Evaluation of PCR as a diagnostic mass-screening tool to detect Leishmania (Viannia) spp. in domestic dogs (Canis familiaris).

Several studies have suggested that the PCR could be used in epidemiological mass-screening surveys to detect Leishmania (Viannia) spp. infection in human and animal hosts. Dogs from an area of Leishmania braziliensis and Leishmania peruviana endemicity were screened for American cutaneous leishmaniasis (ACL) infection by established PCR-based and enzyme-linked immunosorbent antibody test (ELISA) protocols. PCR detected Leishmania (Viannia) infection in a total of 90 of 1,066 (8.4%) dogs: 32 of 368 (8.7%), 65 of 769 (8.5%), and 7 of 42 (16.7%) dogs were PCR positive by testing of whole blood, buffy coat, and bone marrow aspirates, respectively. ELISA detected infection in 221 of 1,059 (20.9%) tested dogs. The high prevalence of Leishmania (Viannia) detected by PCR and ELISA in both asymptomatic (7.5 and 19.2%, respectively) and symptomatic (32 and 62.5%, respectively) dogs is further circumstantial evidence for their suspected role as reservoir hosts of ACL. However, the low sensitivity of PCR (31%) compared to ELISA (81%) indicates that PCR cannot be used for mass screening of samples in ACL epidemiological studies. Unless more-sensitive PCR protocols were to be developed, its use should be restricted to the diagnosis of active (canine and human) cases and to the parasitological monitoring of patients after chemotherapy

Advances toward diagnostic tools for managing zoonotic visceral leishmaniasis

Visceral leishmaniasis (VL) is a life-threatening outcome of Leishmania infantum or Leishmania donovani infection. Dogs are the primary domestic reservoir of L. infantum parasites, and ownership of infected dogs increases the risk of human VL. Controlling infection within dog populations is regarded as critical to VL management in endemic countries, both preventing progression of canine disease and limiting parasite transmission to humans and dogs. Here we discuss various strategies that are used to diagnose canine visceral leishmaniasis (CVL) and the possibilities of adapting these for use within population screening and control programs. In addition, given the variable transmissibility of L. infantum to the sand fly vector, we outline some possibilities for the preferential identification of ‘super-spreader’ dogs among the overall infected population

Increasing incidence of zoonotic visceral leishmaniasis on Crete, Greece

To determine whether the incidence of canine leishmaniasis has increased on Crete, Greece, we fitted infection models to serodiagnostic records of 8,848 dog samples for 1990–2006. Models predicted that seroprevalence has increased 2.4% (95% confidence interval 1.61%–3.51%) per year and that incidence has increased 2.2- to 3.8-fold over this 17-year period

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control.

Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions

Modelling Sand Fly Lutzomyia longipalpis Attraction to Host Odour: Synthetic Sex-Aggregation Pheromone Dominates the Response.

Zoontic visceral leishmaniasis (ZVL) due to Leishmania infantum is a potentially fatal protozoan parasitic disease of humans and dogs. In the Americas, dogs are the reservoir and the sand fly, Lutzomyia longipalpis, the principal vector. A synthetic version of the male sand fly produced sex-aggregation pheromone attracts both female and male conspecifics to co-located insecticide, reducing both reservoir infection and vector abundance. However the effect of the synthetic pheromone on the vector's "choice" of host (human, animal reservoir, or dead-end host) for blood feeding in the presence of the pheromone is less well understood. In this study, we developed a modelling framework to allow us to predict the relative attractiveness of the synthetic pheromone and potential alterations in host choice. Our analysis indicates that the synthetic pheromone can attract 53% (95% CIs: 39%-86%) of host-seeking female Lu. longipalpis and thus it out-competes competing host odours. Importantly, the results suggest that the synthetic pheromone can lure vectors away from humans and dogs, such that when co-located with insecticide, it provides protection against transmission leading to human and canine ZVL

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 faeces. 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

A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis

Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) and Leishmania homologue of the mammalian receptor for activated C kinase (LACK) against Leishmania major challenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host of Leishmania infantum, followed-up for 4 months post-vaccination. In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000 μg (high dose) or 100 μg (low dose) DNA prime (day 0) and 1 × 108 pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-γ than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge with Leishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positive in vivo intradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-γ in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/Treg response, as expected in the absence of in vivo restimulation or natural/experimental challenge with Leishmania. These collective results indicate significant antigen-specific type-1 responses and in vivo memory phase cellular immune responses, consistent with superior potential for protective vaccine immunogenicity of DNA/MVA TRYP over LACK

Quantification of the natural history of visceral leishmaniasis and consequences for control

Background Visceral leishmaniasis has been targeted for elimination as a public health problem (less than 1 case per 10,000 people per year) in the Indian sub-continent by 2017. However, there is still a high degree of uncertainty about the natural history of the disease, in particular about the duration of asymptomatic infection and the proportion of asymptomatically infected individuals that develop clinical visceral leishmaniasis. Quantifying these aspects of the disease is key for guiding efforts to eliminate visceral leishmaniasis and maintaining elimination once it is reached. Methods Data from a detailed epidemiological study in Bangladesh in 2002–2004 was analysed to estimate key epidemiological parameters. The role of diagnostics in determining the probability and rate of progression to clinical disease was estimated by fitting Cox proportional hazards models. A multi-state Markov model of the natural history of visceral leishmaniasis was fitted to the data to estimate the asymptomatic infection period and the proportion of asymptomatic individuals going on to develop clinical symptoms. Results At the time of the study, individuals were taking several months to be diagnosed with visceral leishmaniasis, leading to many opportunities for ongoing transmission. The probability of progression to clinical disease was strongly associated with initial seropositivity and even more strongly with seroconversion, with most clinical symptoms developing within a year. The estimated average durations of asymptomatic infection and symptomatic infection for our model of the natural history are 147 days (95 % CI 130–166) and 140 days (95 % CI 123–160), respectively, and are significantly longer than previously reported estimates. We estimate from the data that 14.7 % (95 % CI 12.6-20.0 %) of asymptomatic individuals develop clinical symptoms—a greater proportion than previously estimated. Conclusions Extended periods of asymptomatic infection could be important for visceral leishmaniasis transmission, but this depends critically on the relative infectivity of asymptomatic and symptomatic individuals to sandflies. These estimates could be informed by similar analysis of other datasets. Our results highlight the importance of reducing times from onset of symptoms to diagnosis and treatment to reduce opportunities for transmission

Performance of an environmental test to detect Mycobacterium bovis infection in badger social groups

A study by Courtenay and others (2006) demonstrated that the probability of detecting Mycobacterium bovis by PCR in soil samples from the spoil heaps of main badger setts correlated with the prevalence of excretion (infectiousness) of captured badgers belonging to the social group. It has been proposed that such a test could be used to target badger culling to setts containing infectious animals (Anon 2007). This short communication discusses the issues surrounding this concept, with the intention of dispelling any misconceptions among relevant stakeholders (farmers, policy makers and conservationists)

Insecticide-impregnated dog collars reduce infantile clinical visceral leishmaniasis under operational conditions in NW Iran: A community-wide cluster randomised trial.

OBJECTIVE: To assess the effectiveness of community-wide deployment of insecticide-impregnated collars for dogs- the reservoir of Leishmania infantum-to reduce infantile clinical visceral leishmaniasis (VL). METHODS: A pair matched-cluster randomised controlled trial involving 40 collared and 40 uncollared control villages (161 [95% C.L.s: 136, 187] children per cluster), was designed to detect a 55% reduction in 48 month confirmed VL case incidence. The intervention study was designed by the authors, but implemented by the Leishmaniasis Control Program in NW Iran, from 2002 to 2006. RESULTS: The collars provided 50% (95% C.I. 17·8%-70·0%) protection against infantile VL incidence (0·95/1000/yr compared to 1·75/1000/yr). Reductions in incidence were observed across 76% (22/29) of collared villages compared to pair-matched control villages, with 31 fewer cases by the end of the trial period. In 11 paired villages, no further cases were recorded post-intervention, whereas in 7 collared villages there were 9 new clinical cases relative to controls. Over the trial period, 6,835 collars were fitted at the beginning of the 4 month sand fly season, of which 6.9% (95% C.I. 6.25%, 7.56%) were lost but rapidly replaced. Collar coverage (percent dogs collared) per village varied between 66% and 100%, with a mean annual coverage of 87% (95% C.I. 84·2, 89·0%). The variation in post-intervention clinical VL incidence was not associated with collar coverage, dog population size, implementation logistics, dog owner compliance, or other demographic variables tested. Larger reductions and greater persistence in incident case numbers (indicative of transmission) were observed in villages with higher pre-existing VL case incidence. CONCLUSION: Community-wide deployment of collars can provide a significant level of protection against infantile clinical VL, achieved in this study by the local VL Control Program, demonstrating attributes desirable of a sustainable public health program. The effectiveness is not dissimilar to the community-level protection provided against human and canine infection with L. infantum