https://doi.org/10.52973/rcfcv-e33201
Received: 17/09/2022 Accepted: 24/10/2022 Published: 29/12/2022
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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
identicar, 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. _______________________________________________
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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 classied 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 typied by CAAT
Zimbabwe [18]
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33201, 1 - 9
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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 Immunouorescence (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 identication
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. _______________________________________________
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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 Scientic, 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 Scientic, 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 Scientic,
4375305, Massachusetts,USA) that amplied 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 amplication 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 puried 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% condence intervals (CIs);
the general frequency (of anti-Leptospira antibodies) and specicity
(by serovar) were determined with the statistical program STATA V.14.
RESULTS AND DISCUSSION
Serological assays identied 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, Condence 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 amplied 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).