committee
of the University of Veracruz, School of Veterinary Medicine and
Animal Husbandry in Veracruz, Veracruz, Mexico.
BIBLIOGRAPHICS REFERENCES
[1] AJAYI, O.L.; ANTIA, R.E.; AWOYOMI, O.J.; OLADIPO,
T.M.; ADEBOWALE, O.O. Cultural, histochemical, and
immunohistochemical detection of pathogenic Leptospira
species in the kidneys of cattle slaughtered in two abattoirs in
Southwest Nigeria. J. Immunoass. Immunochem. 41(3): 337–353.
2020. https://doi.org/jqrv.
[2] ALINAITWE, L.; KANKYA, C.; ALLAN, K. J.; RODRIGUEZ-CAMPOS,
S.; TORGERSON, P.; DREYFUS, A. Bovine leptospirosis in
abattoirs in Uganda: Molecular detection and risk of exposure
among workers. Zoon. Publ. Health. 66(6): 636–646. 2019.
https://doi.org/jqrw.
[3] AMATREDJO, A.; CAMPBELL, R.S., TRUEMAN, K.F. A study of
nephritis of beef cattle in North Queensland. Austr. Vet. J. 52(9):
398–402. 1976. https://doi.org/d4nnvv.
[4] AZIZI, S.; TAJBAKHSH, E.; HAJIMIRZAEI, M.R.; GHOLAMI –
VARNAMKHAST, M.; SADEGHIAN, H.; ORYAN, A. Evaluation of ‘white-
spotted kidneys’ associated with leptospirosis by polymerase chain
reaction based LipL32 gene in slaughtered cows.J. South Afric.
Vet. Assoc. 83(1): 69. 2012. https://doi.org/jqrx.
[5] BROWN, P.D.; MCKENZIE, M.; PINNOCK, M.; MCGROWDER,
D. Environmental risk factors associated with leptospirosis
among butchers and their associates in Jamaica. The Intern.
J. Occupat. Environm. Med. 2(1): 47–57. 2011.
[6] CABRAL-PIRES, B.; BERZIN-GRAPIGLIA, J.; MOREIRA, L.;
JAEGER, L. H.; CARVALHO-COSTA, F. A.; LILENBAUM, W.
Occurrence of uterine carriers for Leptospira interrogans on
slaughtered cows. Microb. Pathog. 114: 163–165. 2018. https://
doi.org/gc52vg.
[7] COLÍN-ORTIZ, J.; PÉREZ-SERNA, J.; CABALLERO-SERVÍN, A.;
GARCÍA-ROMERO, J.; IBARRA-LÓPEZ, L.; CUELLAR-ESPINOZA,
J.; BERNAL-VÉLEZ, C. Seroprevalencia a leptospiras en grupos
de riesgo de Guadalajara, Jalisco. Enferm. Infecc. Microbiol.
24(2): 134–137. 2004.
[8] CAMPOS-HERNANDEZ, E.; GUTIERREZ, I.; DIAZ, R. Serologic
frequency of bovine leptosirosis in the Cd. Altamirano, Guerrero
State, Mexico abattoir. Vet. Mex. 26(3): 175–182.1995.
[9] COSTA, F.; HAGAN, J.E.; CALCAGNO, J.; KANE, M.; TORGERSON,
P.; MARTINEZ-SILVEIRA, M.S.; STEIN, C.; ABELA-RIDDER, B.; KO,
A.I. Global Morbidity and Mortality of Leptospirosis: A Systematic
Review. Plos Neglect. Trop. Dis. 9(9): e0003898. 2015. https://
doi.org/b7sd.
[10] DOGONYARO, B.B.; VAN HEERDEN, H.; POTTS, A.D.; KOLO, B.F.;
LOTTER, C.; KATSANDE, C.; FASINA, F.O.; KO, A.I.; WUNDER, E.A.
JR.; ADESIYUN, A.A. Seroepidemiology of Leptospira infection
in slaughtered cattle in Gauteng Province, South Africa. Trop.
Anim. Health Prod. 52(6): 3789–3798. 2020. https://doi.org/jqr3.
[11] ELLIS, W.A. Animal leptospirosis. Current Topics Microbiol.
Immunol. 387: 99–137. 2015. https://doi.org/jqr4.
[12] ELLIS, W.A.; NEILL, S.D.; O’BRIEN, J.J.; CASSELLS, J.A.; HANNA,
J. Bovine leptospirosis: microbiological and serological ndings
in normal fetuses removed from the uteri after slaughter. The
Vet. Rec. 110(9): 192–194. 1982. https://doi.org/c547rj.
[13] ELLIS, W.A.; SONGER, J.G.; MONTGOMERY, J.; CASSELLS,
J.A. Prevalence of Leptospira interrogans serovar Hardjo in
the genital and urinary tracts of non-pregnant cattle. The Vet.
Rec. 118(1): 11–13. 1986. https://doi.org/bhhff7.
[14] ELLIS, W.A.; THIERMANN, A.B.; MONTGOMERY, J.; HANDSAKER,
A.; WINTER, P.J.; MARSHALL, R.B. Restriction endonuclease
analysis of Leptospira interrogans serovar Hardjo isolates from
cattle. Res. Vet. Sci. 44(3): 375–379. 1988. https://doi.org/jqr5.
[15] FANG, F.; COLLINS-EMERSON, J.M.; CULLUM, A.; HEUER, C.;
WILSON, P. R.; BENSCHOP, J. Shedding and seroprevalence of
pathogenic Leptospira spp. in sheep and cattle at a New Zealand
Abattoir. Zoon. Public. Health. 62(4): 258–268. 2015. https://doi.
org/f68kb5.
[16] FANG, F.; COLLINS-EMERSON, J.M.; HEUER, C.; HILL, F.I.;
TISDALL, D.J.; WILSON, P.R.; BENSCHOP, J. Interlaboratory
and between-specimen comparisons of diagnostic tests for
leptospirosis in sheep and cattle. J. Vet. Diag. Invest. Oc.
Publicat. Ame. Assoc. Vet. Lab. Diagnost. 26(6): 734–747. 2014.
https://doi.org/jqr6.
[17] FERESU, S.B. Isolation of Leptospira interrogans from kidneys
of Zimbabwe beef cattle. The Vet. Rec. 130(20): 446–448. 1992.
https://doi.org/d8r78r.
[18] FERESU, S.B.; BOLIN, C.A.; KORVER, H. A new leptospiral serovar,
ngavi, in the Tarassovi serogroup isolated from Zimbabwe oxen.
Intern. J. Systemat. Bacteriol. 48 (1): 207–213. 1992. https://doi.
org/fw6zwf.
[19] FERESU, S.B.; KORVER, H.; RIQUELME, N.; BARANTON, G.;
BOLIN, C.A. Two new leptospiral serovars in the Hebdomadis
serogroup isolated from Zimbabwe cattle. Intern. J. Systemat.
Bacteriol. 46(3): 694–698. 1996. https://doi.org/b74f7b.
[20] GAMAGE, C.D.; KOIZUMI, N.; PERERA, A.K.; MUTO, M.; NWAFOR-
OKOLI, C.; RANASINGHE, S.; KULARATNE, S.A.; RAJAPAKSE,
R.P.; KANDA, K.; LEE, R.B.; OBAYASHI, Y.; OHNISHI, M.;
TAMASHIRO, H. Carrier status of leptospirosis among cattle
in Sri Lanka: a zoonotic threat to public health.Transbound.
Emerg. Dis. 61(1): 91–96. 2014. https://doi.org/f5npb8.
[21] GONZALEZ, H.E.; CROWELL, W.A.; CAUDLE, A.B.; THOMPSON,
F.N. Morphometric studies of the bovine uterus: microscopic
lesions and retrospective reproductive history. Ame. J. Vet.
Res. 46(12): 2588–2595. 1985.
[22] GRÉGOIRE, N.; HIGGINS, R.; ROBINSON, Y. Isolation of leptospires
from nephritic kidneys of beef cattle at slaughter. Ame. J. Vet.
Res. 48(3): 370–371. 1987.
[23] GUEDES, I.B.; ARAÚJO, S.; DE SOUZA, G.O.; DE SOUZA-SILVA, S.O.;
TANIWAKI, S.A.; CORTEZ, A.; BRANDÃO, P.E.; HEINEMANN, M.B.
Circulating Leptospira species identied in cattle of the Brazilian
Amazon. Acta Trop. 191: 212–216. 2019. https://doi.org/jqr7.
Leptospira species in cattle from slaughterhouses in Mexico / Ochoa-Valencia et al. _______________________________________________
8 of 9
[24] HAAKE, D.A.; LEVETT, P.N. Leptospirosis in humans. Curr. Trop.
Microbiol. Immunol. 387: 65–97. 2015. https://doi.org/bmhq.
[25] HORTON, K.C.; WASFY, M.; SAMAHA, H.; ABDEL-RAHMAN, B.;
SAFWAT, S.; ABDEL-FADEEL, M.; MOHAREB, E.; DUEGER, E.
Serosurvey for zoonotic viral and bacterial pathogens among
slaughtered livestock in Egypt. Vect. Borne Zoon. Dis. 14(9):
633–639. 2014. https://doi.org/f6hq5c.
[26] HORNSBY, R.L.; ALT, D.P.; NALLY, J.E. Isolation and propagation
of leptospires at 37 degrees C directly from the mammalian
host.Sci. Rep. 10:9620. 2020. https://doi.org/jqsb.
[27] KINGSCOTE, B.F. Leptospira interrogans serovar Hardjo Infection
in Cattle in the South Okanagan District of British Columbia.The
Canada. Vet. J. = La Revue Vet. Canada. 26(10): 328–332. 1985.
[28] KORBA, A.A.; LOUNICI, H.; KAINIU, M.; VINCENT, A.T.; MARIET,
J.F.; VEYRIER, F.J.; GOARANT, C.; PICARDEAU, M. Leptospira
ainlahdjerensissp. nov., Leptospira ainazelensissp. nov.,Leptospira
abararensissp. nov. andLeptospira chreensissp. nov., four new
species isolated from water sources in Algeria. Intern. J. System.
Evolut. Microbiol. 71(12): 005148. 2021. https://doi.org/jqsc.
[29] LOUREIRO, A.P.; JAEGER, L.H.; DI AZEVEDO, M.; MIRAGLIA,
F.; MORENO, L.Z.; MORENO, A.M.; PESTANA, C.P.; CARVALHO-
COSTA, F.A.; MEDEIROS, M.A.; LILENBAUM, W. Molecular
epidemiology of Leptospira noguchii reveals important insights
into a One Health context. Transboun. Emerg. Dis. 67(1): 276–283.
2020. https://doi.org/jqsd.
[30] LOUREIRO, A.P.; PESTANA, C.; MEDEIROS, M.A.; LILENBAUM, W.
High frequency of leptospiral vaginal carriers among slaughtered
cows. Anim. Reprod. Sci. 178: 50–54. 2017. https://doi.org/f9v84z.
[31] MILLER, D.A.; WILSON, M.A.; BERAN, G.W. The effect of storage
time on isolation of Leptospira interrogans from bovine kidneys.
Journal of veterinary diagnostic investigation: J. Vet. Diagn.
Invest. Oc. Publ. Ame. Assoc. Vet. Lab. 2(1): 63–65. 1990.
https://doi.org/bptqxr.
[32] MILLER, D.A.; WILSON, M.A.; BERAN, G.W. Survey to estimate
prevalence of Leptospira interrogans infection in mature cattle
in the United States. Ame. J. Vet. Res. 52(11): 1761–1765. 1991.
[33] MILNER, A.R.; WILKS, C.R.; MORGAN, I.R.; ROSEN, N.E. Leptospira
serogroup Hebdomadis infection as an Australian zoonosis.Austr.
Vet. J. 56(2): 70–73. 1980. https://doi.org/dggsr7.
[34] MYERS, D.M.; JELAMBI, F. Isolation and identification of
Leptospira Hardjo from cattle in Argentina. Trop. Geograf. Med.
27(1): 63–70. 1975.
[35] NALLY, J.E.; HORNSBY, R.L.; ALT, D.P.; BAYLES, D.; WILSON-
WELDER, J.H.; PALMQUIST, D.E.; BAUER, N.E. Isolation and
characterization of pathogenic leptospires associated with
cattle. Vet. Microbiol. 218: 25–30. 2018. https://doi.org/gdmqn8.
[36] NGBEDE, E.O.; RAJI, M.A.; KWANASHIE, C.N.; OKOLOCHA,
E.C.; GUGONG, V.T.; HAMBOLU, S.E. Serological prevalence of
leptospirosis in cattle slaughtered in the Zango abattoir in Zaria,
Kaduna State, Nigeria. Vet. Ital. 48(2): 179–184. 2012.
[37] NODA, A.; RODRÍGUEZ, I. DNA isolation by Chelex-100: an
ecient approach to consider in leptospirosis early stages. J.
Coast. Life Med. 2(6): 501–504. 2018. https://doi.org/jqsf.
[38] ORGANIZACIÓN MUNDIAL DE SANIDAD ANIMAL (OIE). Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals (mammals,
birds and bees). 8
th
. Ed. Volume 3. Chapter 3.1.12. Leptospirosis.
2018. On Line: https://bit.ly/3FFkbgs. 03/07/2022.
[39] PICARDEAU, M. Virulence of the zoonotic agent of leptospirosis:
still terra incognita?. Nat. Rev. Microbiol. 15 (5): 297–307. 2017.
https://doi.org/fjb7.
[40] PINNA, M.H.; MARTINS, G.; LOUREIRO, A.P.; LILENBAUM,
W. Detection of bovine carriers of Leptospira by serological,
bacteriological, and molecular tools. Trop. Anim. Health Prod.
50(4): 883–888. 2018. https://doi.org/gdbjg5.
[41] PINTO, P.S.; PESTANA, C.; MEDEIROS, M.A.; LILENBAUM, W.
Plurality of Leptospira strains on slaughtered animals suggest
a broader concept of adaptability of leptospires to cattle. Acta
Trop. 172: 156–159. 2017. https://doi.org/gbjw27.
[42] PRESCOTT, J.F.; MILLER, R.B.; NICHOLSON, V.M. Isolation of
Leptospira hardjo from kidneys of Ontario cattle at slaughter.Can.
J. Vet. Res. = Revue Canada. Res. Vet. 51(2): 229–231. 1987.
[43] RAHELINIRINA, S.; MOSELEY, M.H.; ALLAN, K.J.;
RAMANOHIZAKANDRAINY, E.; RAVAOARINORO, S.; RAJERISON,
M.; RAKOTOHARINOME, V.; TELFER, S. Leptospira in livestock
in Madagascar: uncultured strains, mixed infections and
small mammal-livestock transmission highlight challenges in
controlling and diagnosing leptospirosis in the developing world.
Parasitol. 146(14): 1707–1713. 2019. https://doi.org/jqsg.
[44] RAJEEV, S.; ILHA, M.; WOLDEMESKEL, M.; BERGHAUS, R.D.;
PENCE, M.E. Detection of asymptomatic renal Leptospira infection
in abattoir slaughtered cattle in southeastern Georgia, United
States. SAGE Open Med. 30(2): 1–5. 2014. https://doi.org/jqsh.
[45] SHAFIGHI, T.; ABDOLLAHPOUR, G.; SALEHI, T.Z.; TADJBAKHSH,
H. Serological and bacteriological study of leptospirosis in
slaughtered cattle in north of Iran (Rasht). Afric. J. Microbiol.
Res. 4(20): 2118–2121. 2010.
[46] SHAFIGHI, T.; ZAHRAEI-SALEHI, T.; ABDOLLAHPOUR, G.;
ASADPOUR, L.; AKBAREIN, H.; SALEHZADEH, A. Molecular
detection of Leptospira spp. in the urine of cattle in northern
Iran. Iran.J. Vet. Res. 15(4): 402–405. 2014.
[47] SHIOKAWA, K.; WELCOME, S.; KENIG, M.; LIM, B.; RAJEEV, S.
Epidemiology of Leptospira infection in livestock species in Saint
Kitts. Trop. Anim. Health Prod. 51(6): 1645–1650. 2019. https://
doi.org/jqsj.
[48] SKILBECK, N.W.; FORSYTH, W.M.; DOHNT, M. Bovine leptospirosis:
microbiological and histological ndings in cattle at slaughter.
Austr. Vet. J. 65(3): 73–75. 1988. https://doi.org/d8f8tr.
[49] SWAI, E.S.; SCHOONMAN, L. A survey of zoonotic diseases in
trade cattle slaughtered at Tanga city abattoir: a cause of Public
Health concern. Asian Pacif. J. Trop. Biomed. 2(1): 55–60. 2012.
https://doi.org/dp8xzj.
[50] TAGHADOSI, V.; HOSSEINZADEH, S.; SHEKARFOROUSH, S.S.;
SAMIEI, A. Prevalence of renal lesions in slaughtered cattle
in Shiraz, Iran, and detection of Leptospira in them by nested
PCR-RFLP. Trop. Anim. Health Prod. 48(8): 1691–1696. 2016.
https://doi.org/f9fhtv.
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
9 of 9
[51] THIBEAUX, R.; GIRAULT, D.; BIERQUE, E.; SOUPÉ-GILBERT,
M.E.; RETTINGER, A.; DOUYÈRE, A.; MEYER, M.; IRAOLA, G.;
PICARDEAU, M.; GOARANT, C. Biodiversity of Environmental
Leptospira: Improving Identification and Revisiting the
Diagnosis. Front. Microbiol. 9: 816. 2018. https://doi.org/gdjk3z.
[52] THIERMANN, A.B. Bovine leptospirosis: bacteriologic versus
serologic diagnosis of cows at slaughter.Am. J. Vet. Res. 44(12):
2244–2245. 1983.
[53] TREML, F.; NESNALOVÁ, E. Leptospiróza jatecného skotu
sérologické a bakteriologické vysetrení [Leptospirosis in
slaughter cattle serologic and bacterial study]. Vet. Med. 40(10):
305–309. 1995.
[54] UZAL, F.A.; DOBRENOV, B.; SMYTHE, L.; NORRIS, M.; DOHNT,
M.; SYMONDS, M.; O’BOYLE, D.; SCHOUTEN, F.; KELLY, W.R. A
study of “white spotted kidneys” in cattle. Vet. Microbiol. 86(4):
369–375. 2002. https://doi.org/fs8j2p.
[55] VINCENT, A.T.; SCHIETTEKATTE, O.; GOARANT, C.; NEELA, V. K.;
BERNET, E.; THIBEAUX, R.; ISMAIL, N.; MOHD-KHALID, M.; AMRAN,
F.; MASUZAWA, T.; NAKAO, R.; AMARA-KORBA, A.; BOURHY, P.;
VEYRIER, F.J.; PICARDEAU, M. Revisiting the taxonomy and
evolution of pathogenicity of the genus Leptospira through the
prism of genomics. PLoS Negl Trop Dis. 13(5): e0007270. 2019.
https://doi.org/ghg4zf.
[56] VITAL-BRAZIL, J.M.; BALASSIANO, I.T.; OLIVEIRA, F.S.; COSTA,
A.D.; HILLEN, L.; PEREIRA, M.M. Multiplex PCR-based detection
of Leptospira in environmental water samples obtained from
a slum settlement. Mem. Inst. Oswaldo Cruz. 105(3): 353–355.
2010. https://doi.org/dn8bfp.
[57] GANGULA, R.S., PRABHU, M.M., STANLEY, W. Weil syndrome
causing autoimmune haemolytic anaemia. Natl. Med. J. India.
32(2): 88–89. 2019. https://doi.org/jqsk.
[58] BAL, A.E., GRAVEKAMP, C., HARTSKEERL, R.A., DE MEZA-
BREWSTER, J., KORVER, H.; TERPSTRA, W.J. Detection of
leptospires in urine by PCR for early diagnosis of leptospirosis.J.
Clin. Microbiol. 32(8): 1894–1898. 1994. https://doi.org/jqsm.
[59] NARKKUL, U.; THAIPADUNGPANIT, J.; SRILOHASIN, P.; SINGKHAIMUK,
P.; THONGDEE, M.; CHAIWATTANARUNGRUENGPAISAN, S.;
KRAIROJANANAN, P.; PAN-NGUM, W. Optimization of Culture
Protocols to Isolate Leptospira spp. from Environmental Water,
Field Investigation, and Identication of Factors Associated with the
Presence of Leptospira spp. in the Environment.Trop. Med. Infect.
Dis.5(2): 94. 2020. https://doi.org/jqsn.