• View in gallery

    Schematic map showing the location of capture and respective rodent species distribution.

  • View in gallery

    Phylogenetic tree based on partial hap1 gene sequences from the isolates and the reference strains available in GenBank. Genospecies Leptospira biflexa was used as the out-group. Accession numbers of Leptospira spp. isolates: LC002658–LC002661.

  • 1.

    WHO, 2011. Report of the Second Meeting of the Leptospirosis Burden Epidemiology Reference Group. Geneva, Switzerland: WHO.

  • 2.

    Collares-Pereira M, Mathias ML, Santos-Reis M, Ramalhinho MG, Duarte-Rodrigues P, 2000. Rodents and Leptospira transmission risk in Terceira island (Azores). Eur J Epidemiol 16: 11511157.

    • Search Google Scholar
    • Export Citation
  • 3.

    Faine S, Adler B, Bolin C, Perolat P, 1999. Leptospira and Leptospirosis. Melbourne, Australia: MediSci, 272.

  • 4.

    Collares-Pereira M, Korver H, Terpstra WJ, Santos-Reis M, Ramalhinho MG, Mathias ML, Oom MM, Fons R, Libois R, Petrucci-Fonseca F, 1997. First epidemiological data on pathogenic leptospires isolated on the Azorean islands. Eur J Epidemiol 13: 435441.

    • Search Google Scholar
    • Export Citation
  • 5.

    De Vries SG, Visser BJ, Nagel IM, Goris MG, Hartskeerl RA, Grobusch MP, 2014. Leptospirosis in sub-Saharan Africa: a systematic review. Int J Infect Dis 28c: 4764.

    • Search Google Scholar
    • Export Citation
  • 6.

    Herbreteau V, Jittapalapong S, Rerkamnuaychoke W, Chaval Y, Cosson J-F, Morand S, 2011. Protocols for Field and Laboratory Rodent Studies. Bangkok, Thailand: Kasetsart University Press, 2014.

    • Search Google Scholar
    • Export Citation
  • 7.

    Johnson RC, Rogers P, 1964. Differentiation of pathogenic and saprophytic Leptospires with 8-azaguanine. J Bacteriol 88: 16181623.

  • 8.

    Johnson RC, Harris VG, 1967. Differentiation of pathogenic and saprophytic leptospires. I. Growth at low temperatures. J Bacteriol 94: 2731.

    • Search Google Scholar
    • Export Citation
  • 9.

    Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, André-Fontaine G, 2005. Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett 243: 437445.

    • Search Google Scholar
    • Export Citation
  • 10.

    Barocchi MA, Ko AI, Ferrer SR, Faria MT, Reis MG, Riley LW, 2001. Identification of new repetitive element in Leptospira interrogans serovar Copenhageni and its application to PCR-based differentiation of Leptospira serogroups. J Clin Microbiol 39: 191195.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 12.

    Holt J, Davis S, Leirs H, 2006. A model of leptospirosis infection in an African rodent to determine risk to humans: seasonal fluctuations and the impact of rodent control. Acta Trop 99: 218225.

    • Search Google Scholar
    • Export Citation
  • 13.

    Jobbins SE, Sanderson CE, Alexander KA, 2013. Leptospira interrogans at the human-wildlife interface in northern Botswana: a newly identified public health threat. Zoonoses Public Health 61: 113123.

    • Search Google Scholar
    • Export Citation
  • 14.

    Halliday JE, Knobel DL, Allan KJ, de C Bronsvoort BM, Handel I, Agwanda B, Cutler SJ, Olack B, Ahmed A, Hartskeerl RA, Njenga MK, Cleaveland S, Breiman RF, 2013. Urban leptospirosis in Africa: a cross-sectional survey of Leptospira infection in rodents in the Kibera urban settlement, Nairobi, Kenya. Am J Trop Med Hyg 89: 10951102.

    • Search Google Scholar
    • Export Citation
  • 15.

    Houemenou G, Ahmed A, Libois R, Hartskeerl RA, 2013. Leptospira spp. prevalence in small mammal populations in Cotonou, Benin. ISRN Epidemiol 2013: 18.

    • Search Google Scholar
    • Export Citation
  • 16.

    Haake DA, Suchard MA, Kelley MM, Dundoo M, Alt DP, Zuerner RL, 2004. Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol 186: 28182828.

    • Search Google Scholar
    • Export Citation

 

 

 

 

First Isolates of Leptospira spp., from Rodents Captured in Angola

View More View Less
  • Grupo de Leptospirose e Borreliose de Lyme, Unidade de Microbiologia Médica, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisbon, Portugal

Rodents play an important role in the transmission of pathogenic Leptospira spp. However, in Angola, neither the natural reservoirs of these spirochetes nor leptospirosis diagnosis has been considered. Regarding this gap, we captured rodents in Luanda and Huambo provinces to identify circulating Leptospira spp. Rodent kidney tissue was cultured and DNA amplified and sequenced. Culture isolates were evaluated for pathogenic status and typing with rabbit antisera; polymerase chain reaction (PCR) and sequencing were also performed. A total of 37 rodents were captured: Rattus rattus (15, 40.5%), Rattus norvegicus (9, 24.3%), and Mus musculus (13, 35.2%). Leptospiral DNA was amplified in eight (21.6%) kidney samples. From the cultures, we obtained four (10.8%) Leptospira isolates belonging to the Icterohaemorrhagiae and Ballum serogroups of Leptospira interrogans and Leptospira borgpetersenii genospecies, respectively. This study provides information about circulating leptospires spread by rats and mice in Angola.

Leptospirosis is a widespread zoonosis caused by spirochetes of the genus Leptospira, with a recognized high incidence in tropical countries.1 Rodents are the natural reservoirs of pathogenic leptospires and the main host responsible for its transmission to humans.24 Outbreaks of leptospirosis have been associated with high-level infestation of rodents, floods, and occupational activities. However, in many countries, morbidity and mortality due to leptospirosis are underestimated.14

In Africa, few reports about human leptospirosis are available.5 In Angola, a serological survey was conducted by Baptista in 1991 in Huila province among cattle; this study revealed the presence of antibodies against Leptospira interrogans sensu lato in 35% of 1,518 animals analyzed (unpublished data). We recently performed a serological and epidemiological survey, in 650 febrile patients in Luanda and Huambo provinces. We found evidence of infection by Leptospira serovars belonging to Icterohaemorrhagiae, Pomona, and Ballum serogroups (E. Fortes-Gabriel, 2013, personal communication). Responses to questionnaires revealed that more than 50% of individuals surveyed had seen rodents near their houses. We now report a characterization of rodent Leptospira species in the Luanda and Huambo provinces.

We trapped rodents in February and April 2013 during the rainy season in Luanda (8°40′S and 13°40′E) and Huambo (12°45′S and 15°45′E) provinces, respectively (Figure 1). About 50 handmade live traps (Tomahawk type) were distributed randomly in 20 different urban and rural neighborhoods. Morphometric parameters of rodents were recorded to identify the species. Rodent kidneys were aseptically collected following biosafety and animal welfare guidelines.6

Figure 1.
Figure 1.

Schematic map showing the location of capture and respective rodent species distribution.

Citation: The American Society of Tropical Medicine and Hygiene 94, 5; 10.4269/ajtmh.15-0027

For culture, one kidney from each animal was homogenized in 10 mL Ellinghausen, McCullough, Johnson, and Harris (EMJH, Difco, BD Diagnostics, Sparks, MD) liquid medium, and 0.5 mL of the suspension was inoculated in EMJH semisolid medium following incubation at 29°C. The other kidney was frozen at −20°C and stored for molecular assays.

Cultures and kidneys were transported to the Reference Laboratory for Leptospirosis at the Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (UNL). Cultivation was continued for 2 months at 29°C, and culture tubes were assessed every week by dark field microscopy.

Culture isolates were differentiated according to previously described methods,7,8 using two reference Leptospira serovars [Copenhageni (strain M20) and Patoc (strain PatocI)] as pathogenic and saprophytic controls, respectively. Cultures (108 cells/mL) of each isolate were analyzed for: 1) growth at 13°C and 29°C; 2) growth in EMJH with and without 8-azaguanine 225 μg/mL; and 3) morphological modification to spherical forms in the presence of 1M NaCl. A microscopic agglutination test with six rabbit antisera for two of the major Leptospira serogroups (Icterohaemorrhagiae and Ballum), was also performed.

Genomic DNA from Leptospira isolates and kidney samples was extracted with a kit (Citogene®, Citomed, Lisbon, Portugal) in accordance with the manufacturer's instructions. Polymerase chain reaction (PCR) was performed targeting the hap1 gene (also designated lipL32), which encodes a hemolysis-associated protein,9 and with the iRep1 primer (5′-AGC GGG TAT GAGTCC GC-3′),10 to compare DNA fingerprints patterns, with the 19 serogroups represented by 23 pathogenic serovars (L. interrogans sensu lato) and one saprophytic (Leptospira biflexa). Clustering and alignment of DNA sequences from the isolates was performed with ClustalW2/EMB/EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/). A phylogenetic tree was constructed with Molecular Evolutionary Genetics Analysis (MEGA) version 6,11 using the neighbor-joining method, Jukes-Cantal model, and represents 1000 replicates with confidence greater than 50%.

Our study included 37 rodents collected at eight sites from urban and rural areas near households and garbage dumps (Figure 1) in Luanda and Huambo provinces. The rodents were predominantly adult females and identified as Rattus rattus (15, 40.5%), Rattus norvegicus (9, 24.3%), and Mus musculus (13, 35.2%).

Leptospires were successfully isolated from four (10.8%) rodents. These isolates, LDA02, LDA05, LDA10, and HBO34, were from R. norvegicus and M. musculus species, captured in Luanda and Huambo provinces, respectively. Pathogenic characteristics were demonstrated by phenotypic tests. All four isolates exhibited an agglutination titer of 1:3200 with rabbit antisera (Table 1).

Table 1

Summary of isolates characterization by phenotypic, serological, and molecular tests

Leptospira isolates (rodent species)Pathogenic statusRabbit antisera serogroup (reciprocal titer)iRep-PCR serogroupPCR-hap1 genospecies (GenBank accession no.)
LDA02 (Rattus norvegicus)YesIcterohaemorrhagiae (1:3200)IcterohaemorrhagiaeLeptospira interrogans (LC006258)
LDA05 (R. norvegicus)YesIcterohaemorrhagiae (1:3200)IcterohaemorrhagiaeL. interrogans (LC006259)
LDA10 (R. norvegicus)YesIcterohaemorrhagiae (1:3200)IcterohaemorrhagiaeL. interrogans (LC006260)
HBO34 (Mus musculus)YesBallum (1:3200)BallumL. borgpetersenii (LC006261)

PCR = polymerase chain reaction.

DNA from the four isolates was amplified, and the nucleotide sequences revealed a similarity of 100% with either L. interrogans or L. borgpetersenii (Table 1). DNA from the kidney samples was also amplified in eight (21.62%) out of a total (N = 37) of the samples.

When compared with 24 reference serovars, iRep1-PCR results for our isolates showed a similarity between fingerprint patterns to either the Icterohaemorrhagiae or Ballum serogroups.

The four rodent isolates were assigned GenBank accession numbers LC006258, LC006259, LC006260, and LC006261. A phylogenetic tree (Figure 2) demonstrates consensus between the genospecies L. interrogans and L. borgpetersenii (reference strains and/or homologous genospecies available in GenBank), and the nucleotide sequences from our isolates.

Figure 2.
Figure 2.

Phylogenetic tree based on partial hap1 gene sequences from the isolates and the reference strains available in GenBank. Genospecies Leptospira biflexa was used as the out-group. Accession numbers of Leptospira spp. isolates: LC002658–LC002661.

Citation: The American Society of Tropical Medicine and Hygiene 94, 5; 10.4269/ajtmh.15-0027

This study offers the first characterization of leptospires in rodents captured in Angola. Our findings confirm data from other countries, suggesting a close relationship between rodents in the environment and the occurrence of human leptospirosis.1215 Our field work was carried out during the rainy season, appreciating the importance of seasonality in predicting human infection risk due to an increase in leptospire survival during the rainy season.12

Our isolates of Leptospira spp., obtained from two of the three most abundant rodent species (R. norvegicus and M. musculus) in Angola, suggest that these small mammals are a primary infection source of leptospires among the human population. The isolates obtained were classified according to molecular assays as L. interrogans and L. borgpetersenii genospecies and as belonging to Ballum and Icterohaemorrhagiae serogroups based on rabbit antisera. Results were consistent across all tests (phenotypic, serological, and genotypic).

Phylogenetic analysis revealed six Leptospira species partitioned into two clusters showing monophyletic groups. Similar results were describing of the lipL32 gene.16 Therefore, the present study opens new perspectives on the knowledge of the patterns of Leptospira infection among rodents in Angola. Further studies of serovar diversity and rodent reservoirs in this country will be helpful.

ACKNOWLEDGMENTS

We acknowledge the following Angolan institutions: Military Health Service/Angolan Armed Forces, Malaria Program/Ministry of Health, Institute of Veterinary Research/Ministry of Agriculture.

  • 1.

    WHO, 2011. Report of the Second Meeting of the Leptospirosis Burden Epidemiology Reference Group. Geneva, Switzerland: WHO.

  • 2.

    Collares-Pereira M, Mathias ML, Santos-Reis M, Ramalhinho MG, Duarte-Rodrigues P, 2000. Rodents and Leptospira transmission risk in Terceira island (Azores). Eur J Epidemiol 16: 11511157.

    • Search Google Scholar
    • Export Citation
  • 3.

    Faine S, Adler B, Bolin C, Perolat P, 1999. Leptospira and Leptospirosis. Melbourne, Australia: MediSci, 272.

  • 4.

    Collares-Pereira M, Korver H, Terpstra WJ, Santos-Reis M, Ramalhinho MG, Mathias ML, Oom MM, Fons R, Libois R, Petrucci-Fonseca F, 1997. First epidemiological data on pathogenic leptospires isolated on the Azorean islands. Eur J Epidemiol 13: 435441.

    • Search Google Scholar
    • Export Citation
  • 5.

    De Vries SG, Visser BJ, Nagel IM, Goris MG, Hartskeerl RA, Grobusch MP, 2014. Leptospirosis in sub-Saharan Africa: a systematic review. Int J Infect Dis 28c: 4764.

    • Search Google Scholar
    • Export Citation
  • 6.

    Herbreteau V, Jittapalapong S, Rerkamnuaychoke W, Chaval Y, Cosson J-F, Morand S, 2011. Protocols for Field and Laboratory Rodent Studies. Bangkok, Thailand: Kasetsart University Press, 2014.

    • Search Google Scholar
    • Export Citation
  • 7.

    Johnson RC, Rogers P, 1964. Differentiation of pathogenic and saprophytic Leptospires with 8-azaguanine. J Bacteriol 88: 16181623.

  • 8.

    Johnson RC, Harris VG, 1967. Differentiation of pathogenic and saprophytic leptospires. I. Growth at low temperatures. J Bacteriol 94: 2731.

    • Search Google Scholar
    • Export Citation
  • 9.

    Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, André-Fontaine G, 2005. Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett 243: 437445.

    • Search Google Scholar
    • Export Citation
  • 10.

    Barocchi MA, Ko AI, Ferrer SR, Faria MT, Reis MG, Riley LW, 2001. Identification of new repetitive element in Leptospira interrogans serovar Copenhageni and its application to PCR-based differentiation of Leptospira serogroups. J Clin Microbiol 39: 191195.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 12.

    Holt J, Davis S, Leirs H, 2006. A model of leptospirosis infection in an African rodent to determine risk to humans: seasonal fluctuations and the impact of rodent control. Acta Trop 99: 218225.

    • Search Google Scholar
    • Export Citation
  • 13.

    Jobbins SE, Sanderson CE, Alexander KA, 2013. Leptospira interrogans at the human-wildlife interface in northern Botswana: a newly identified public health threat. Zoonoses Public Health 61: 113123.

    • Search Google Scholar
    • Export Citation
  • 14.

    Halliday JE, Knobel DL, Allan KJ, de C Bronsvoort BM, Handel I, Agwanda B, Cutler SJ, Olack B, Ahmed A, Hartskeerl RA, Njenga MK, Cleaveland S, Breiman RF, 2013. Urban leptospirosis in Africa: a cross-sectional survey of Leptospira infection in rodents in the Kibera urban settlement, Nairobi, Kenya. Am J Trop Med Hyg 89: 10951102.

    • Search Google Scholar
    • Export Citation
  • 15.

    Houemenou G, Ahmed A, Libois R, Hartskeerl RA, 2013. Leptospira spp. prevalence in small mammal populations in Cotonou, Benin. ISRN Epidemiol 2013: 18.

    • Search Google Scholar
    • Export Citation
  • 16.

    Haake DA, Suchard MA, Kelley MM, Dundoo M, Alt DP, Zuerner RL, 2004. Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol 186: 28182828.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Elsa Fortes-Gabriel, Grupo de Leptospirose e Borreliose de Lyme, Unidade de Microbiologia Médica, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira No. 100, 1349-008, Lisboa, Portugal. E-mail: elsa.gabriel@ihmt.unl.pt

Financial support: The first author was supported by Angolan Armed Forces and through a fellowship awarded by the Calouste Gulbenkian Foundation (Portugal).

Authors' addresses: Elsa Fortes-Gabriel, Medical Microbiology Unit, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisbon, Portugal, and Angolan Armed Forces, Military Health Service, Luanda, Angola, E-mail: elsa.gabriel@ihmt.unl.pt. Teresa Carreira and Maria Luísa Vieira, Medical Microbiology Unit, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisbon, Portugal, E-mails: t.carreira@ihmt.unl.pt and vieira@ihmt.unl.pt.

Save