Survey of Leptospira Spp in Pampas Deer
Total Page：16
File Type：pdf, Size：1020Kb
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(6): 763-768, September 2011 763 Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction Anahí Souto Vieira1/+, Grácia Maria Soares Rosinha2, Carina Elisei de Oliveira2, Silvio Arruda Vasconcellos3, Paulo Andre Lima-Borges4, Walfrido Moraes Tomás5, Guilherme Miranda Mourão5, Ana Cristina Reis Lacerda6, Cleber Oliveira Soares2, Flábio Ribeiro de Araújo2, Ubiratan Piovezan5, Carlos Andre Zucco7, Aiesca Oliveira Pellegrin5 1Programa de Pós-Graduação em Ciência Animal 7Programa de Pós-Graduação em Ecologia e Conservação, Universidade Federal do Mato Grosso do Sul, 79070-900 Campo Grande, MS, Brasil 2Embrapa Gado de Corte, Campo Grande, MS, Brasil 3Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil 4Independent Consultant on Veterinary, Brasília, DF, Brasil 5Embrapa Pantanal, Corumbá, MS, Brasil 6Programa de Pós-graduação em Biologia Animal, Universidade de Brasília, Brasília, DF, Brasil This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed. Key words: pampas deer - Leptospira spp - MAT - PCR - Pantanal Leptospirosis is a worldwide disease (Scarcelli et al. et al. 2002). The region of Nhecolândia has the high- 2003) responsible for important reproductive losses in cat- est population density of pampas deer in the Pantanal, tle and severe zoonosis in man and domestic animals. Re- estimated to be approximately 60 thousand individuals ports of leptospirosis in wildlife that share habitats with (Mourão et al. 2000). cattle are increasing (Mathias et al. 1999, Bengis et al. In the Pantanal, ecological conditions are highly 2002, Girio et al. 2003, Scarcelli et al. 2003, Deem et al. suitable for Leptospira spp, mainly because of the high 2004). Because of the expansion of cattle ranching into ar- temperatures and pluviometric index (1,200 mm/year) eas traditionally occupied by wildlife, such as the Pantanal (Garcia & Castro 1986, Lins et al. 1986). wetlands in the state of Mato Grosso do Sul (MS), Brazil, Leptospirosis is diagnosed by the detection of specif- interest in pathogens that affect native and livestock popu- ic antibodies using microscopic agglutination test (MAT) lations simultaneously has become more important. and the isolation of the Leptospira pathogen in culture The cervids belong to the order Artiodactyla, family media. However, both techniques are laborious and time Cervidae (Emmons & Feer 1990, Eisemberg & Redford consuming. MAT requires the use of live antigens and 1999). In the Pantanal wetlands there are four species of the culture technique demands freshly collected samples cervids, Blastocerus dichotomus (marsh deer), Ozotoceros (Faine et al. 1999, Cerqueira & Picardeu 2009). bezoarticus (pampas deer), Mazama americana (red brock- Polymerase chain reaction (PCR) has been used for et deer) and Mazama gouazoubira (brown brocket deer), the diagnosis of leptospirosis in humans and domestic which have distinct habitats (Vaughan 1986, Rodrigues et animals, demonstrating high accuracy and sensitivity al. 2002, Tomás et al. 2004, Tiepolo & Tomás 2006). when compared with MAT (Van Eys et al. 1989, Gerrist- Populations of pampas deer are distributed all over sen et al. 1991, Radstone & Woodward 1996). the Pantanal, with higher densities in the fields and ebbs The objectives of this work were to estimate the (Mourão et al. 2000, Tomás et al. 2001, 2004). There are prevalence of pampas deer infected with Leptospira spp more pampas deer in the Pantanal than in the Cerrado in the Pantanal wetlands of MS, to identify the infective biome; these two populations are sympatric (Rodrigues serovars and to evaluate a PCR method to detect these pathogens, assessing the analytic sensitivity. The study was conducted in four contiguous ranches located in the central area of the Pantanal, known as Nhecolândia (18º59’115’’S 56º37’63’’N), over an area of approximately 40,000 ha. The captures were performed + Corresponding author: [email protected] from May-November 2006. Nhecolândia has the high- Received 13 September 2010 est density population of pampas deer in the Pantanal; Accepted 13 June 2011 its population is estimated to be approximately 60,000 online | memorias.ioc.fiocruz.br 764 Leptospira spp in pampas deer • Anahí Souto Vieira et al. animals (Mourão et al. 2000). The surveyed area includ- volume of antigen suspension was added to each well ed private beef cattle ranches with land areas between and mixed by agitation. The suspension in each well was 4,000-20,000 ha. In these ranches, cow-calf operations examined microscopically for agglutination. The end were based on extensive breeding on natural pastures. point was the final dilution of serum at which 50% of the Pampas deer were captured using anaesthetic darts leptospires were agglutinated and the cut-off considered shot with CO2 guns with adjustable pressure (Distinject, to be a positive reaction was a titre greater or equal to model 35) or a blowpipe of 2 m length x 11 mm diam- 100 (Faine et al. 1999). eter (Zootech). Chemical anaesthesia with tiletamine/ Purification of genomic DNA was performed ac- zolazepam or zolazepam/xylazine was administered to cording to Araujo et al. (2009) and a positive control 42 animals (Piovezan et al. 2006) and a combination of of Leptospira interrogans, serovar hardjoprajitno was zolazepam, tiletamine, xylazine and atropine was ad- obtained from culture in semi-solid media (Fletcher, ministered to 60 animals (Lima-Borges et al. 2006). Difco). The primers used in this study were described The environmental licenses for captures were provid- by Mérien et al. (1992). Reactions were prepared in a ed by Brazilian Institute of Environment and Renewable total volume of 20 µL and the parameters used in the Natural Resources (032/2005-0214.0022008/05-00 and amplification were standardised in this work: 95ºC for 005/2007-02014.000382/2007-22). Blood samples were 4 min, followed by 30 cycles at 95ºC for 30 sec, anneal- taken from the jugular vein into tubes with or without ing at 58ºC for 30 sec and extension at 72ºC for 60 sec anticoagulant ethylenediamine tetraacetic acid. After and a final extension at 72ºC for 10 min. PCR products centrifugation, serum and erythrocyte layers were used were submitted to electrophoresis on a 1% agarose gel for MAT and PCR, respectively. stained with SYBR Gold (Invitrogen) and visualised Antibodies against Leptospira spp were detected under ultraviolet light. by MAT according to Cole et al. (1973) using a routine For the assessment of analytical sensitivity, the ge- panel of 19 live antigens of Leptospira spp and nine field nomic DNA of L. interrogans serovar was diluted se- isolates from domestic and wild animals. The panel rially (10-fold) to 1:10-7 starting from 280 ng/µL and represented the following serovars and strains of Lep- each dilution was analysed by PCR using same protocol tospira spp: Australis (Ballico), Bratislava (Jez Bratis- described above. The specificity of the PCR technique lava), Autumnalis (Akiyami A), Castellonis (Castellon), was evaluated by testing all the antigens used in MAT. Batavie (van Tienen), Butembo (Butembo), Canicola PCR products were cloned into the pGEM-T Easy plas- (Hond Utrecht IV), Whitcombi (Whitcombi), Cynop- mid (Promega) using Escherichia coli TOP10 F’ chemi- teri (3522-C), Grippotyphosa (Moskva V), Hebdomadis cally competent host cells. Purification of recombinant (Hebdomadis), Copenhageni (M-20), Icterohaemorrga- plasmids was performed with the Wizard Miniprep kit giae (RGA), Javanica (Veldrat Batavia 46), Panamá (CZ (Promega). The resulting clones were sequenced in both 214), Pomona (Pomona), Pyrogenes (Salinem), Hardjo directions using the BigDye Terminator Cycle Sequenc- (Hardjoprajitno), Wolffi (3705), Hardjo (Hardjobovis), ing Ready Reaction kit (Applied Biosystems). Sequences Shermani (1342 K), Tarassovi (Perpelitsin), Sentot (Sen- were submitted to a homology search by the BLASTn tot) and Patoc (Patoc 1). The panel of field isolates used algorithm (blast.ncbi.nlm.nih.gov/Blast.cgi). as MAT antigens were represented by serovars Brasi- The phylogenetic analyses were conducted with DNA liensis (isolated in Didelphis marsupialis), Pomona (Sus sequences of Leptospira using the MEGA software pro- scrofa), Guaricura (Bubalus bubalis), Copenhageni gram, ver sion 4.0 (Tamura et al. 2007) and a phylogenetic (Rattus norvegicus), Canicola (Canis familiaris), Cani- tree was generated using the neighbour-joining method cola (Bos taurus), Grippothyphosa (Hidrochaeris hidro- (Saitou & Nei 1987) and a bootstrap resampling technique chaeris) and a non-characterised isolate of Cerdocyum of 1,000 replications to statistically support the reliabilities