Molecular and serological characterization of Leptospira interrogans ...

Trop Anim Health Prod (2013) 45:117–121 DOI 10.1007/s11250-012-0181-6

ORIGINAL RESEARCH

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil Fabiana Miraglia & Zenaide M. de Morais & Odir A. Dellagostin & Fabiana K. Seixas & Julio C. Freitas & Francielle G. S. Zacarias & Ádina C. Delbem & Thaís S. P. Ferreira & Gisele O. Souza & Rudy A. Hartskeerl & Silvio A. Vasconcellos & Andrea M. Moreno

Accepted: 9 May 2012 / Published online: 20 May 2012 # Springer Science+Business Media B.V. 2012

Abstract The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of F. Miraglia : Z. M. de Morais : T. S. P. Ferreira : G. O. Souza : S. A. Vasconcellos : A. M. Moreno (*) Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária, CEP 05508 270 São Paulo/ SP, Brazil e-mail: [email protected] O. A. Dellagostin : F. K. Seixas Centro de Desenvolvimento Tecnológico - Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil J. C. Freitas : F. G. S. Zacarias : Á. C. Delbem Laboratório de Leptospirose Animal, Universidade Estadual de Londrina, Londrina, PR, Brazil R. A. Hartskeerl WHO/FAO/OIE and National Leptospirosis Reference Centre, KIT Biomedical Research, Royal Tropical Institute, Amsterdam, Netherlands

serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries. Keywords Leptospirosis . Canicola . Dog . Swine . Bovine . Genotyping

Introduction Leptospirosis, the most widespread and prevalent zoonosis in the world, is an emerging major public health issue; however, the disease is frequently unrecognized, and consequently, severely neglected. The disease occurs in both rural and urban areas of tropical, subtropical, and temperate regions, and its outbreaks are usually associated with occupational exposure, tourism, or sports events. Developing countries carry the major burden of the disease; it significantly impacts the most vulnerable populations served by inadequate sanitation, with half a million cases reported yearly, and a mortality rate ranging from 5 to 10 % (Evangelista and Coburn 2010; Hartskeerl et al. 2011). Since they host activities involving high risk of infection, such as rafting, jungle tracking, and caving, tropical countries are growing in popularity as holiday destinations (Hartskeerl et al. 2011). Although also getting particular attention in Europe in the context of the climatic change, specific surveillance and control measures have been recommended for this zoonosis by the World Organization for Animal Health (OIE; Dufour et al. 2008).

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The disease is usually transmitted by the urine of an infected animal, and is contagious as long as it remains moist. Although rats, mice, and moles are important primary hosts, a wide range of other mammals, including dogs, deer, rabbits, hedgehogs, cows, sheep, raccoons, possums, skunks, as well as certain marine mammals, may carry and transmit the disease as secondary hosts. The types of habitats most likely to be contaminated by infective bacteria are muddy riverbanks, ditches, gullies, and muddy livestock rearing areas, where there is a regular passage of either wild or farm mammals. Clinical signs and symptoms are highly variable in humans and domestic animals, which may include fever, renal, and hepatic insufficiency, pulmonary manifestations, and reproductive failure. There is a wide range of animal hosts that maintain Leptospira organisms in their renal tubules and genital tracts, contaminating the environment via urine shedding (Adler and Mocteazuma 2010; Evangelista and Coburn 2010; Galloway and Levett 2010; Hartskeerl et al. 2011). Serovar identification of Leptospira clinical isolates is very important for the epidemiology of leptospirosis. It contributes for the detection of carrier mammals and facilitates targeted prevention methods for both endemic and epidemic leptospirosis. Additionally, it recognizes new species and serovars. Since 1980, it has been verified that molecular fingerprint methods may be in agreement with serotyping, so various techniques have been proposed for serovar identification (Galloway and Levett 2010; Turk et al. 2009). In this study, 14 Leptospira isolates were evaluated by means of single enzyme amplified length polymorphism (SEAFLP), variable number of tandem repeat analysis (VNTR), pulsed field gel electrophoresis (PFGE) and serotyping, and the results were then compared and discussed (Fig. 1). Fig. 1 Dendrogram showing the relationship among AFLP patterns from Leptospira interrogans, serogroup Canicola, serovar Canicola isolates from different animal species (bovine, canine, and swine). All strains were from Paraná State except M12-90 and M5/91 that were from Sao Paulo State

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Materials and methods Bacterial isolates and culture conditions Fourteen Leptospira isolates from the states of São Paulo and Paraná, collected between 1990 and 2001, obtained from bovine (n01), dogs (n011), and swine (n02) in kidney culture, were grown in 5 mL EMJH semisolid medium at 30°C (Ellinghausen and Culloug 1965), and subsequently kept in EMJH liquid medium until serotyping and molecular analysis. The Leptospira reference strain adopted in this study came from the Collection at the Bacterial Zoonoses Laboratory, Department of Veterinary Preventive and Animal Health of School of Veterinary Medicine and Animal Science, São Paulo University, Brazil. Polyclonal and monoclonal antibodies Isolates were first typed at the WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). In order to determinate their presumptive serogroups, Leptospira isolates were subjected to the microscopic agglutination test (MAT) using a panel of 43 rabbit anti-Leptospira sera, which were representative of all pathogenic and saprophytic serogroups. Rabbit antisera (polyclonal) were prepared as described by Miraglia et al. (2008). MAT was carried out in a microtiter plate with serial twofold dilutions of the rabbit antisera, starting with a serum dilution of 1:10. Equal volumes of viable leptospiral strains and antiserum dilutions were mixed together. After incubation at 30°C for 2 h, the mixtures were investigated for agglutination by means of dark field microscopy. Titers represent the highest serum dilution, showing 50 % agglutination of leptospiral cells in suspension.


Isolates were further typed at serovar level by performing MAT with panels of monoclonal antibodies that characteristically agglutinate serovars belonging to a specific serogroup, as described by Korver et al. (1988) and Terpstra et al. (1985). Pulsed field gel electrophoresis PFGE was performed as described by Galloway and Levett (2010) at the Center for Disease Control and Prevention, USA. Variable number tandem-repeat analysis VNTR analysis was performed by means of eight discriminatory markers (VNTR4, VNTR7, VNTR9, VNTR10, VNTR11, VNTR19, VNTR23 and VNTR31), as described by Majed et al. (2005). Single enzyme amplified fragment length polymorphism Purified DNA was recovered according to the protocol for DNA extraction of Boom et al. (1990), and stored at −20°C. SE-AFLP protocol was performed according to McLauchlin et al. (2000). A 24 U of HindIII (Invitrogen, Inc.) were added to 10 μL of the extracted DNA, and then supplemented with ultrapure water to a final volume of 20 μL, followed by an overnight incubation at 37°C. An aliquot of digested DNA (5 μL) was added to 0.2 μg of each ADH1 (5′ ACGGTATGCGACAG) and ADH2 (5′ AGCTCTGTCG CATACCGTGAG; LGC Biotecnologia, São Paulo, Brazil) adapter, 1 U of T4 DNA ligase (Invitrogen, Inc), ultrapure water to a final volume of 20 μL, and this reaction was incubated at room temperature for 3 h. The ligated DNA was heated to 80°C for 10 min, and 5 μL were used for each PCR reaction. PCR reactions were performed in a final volume of 50 μL, containing 5 μL of ligated DNA, 2.5 mM MgCl2, 30 pmol of primer (HI-G; LGC Biotecnologia, São Paulo, Brazil), and 1 U of Taq DNA polymerase (LGC Biotecnologia, São Paulo, Brazil) in a 1× PCR buffer. The mixture was submitted to an initial denaturing step at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C, 1 min at 60°C, and 2.5 min at 72°C. The selective primer sequences were: HI-G-5′ GGTATGCGACAGAGCTTG 3′. Electrophoresis was conducted on 1.5 % agarose gel at 22 V for 24 hs. The amplified products were visualized with Blue Green® (LGC Biotecnologia, São Paulo, Brazil) staining, and then compared to a 100 bp DNA ladder (LGC Biotecnologia, São Paulo, Brazil). The levels of relatedness of the isolates were determined by comprehensive pairwise comparison of restriction fragment sizes, using Pearson correlation coefficient. The mean values obtained were employed for UPGMA, using BioNumerics 6.6 software (Applied Maths, Belgium) to generate a dendrogram.

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Results Fourteen Lesptospira isolates obtained from dogs (11), bovine (one), and swine (two) were submitted to four different characterization techniques. First, the isolates were evaluated by means of MAT with polyclonal and monoclonal antibodies, and were all identified as Leptospira interrogans, serogroup Canicola, serovar Canicola. This identification was fully supported by all three molecular methods subsequently applied. PFGE separated the 14 isolates in six profiles with a similarity level higher than 85 %, which was consistent with serovar Canicola. Additionally, VNTR also confirmed all 14 isolates as serovar Canicola (Majed et al. 2005). SE-AFLP applied to the 14 isolates, showed patterns consisting of 10–15 bands. These patterns were then separated into four groups, differing by one to four bands. The dendrogram deduced showed that 11 isolates (LO1–LO13) were identical, while isolate LO14 showed 95 % of similarity. Isolates from swine could not be distinguished from canine isolates using SE-AFLP, but bovine isolate (LO14) and canine isolates (M12/90 and M5/91) were discriminated from canine isolates from Paraná state, dating to 2000 and 2001.

Discussion To date, serovar identification is not routinely performed in laboratories, due to the difficulties involved in performing serological identification, which is considered its reference method (Galloway and Levett 2010). Multiple molecular techniques have been described to characterize Leptospira; nonetheless, most of them can only identify down to the species level, while others are often limited to a few species and are not appropriate to all pathogenic species (Galloway and Levett 2010). DNA-based typing by means of PFGE has been widely used as a molecular fingerprinting technique for the epidemiological analysis of bacterial infections. PFGE analysis of genomic DNA is recognized as the “gold standard” molecular tool for epidemiological studies for various bacterial species, and it has been successfully used to differentiate among closely related serovars of the Leptospiraceae family (Romero et al. 2009). This method is applicable to the whole Leptospira genus, and profiles seem to frequently coincide with serovar status. PFGE analysis of 14 isolates from animals revealed an identical pattern for all 11 isolates from dogs, consistent with serovar Canicola. Galloway and Levett (2010) presented fingerprint patterns revealed by PFGE, for 175 clinical Leptospira isolated from animals and humans, in eight different countries. In Brazil, 16 of the 41 strains examined with PFGE were isolated from dogs (11), humans (two), swine (two), and cows (one), all associated with serovar Canicola. The comparison of these profiles with those previously generated in two human isolates from

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Brazil by Galloway et al. (2010) revealed that human and dog isolates were identical, thus reinforcing the potential transmission between species indicated by Romero et al. (2009) and Galloway and Levett (2010). AFLP is a widespread technique, which is not only recommended for Leptospira spp. genotyping (Vijayachari et al. 2008; Nalam et al. 2010), but also seems to be well applicable to clonality studies in outbreaks. AFLP using a single enzyme has been successfully employed in the epidemiological typing of many Gram-positive and Gram-negative bacteria, but has not yet been described to evaluate Leptospira strains (Coutinho et al. 2011). By using the SE-AFLP technique, it was possible to characterize isolates collected from different sources, different times, and different states in Brazil. In this study, we demonstrated the high similarity of serovar Canicola isolates from porcine, bovine, and canine origin from the state of Paraná, collected in 2001 (LO1–LO8 and LO11–LO14). While isolates from São Paulo State collected in 1990 and 1991 (M12/90 and M5/91), presented less than 85 % of similarity, in Paraná State all strains isolated showed identical profiles using the VNTR method. Molecular typing results were in perfect accordance with the results obtained by serological typing, identifying the isolate as serovar Canicola. A reasonably good conformity between serovar determination and molecular characterization of isolates from different animal species and humans has been previously demonstrated by other authors (Turk et al. 2009). These methods are a potentially useful tool for the identification of leptospires, being fairly helpful for the rapid identification of strains, a required process for the development of strategies targeted to preventing the disease. Historically, leptospirosis was recognized as a canine disease before being detected in any other animal. Serovars Canicola and Icterohaemorrhagiae have been described as the commonest serovars infecting dogs, which are generally recognized as the global maintenance host for serovar Canicola, and in urban regions, stray dogs probably represent the main infection reservoir of Canicola infections (Hartskeerl et al. 2011). Cattle may be infected by serovars Hardjo, Pomona, and Grippotyphosa. Infections by Icterohaemorrhagiae, Bratislava, Hebdomadis, Autumnalis, Australis, Sejroe, Canicola, and Bataviae have also been described. Pigs are commonly infected by serovars Pomona, Tarassovi, Grippotyphosa, Bratislava, Sejroe, Icterohaemorrhagiae, and Canicola (Vijayachari et al. 2008). Isolation of Leptospira strains, serovar Canicola, from bovine and swine in Brazil is not in conformity with the serological results previously obtained, in which the occurrence of serovar Hardjo is most common in cattle, and serovar Pomona and Bratislava are most frequent in swine. Ganoza et al. (2006) conclude that it is well conceivable that the excretion of highly pathogenic serovars—such as Canicola—by livestook animals, represents an increasing risk for severe leptospirosis incidence in


large populations, especially in rural areas. Adult nonpregnant infected pigs and cows are usually symptom-free and become chronic carriers (Vijayachari et al. 2008). Urban leptospirosis in humans is often associated with brown rats and dogs, mainly affecting populations living in slum areas. In contrast, leptospirosis acquired through contact with infected cattle and pigs is considered to be an occupational risk, affecting farmers, and meat workers (Hartskeerl et al. 2011). In conclusion, bovine, dogs, and pigs may be an infection source of Canicola serovar for serious, potentially fatal disease, which bears enormous public health implications that require more research, surveillance, and attention of veterinary and public health authorities. As each leptospires, serovar is usually associated with a particular host, the identification of serovars is essential for epidemiological studies and the development of appropriate prevention strategies. In contrast, dogs are a lot more exposed to the infection than humans since they have free access to contaminated environments. The use of genomic and proteomic knowledge can aid in epidemiological studies, thus contributing to public health practices, in order to decrease illnesses and outbreaks associated with leptospirosis, which is an infectious disease with a considerable impact on veterinary and public health. Acknowledgments We would like to express our gratitude for the help of Dr Paul Levett in performing the PFGE reactions. This study was supported by FAPESP-Fundação de Amparo a Pesquisa do Estado de São Paulo—research projects 2009/52925-2 and 2011/18290-0.

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