- Email: [email protected]
Human and animal leptospirosis in Southern Brazil: A five-year retrospective study
Accepted Manuscript Human and animal leptospirosis in Southern Brazil: A five-year retrospective study Sérgio Jorge, Rodrigo Andrade Schuch, Natasha Rodrigues de Oliveira, Carlos Eduardo Pouey da Cunha, Charles Klazer Gomes, Thais Larré Oliveira, Caroline Rizzi, Aisha Farid Abdel Aziz Yousef Bakry, Violetta Dias Pacce, Ana Lúcia Coelho Recuero, Claudiomar Soares Brod, Odir Antônio Dellagostin PII:
S1477-8939(17)30121-7
DOI:
10.1016/j.tmaid.2017.07.010
Reference:
TMAID 1150
To appear in:
Travel Medicine and Infectious Disease
Received Date: 9 March 2017 Revised Date:
27 June 2017
Accepted Date: 21 July 2017
Please cite this article as: Jorge Sé, Schuch RA, de Oliveira NR, da Cunha CEP, Gomes CK, Oliveira ThaisLarré, Rizzi C, Abdel Aziz Yousef Bakry AF, Pacce VD, Coelho Recuero AnaLú, Soares Brod C, Dellagostin OdirAntô, Human and animal leptospirosis in Southern Brazil: A five-year retrospective study, Travel Medicine and Infectious Disease (2017), doi: 10.1016/j.tmaid.2017.07.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Human and animal leptospirosis in Southern Brazil: A five-year
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retrospective study
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Sérgio Jorgea* Rodrigo Andrade Schucha, Natasha Rodrigues de Oliveiraa,
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Carlos Eduardo Pouey da Cunhaa, Charles Klazer Gomesa, Thais Larré
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Oliveiraa, Caroline Rizzia, Aisha Farid Abdel Aziz Yousef Bakrya, Violetta Dias
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Paccea, Ana Lúcia Coelho Recuerob, Claudiomar Soares Brodb, Odir Antônio
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Dellagostina
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a
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Pelotas, Brazil.
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b
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Universidade Federal de Pelotas, Pelotas, Brazil.
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de
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Departamento
Veterinária
Preventiva,
Faculdade
de
Veterinária,
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Centro de Desenvolvimento Tecnológico. Universidade Federal de Pelotas,
* Corresponding author:
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Dr. S. Jorge, Laboratório de Vacinologia, Centro de Desenvolvimento
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Tecnológico. Universidade Federal de Pelotas, Caixa Postal 354, 96010-900,
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Pelotas, Rio Grande do Sul, Brazil.
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(E-mail: [email protected])
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ACCEPTED MANUSCRIPT Abstract
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Background: Leptospirosis is an emerging zoonosis attributed to multiple
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reservoirs. Climatic conditions influence the transmission of pathogenic
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leptospires, which require warm and humid conditions for survival. The
30
influence of seasonality in human and animal leptospirosis in the subtropical
31
region of Brazil remains poorly understood.
32
Methods: We performed a retrospective study to describe the patterns of
33
human and animal exposure to leptospirosis and their association with
34
precipitation events in Southern Brazil. Rainfall data were obtained from satellite
35
images. Serum samples were tested using the microscopic agglutination test
36
(MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression
37
and Pearson’s correlation were performed to assess whether there is a
38
relationship between these variables.
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Results: We found that precipitation events were not significantly associated
40
with the exposure to leptospirosis in humans or animal species, except for dogs.
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The interspecies analysis revealed an association between canine and human
42
exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup
43
Autumnalis) presented the highest seroreactivity in humans.
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Conclusion: This study provides valuable insights in human and animal
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leptospirosis in Southern Brazil. These insights will be essential to design
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intervention measures directed to reduce disease dissemination.
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Keywords:
risk
factors,
epidemiology,
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zoonotic
disease
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1. Introduction Leptospirosis is one of the most widespread zoonosis in the world, and it
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is particularly prevalent in developing countries, where the climatic and
52
environmental conditions and lack of sanitation are favorable for the survival
53
and transmission of pathogenic Leptospira spp [1,2]. In tropical regions,
54
including South America, leptospirosis is recognized as an important cause of
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human febrile illness [3]. The transmission cycle of leptospirosis begins with an
56
infected reservoir host excreting leptospires in their urine. Infection occurs via
57
direct contact with the urine of an infected host or via indirect contact with
58
contaminated soil or water. Leptospira infection in farm animals can lead to
59
abortion, infertility, and milk and meat production losses [4,5]. As a zoonotic
60
disease, the epidemiology of human and animal leptospirosis are intimately
61
linked, with humans acting as accidental hosts [1]. However, determining the
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sources and transmission routes for human infection can be challenging,
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especially in environments in which multiple potential host animals co-exist [6].
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The genus Leptospira is serologically divided into more than 300
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serovars. This basic taxon is based on the structural heterogeneity of the
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lipopolysaccharide (LPS) and is used to classify these bacteria [7,8]. Serovars
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that are antigenically related have traditionally been grouped into serogroups.
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While serogroups have no taxonomic standing, they have proved useful for
69
epidemiological understanding [2]. Leptospires persist in the host’s renal
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tubules and are excreted in urine, often in increasing amounts [3]. Mammalian
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species can adapt and become associated with certain serovars. They can
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exhibit no symptoms or only mild clinical manifestations (e.g., rats and serovar
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Icterohaemorrhagiae serogroup Icterohaemorrhagiae); however, the basis for
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(serogroup Sejroe), which predominates in livestock in several regions [2,10];
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horses are considered adapted hosts for serovar Bratislava (serogroup
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Australis) [11]; and dogs for serovar Canicola (serogroup Canicola) [2,8].
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Distinct variations in the hosts and the serovars they carry occur throughout the
79
world [12,13]. Knowledge of the prevalent serovars/serogroups could provide
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meaningful insights into the possible animal reservoir and is essential to
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developing an understanding of the epidemiology of the disease in any given
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region [2].
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Climatic conditions strongly influence the transmission of pathogenic
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leptospires, which require warm and humid conditions for survival. These
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favorable conditions are characteristic of tropical countries. Rainfall and floods
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have been linked with a higher number of cases of leptospirosis around the
87
world [1,3,14,15]; however, the influence of precipitation events and the
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seasonality in the human and animal leptospirosis prevalence in the subtropical
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region of Brazil remains poorly understood. Urban outbreaks of human
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leptospirosis occur annually in Brazil and are mainly associated with intense
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periods of heavy rainfall, floods, favorable temperature, lack of sanitation, and
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poor living conditions [16]. Southern Brazil, a region characterized by a
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subtropical climate, does not have a well-defined rainy season [17]. As such,
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precipitation events are well distributed over the year [18]. Unlike other regions
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of Brazil, which have a tropical climate, the climatic conditions in Southern
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Brazil can reduce the impact rainfall has on the seasonal prevalence of
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leptospirosis.
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ACCEPTED MANUSCRIPT In the current study, we investigated human and animal leptospirosis in a
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Brazilian subtropical area between 2003 and 2007. The objectives of this study
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were to (1) describe seasonal patterns of Leptospira exposure reported in the
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study area; (2) determine whether seroreactive samples were associated with
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rainfall in the study area; (3) quantify human and domestic animal exposure;
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and (4) identify prevalent Leptospira serovars/serogroups in humans and
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domestic animal species. Within this paper, we discuss the outcomes of these
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approaches and their implication for leptospirosis control in Southern Brazil.
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2. Material and Methods
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2.1 Study area
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Serological data were collected between January 1st, 2003, and December 31st,
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2007 in the Pelotas area (31o 46’S, 52o 20’W) of Southern Brazil. A wide range
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of domestic animals exist in both the urban and rural communities of this region.
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The data were aggregated and represent the whole municipality.
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2.2 Sample collection and serological diagnosis
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The serologic data were obtained from the Centro de Controle de
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Zoonoses (CCZ) of Universidade Federal de Pelotas (UFPel). Human blood
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samples (n = 997) were obtained from members of the population within the
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study area that were suspected of being infected with leptospirosis. A
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suspected
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epidemiological criteria defined by the World Health Organization and consisted
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of the clinical signs compatible with leptospirosis, or/and a contact history of
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exposure to water or soil contaminated with the urine of infected animals [19].
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The data from the human samples employed in this research did not include
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names or personal information that could be used to identify the individual
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diagnosis
of
leptospirosis
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was
defined
according
to
the
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animals that were believed to have contracted leptospirosis. In cattle (n =
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1.484), clinical suspicion was based on the identification of reproductive
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problems: Repeated failure after artificial insemination or natural breeding,
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spontaneous abortion, birth of weak calves or mummified fetuses. In horses (n
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= 240), clinical signs included uveitis, mid-to-late-term abortion, fever, lethargy,
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and kidney and liver failure. Dogs (n = 1.176) were also sampled with veterinary
130
suspicion from different localities in Pelotas, including peri-urban and urban
131
areas. Serum samples were evaluated using MAT according to the methods
132
previously described [20]. Sixty-six serovars (Table A.1) were selected in this
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study based on the laboratory’s expertise on the prevalence of serovars and
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serogroups in Southern Brazil. Serum samples were tested at dilutions ranging
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from 1:100 to 1:3200 for all species except cattle, for which the samples used
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had an initial dilution of 1:200. Leptospirosis exposition was defined as a MAT
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titer ≥ 100 (Cattle ≥ 200). For samples that reacted to multiple serovars within a
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serogroup, the serovar/serogroup with the highest titer was considered the
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reactive.
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2.3 Estimation of the precipitation events
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Satellite rainfall data from the city of Pelotas were obtained from Centro
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de Previsão de Tempo e Estudos Climáticos/Instituto Nacional de Pesquisas
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Espaciais (CPTEC/INPE)/Ministério de Ciência, Tecnologia e Inovação,
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Brazilian government. Briefly, the hydro-estimator uses infrared data from
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CPTEC/INPE Geostationary Operational Environmental Satellites (GOES) to
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estimate rainfall rates [21]. In this study, we evaluated the precipitation events
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between January 1st, 2003 and December 31st, 2007 in three-month periods.
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Precipitation events were measured in cubic centimeter per square meter
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(cm3/m2).
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2.4 Data analysis Prevalence of leptospiral antibodies was computed using Epi-Info version
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7 (CDC Atlanta, USA). Linear regression analyses and Pearson’s correlation
153
were performed with GraphPad Prism Version 5.01 (SPSS Inc., Illinois, USA).
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Microsoft Office Excel® 2007 (Microsoft Corporation, Redmond, 98052-7329,
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USA) was used to store data and draw graphs. A p < 0.05 was considered to be
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statistically significant.
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3. Results
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3.1 Estimated quarterly rainfall
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The satellite estimation showed precipitation events ranging from 115.08
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cm3/m2 in the first quarter of 2004 to 693.38 cm3/m2 in the second quarter of
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2004. In 2005, rainfall ranged from 290.92 cm3/m2 in the first quarter to 462.98
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cm3/m2 in the fourth quarter; in 2006, rainfall ranged from 499.74 cm3/m2 in the
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second quarter to 339.27 cm3/m2 in the fourth quarter; and, in 2007, rainfall
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ranged from 184.30 cm3/m2 in the first quarter to 462.98 cm3/m2 in the second
165
quarter (Fig. 1).
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3.2 Association between precipitation events and leptospirosis prevalence
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The human and animal leptospirosis exposition between 2003 and 2007
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are shown in Figure 1. Human leptospirosis occurred repeatedly during the
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study period. The canine leptospirosis exposition showed a positive correlation
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with precipitation events (r=0,6367; p=0,0045) (Fig 2.a). The correlation
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between precipitation events and human, cattle, and equine leptospirosis was
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not significant (Fig. 2b, c, and d, respectively).
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3.3 Prevalent Leptospira serovars/serogroups The highest titer reached was used to suggest the infective serogroup.
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Since the reliability of MAT for discriminating among serovars of the same
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serogroup has been reported [22], in the present study, we refer to the
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serological results by serovar and its serogroups. The MAT results revealed that
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L. kirschneri serovar Butembo (serogroup Autumnalis) had the highest
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prevalence rates in human seroreactive samples (19.41%) for pathogenic
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serovars, followed by L. interrogans serovar Sentot (serogroup Djasiman)
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(11.17%). Seroconversion from saprophytic strains was detected in 69.41% of
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the human samples. The prevalent serovars in canine samples were Canicola
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(serogroup Canicola) strains Tande (27.96%), Kito (22.60%), and Hond Utrecht
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IV (18.34%), followed by Copenhageni (serogroup Icterohaemorrhagiae)
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(12.75%), Ballum (serogroup Ballum) (9.84%), and Butembo (serogroup
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Autumnalis) (8.50%). In cattle, the most prevalent serovars were serovar Hardjo
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strain Hardjoprajitno (serogroup Sejroe) (31.07%), followed by Wolffi (serogroup
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Sejroe) (25.23%), Hardjo strain Lely (serogroup Sejroe) (17.52%), Sejroe
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(serogroup Sejroe) (9.57%), Tarassovi (serogroup Tarassovi) (9.34%), and
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Shermani (serogroup Shermani) (7.24%). Bratislava, serogroup Australis, was
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the main horse-infecting Leptospira serovar (32.72%), followed by serovars
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Copenhageni (serogroup Icterohaemorrhagiae) (24.54%), Canicola strain
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Tande (serogroup Canicola) (21.82%), and Bataviae (serogroup Bataviae)
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(20.90%) (Fig. 3).
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3.4 Association between human and domestic animal leptospirosis exposition
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The canine leptospirosis exposition showed a positive correlation with
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human exposition (r=0,5764; p=0,0078) (Fig.4a). The association between the
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ACCEPTED MANUSCRIPT human and cattle and equine leptospirosis prevalence was not consistent (Fig.
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4b and c respectively). The association between equine and bovine
200
leptospirosis prevalence exhibited a positive correlation (r=0,6885; p=0,0008)
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(Fig. 5a). The association between cattle vs. canine and equine vs. canine
202
correlations were not significant (Fig. 5b and c respectively).
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4. Discussion
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Several studies have hypothesized that high levels of rainfall may be one
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of the main risk factors of leptospirosis in developing countries. In these
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countries, an increased number of leptospirosis cases after precipitation events
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have been used as evidence of this correlation [15,23-25]. These conditions
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lead to augmented exposure of humans to surface water, the increased survival
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of the bacteria in the environment [26], and larger rodent populations, which
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further contribute to environmental contamination [27].
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In the current study, the precipitation events showed a wide variation
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during the studied years, with rainfall peaks present in almost all seasons. This
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zone refers to the areas that are just outside the formal tropical zones. In Brazil,
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the subtropical zone experiences different levels of rainfall than the other zones.
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Here, we showed that human leptospirosis exposition occurred repeatedly over
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the study period. Overall, rainfall was not related to the number of positive
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human samples detected using MAT during the study period. In developing
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countries, populations are exposed to leptospirosis risk factors other than
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rainfall such as lack of basic sanitation and occupational exposure (e.g., rice
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farming and other agricultural activities) [3]. Interactions between humans and
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animals can lead to interspecies transmission of distinct serovars. The
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prevalence of different leptospiral serovars within a human population depends
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on the reservoir animals present and the serovars they carry [3]. Lepstospira interrogans serovars Copenhageni and Icterohaemorrhagiae
225
(serogroup Icterohaemorrhagiae) are frequently considered to be the major
226
agents of human infection worldwide [1, 28]. However, in this study, we found L.
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kirschneri serovar Butembo (serogroup Autumnalis) had the highest prevalence
228
rates in humans for pathogenic serovars, followed by L. interrogans serovar
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Sentot (serogroup Djasiman). These findings were quite unexpected since
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serovar Butembo is not frequently detected in human infections, especially in
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the study area [29]. Leptospira kirschneri serovar Butembo was originally
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isolated from the blood of a human patient with leptospirosis in the Congo [30]
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and no specific animal reservoir has been established for this serovar. To
234
improve our understanding of the prevalence of Leptospira ssp. in distinct
235
habitats, we included saprophytic strains in the MAT. The majority of human
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seroreactive samples (69.41%) reacted for saprophytic strains; however, with
237
no clinical importance. This is expected due to the ability of saprophytic strains
238
to survive in environmental niches.
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Anti-Butembo antibodies have been detected in cattle in Brazil [31];
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however, the bovine sera evaluated in this study presented antibodies titers
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only against the serovars Hardjo strain Hardjoprajtino (serogroup Sejroe),
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Wolffi, (serogroup Sejroe), Hardjo strain Lely (serogroup Sejroe), Serjoe
243
(serogroup Sejroe), Tarassovi (serogroup Tarassovi) and Shermani (serogroup
244
Shermani). These findings indicate that bovine specie are not involved in the
245
epidemiology of human leptospirosis in the study area, since the serovars
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prevalent in human infection are not directly related to the serovars detected in
247
bovine sera. Rainfall was related to the number of canine leptospirosis seroreactive
249
samples. Moreover, we detected an association between canine and human
250
leptospirosis. The most prevalent serovar in dogs was Canicola, serogroup
251
Canicola (strains Tande, Kito, and Hond Utrecht IV, respectively). Serovars
252
Canicola and Icterohaemorrhagiae have often been detected in convalescence
253
canine populations worldwide [32]. Moreover, we detected seroconvertion for
254
Butembo serovar (serogroup Autumnalis) in 8.5% of the canine seroreactive
255
samples, suggesting that dogs can act as sentinels for human leptospirosis.
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Dogs are more frequently exposed to known risk factors of disease and, thus,
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may act as sentinels of environmental contamination [33]. The specific serovars
258
prevalent in human infection detected in this study indicated that the role of
259
dogs in human infection may be limited; however, the correlation between
260
canine and human seroreactive samples was significant and specific antibodies
261
against Butembo serovar were detected in both species.
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Contacts with livestock have previously been found to be strongly
263
correlated with human occupational leptospirosis in epidemiological studies in
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subtropical regions of South America [34]. However, the bovine sera evaluated
265
in the current study presented antibodies anti-serovars absent in humans,
266
indicating that herds were not involved in the epidemiology of human
267
leptospirosis in the study area during that period. Cattle also did not act as
268
hosts for pathogenic serovars for other domestic animals because the serovars
269
prevalent in cattle serum samples were not detected in MAT results of any other
270
hosts. However, we detected a correlation between bovine and equine
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seroreactivity in the study period. There is a distinct probability that this
272
association occurred due to the common environmental conditions. We found that precipitation events were not significantly associated with
274
the prevalence of leptospirosis in bovines during the study period. Leptospirosis
275
in cattle is frequently due to infection with the serogroup Sejroe, Serovar Hardjo
276
(strains Hardjobovis or Hardjoprajitno) [35]. However, other serovars may be
277
associated with leptospiral infection in cattle, including Icterohaemorrhagiae,
278
Bratislava, Pomona, Canicola, and Grippotyphosa. There is seroepidemiological
279
evidence that serovar Hardjo is prevalent in cattle worldwide, which also seems
280
to be true for countries within Latin America [36]. As expected, our study shows
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that the most prevalent serovars in cattle were serovar Hardjo strain
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Hardjoprajitno, followed by Wolffi and Hardjo strain Lely (serogroup Sejroe).
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Serological surveys performed worldwide indicate that horses are
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susceptible to a wide variety of Leptospira spp. serovars infection [37]. Strains
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of L. interrogans belonging to two closely related serovars, Bratislava and
286
Muenchen, are known to cause widespread infection in horse populations in
287
many parts of the world [38]. This study corroborates our data, which shows
288
that Bratislava was the main horse-infecting Leptospira serovar (serogroup
289
Australis), followed by serovars Copenhageni (serogroup Icterohaemorrhagiae),
290
Canicola strain Tande (serogroup Canicola), and Bataviae (serogroup
291
Bataviae). There was no correlation between equine leptospirosis seroreactive
292
samples and rainfall. There was also no correlation between equine and canine
293
leptospirosis seroreactive samples, even though serovars Canicola strain
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Tande
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Icterohaemorrhagiae) were present in the serum samples extracted from both
296
animals. The MAT is the serological test recommended for the diagnosis of
298
leptospirosis for both human beings [19] and animals [39]. Although widely
299
utilized and recommended, MAT has important and well-known limitations. With
300
respect to the ability to identify the infectious serovars, there is a general
301
consensus that MAT can reliably identify the presumptive serogroup; however,
302
due to the high degree of cross-reaction among different serovars in each
303
serogroup, it cannot be considered to be purely serovar-specific [2,40,41]. Here,
304
the serovar/serogroup with the highest titer was considered to be reactive. MAT
305
detects antibodies from both M and G classes, and cannot differentiate between
306
current, recent, or past infections [42]. The current interpretive criteria indicative
307
of active infection for MAT requires a fourfold rise in titers between the acute
308
and convalescent sera. Although it is well recognized that seroconversion or
309
increasing antibody titers in paired serum specimens provides strong evidence
310
of true infection, paired serology is not practical in the clinical or veterinary
311
setting [43]. In this work, leptospirosis exposition was defined as a MAT titer ≥
312
100 (Cattle ≥ 200) for any pathogenic serovar.
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studies and the development and implementation of preventive strategies.
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Although our MAT live antigen panel was quite representative of locally relevant
316
serovars, it is noteworthy that new serovars may be introduced into human
317
and/or animal populations. Therefore, we suggest that the MAT battery should
318
be regularly updated and Leptospira isolation studies continually carried out.
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This kind of study should also be carried out in a continual basis to overcome
320
the issues described above. In conclusion, this study provides valuable insights into the circulating
322
Leptospira serovars/serogroups in human and animal species in Southern
323
Brazil. These insights will be essential in any studies that seek to design
324
intervention measures that can effectively reduce the risk of disease
325
transmission, including the identification and addition of prevalent local serovars
326
in vaccine formulations. Moreover, understanding the impact of seasonality and
327
precipitation events on human and animal leptospirosis prevalence is an
328
important step in leptospirosis control. Future epidemiological studies should
329
address
330
relations, and climatic event components for a more detailed understanding of
331
environmental contamination in the study area, which will enable the design of
332
optimal prevention and control strategies against leptospirosis. We believe that
333
the identification of infecting serovars in the man and domestic animals species
334
(i.e., Butembo in this case) is of crucial importance to establishing the source of
335
serovars transmission.
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Funding Sources
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host-adapted
serovars,
human-environment
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The authors are grateful to Centro de Previsão de Tempo e Estudos
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Climáticos/Instituto Nacional de Pesquisas Espaciais (CPTEC/INPE)/Ministério
339
da Ciência, Tecnologia e Inovação, Brazilian Government, for the precipitation
340
data used in this research. This work was supported by Coordenacão de
341
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho
342
Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
343
Conflict of Interest
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The authors certify that they have no potential conflicts of interest.
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References
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Leptospirosis: a zoonotic disease of global importance. Lancet 2003;3:757-71.
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Figure Captions
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Figure 1. Total human and animal leptospirosis seroreactive samples by
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season between January 1st, 2003, and December 31st, 2007, in three-month
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periods.
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serologically seroreactive samples of leptospirosis in humans, bovines, canines,
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equines, and rainfall (continuous line) in Pelotas, Southern Brazil.
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Figure 2. Association between rainfall and leptospirosis seroreactivity in human
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and animal species. (a) canine seroreactiive samples (n=1.176); (b) human
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(n=997); (c) bovine (n=1.484) and (d) equine (n=240). A *p < 0.05 was
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considered to be statistically significant.
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Figure 3. The distribution of prevalent Leptospira serovars and serogroups (in
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parentheses)
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serovars/serogroups were detected on MAT. str.: strain.
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Figure 4. Association between human and animal leptospirosis seroreactive
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samples. (a) human vs. canine samples; (b) human vs. bovine and (c) human
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vs. equine. A *p < 0.05 was considered to be statistically significant.
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Figure 5. Leptospirosis association between animal species. (a) equine vs.
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bovine seroreactive samples; (b) bovine vs. canine and (c) equine vs. canine. A
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*p < 0.05 was considered to be statistically significant.
distribution
of
precipitation
(intermittent
lines)
and
distinct
animal
species.
Antibodies
against
specific-
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S1477-8939(17)30121-7
DOI:
10.1016/j.tmaid.2017.07.010
Reference:
TMAID 1150
To appear in:
Travel Medicine and Infectious Disease
Received Date: 9 March 2017 Revised Date:
27 June 2017
Accepted Date: 21 July 2017
Please cite this article as: Jorge Sé, Schuch RA, de Oliveira NR, da Cunha CEP, Gomes CK, Oliveira ThaisLarré, Rizzi C, Abdel Aziz Yousef Bakry AF, Pacce VD, Coelho Recuero AnaLú, Soares Brod C, Dellagostin OdirAntô, Human and animal leptospirosis in Southern Brazil: A five-year retrospective study, Travel Medicine and Infectious Disease (2017), doi: 10.1016/j.tmaid.2017.07.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Human and animal leptospirosis in Southern Brazil: A five-year
2
retrospective study
3 4
Sérgio Jorgea* Rodrigo Andrade Schucha, Natasha Rodrigues de Oliveiraa,
6
Carlos Eduardo Pouey da Cunhaa, Charles Klazer Gomesa, Thais Larré
7
Oliveiraa, Caroline Rizzia, Aisha Farid Abdel Aziz Yousef Bakrya, Violetta Dias
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Paccea, Ana Lúcia Coelho Recuerob, Claudiomar Soares Brodb, Odir Antônio
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Dellagostina
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a
14
Pelotas, Brazil.
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b
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Universidade Federal de Pelotas, Pelotas, Brazil.
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de
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Departamento
Veterinária
Preventiva,
Faculdade
de
Veterinária,
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Centro de Desenvolvimento Tecnológico. Universidade Federal de Pelotas,
* Corresponding author:
20
Dr. S. Jorge, Laboratório de Vacinologia, Centro de Desenvolvimento
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Tecnológico. Universidade Federal de Pelotas, Caixa Postal 354, 96010-900,
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Pelotas, Rio Grande do Sul, Brazil.
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(E-mail: [email protected])
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ACCEPTED MANUSCRIPT Abstract
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Background: Leptospirosis is an emerging zoonosis attributed to multiple
28
reservoirs. Climatic conditions influence the transmission of pathogenic
29
leptospires, which require warm and humid conditions for survival. The
30
influence of seasonality in human and animal leptospirosis in the subtropical
31
region of Brazil remains poorly understood.
32
Methods: We performed a retrospective study to describe the patterns of
33
human and animal exposure to leptospirosis and their association with
34
precipitation events in Southern Brazil. Rainfall data were obtained from satellite
35
images. Serum samples were tested using the microscopic agglutination test
36
(MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression
37
and Pearson’s correlation were performed to assess whether there is a
38
relationship between these variables.
39
Results: We found that precipitation events were not significantly associated
40
with the exposure to leptospirosis in humans or animal species, except for dogs.
41
The interspecies analysis revealed an association between canine and human
42
exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup
43
Autumnalis) presented the highest seroreactivity in humans.
44
Conclusion: This study provides valuable insights in human and animal
45
leptospirosis in Southern Brazil. These insights will be essential to design
46
intervention measures directed to reduce disease dissemination.
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Keywords:
risk
factors,
epidemiology,
2
zoonotic
disease
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1. Introduction Leptospirosis is one of the most widespread zoonosis in the world, and it
51
is particularly prevalent in developing countries, where the climatic and
52
environmental conditions and lack of sanitation are favorable for the survival
53
and transmission of pathogenic Leptospira spp [1,2]. In tropical regions,
54
including South America, leptospirosis is recognized as an important cause of
55
human febrile illness [3]. The transmission cycle of leptospirosis begins with an
56
infected reservoir host excreting leptospires in their urine. Infection occurs via
57
direct contact with the urine of an infected host or via indirect contact with
58
contaminated soil or water. Leptospira infection in farm animals can lead to
59
abortion, infertility, and milk and meat production losses [4,5]. As a zoonotic
60
disease, the epidemiology of human and animal leptospirosis are intimately
61
linked, with humans acting as accidental hosts [1]. However, determining the
62
sources and transmission routes for human infection can be challenging,
63
especially in environments in which multiple potential host animals co-exist [6].
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The genus Leptospira is serologically divided into more than 300
65
serovars. This basic taxon is based on the structural heterogeneity of the
66
lipopolysaccharide (LPS) and is used to classify these bacteria [7,8]. Serovars
67
that are antigenically related have traditionally been grouped into serogroups.
68
While serogroups have no taxonomic standing, they have proved useful for
69
epidemiological understanding [2]. Leptospires persist in the host’s renal
70
tubules and are excreted in urine, often in increasing amounts [3]. Mammalian
71
species can adapt and become associated with certain serovars. They can
72
exhibit no symptoms or only mild clinical manifestations (e.g., rats and serovar
73
Icterohaemorrhagiae serogroup Icterohaemorrhagiae); however, the basis for
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3
ACCEPTED MANUSCRIPT this association is still unknown [9]. Cattle often harbor serovar Hardjo
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(serogroup Sejroe), which predominates in livestock in several regions [2,10];
76
horses are considered adapted hosts for serovar Bratislava (serogroup
77
Australis) [11]; and dogs for serovar Canicola (serogroup Canicola) [2,8].
78
Distinct variations in the hosts and the serovars they carry occur throughout the
79
world [12,13]. Knowledge of the prevalent serovars/serogroups could provide
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meaningful insights into the possible animal reservoir and is essential to
81
developing an understanding of the epidemiology of the disease in any given
82
region [2].
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Climatic conditions strongly influence the transmission of pathogenic
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leptospires, which require warm and humid conditions for survival. These
85
favorable conditions are characteristic of tropical countries. Rainfall and floods
86
have been linked with a higher number of cases of leptospirosis around the
87
world [1,3,14,15]; however, the influence of precipitation events and the
88
seasonality in the human and animal leptospirosis prevalence in the subtropical
89
region of Brazil remains poorly understood. Urban outbreaks of human
90
leptospirosis occur annually in Brazil and are mainly associated with intense
91
periods of heavy rainfall, floods, favorable temperature, lack of sanitation, and
92
poor living conditions [16]. Southern Brazil, a region characterized by a
93
subtropical climate, does not have a well-defined rainy season [17]. As such,
94
precipitation events are well distributed over the year [18]. Unlike other regions
95
of Brazil, which have a tropical climate, the climatic conditions in Southern
96
Brazil can reduce the impact rainfall has on the seasonal prevalence of
97
leptospirosis.
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ACCEPTED MANUSCRIPT In the current study, we investigated human and animal leptospirosis in a
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Brazilian subtropical area between 2003 and 2007. The objectives of this study
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were to (1) describe seasonal patterns of Leptospira exposure reported in the
101
study area; (2) determine whether seroreactive samples were associated with
102
rainfall in the study area; (3) quantify human and domestic animal exposure;
103
and (4) identify prevalent Leptospira serovars/serogroups in humans and
104
domestic animal species. Within this paper, we discuss the outcomes of these
105
approaches and their implication for leptospirosis control in Southern Brazil.
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2. Material and Methods
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2.1 Study area
108
Serological data were collected between January 1st, 2003, and December 31st,
109
2007 in the Pelotas area (31o 46’S, 52o 20’W) of Southern Brazil. A wide range
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of domestic animals exist in both the urban and rural communities of this region.
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The data were aggregated and represent the whole municipality.
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2.2 Sample collection and serological diagnosis
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The serologic data were obtained from the Centro de Controle de
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Zoonoses (CCZ) of Universidade Federal de Pelotas (UFPel). Human blood
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samples (n = 997) were obtained from members of the population within the
116
study area that were suspected of being infected with leptospirosis. A
117
suspected
118
epidemiological criteria defined by the World Health Organization and consisted
119
of the clinical signs compatible with leptospirosis, or/and a contact history of
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exposure to water or soil contaminated with the urine of infected animals [19].
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The data from the human samples employed in this research did not include
122
names or personal information that could be used to identify the individual
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diagnosis
of
leptospirosis
5
was
defined
according
to
the
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animals that were believed to have contracted leptospirosis. In cattle (n =
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1.484), clinical suspicion was based on the identification of reproductive
126
problems: Repeated failure after artificial insemination or natural breeding,
127
spontaneous abortion, birth of weak calves or mummified fetuses. In horses (n
128
= 240), clinical signs included uveitis, mid-to-late-term abortion, fever, lethargy,
129
and kidney and liver failure. Dogs (n = 1.176) were also sampled with veterinary
130
suspicion from different localities in Pelotas, including peri-urban and urban
131
areas. Serum samples were evaluated using MAT according to the methods
132
previously described [20]. Sixty-six serovars (Table A.1) were selected in this
133
study based on the laboratory’s expertise on the prevalence of serovars and
134
serogroups in Southern Brazil. Serum samples were tested at dilutions ranging
135
from 1:100 to 1:3200 for all species except cattle, for which the samples used
136
had an initial dilution of 1:200. Leptospirosis exposition was defined as a MAT
137
titer ≥ 100 (Cattle ≥ 200). For samples that reacted to multiple serovars within a
138
serogroup, the serovar/serogroup with the highest titer was considered the
139
reactive.
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2.3 Estimation of the precipitation events
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Satellite rainfall data from the city of Pelotas were obtained from Centro
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de Previsão de Tempo e Estudos Climáticos/Instituto Nacional de Pesquisas
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Espaciais (CPTEC/INPE)/Ministério de Ciência, Tecnologia e Inovação,
144
Brazilian government. Briefly, the hydro-estimator uses infrared data from
145
CPTEC/INPE Geostationary Operational Environmental Satellites (GOES) to
146
estimate rainfall rates [21]. In this study, we evaluated the precipitation events
147
between January 1st, 2003 and December 31st, 2007 in three-month periods.
6
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Precipitation events were measured in cubic centimeter per square meter
149
(cm3/m2).
150
2.4 Data analysis Prevalence of leptospiral antibodies was computed using Epi-Info version
152
7 (CDC Atlanta, USA). Linear regression analyses and Pearson’s correlation
153
were performed with GraphPad Prism Version 5.01 (SPSS Inc., Illinois, USA).
154
Microsoft Office Excel® 2007 (Microsoft Corporation, Redmond, 98052-7329,
155
USA) was used to store data and draw graphs. A p < 0.05 was considered to be
156
statistically significant.
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3. Results
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3.1 Estimated quarterly rainfall
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The satellite estimation showed precipitation events ranging from 115.08
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cm3/m2 in the first quarter of 2004 to 693.38 cm3/m2 in the second quarter of
161
2004. In 2005, rainfall ranged from 290.92 cm3/m2 in the first quarter to 462.98
162
cm3/m2 in the fourth quarter; in 2006, rainfall ranged from 499.74 cm3/m2 in the
163
second quarter to 339.27 cm3/m2 in the fourth quarter; and, in 2007, rainfall
164
ranged from 184.30 cm3/m2 in the first quarter to 462.98 cm3/m2 in the second
165
quarter (Fig. 1).
166
3.2 Association between precipitation events and leptospirosis prevalence
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The human and animal leptospirosis exposition between 2003 and 2007
168
are shown in Figure 1. Human leptospirosis occurred repeatedly during the
169
study period. The canine leptospirosis exposition showed a positive correlation
170
with precipitation events (r=0,6367; p=0,0045) (Fig 2.a). The correlation
171
between precipitation events and human, cattle, and equine leptospirosis was
172
not significant (Fig. 2b, c, and d, respectively).
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3.3 Prevalent Leptospira serovars/serogroups The highest titer reached was used to suggest the infective serogroup.
175
Since the reliability of MAT for discriminating among serovars of the same
176
serogroup has been reported [22], in the present study, we refer to the
177
serological results by serovar and its serogroups. The MAT results revealed that
178
L. kirschneri serovar Butembo (serogroup Autumnalis) had the highest
179
prevalence rates in human seroreactive samples (19.41%) for pathogenic
180
serovars, followed by L. interrogans serovar Sentot (serogroup Djasiman)
181
(11.17%). Seroconversion from saprophytic strains was detected in 69.41% of
182
the human samples. The prevalent serovars in canine samples were Canicola
183
(serogroup Canicola) strains Tande (27.96%), Kito (22.60%), and Hond Utrecht
184
IV (18.34%), followed by Copenhageni (serogroup Icterohaemorrhagiae)
185
(12.75%), Ballum (serogroup Ballum) (9.84%), and Butembo (serogroup
186
Autumnalis) (8.50%). In cattle, the most prevalent serovars were serovar Hardjo
187
strain Hardjoprajitno (serogroup Sejroe) (31.07%), followed by Wolffi (serogroup
188
Sejroe) (25.23%), Hardjo strain Lely (serogroup Sejroe) (17.52%), Sejroe
189
(serogroup Sejroe) (9.57%), Tarassovi (serogroup Tarassovi) (9.34%), and
190
Shermani (serogroup Shermani) (7.24%). Bratislava, serogroup Australis, was
191
the main horse-infecting Leptospira serovar (32.72%), followed by serovars
192
Copenhageni (serogroup Icterohaemorrhagiae) (24.54%), Canicola strain
193
Tande (serogroup Canicola) (21.82%), and Bataviae (serogroup Bataviae)
194
(20.90%) (Fig. 3).
195
3.4 Association between human and domestic animal leptospirosis exposition
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196
The canine leptospirosis exposition showed a positive correlation with
197
human exposition (r=0,5764; p=0,0078) (Fig.4a). The association between the
8
ACCEPTED MANUSCRIPT human and cattle and equine leptospirosis prevalence was not consistent (Fig.
199
4b and c respectively). The association between equine and bovine
200
leptospirosis prevalence exhibited a positive correlation (r=0,6885; p=0,0008)
201
(Fig. 5a). The association between cattle vs. canine and equine vs. canine
202
correlations were not significant (Fig. 5b and c respectively).
203
4. Discussion
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Several studies have hypothesized that high levels of rainfall may be one
205
of the main risk factors of leptospirosis in developing countries. In these
206
countries, an increased number of leptospirosis cases after precipitation events
207
have been used as evidence of this correlation [15,23-25]. These conditions
208
lead to augmented exposure of humans to surface water, the increased survival
209
of the bacteria in the environment [26], and larger rodent populations, which
210
further contribute to environmental contamination [27].
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204
In the current study, the precipitation events showed a wide variation
212
during the studied years, with rainfall peaks present in almost all seasons. This
213
zone refers to the areas that are just outside the formal tropical zones. In Brazil,
214
the subtropical zone experiences different levels of rainfall than the other zones.
215
Here, we showed that human leptospirosis exposition occurred repeatedly over
216
the study period. Overall, rainfall was not related to the number of positive
217
human samples detected using MAT during the study period. In developing
218
countries, populations are exposed to leptospirosis risk factors other than
219
rainfall such as lack of basic sanitation and occupational exposure (e.g., rice
220
farming and other agricultural activities) [3]. Interactions between humans and
221
animals can lead to interspecies transmission of distinct serovars. The
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prevalence of different leptospiral serovars within a human population depends
223
on the reservoir animals present and the serovars they carry [3]. Lepstospira interrogans serovars Copenhageni and Icterohaemorrhagiae
225
(serogroup Icterohaemorrhagiae) are frequently considered to be the major
226
agents of human infection worldwide [1, 28]. However, in this study, we found L.
227
kirschneri serovar Butembo (serogroup Autumnalis) had the highest prevalence
228
rates in humans for pathogenic serovars, followed by L. interrogans serovar
229
Sentot (serogroup Djasiman). These findings were quite unexpected since
230
serovar Butembo is not frequently detected in human infections, especially in
231
the study area [29]. Leptospira kirschneri serovar Butembo was originally
232
isolated from the blood of a human patient with leptospirosis in the Congo [30]
233
and no specific animal reservoir has been established for this serovar. To
234
improve our understanding of the prevalence of Leptospira ssp. in distinct
235
habitats, we included saprophytic strains in the MAT. The majority of human
236
seroreactive samples (69.41%) reacted for saprophytic strains; however, with
237
no clinical importance. This is expected due to the ability of saprophytic strains
238
to survive in environmental niches.
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224
Anti-Butembo antibodies have been detected in cattle in Brazil [31];
240
however, the bovine sera evaluated in this study presented antibodies titers
241
only against the serovars Hardjo strain Hardjoprajtino (serogroup Sejroe),
242
Wolffi, (serogroup Sejroe), Hardjo strain Lely (serogroup Sejroe), Serjoe
243
(serogroup Sejroe), Tarassovi (serogroup Tarassovi) and Shermani (serogroup
244
Shermani). These findings indicate that bovine specie are not involved in the
245
epidemiology of human leptospirosis in the study area, since the serovars
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prevalent in human infection are not directly related to the serovars detected in
247
bovine sera. Rainfall was related to the number of canine leptospirosis seroreactive
249
samples. Moreover, we detected an association between canine and human
250
leptospirosis. The most prevalent serovar in dogs was Canicola, serogroup
251
Canicola (strains Tande, Kito, and Hond Utrecht IV, respectively). Serovars
252
Canicola and Icterohaemorrhagiae have often been detected in convalescence
253
canine populations worldwide [32]. Moreover, we detected seroconvertion for
254
Butembo serovar (serogroup Autumnalis) in 8.5% of the canine seroreactive
255
samples, suggesting that dogs can act as sentinels for human leptospirosis.
256
Dogs are more frequently exposed to known risk factors of disease and, thus,
257
may act as sentinels of environmental contamination [33]. The specific serovars
258
prevalent in human infection detected in this study indicated that the role of
259
dogs in human infection may be limited; however, the correlation between
260
canine and human seroreactive samples was significant and specific antibodies
261
against Butembo serovar were detected in both species.
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248
Contacts with livestock have previously been found to be strongly
263
correlated with human occupational leptospirosis in epidemiological studies in
264
subtropical regions of South America [34]. However, the bovine sera evaluated
265
in the current study presented antibodies anti-serovars absent in humans,
266
indicating that herds were not involved in the epidemiology of human
267
leptospirosis in the study area during that period. Cattle also did not act as
268
hosts for pathogenic serovars for other domestic animals because the serovars
269
prevalent in cattle serum samples were not detected in MAT results of any other
270
hosts. However, we detected a correlation between bovine and equine
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seroreactivity in the study period. There is a distinct probability that this
272
association occurred due to the common environmental conditions. We found that precipitation events were not significantly associated with
274
the prevalence of leptospirosis in bovines during the study period. Leptospirosis
275
in cattle is frequently due to infection with the serogroup Sejroe, Serovar Hardjo
276
(strains Hardjobovis or Hardjoprajitno) [35]. However, other serovars may be
277
associated with leptospiral infection in cattle, including Icterohaemorrhagiae,
278
Bratislava, Pomona, Canicola, and Grippotyphosa. There is seroepidemiological
279
evidence that serovar Hardjo is prevalent in cattle worldwide, which also seems
280
to be true for countries within Latin America [36]. As expected, our study shows
281
that the most prevalent serovars in cattle were serovar Hardjo strain
282
Hardjoprajitno, followed by Wolffi and Hardjo strain Lely (serogroup Sejroe).
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Serological surveys performed worldwide indicate that horses are
284
susceptible to a wide variety of Leptospira spp. serovars infection [37]. Strains
285
of L. interrogans belonging to two closely related serovars, Bratislava and
286
Muenchen, are known to cause widespread infection in horse populations in
287
many parts of the world [38]. This study corroborates our data, which shows
288
that Bratislava was the main horse-infecting Leptospira serovar (serogroup
289
Australis), followed by serovars Copenhageni (serogroup Icterohaemorrhagiae),
290
Canicola strain Tande (serogroup Canicola), and Bataviae (serogroup
291
Bataviae). There was no correlation between equine leptospirosis seroreactive
292
samples and rainfall. There was also no correlation between equine and canine
293
leptospirosis seroreactive samples, even though serovars Canicola strain
294
Tande
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(serogroup
Canicola)
and
12
Copenhageni
(serogroup
ACCEPTED MANUSCRIPT 295
Icterohaemorrhagiae) were present in the serum samples extracted from both
296
animals. The MAT is the serological test recommended for the diagnosis of
298
leptospirosis for both human beings [19] and animals [39]. Although widely
299
utilized and recommended, MAT has important and well-known limitations. With
300
respect to the ability to identify the infectious serovars, there is a general
301
consensus that MAT can reliably identify the presumptive serogroup; however,
302
due to the high degree of cross-reaction among different serovars in each
303
serogroup, it cannot be considered to be purely serovar-specific [2,40,41]. Here,
304
the serovar/serogroup with the highest titer was considered to be reactive. MAT
305
detects antibodies from both M and G classes, and cannot differentiate between
306
current, recent, or past infections [42]. The current interpretive criteria indicative
307
of active infection for MAT requires a fourfold rise in titers between the acute
308
and convalescent sera. Although it is well recognized that seroconversion or
309
increasing antibody titers in paired serum specimens provides strong evidence
310
of true infection, paired serology is not practical in the clinical or veterinary
311
setting [43]. In this work, leptospirosis exposition was defined as a MAT titer ≥
312
100 (Cattle ≥ 200) for any pathogenic serovar.
SC
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EP
Identifying infecting serovars/serogroups is essential for epidemiological
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313
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314
studies and the development and implementation of preventive strategies.
315
Although our MAT live antigen panel was quite representative of locally relevant
316
serovars, it is noteworthy that new serovars may be introduced into human
317
and/or animal populations. Therefore, we suggest that the MAT battery should
318
be regularly updated and Leptospira isolation studies continually carried out.
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This kind of study should also be carried out in a continual basis to overcome
320
the issues described above. In conclusion, this study provides valuable insights into the circulating
322
Leptospira serovars/serogroups in human and animal species in Southern
323
Brazil. These insights will be essential in any studies that seek to design
324
intervention measures that can effectively reduce the risk of disease
325
transmission, including the identification and addition of prevalent local serovars
326
in vaccine formulations. Moreover, understanding the impact of seasonality and
327
precipitation events on human and animal leptospirosis prevalence is an
328
important step in leptospirosis control. Future epidemiological studies should
329
address
330
relations, and climatic event components for a more detailed understanding of
331
environmental contamination in the study area, which will enable the design of
332
optimal prevention and control strategies against leptospirosis. We believe that
333
the identification of infecting serovars in the man and domestic animals species
334
(i.e., Butembo in this case) is of crucial importance to establishing the source of
335
serovars transmission.
336
Funding Sources
SC
M AN U
about
host-adapted
serovars,
human-environment
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information
The authors are grateful to Centro de Previsão de Tempo e Estudos
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337
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321
338
Climáticos/Instituto Nacional de Pesquisas Espaciais (CPTEC/INPE)/Ministério
339
da Ciência, Tecnologia e Inovação, Brazilian Government, for the precipitation
340
data used in this research. This work was supported by Coordenacão de
341
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho
342
Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
343
Conflict of Interest
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ACCEPTED MANUSCRIPT 344
The authors certify that they have no potential conflicts of interest.
345
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doi:10.4269/ajtmh.13-0041.
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Figure Captions
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Figure 1. Total human and animal leptospirosis seroreactive samples by
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season between January 1st, 2003, and December 31st, 2007, in three-month
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periods.
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serologically seroreactive samples of leptospirosis in humans, bovines, canines,
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equines, and rainfall (continuous line) in Pelotas, Southern Brazil.
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Figure 2. Association between rainfall and leptospirosis seroreactivity in human
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and animal species. (a) canine seroreactiive samples (n=1.176); (b) human
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(n=997); (c) bovine (n=1.484) and (d) equine (n=240). A *p < 0.05 was
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considered to be statistically significant.
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Figure 3. The distribution of prevalent Leptospira serovars and serogroups (in
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parentheses)
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serovars/serogroups were detected on MAT. str.: strain.
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Figure 4. Association between human and animal leptospirosis seroreactive
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samples. (a) human vs. canine samples; (b) human vs. bovine and (c) human
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vs. equine. A *p < 0.05 was considered to be statistically significant.
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Figure 5. Leptospirosis association between animal species. (a) equine vs.
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bovine seroreactive samples; (b) bovine vs. canine and (c) equine vs. canine. A
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*p < 0.05 was considered to be statistically significant.
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