Laboratory evidence that dinotefuran, pyriproxyfen and permethrin combination abrogates Leishmania infantum transmissibility by sick dogs.

Dogs are reservoir hosts of leishmaniasis caused by Leishmania infantum and transmitted by phlebotomine vectors. The effect of dinotefuran, pyriproxyfen and permethrin spot-on solution (Vectra®3D, Ceva Santé Animale, Libourne, France) on Leishmania transmissibility by naturally infected dogs via reared Phlebotomus perniciosus, was assessed. Dogs affected by leishmaniasis were submitted to xenodiagnosis and 6 infecting >10% of insects were treated topically on day 0. Antifeeding, insecticidal and anti-transmissibility effects were evaluated through xenodiagnoses performed on days 1, 7 and 28, using individual pre-treatment parameters as control. Feeding and mortality rates were assessed at 24 h, whereas promastigote infection, maturation and burden were assessed up to 96 h post blood meal (potentially infectious rate). On day 1, the anti-feeding efficacy was >95% in 4 dogs, insecticidal efficacy 100% in 4 dogs, and anti-transmissibility effect 100% in 6 dogs. Efficacy rates recorded on day 7 were very similar to day 1. On day 28, anti-feeding and insecticidal efficacy values were much broader, ranging 32.6-100% and 7.7-94.4%, respectively. Potentially infectious insects were recorded from two dogs, with sharp decrease in transmissibility rate as compared with pre-treatment condition. Altogether, Vectra®3D abrogated by >98% the potential Leishmania transmissibility by the examined pool of infected dogs over 1 month.

Elevated and sustained anti-feeding effect of Scalibor® deltamethrin collar against the sand fly Phlebotomus perniciosus in dogs confirmed for 1 year following treatment.

Dogs are reservoir hosts for Leishmania infantum, a protozoan parasite transmitted by phlebotomine sand flies. The anti-feeding and fast-killing efficacy of Scalibor® deltamethrin collars against experimental Phlebotomus perniciosus challenges on dogs was determined over 1 year. Two groups of 8 dogs each were fitted with placebo (control) or deltamethrin collars (treated) on Day 0 and exposed to sand flies approximately every 28 days up to Day 364. After each exposure, anti-feeding and fast-killing efficacy rates were determined by comparing blood-fed or live insects, respectively, in the treated vs. the control group. Blood-fed and live sand flies were significantly less in treated dogs as compared to control dogs at each assessment. The anti-feeding efficacy rate exceeded 90% except on Day 337 (89%) but increased again (96%) on Day 364. Fast killing efficacy was <74% over the study when considering all flies. However, this value increased cumulatively to 98% when only blood-fed flies were compared between groups. Scalibor® collars are highly effective at preventing P. perniciosus blood-feeding and in fast-killing flies taking a blood meal for up to 1 year after application. These strong and long-lasting effects are an important strategic component for L. infantum transmission control.

A single oral dose of fluralaner (Bravecto®) in dogs rapidly kills 100% of blood-fed Phlebotomus perniciosus, a main visceral leishmaniasis vector, for at least 1 month after treatment.

Dogs are the reservoir host of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). Both subclinically-infected and sick animals can be infectious to competent phlebotomine vectors. The degree and duration of insecticidal efficacy of an oral dose of fluralaner (Bravecto®; Merck Animal Health) was determined in dogs exposed to bites of Phlebotomus perniciosus (Diptera: Psychodidae), a main Mediterranean vector of VL. Twelve dogs allocated to two groups of six animals each were included in a parallel-group designed, negative-controlled, randomized, blinded, single-centre efficacy study. Group 2 was treated with fluralaner on day 0, and sand-fly exposure of both groups was performed on days 1, 28 and 84. Viability of blood-fed females was assessed up to 96 h after exposure and efficacy was measured as the survival rate of specimens fed on Group 2 versus those fed on Group 1. A mortality of 100% was recorded at 24 h in females fed on Group 2 at both days 1 and 28. Significant insecticidal efficacy was still observed on day 84, with > 50% mortality recorded by 48 h post blood meal in Group 2. Fluralaner treatment of dogs represents a promising and affordable method for reducing the pool of infected vectors in endemic settings of zoonotic VL.

Visceral leishmaniasis in hematopoietic cell transplantation: Case report and review of the literature.

Visceral leishmaniasis has been recognized as an opportunistic infection affecting people with cellular-immunity impairment, including hematopoietic cell transplantation (HCT) recipients. We describe the case of a young Italian man with Hodgkin lymphoma, who developed visceral leishmaniasis after multiple lines of chemotherapy and allogenic HCT. Literature review of visceral leishmaniasis in HCT recipients was also performed. Eleven patients (median age 50 years, 9 male) developed visceral leishmaniasis after allogenic (n = 9) and autologous (n = 2) HCT. Most of them presented with fever and pancytopenia. Bone marrow examination was the main diagnostic technique; liposomal amphotericin B was the treatment of choice. Four out of eight patients (for whom data are available) experienced visceral leishmaniasis relapse. Visceral leishmaniasis in HCT recipients is a rare event that should be suspected in patients with persistent fever, pancytopenia and possible exposure to Leishmania spp., remembering that - as well as South-East Asia, East Africa and South America - it is endemic in several European regions.

Efficacy of a novel neem oil formulation (RP03™) to control the poultry red mite Dermanyssus gallinae.

Dermanyssus gallinae (Mesostigmata: Dermanyssidae) is the most harmful ectoparasite of laying hens, represents an occupational hazard for poultry workers, and a growing threat to medical science per se. There is increasing demand for alternative products, including plant-derived acaricides, with which to control the mite. The present study investigated the efficacy of neem oil against D. gallinae on a heavily infested commercial laying hen farm. A novel formulation of 20% neem oil, diluted from a 2400-p.p.m. azadirachtin-concentrated stock (RP03™), was administered by nebulization three times in 1 week. Using corrugated cardboard traps, mite density was monitored before, during and after treatment and results were statistically analysed. Mite populations in the treated block showed 94.65%, 99.64% and 99.80% reductions after the first, second and third product administrations, respectively. The rate of reduction of the mite population was significantly higher in the treated block (P < 0.001) compared with the control and buffer blocks. The results suggest the strong bioactivity of neem, and specifically of the patented neem-based formulation RP03™, against D. gallinae. The treatment was most effective in the 10 days following the first application and its effects persisted for over 2 months. Further studies will aim to overcome observed side effects of treatment represented by an oily layer on equipment and eggs.

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.

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus, displaying the killer phenotype.

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory-reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito-isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp.

De novo assembly and sex-specific transcriptome profiling in the sand fly Phlebotomus perniciosus (Diptera, Phlebotominae), a major Old World vector of Leishmania infantum.

BACKGROUND: The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly. RESULTS: We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 981 transcripts were classified as putative long non-coding RNAs and 244 transcripts encoded for putative novel proteins specific of the Phlebotominae sub-family. Differential expression analysis identified 8590 transcripts significantly biased between sexes. Among them, some show relaxation of selective constraints when compared to their orthologs of the New World sand fly species Lutzomyia longipalpis. CONCLUSIONS: In this paper, we present a comprehensive transcriptome resource for the sand fly species P. perniciosus built from short-read RNA-seq and we provide insights into sex-specific gene expression at adult stage. Our analysis represents a first step towards the identification of sex-specific genes and pathways and a foundation for forthcoming investigations into this important vector species, including the study of the evolution of sex-biased genes and of the sexual differentiation in phlebotomine sand flies.

Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013.

An increased number of autochthonous visceral leishmaniasis (VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0-8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15-120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe.

The burden of visceral leishmaniasis in Italy from 1982 to 2012: a retrospective analysis of the multi-annual epidemic that occurred from 1989 to 2009.

Starting from 1989 Italy experienced an increase of visceral leishmaniasis (VL) cases over a baseline of 10 to 30 cases reported annually. The number of cases peaked in 2000 and 2004 with more than 200 cases/year, and subsequently declined to reach on average one third of the 2000 peak value in the period after 2010. A retrospective analysis from 1982 to 2012 showed that the multi-annual epidemic consisted of major components including (i) an outbreak involving infants and immunocompetent adults in parts of the Campania region (southern peninsular Italy) and that appears to have declined naturally, (ii) a second outbreak affecting human immunodeficiency virus (HIV)-infected individuals throughout the country, that declined owing to the use of highly active antiretroviral therapies (HAART), (iii) a generalised increase of VL cases in immunocompetent individuals and patients affected by associated conditions other than HIV from endemic regions of peninsular and insular Italy (other than Campania), which was due to a geographical spreading of VL foci, with no major case-clusters or outbreak features. A minor component consisted in the appearance of a few autochthonous cases in formerly non-endemic areas, starting from the early 1990s. Epidemic determinants and reasons for VL decline in the Campania region remain largely unexplained, despite the information available on canine reservoir and phlebotomine vectors in Italy.

Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern.

Phlebotomine sandflies transmit pathogens that affect humans and animals worldwide. We review the roles of phlebotomines in the spreading of leishmaniases, sandfly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Carrión's disease. Among over 800 species of sandfly recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae). Based on incrimination criteria, we provide an updated list of proven or suspected vector species by endemic country where data are available. Increases in sandfly diffusion and density resulting from increases in breeding sites and blood sources, and the interruption of vector control activities contribute to the spreading of leishmaniasis in the settings of human migration, deforestation, urbanization and conflict. In addition, climatic changes can be expected to affect the density and dispersion of sandflies. Phlebovirus infections and diseases are present in large areas of the Old World, especially in the Mediterranean subregion, in which virus diversity has proven to be higher than initially suspected. Vesiculovirus diseases are important to livestock and humans in the southeastern U.S.A. and Latin America, and represent emerging human threats in parts of India. Carrión's disease, formerly restricted to regions of elevated altitude in Peru, Ecuador and Colombia, has shown recent expansion to non-endemic areas of the Amazon basin.

Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability.

The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs.

Aspects of the bionomics of Phlebotomus sergenti sandflies from an endemic area of anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria.

Aspects of the bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied from June to November 2005 in three foci of anthroponotic cutaneous leishmaniasis (ACL) in Aleppo Governorate, Syria, where the agent Leishmania tropica (Wright) (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus sergenti Parrot. Syria has been designated by the World Health Organization as one of four countries in the Old World where cutaneous leishmaniasis is hyperendemic, but little is known about the biology of local vector populations. Standard collections by sticky traps showed two peaks in density, in June and late August. In total, 1840 sandflies were caught, comprising five species: Phlebotomus papatasi (Scopoli) (68.0%); P. sergenti (25.4%); Sergentomyia minuta (Rondani) (6.4%); Phlebotomus tobbi Adler & Theodor (0.1%), and Phlebotomus mascittii canaaniticus Adler & Theodor (0.1%). Similar numbers of P. sergenti were caught indoors (246 specimens) and outdoors (222), whereas P. papatasi was significantly more abundant indoors (1096 specimens) than outdoors (156) (chi(2) = 241, P < 0.01). In total, 212 blood-fed females were tested for host blood determination, of which 176 (83.0%) reacted with anti-species reagent. Results from 20 P. sergenti suggest that this species is an opportunistic feeder, imbibing human, ovine, avian, bovine and feline blood, although more bloodmeals were taken from humans and cattle than expected in relation to the relative proportions of potential hosts present (the forage ratio, FR). The bionomics of P. sergenti are discussed in relation to the inefficacy of control campaigns based on indoor spraying with residual insecticides that have been implemented by the Syrian Ministry of Health to control the epidemics of ACL in the Aleppo Governorate.

Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus (Diptera, Psychodidae) in a high-endemic focus of canine leishmaniasis in Rome province, Italy.

A 2-year survey aimed to study seasonal phenology, host-blood feeding preferences and Leishmania infections of Phlebotomus perniciosus, was carried out in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Sandfly densities were monitored by sticky traps, while CDC light traps and hand collections were used for individual sandfly analyses. Four species were identified, three belonging to Phlebotomus (P. perniciosus, Phlebotomus mascittii and Phlebotomus papatasi) and one to Sergentomyia (Sergentomyia minuta) genera. In sticky traps, P. perniciosus (53.5%) and S. minuta (46.0%) were the prevalent species, whereas specimens collected by CDC light traps and hand collections consisted mostly of P. perniciosus. The study showed a markedly different sandfly density between 2003 and 2002 collections (5024 specimens versus 644 specimens, respectively), which was attributed to different climatic conditions. In 2003 adults were active during 7 months (May-November) with a typical bimodal distribution, while in 2002 their activity was shorter (5 months, June-October) without clear density peaks. In this low-density year, the prevalence of P. perniciosus was significantly higher in domestic than in wild resting sites. Blood meal tests showed that P. perniciosus had no preferences for any of the two Leishmania susceptible hosts, being the forage ratio rate 0.7 and 1.0 for man and dog, respectively. A significant number of P. perniciosus was found with avian (60.3%) or ovine (24.2%) blood, that was in relation with the habitats where fed specimens were collected. The rate of specimens of P. perniciosus without blood and found positive for Leishmania DNA, detected by a sensitive nested-PCR technique, was much higher (27.6%) than the promastigote infection rate determined by microscopy in the dissected females (1.4%). The intense Leishmania transmission in the study area was confirmed by the high prevalence of anti-leishmanial antibodies in dogs (33.3%).

Insecticide-treated bednets to prevent anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria: results from two trials.

The increasing incidence of anthroponotic cutaneous leishmaniasis (ACL) in Syria, coupled with the inefficacy of residual insecticide spraying to control the disease, have led to the further evaluation of the preventive efficacy of insecticide-treated bednets (ITNs) through large-scale field studies. Two trials were performed in the historical ACL-endemic areas of the Aleppo Governorate. In 1997-1999, a matched-cluster randomized trial was conducted in five intervention (ITNs) and five control (untreated bednets) villages, which involved a population of 10354 in 1321 households. In 2001-2003, a second study was performed in four villages (a population of 9325 in 858 households), which allowed the evaluation of the impact of the interruption of ITN intervention on ACL incidence rates. Both studies not only confirmed the high efficacy of ITNs in preventing ACL during 1 year post-intervention [about 85% (95% CL 76-98%) in the 1997-1999 trial], but also suggested that the interruption of this control measure might restore the pre-intervention disease incidence within 1-2 years. These findings underline the need for a sustainable and durable implementation of ITN-based control of the disease.

Deltamethrin-impregnated collars for the control of canine leishmaniasis: evaluation of the protective effect and influence on the clinical outcome of Leishmania infection in kennelled stray dogs.

A 2-year field study on kennelled stray dogs living in a highly endemic area of leishmaniasis was designed to evaluate whether deltamethrin-impregnated collars (Scalibor) Protector Band) could confer protection against leishmaniasis in this peculiar setting, and to assess differences in clinical outcomes between collared and uncollared dogs. A cohort of 120 clinically healthy and Leishmania-seronegative dogs was enrolled, 50% of which were collared before the 2003 transmission season, and then re-collared before the subsequent season. Collared and uncollared animals were allowed to live with infected dogs in same groups within the kennel. Follow-up included serological (IFAT) assessment twice a year with parasitological Leishmania confirmation, and clinical evaluation performed every 3 months on seroconverted dogs from both groups. Collar losses during the two seasons were high (35%). About 50% of enrolled dogs were lost at follow-up because of death or they were moved to other locations. After the 2003 season, cross-sectional serological examinations tested positive in 5 out of 44 collared animals (11.4%) and in 14 out of 34 controls (41.2%), with 72.3% estimated protection (P<0.005). After the 2004 season, 7/31 seronegative collared dogs seroconverted (22.6%) compared with 7/17 seronegative controls (41.2%), with 45.1% protection (P=0.15). At the end of the study, the cumulative rate of protection was 50.8% (P=0.005). At the clinical evaluation of 21 seroconverted dogs from both groups, canine leishmaniasis signs were significantly more frequent (90% versus 36%, P=0.017) and rapidly progressive in uncollared than in collared dogs. Reasons for such partial clinical protection in collared dogs may be found in the vector anti-feeding effect of protector bands, resulting in a lower number of infectious bites and, probably, in the reduction of antigenic stimuli necessary to shift toward a non-protective immune response.

Pathological changes in the bone marrow of dogs with leishmaniosis.

Bone marrow aspiration smears from 15 dogs naturally infected with leishmania were evaluated. Three of the dogs showed no clinical signs, six had up to three clinical signs and six had more than three. The most common pathological features of the bone marrow were megakaryocytic dysplasia in 10 of the dogs, erythrophagocytosis in eight, erythroid dysplasia in two and emperipolesis in two. The megakaryocytic and erythroid dysplasia were probably related to an increased number of marrow macrophages producing high levels of tumour necrosis factor alpha and interferon gamma. Six of the dogs with clinical signs showed bone marrow dysplastic features and erythrophagocytosis, suggesting that leishmaniosis could be the unique cause of both conditions.