G Model VETPAR-7488; No. of Pages 7
ARTICLE IN PRESS Veterinary Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Trypanosoma from rodents as potential source of infection in human-shaped landscapes of South-East Asia Pornpan Pumhom a,b,c , Serge Morand d,e , Annelise Tran d,f , Sathaporn Jittapalapong b,c,∗ , Marc Desquesnes c,g a
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, Thailand Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand d CIRAD, UPR Animal et Gestion Intégrée des Risques, F-34398 Montpellier, France e CNRS-CIRAD, Centre d’Infectiologie Christophe Mérieux du Laos, Vientiane, Lao Democratic People’s Republic f CIRAD, UMR Territoires Environnement Télédétection et Information Spatiale, F-34093 Montpellier, France g CIRAD-Bios, UMR17 InterTryp, Montpellier F-34000, France b c
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 18 December 2014 Accepted 18 December 2014 Keywords: Trypanosoma lewisi Trypanosoma evansi Rodents Habitat Land cover Southeast Asia
a b s t r a c t Reports of atypical human cases of Trypanosoma lewisi or T. lewisi-like and Trypanosoma evansi infections have increased in South-East Asia, urging to investigate the possible links between humans, animal reservoirs and habitats. We tested how habitat structure affects the infection by Trypanosoma species of common murine rodents, inhabiting humandominated landscapes in South East Asia. For this, we used geo-referenced data of rodents investigated for Trypanosoma infection and land cover maps produced for seven study sites in Thailand, Cambodia and Lao PDR. High prevalence of infection by T. lewisi was observed in rodents living near human settlement and in areas with high cover of built-up habitat, while the infection of rodents by T. evansi was explained by increased landscape patchiness and high cover of rain-fed agriculture lands. These results suggest a likely role of wild rodents as reservoir and possible source of atypical human infection by animal trypanosomes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosomosis is a disease of medical and veterinary importance, occurring mainly in tropical areas all around the world. The disease is caused by haemoflagellate protozoan parasites transmitted by biting insects (Hoare, 1972). In the past decade, the number of reports on atypical human cases of Trypanosoma lewisi or T. lewisi-like (Howie et al., 2006; Shrivastava and Shrivastava, 1974; Sarataphan
∗ Corresponding author at: Faculty of Veterinary Medicine, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. Tel.: +66 08 49 40 72 68; fax: +66 029 42 86 84. E-mail address: [email protected] (S. Jittapalapong).
et al., 2007) and Trypanosoma evansi (Joshi et al., 2006; Shegokar et al., 2006) infections strongly increased notably in Malaysia, Sri Lanka, India and Thailand (Truc et al., 2013). As stressed by Truc et al. (2013), although there is an increase of detection due to improvement in molecular techniques, the number of cases of atypical human trypanosomoses seems greatly underestimated. Thus, there is a need for a better investigation of the transmission of these Trypanosoma species particularly the role of rodents as reservoirs. Indeed, forty-four Trypanosoma species have been described in 144 rodent species (Hoare, 1972; Milocco et al., 2012) including the Salivaria T. evansi. However most of them belong to the Stercoraria section. In a recent study, Pumhom et al. (2014) investigated the molecular prevalence of T. lewisi and T. evansi in wild
http://dx.doi.org/10.1016/j.vetpar.2014.12.027 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Pumhom, P., et al., Trypanosoma from rodents as potential source of infection in human-shaped landscapes of South-East Asia. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2014.12.027
G Model VETPAR-7488; No. of Pages 7
ARTICLE IN PRESS P. Pumhom et al. / Veterinary Parasitology xxx (2015) xxx–xxx
2
rodents and shrews from Cambodia, Lao PDR and Thailand. Rodents were trapped in several locations and were tested using three markers: TRYP1 (amplifying ITS1 of ribosomal DNA of all trypanosomes), TBR (amplifying satellite genomic DNA of Trypanozoon parasites) and LEW1 (amplifying ITS1 of ribosomal DNA of T. lewisi) (see Desquesnes et al., 2011). Using LEW1, T. lewisi was found in several rodent species: Bandicota savilei, Berylmys berdomorei, Mus cervicolor, Maxomys surifer, Rattus tanezumi, Rattus exulans and Rattus norvegicus, and in the shrew Suncus murinus. Using TBR, T. evansi was detected in R. tanezumi and R. exulans, two synanthropic species likely found in human settlement (Pumhom et al., 2014). These observations suggest a role for rodents living closely to humans as reservoirs and thus as potential sources of trypanosomes for human infection. The objective of this study was to better evaluate the landscape features associated with Trypanosoma infection in rodents from mainland South-East Asia. For this, we used the geo-referenced data of the rodents investigated for Trypanosoma infection from Pumhom et al. (2014) and the land cover layers developed for seven study sites in Thailand, Cambodia and Lao PDR (Dupuy et al., 2012; Bordes et al., 2013), where most of these rodents were trapped. Sixteen species of rodents and one species of shrew from Cambodia (202 individuals in 2 study sites), Lao PDR (138 individuals in 2 study sites) and Thailand (151 individuals in 3 study sites) were found infected by T. lewisi (48 individuals in total) and T. evansi (4 individuals in total) (but see Pumhom et al., 2014). We explored if and how the infection of rodents is related to the land cover and landscape structure and then to confirm the likely association of rodent infection with the level of anthropization such as the importance of built-up areas. 2. Materials and methods 2.1. Rodents Rodents were trapped in the Cambodian provinces of Preah Sihanouk and Mondolkiri, the Thai provinces of Loei, Buriram and Nan, and the Laotian provinces of Champasak and Luang Prabang within the framework of the CERoPath project (www.ceropath.org) (see map in Pumhom et al., 2014). The sampling effort corresponded to a total of 1200 night-traps per trapping session. At each locality, 30 lines of 10 traps were placed during four nights in three different habitats: (1) forests and mature plantations; (2) non-flooded lands or fields (shrubby waste land, young plantations, orchards), (3) rain-fed lowland paddy rice fields (cultivated floodplain). Villages and isolated houses, which correspond to a fourth habitat category (4), the human settlement, were also sampled using cage-traps distributed to residents. We used locally made cage live-traps. We sampled each locality twice i.e. during the rainy and dry seasons. In both seasons, trap lines were located in the same place with a Global Positioning System (GPS) receiver. The 30 lines within a locality were located within an area of about 10 km × 10 km. Geographical coordinates of trap line devices and households were systematically recorded with a GPS
receiver and the surrounding landscape was described by field observation. Habitat description and coordinates of trap lines, accompanied by photographs of research sites are available in the CERoPath project web site (www.ceropath.org). Rodents were identified on the basis of their morphology or using species-specific primer and/or barcoding assignment (Chaval et al., 2010; Pagès et al., 2010, 2013; Aplin et al., 2011; Galan et al., 2012). Complete data for animals used as reference for barcoding assignment are available on the CERoPath project web site (www.ceropath.org). Voucher specimens (skulls) were deposited at the Centre de Biologie et de Gestion des Population (Montpellier, France), rodent tissues (liver) were deposited at Kasetsart University (Department of Veterinary Medicine, Bangkok, Thailand). 2.2. Trypanosome infection Rodent blood was deposited in sodium citrate in a micro-tube and centrifuged at 12,000 rpm (9000 × g). Plasma was collected in a microtube for further serological tests. Trypanosoma species identification was based on microscopic observation of the morphology and morphometry of the parasites, and the results obtained by PCR, followed by sequencing and sequence-analysis. Three sets of primers were used systematically, namely TRYP1, TBR and LEW1 primers (see Pumhom et al., 2014). 2.3. Land-cover classification of the seven study sites using SPOT 5 imagery and DEM For each locality, four main land cover types (forests, flat agriculture lands, steep agriculture lands, and builtup areas) were mapped using recent (years 2007–2008) high spatial resolution SPOT 5 satellite images and Digital Elevation Model (DEM) (Dupuy et al., 2012). Images characteristics, classification and validation procedures are detailed in Supplementary Material. Then, the land cover maps were integrated into a Geographical Information System (GIS) in order to compute landscape metrics for each trapping site (located with GPS accuracy
ARTICLE IN PRESS Veterinary Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Trypanosoma from rodents as potential source of infection in human-shaped landscapes of South-East Asia Pornpan Pumhom a,b,c , Serge Morand d,e , Annelise Tran d,f , Sathaporn Jittapalapong b,c,∗ , Marc Desquesnes c,g a
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, Thailand Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand d CIRAD, UPR Animal et Gestion Intégrée des Risques, F-34398 Montpellier, France e CNRS-CIRAD, Centre d’Infectiologie Christophe Mérieux du Laos, Vientiane, Lao Democratic People’s Republic f CIRAD, UMR Territoires Environnement Télédétection et Information Spatiale, F-34093 Montpellier, France g CIRAD-Bios, UMR17 InterTryp, Montpellier F-34000, France b c
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 18 December 2014 Accepted 18 December 2014 Keywords: Trypanosoma lewisi Trypanosoma evansi Rodents Habitat Land cover Southeast Asia
a b s t r a c t Reports of atypical human cases of Trypanosoma lewisi or T. lewisi-like and Trypanosoma evansi infections have increased in South-East Asia, urging to investigate the possible links between humans, animal reservoirs and habitats. We tested how habitat structure affects the infection by Trypanosoma species of common murine rodents, inhabiting humandominated landscapes in South East Asia. For this, we used geo-referenced data of rodents investigated for Trypanosoma infection and land cover maps produced for seven study sites in Thailand, Cambodia and Lao PDR. High prevalence of infection by T. lewisi was observed in rodents living near human settlement and in areas with high cover of built-up habitat, while the infection of rodents by T. evansi was explained by increased landscape patchiness and high cover of rain-fed agriculture lands. These results suggest a likely role of wild rodents as reservoir and possible source of atypical human infection by animal trypanosomes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosomosis is a disease of medical and veterinary importance, occurring mainly in tropical areas all around the world. The disease is caused by haemoflagellate protozoan parasites transmitted by biting insects (Hoare, 1972). In the past decade, the number of reports on atypical human cases of Trypanosoma lewisi or T. lewisi-like (Howie et al., 2006; Shrivastava and Shrivastava, 1974; Sarataphan
∗ Corresponding author at: Faculty of Veterinary Medicine, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. Tel.: +66 08 49 40 72 68; fax: +66 029 42 86 84. E-mail address: [email protected] (S. Jittapalapong).
et al., 2007) and Trypanosoma evansi (Joshi et al., 2006; Shegokar et al., 2006) infections strongly increased notably in Malaysia, Sri Lanka, India and Thailand (Truc et al., 2013). As stressed by Truc et al. (2013), although there is an increase of detection due to improvement in molecular techniques, the number of cases of atypical human trypanosomoses seems greatly underestimated. Thus, there is a need for a better investigation of the transmission of these Trypanosoma species particularly the role of rodents as reservoirs. Indeed, forty-four Trypanosoma species have been described in 144 rodent species (Hoare, 1972; Milocco et al., 2012) including the Salivaria T. evansi. However most of them belong to the Stercoraria section. In a recent study, Pumhom et al. (2014) investigated the molecular prevalence of T. lewisi and T. evansi in wild
http://dx.doi.org/10.1016/j.vetpar.2014.12.027 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Pumhom, P., et al., Trypanosoma from rodents as potential source of infection in human-shaped landscapes of South-East Asia. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2014.12.027
G Model VETPAR-7488; No. of Pages 7
ARTICLE IN PRESS P. Pumhom et al. / Veterinary Parasitology xxx (2015) xxx–xxx
2
rodents and shrews from Cambodia, Lao PDR and Thailand. Rodents were trapped in several locations and were tested using three markers: TRYP1 (amplifying ITS1 of ribosomal DNA of all trypanosomes), TBR (amplifying satellite genomic DNA of Trypanozoon parasites) and LEW1 (amplifying ITS1 of ribosomal DNA of T. lewisi) (see Desquesnes et al., 2011). Using LEW1, T. lewisi was found in several rodent species: Bandicota savilei, Berylmys berdomorei, Mus cervicolor, Maxomys surifer, Rattus tanezumi, Rattus exulans and Rattus norvegicus, and in the shrew Suncus murinus. Using TBR, T. evansi was detected in R. tanezumi and R. exulans, two synanthropic species likely found in human settlement (Pumhom et al., 2014). These observations suggest a role for rodents living closely to humans as reservoirs and thus as potential sources of trypanosomes for human infection. The objective of this study was to better evaluate the landscape features associated with Trypanosoma infection in rodents from mainland South-East Asia. For this, we used the geo-referenced data of the rodents investigated for Trypanosoma infection from Pumhom et al. (2014) and the land cover layers developed for seven study sites in Thailand, Cambodia and Lao PDR (Dupuy et al., 2012; Bordes et al., 2013), where most of these rodents were trapped. Sixteen species of rodents and one species of shrew from Cambodia (202 individuals in 2 study sites), Lao PDR (138 individuals in 2 study sites) and Thailand (151 individuals in 3 study sites) were found infected by T. lewisi (48 individuals in total) and T. evansi (4 individuals in total) (but see Pumhom et al., 2014). We explored if and how the infection of rodents is related to the land cover and landscape structure and then to confirm the likely association of rodent infection with the level of anthropization such as the importance of built-up areas. 2. Materials and methods 2.1. Rodents Rodents were trapped in the Cambodian provinces of Preah Sihanouk and Mondolkiri, the Thai provinces of Loei, Buriram and Nan, and the Laotian provinces of Champasak and Luang Prabang within the framework of the CERoPath project (www.ceropath.org) (see map in Pumhom et al., 2014). The sampling effort corresponded to a total of 1200 night-traps per trapping session. At each locality, 30 lines of 10 traps were placed during four nights in three different habitats: (1) forests and mature plantations; (2) non-flooded lands or fields (shrubby waste land, young plantations, orchards), (3) rain-fed lowland paddy rice fields (cultivated floodplain). Villages and isolated houses, which correspond to a fourth habitat category (4), the human settlement, were also sampled using cage-traps distributed to residents. We used locally made cage live-traps. We sampled each locality twice i.e. during the rainy and dry seasons. In both seasons, trap lines were located in the same place with a Global Positioning System (GPS) receiver. The 30 lines within a locality were located within an area of about 10 km × 10 km. Geographical coordinates of trap line devices and households were systematically recorded with a GPS
receiver and the surrounding landscape was described by field observation. Habitat description and coordinates of trap lines, accompanied by photographs of research sites are available in the CERoPath project web site (www.ceropath.org). Rodents were identified on the basis of their morphology or using species-specific primer and/or barcoding assignment (Chaval et al., 2010; Pagès et al., 2010, 2013; Aplin et al., 2011; Galan et al., 2012). Complete data for animals used as reference for barcoding assignment are available on the CERoPath project web site (www.ceropath.org). Voucher specimens (skulls) were deposited at the Centre de Biologie et de Gestion des Population (Montpellier, France), rodent tissues (liver) were deposited at Kasetsart University (Department of Veterinary Medicine, Bangkok, Thailand). 2.2. Trypanosome infection Rodent blood was deposited in sodium citrate in a micro-tube and centrifuged at 12,000 rpm (9000 × g). Plasma was collected in a microtube for further serological tests. Trypanosoma species identification was based on microscopic observation of the morphology and morphometry of the parasites, and the results obtained by PCR, followed by sequencing and sequence-analysis. Three sets of primers were used systematically, namely TRYP1, TBR and LEW1 primers (see Pumhom et al., 2014). 2.3. Land-cover classification of the seven study sites using SPOT 5 imagery and DEM For each locality, four main land cover types (forests, flat agriculture lands, steep agriculture lands, and builtup areas) were mapped using recent (years 2007–2008) high spatial resolution SPOT 5 satellite images and Digital Elevation Model (DEM) (Dupuy et al., 2012). Images characteristics, classification and validation procedures are detailed in Supplementary Material. Then, the land cover maps were integrated into a Geographical Information System (GIS) in order to compute landscape metrics for each trapping site (located with GPS accuracy
