Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis.

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

Progress in the Mathematical Modelling of Visceral Leishmaniasis.

The leishmaniases comprise a complex of diseases characterized by clinical outcomes that range from self-limiting to chronic, and disfiguring and stigmatizing to life threatening. Diagnostic methods, treatments, and vector and reservoir control options exist, but deciding the most effective interventions requires a quantitative understanding of the population level infection and disease dynamics. The effectiveness of any set of interventions has to be determined within the context of operational conditions, including economic and political commitment. Mathematical models are the best available tools for studying quantitative systems crossing disciplinary spheres (biology, medicine, economics) within environmental and societal constraints. In 2005, the World Health Assembly and government health ministers of India, Nepal, and Bangladesh signed a Memorandum of Understanding to eliminate the life threatening form of leishmaniasis, visceral leishmaniasis (VL), on the Indian subcontinent by 2015 through a combination of early case detection, improved treatments, and vector control. The elimination target is <1 case/10,000 population at the district or subdistrict level compared to the current 20/10,000 in the regions of highest transmission. Towards this goal, this chapter focuses on mathematical models of VL, and the biology driving those models, to enable realistic predictions of the best combination of interventions. Several key issues will be discussed which have affected previous modelling of VL and the direction future modelling may take. Current understanding of the natural history of disease, immunity (and loss of immunity), and stages of infection and their durations are considered particularly for humans, and also for dogs. Asymptomatic and clinical infection are discussed in the context of their relative roles in Leishmania transmission, as well as key components of the parasite-sandfly-vector interaction and intervention strategies including diagnosis, treatment and vector control. Gaps in current biological knowledge and potential avenues to improve model structures and mathematical predictions are identified. Underpinning the marriage between biology and mathematical modelling, the content of this chapter represents the first step towards developing the next generation of models for VL.

Correlations between peripheral parasite load and common clinical and laboratory alterations in dogs with visceral leishmaniasis.

Intensity of peripheral parasite infection has an important role in the transmission of Leishmania spp. from one host to another. As parasite load quantification is still an expensive procedure to be used routinely in epidemiological surveillance, the use of surrogate predictors may be an important asset in the identification of dogs with high transmitting ability. The present study examined whether common clinical and laboratory alterations can serve as predictors of peripheral parasitism in dogs naturally infected with Leishmania spp. Thirty-seven dogs were examined in order to establish correlations between parasite load (PL) in multiple peripheral tissues and common clinical and laboratory findings in canine visceral leishmaniasis (CVL). Quantitative polymerase chain reaction was employed to determine PL in conjunctival swabs, ear skin, peripheral blood and buffy coat. Additionally, a series of hematological, biochemical and oxidative stress markers were quantified. Correlations between net peripheral infection and severity of clinical alterations and variation in laboratory parameters were assessed through a new analytical approach, namely Compressed Parasite Load Data (CPLD), which uses dimension reduction techniques from multivariate statistics to summarize PL across tissues into a single variable. The analysis revealed that elevation in PL is positively correlated with severity of clinical sings commonly observed in CVL, such as skin lesions, ophthalmic alterations, onycogriphosis, popliteal lymphadenomegaly and low body mass. Furthermore, increase in PL was found to be followed by intensification of non-regenerative anemia, neutrophilia, eosinopenia, hepatic injury and oxidative imbalance. These results suggest that routinely used clinical and laboratory exams can be predictive of intensity of peripheral parasite infection, which has an important implication in the identification of dogs with high transmitting ability.

Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis.

Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is an important disease of humans and dogs. Here we review aspects of the transmission and control of ZVL. Whilst there is clear evidence that ZVL is maintained by sandfly transmission, transmission may also occur by non-sandfly routes, such as congenital and sexual transmission. Dogs are the only confirmed primary reservoir of infection. Meta-analysis of dog studies confirms that infectiousness is higher in symptomatic infection; infectiousness is also higher in European than South American studies. A high prevalence of infection has been reported from an increasing number of domestic and wild mammals; updated host ranges are provided. The crab-eating fox Cerdocyon thous, opossums Didelphis spp., domestic cat Felis cattus, black rat Rattus rattus and humans can infect sandflies, but confirmation of these hosts as primary or secondary reservoirs requires further xenodiagnosis studies at the population level. Thus the putative sylvatic reservoir(s) of ZVL remains unknown. Review of intervention studies examining the effectiveness of current control methods highlights the lack of randomized controlled trials of both dog culling and residual insecticide spraying. Topical insecticides (deltamethrin-impregnated collars and pour-ons) have been shown to provide a high level of individual protection to treated dogs, but further community-level studies are needed.

A long-lasting topical deltamethrin treatment to protect dogs against visceral leishmaniasis.

To develop long-lasting, topical pour-on insecticides for dogs to control zoonotic visceral leishmaniasis, two deltamethrin-based formulations (emulsifiable concentrate [EC] and suspension concentrate [SC]) were tested for their efficacy against the phlebotomine sandfly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae), vector of Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae). The entomological outcomes tested were anti-feeding effect (proportion of female sandflies unfed), lethal effect (24-h female sandfly mortality) and these two effects combined, and the insecticide persistence time at 50% (residual activity, RA50) and 80% (RA80) efficacy. On initial application, the proportions of female flies that demonstrated anti-feeding activity or were killed were similar for both formulations, at 0.93 (95% confidence interval [CI] 0.856-0.977) vs. 0.81 (95% CI 0.763-0.858) (anti-feeding) and 0.86 (95% CI 0.787-0.920) vs. 0.76 (95% CI 0.698-0.817) (24-h mortality) for EC and SC, respectively. The RA(50) rates for anti-feeding and mortality caused by the EC formulation were 4.7 months (95% CI 4.18-5.84) and 2.5 months (95% CI 2.25-2.90), respectively, compared with 1.1 months (95% CI 0.96-1.15) and 0.6 months (95% CI 0.50-0.61), respectively, for the SC formulation. The RA(50) for the combined anti-feeding and mortality effects of EC was 5.2 months (95% CI 4.73-5.96), compared with only 0.9 months (95% CI 0.85-1.00) for the SC formulation. The four- to six-fold superior residual activity of the EC formulation was attributed to the addition of a solvent-soluble resin in the formulation which improved fur adhesion and acted as a reservoir for the slow release of the active ingredient. These results identify the potential of such a low-cost formulation to reduce the inter-intervention interval to 5-6 months, similar to that recommended for deltamethrin-impregnated dog collars or for re-impregnation of conventional bednets, both of which are currently used to combat Leishmania transmission. Finally, a novel bioassay was developed in which sandflies were exposed to fur from treated dogs, revealing no detectable tolerance (24-h mortality) in wild-caught sandflies to the insecticide formulations up to 8 months after the initiation of communitywide application of the insecticides to dogs.

Two Leishmania species circulating in the Kaleybar focus of infantile visceral leishmaniasis, northwest Iran: implications for deltamethrin dog collar intervention.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of a few isolates from patients and domestic dogs, this parasite was considered to predominate in the Kaleybar focus of IVL in northwest Iran. However, in the current investigation only one out of five sandfly infections was found to be L. infantum, based on PCR detection and sequencing of parasite internal transcribed spacer (ITS) rDNA infecting Phlebotomus perfiliewi transcaucasicus. The four other infections were of haplotypes of L. tropica, the causative agent of anthroponotic cutaneous leishmaniasis in the Middle East and a parasite occasionally detected in the viscera of dogs and patients in Iran and elsewhere. The widespread distribution of L. tropica in the Kaleybar focus suggests that this parasite is not a transient introduction. Kaleybar has been used for a deltamethrin dog collar intervention to reduce the biting rates of the vectors of L. infantum and this has significantly reduced the incidence of Leishmania infections both in children and the domestic dog, the usual reservoir host of IVL. The implications of finding L. tropica widespread in the heart of the intervention area are discussed. Extensive and intensive typing of natural Leishmania infections is a characteristic of epidemiological investigations in the Neotropics and the current report indicates that this will also be necessary in some regions of the Old World.

Environmental monitoring of Mycobacterium bovis in badger feces and badger sett soil by real-time PCR, as confirmed by immunofluorescence, immunocapture, and cultivation.

Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

Deltamethrin-impregnated bednets reduce human landing rates of sandfly vector Lutzomyia longipalpis in Amazon households.

The entomological efficacy of using 25% deltamethrin EC insecticide-treated bednets (ITNs) was evaluated against the sandfly Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the principal vector of zoonotic visceral leishmaniasis (ZVL) in Latin America. A crossover field study in Amazon Brazil (Marajó Island, Pará State) demonstrated that, compared with untreated nets, the insecticide increased the barrier effect of nets by 39% (95% confidence interval [CI] 34-44%), reduced human landing rates by 80% (95% CI 62-90%) and increased the 24-h mortality rate from 0% to 98% (95% CI 93-99%) inside ITNs. The presence of an ITN also reduced the human landing rate on unprotected persons outside the net in the same room by 56% (95% CI 52-59%), and increased 24-h mortality to 68% (95% CI 62-73%) compared to 0.4% (0.1-2.0%) in untreated houses. The reduction in human landing rates in ITN rooms was associated with a doubling in the proportion of sandflies alighting on walls compared with that in untreated rooms, which was attributed to insecticide-induced excito-repellency. There was no evidence that sandflies were diverted onto unprotected hosts. Human landing catches inside houses peaked between 19.00 hours and 23.00 hours and declined steadily to zero at 02.00 hours and thereafter. House-to-house questionnaires established that only 34% of households owned at least one net (median two, range 1-8), only 20% of the population slept under a net (33% of 0-5-year-old children), and the majority (73%) of the population slept in hammocks. Combined data pertaining to sleeping times for children and sandfly activity period indicate that > 50% of sandfly bites inside houses, and substantially more outside houses, were taken before a third of children were potentially protected by a net. This study demonstrates the clear entomological efficacy of ITNs against Lu. longipalpis in this endemic region. The effectiveness of ITNs at preventing ZVL infection and disease has still to be evaluated.

Husbandry practices, badger sett density and habitat composition as risk factors for transient and persistent bovine tuberculosis on UK cattle farms.

Bovine tuberculosis (bTB) is a persistent problem in cattle herds in Great Britain and Ireland. Farm management and cattle husbandry practices can influence the risk of transmission of bTB and hence the likelihood of bTB breakdown (>or=1 reactor to the tuberculin skin test). Biological differences are expected in the transmission dynamics, and hence risk factors for bTB breakdown, on farms where infection persists in the herd compared to farms where infection is more sporadic or short-lived. Comparative case-control studies were performed to test farm management practices as potential risk factors for transient (under breakdown restrictions for 6 months) bTB breakdown over 5 years (1995-1999) on 179 and 171 UK cattle farms, respectively. Farms were characterised for badger sett density and farm habitat composition by ground survey, farmers were questioned retrospectively on management practices, and cases and controls were identified from national tuberculin test records. Controlling for routine tuberculin testing interval, log-transformed herd size, regional location, badger sett density and farm habitat complexity, multivariable logistic regression identified increased odds of both transient and persistent breakdown on farms that bought-in cows (odds ratio (OR)>or=4.9; 95% confidence interval (CI)>or=1.1;22.8). In addition, the purchase of >50 head of cattle (OR=4.0, 95% CI=1.0;16.0) and the storage of manure for >or/=6 months (OR=4.4; 95% CI=1.3;15.4) were risk factors for transient breakdown, whereas the use of silage clamps (OR=9.1; 95% CI=2.0;40.8) increased the risk of persistent breakdown. Decreased odds of both transient and persistent breakdown were associated with higher stocking densities (>3cattle/ha) (OR

Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis?

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.

Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples.

AIMS: To adapt an immunomagnetic capture (IMC) technique to concentrate and cultivate Mycobacterium bovis from environmental samples including soil, faeces and urine. METHODS AND RESULTS: Cells of Myco. bovis BCG and wild-type Myco. bovis were successfully isolated and cultured from seeded and naturally infected materials respectively. The IMC cell recovery estimated by colony forming units (CFUs) counts ranged from 0.10% to 0.16% for spiked media, and 0.15-0.36% for naturally infected soil and faeces. Recovery estimated by cell counts calculated using semi-quantitative PCR ranged from 80.3% to 88.6% for spiked and 84.1-88.2% for naturally infected material. The differences in the recovery rates estimated by CFUs compared with pixel intensity is likely to be due to clustering of cells on culture plates, thereby underestimating the true cell count. CONCLUSIONS: The IMC techniques can be applied to isolate viable wild type Myco. bovis from naturally contaminated environmental samples. SIGNIFICANCE AND IMPACT OF STUDY: Cultivation of Myco. bovis from environmental samples using traditional methods is extremely problematic. Here, we demonstrate a novel development of IMC techniques that will greatly facilitate the study of the organism in situ in order to assess its epidemiological importance in bovine tuberculosis persistence.

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.

Low infectiousness of a wildlife host of Leishmania infantum: the crab-eating fox is not important for transmission.

The epidemiological role of the crab-eating fox Cerdocyon thous in the transmission of Leishmania infantum is assessed in a longitudinal study in Amazon Brazil. A total of 37 wild-caught foxes were immunologically and clinically monitored, and 26 foxes exposed to laboratory colonies of the sandfly vector Lutzomyia longipalpis, over a 15-month period. In total 78% (29/37) of foxes were seropositive for anti-Leishmania IgG on at least 1 occasion, and 38% (8/37) had infections confirmed by PCR and/or by culture. Point prevalences were 74% (serology), 15% (PCR), and 26% (culture). No signs of progressive disease were observed. None of the foxes were infectious to the 1469 sandflies dissected from 44 feeds. A conservative estimate of the possible contribution of foxes to transmission was 9% compared to 91% by sympatric domestic dogs. These results show that crab-eating fox populations do not maintain a transmission cycle independently of domestic dogs. The implication is that they are unlikely to introduce the parasite into Leishmania-free dog populations.

Evidence for extensive DLA polymorphism in different dog populations.

Many of the genes within the Canine Major Histocompatibility Complex are highly polymorphic. Most of the alleles defined to date for DLA-DRB1, DQA1 and DQB1 come from the analysis of European or North American pure bred dogs. Little is known about DLA gene polymorphisms in other dog populations. We have studied Alaskan Husky dogs and Brazilian mongrel dogs and compared them with a panel of 568 European dogs and 40 Alaskan gray wolves. DNA sequence based typing was used to characterize a series of 12 Alaskan Huskies and 115 Brazilian mongrels for their DLA-DRB1, DQA1 and DQB1 alleles. Within these dogs, 22 previously undescribed DLA class II alleles were identified: 10 DRB1, 5 DQA1 and 7 DQB1 alleles. All these alleles were found in more than one animal, and, in some cases, as a homozygote. Several alleles initially observed in Alaskan gray wolves were found in these dogs. Each new allele was found in specific haplotypic combinations. Many new DLA class II haplotypes were identified. Several of the new alleles and haplotypes were also identified in the European dogs used for comparison. One new haplotype, containing a previously unknown DLA-DRB1 allele together with DQA1 and DQB1 alleles only seen before in gray wolves, was found in 20 Brazilian dogs, including three homozygous animals. It appears likely that the extent of polymorphism of the DLA genes will increase substantially as dogs from a wider geographic distribution are studied. This has major implications for the study of disease susceptibility and immune responsiveness in dogs.

Contact rates between wild and domestic canids: no evidence of parvovirus or canine distemper virus in crab-eating foxes.

Evaluating the risk of disease spill-over from domestic dogs to wildlife depends on knowledge of inter-specific contact rates and/or exposure to aetiological agents in dog environments. Here, contact rates of crab-eating foxes (Cerdocyon thous) with sympatric domestic dog populations were measured over 25months in Amazon Brazil. Foxes and dogs were serologically and clinically monitored for exposure to canine parvovirus (CPV-2) and canine distemper virus (CDV), pathogens known to have caused wildlife population declines elsewhere. Twenty-two of 24 (92%) tagged foxes visited one or more houses in a median 2 (range 1-3) villages per night where dog densities ranged from 7.2 to 15.4 per km(2) (mean 9.5 per km(2)). Foxes spent an average 6.4% (0-40.3%) of their 10h nocturnal activity period in villages, the equivalent of 38m (range 0-242) per night. The rate of potential exposure to disease agents was thus high, though varied by 3 orders of magnitude for individual foxes. Overall, 46% of the fox population was responsible for 80% of all contacts. None of the 37 monitored foxes however showed serological or clinical evidence of infection with CPV-2 or CDV. Seroprevalences for CPV-2 and CDV antibodies in the local domestic dog population were 13% (3/23) and 9% (2/23), respectively, and 89% of 97 monitored pups born during the study presented clinical signs consistent with active CPV-2 infection (haemorrhagic diarrhoea, vomiting, rapid morbidity and emaciation). Although there was no evidence for infection with either virus in foxes, the high level of contact of foxes with peridomestic habitats suggests that the probability of potential spill-over infections from dogs to foxes is high.

Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs.

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78-88%) 0-135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93-100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

Tissue cytokine responses in canine visceral leishmaniasis.

To elucidate the local tissue cytokine response of dogs infected with Leishmania chagasi, cytokine mRNA levels were measured in bone marrow aspirates from 27 naturally infected dogs from Brazil and were compared with those from 5 uninfected control animals. Interferon-gamma mRNA accumulation was enhanced in infected dogs and was positively correlated with humoral (IgG1) but not with lymphoproliferative responses to Leishmania antigen in infected dogs. Increased accumulation of mRNA for interleukin (IL)-4, IL-10, and IL-18 was not observed in infected dogs, and mRNA for these cytokines did not correlate with antibody or proliferative responses. However, infected dogs with detectable IL-4 mRNA had significantly more severe symptoms. IL-13 mRNA was not detectable in either control or infected dogs. These data suggest that clinical symptoms are not due to a deficiency in interferon-gamma production. However, in contrast to its role in human visceral leishmaniasis, IL-10 may not play a key immunosuppressive role in dogs.

The epidemiology of canine leishmaniasis: transmission rates estimated from a cohort study in Amazonian Brazil.

We estimate the incidence rate, serological conversion rate and basic case reproduction number (R0) of Leishmania infantum from a cohort study of 126 domestic dogs exposed to natural infection rates over 2 years on Marajó Island, Pará State, Brazil. The analysis includes new methods for (1) determining the number of seropositives in cross-sectional serological data, (2) identifying seroconversions in longitudinal studies, based on both the number of antibody units and their rate of change through time, (3) estimating incidence and serological pre-patent periods and (4) calculating R0 for a potentially fatal, vector-borne disease under seasonal transmission. Longitudinal and cross-sectional serological (ELISA) analyses gave similar estimates of the proportion of dogs positive. However, longitudinal analysis allowed the calculation of pre-patent periods, and hence the more accurate estimation of incidence: an infection-conversion model fitted by maximum likelihood to serological data yielded seasonally varying per capita incidence rates with a mean of 8.66 x 10(-3)/day (mean time to infection 115 days, 95% C.L. 107-126 days), and a median pre-patent period of 94 (95% C.L. 82-111) days. These results were used in conjunction with theory and dog demographic data to estimate the basic reproduction number, R0, as 5.9 (95% C.L. 4.4-7.4). R0 is a determinant of the scale of the leishmaniasis control problem, and we comment on the options for control.