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Domestic Animals and Visceral Leishmaniasis, Nepal | CDC EID

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Volume 16, Number 2–February 2010
Domestic Animals and Epidemiology of Visceral Leishmaniasis, Nepal
Narayan Raj Bhattarai, Gert Van der Auwera, Suman Rijal, Albert Picado, Niko Speybroeck, Basudha Khanal, Simonne De Doncker, Murari Lal Das, Bart Ostyn, Clive Davies,1 Marc Coosemans, Dirk Berkvens, Marleen Boelaert, and Jean-Claude Dujardin
Author affiliations: B.P. Koirala Institute of Health Sciences, Dharan, Nepal (N.R. Bhattarai, S. Rijal, B. Khanal, M.L. Das); Institute of Tropical Medicine, Antwerp, Belgium (N.R. Bhattarai, G. Van der Auwera, N. Speybroeck, S. De Doncker, B. Ostyn, M. Coosemans, D. Berkvens, M. Boelaert, J.-C. Dujardin); London School of Hygiene and Tropical Medicine, London, UK (A. Picado, C. Davies); Université Catholique de Louvain, Brussels, Belgium (N.R. Bhattarai); University of Antwerp, Antwerp (M. Coosemans, J.-C. Dujardin); and Prins Leopold Institute, Antwerp (J.-C. Dujardin)

Suggested citation for this article

On the Indian subcontinent, visceral leishmaniasis (VL) is considered an anthroponosis. To determine possible reasons for its persistence during interepidemic periods, we mapped Leishmania infections among healthy persons and animals in an area of active VL transmission in Nepal. During 4 months (September 2007–February 2008), blood was collected from persons, goats, cows, and buffaloes in 1 village. Leishmania infections were determined by using PCR. We found infections among persons (6.1%), cows (5%), buffaloes (4%), and goats (16%). Data were georeferenced and entered into a geographic information system. The bivariate K-function results indicated spatial clustering of Leishmania spp.–positive persons and domestic animals. Classification tree analysis determined that among several possible risk factors for Leishmania infection among persons, proximity of Leishmania spp.–positive goats ranked first. Although our data do not necessarily mean that goats constitute a reservoir host of L. donovani, these observations indicate the need for further investigation of goats' possible role in VL transmission.

Visceral leishmaniasis (VL), also known as kala-azar, is a fatal vector-borne parasitic disease. Worldwide incidence is 500,000 cases per year; ≈90% of cases occur in India, Nepal, Bangladesh, Sudan, and Brazil (1). On the Indian subcontinent, the number of officially reported cases, although only a fraction of the true incidence (2), has increased during the past 5–6 years, and the disease is spreading to new areas (3). A kala-azar elimination program was recently launched by the governments of Bangladesh, India, and Nepal, with the support of the World Health Organization; the goal is to reduce the annual incidence in VL-in endemic regions to <1 case per 10,000 persons by 2015 (4). The program essentially relies on early diagnosis and treatment of persons and on vector control (5). This strategy is based on the assumption that Leishmania donovani, the etiologic agent of VL, is transmitted from person to person (anthroponotic VL).

The possible role of domestic animals in anthroponotic VL has been studied in Bangladesh (6), but no clear conclusions have been drawn with regard to animals as risk factors or reservoir hosts. In contrast, the proximity to a VL-infected person is a major risk factor for VL (6). Thus, persons are still considered the only reservoir host for L. donovani on the Indian subcontinent. However, the reasons for persistence during interepidemic periods are debated, and dermal leishmaniasis after kala-azar has been incriminated (7). Correct identification of the Leishmania reservoir host is crucial for the design of control programs. Molecular tools offer new opportunities to better document and reassess transmission patterns. To explore the potential role of domestic animals in transmission, we performed an extensive study in an area of active VL transmission in Nepal, mapping Leishmania infections among healthy persons and domestic animals.

Materials and Methods
Study Site

Figure 1 (please, see the full-text)

Figure 1. A) Visceral leishmaniasis–endemic area (red) of Nepal under study by KALANET project (; B) satellite picture of Dharan-17, Nepal...

Figure 2 (please, see the full-text)

Figure 2. Distribution of sampled households and domestic animals, by visceral leishmaniasis status as determined by PCR, Dharan-17, Nepal, September 2007–February 2008.

Figure 3 (please, see the full-text)

Figure 3. Classification tree results, showing interplay between risk factors of Leishmania positivity, determined by PCR, for A) humans and B) goats, in Dharan-17, Nepal, September 2007–February 2008.

The study was conducted as part of the KALANET project, a community trial of insecticide-treated bed nets ( in the Terai region of eastern Nepal. In Nepal, each village is divided into several wards. For the KALANET project, 10 wards with active VL transmission were selected. Dharan-17 was 1 such ward; it is a periurban ward in the Dharan municipality, located in the foothills of the Mahabharata hills and along the bank of Sardu River. Dharan-17 covers ≈0.3 km2 (Figure 1) and has 515 inhabitants living in 105 households (Figure 2). A demographic survey conducted in July 2006 showed that 77% of households had at least 1 domestic animal (i.e., cow, goat, dog). Most cows, buffaloes, and goats were kept <10 m from the households at night, although a few goats were kept inside the house. VL was only recently reported in this periurban area; Dharan-17 has an average VL incidence rate of 1.61% per year (for 2004–2006). Furthermore, during a previous study conducted in 2006, we documented a higher rate of VL positivity by PCR among the healthy persons in Dharan-17 compared with those in the 9 other wards in the KALANET trial, possibly suggesting a high transmission rate (8). For a control area, we selected Dhankura-3 in Patlekhola. This ward is ≈60 km from Dharan-17, in a hilly area where no VL cases have yet been reported.

Ethical Aspects
Ethical clearance for the KALANET project was obtained from the Ethical Committee of the B.P. Koirala Institute of Health Sciences (BPKIHS), Dharan, Nepal, and the corresponding bodies at the Institute of Tropical Medicine, Antwerp, Belgium, and the London School of Hygiene and Tropical Medicine, London, UK. A community meeting informed local leaders and village residents about the study purpose; informed consent was obtained from all animal owners before their animals were included in the study. International animal experimentation guidelines were followed. Persons provided written consent before enrollment and providing blood samples, per the human experimentation guidelines approved by BPKIHS and the corresponding body at the Institute of Tropical Medicine, Antwerp. For religious reasons, 10.67% of persons did not provide written consent to donate their blood samples.

Sample Collection
All animal surveys were conducted by experienced veterinarians. In Dharan-17, a house-to-house survey was first conducted in September 2007, among 105 households, to collect information on the number and types of animals present in the ward; only information about bovines (cows and buffaloes) and goats was collected in this first survey. Later, 2 sampling surveys were conducted in this ward. In October 2007, survey I sampled 144 goats, 24 buffaloes, and 20 cows from the 37 households that had >1 bovine or goat. In February 2008, survey II focused on 6 households in which Leishmania spp.–positive animals had been identified during survey I. Although the owners claimed that the goats in surveys I and II were the same goats, we could not confirm this. In February 2008, samples were collected from 25 goats, 17 buffaloes, and 21 cows in the control area. In addition to animal samples, we also collected 278 blood samples from persons, all >5 years of age, who lived in Dharan-17 at the time of the survey and provided consent. The samples (1 mL) were collected by venipuncture from animals and persons into tubes containing molecular biology grade Na2EDTA (240 μg/mL of blood; Sigma-Aldrich, Bornem, Belgium). All tubes were immediately stored in a chilled ice box and transferred on the same day to the laboratory at BPKIHS, where 180 μL of each sample was transferred to a tube containing 180 μL of AS1 buffer (catalog no. 1006243; QIAGEN, Venlo, the Netherlands), mixed well, and stored at room temperature.

DNA Extraction and PCR Amplification
All blood samples stored in AS1 buffer were used to extract the DNA within 1 month. The QIAamp DNA Mini Kit (catalog no. 56301; QIAGEN) was used to extract DNA at BPKIHS, following manufacturer's instructions. All DNA samples were sent at ambient temperature to the Institute of Tropical Medicine, Antwerp, where they were analyzed by PCR specific for small ribosomal genes of Leishmania spp. as described elsewhere (9). To confirm that the amplified DNA corresponded to Leishmania spp., amplicons from a set of positive samples were sequenced. The sequences were compared with those of Leishmania spp. and other trypanosomatids from GenBank.

Spatial Clustering of Leishmania spp.–positive Households
We used the results from survey I to assess the clustering of the Leishmania spp.–positive samples in Dharan-17. Each household, previously georeferenced by a geographic positioning system and mapped by using ArcGIS 9.2 (ESRI, Redlands, CA, USA), was identified as Leishmania spp.–positive or –negative for animal and human samples. Analyses considered all animals (goats, cows, and buffaloes) together. The bivariate K-function was used to determine whether households with Leishmania spp.–positive persons were spatially clustered around households with Leishmania spp.–positive domestic animals in Dharan-17. The following equation was used (10): K(d) = expected no. events B within distance d of arbitrary event A / overall density of events B. For easier interpretation of the results, the bivariate K-function was transformed in an L-function as follows (11):

Suggested Citation for this Article
Bhattarai NR, Van der Auwera G, Rijal S, Picado A, Speybroeck N, Khanal B, et al. Domestic animals and epidemiology of visceral leishmaniasis, Nepal.

DOI: 10.3201/eid1602.090623

1Deceased. This article is dedicated to his memory.

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