Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis.

Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (Ki = 0.2 µM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

Optimization of Potent and Specific Trypanothione Reductase Inhibitors: A Structure-Based Drug Discovery Approach.

Leishmania spp. are responsible for up to 1 million new cases each year. The current therapeutic arsenal against Leishmania is largely inadequate, and there is an urgent need for better drugs. Trypanothione reductase (TR) represents a druggable target since it is essential for the parasite and not shared by the human host. Here, we report the optimization of a novel class of potent and selective LiTR inhibitors realized through a concerted effort involving X-ray crystallography, synthesis, structure-activity relationship (SAR) investigation, molecular modeling, and in vitro phenotypic assays. 5-Nitrothiophene-2-carboxamides 3, 6e, and 8 were among the most potent and selective TR inhibitors identified in this study. 6e and 8 displayed leishmanicidal activity in the low micromolar range coupled to SI > 50. Our studies could pave the way for the use of TR inhibitors not only against leishmaniasis but also against other trypanosomatidae due to the structural similarity of TR enzymes.

Monitoring and detection of new endemic foci of canine leishmaniosis in northern continental Italy: An update from a study involving five regions (2018-2019).

Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission.

Assessment of Circulating Immune Complexes During Natural and Experimental Canine Leishmaniasis.

Canine leishmaniasis (CanL) is a disease characterized by a large variety of clinical alterations, the majority of which being due to immune mediated mechanisms. Sick dogs usually produce high levels of Leishmania-specific immunoglobulins which may give rise to circulating immune complexes (CICs) whose defective clearance by scavenging macrophages induces vasculitis and their deposition in specific organs. The aim of this study was to assess the serum level of CICs in dogs exposed to natural and experimental infection. Fifty-two sera were examined, belonging to untreated groups of naïve beagles previously studied to assess the performance of anti-leishmanial vaccines under natural (no. 22 dogs) or experimental (no. 30 dogs) transmission. Sera were classified in five groups according to the dog's health condition, IFAT titer, and the bone marrow (BM) nested (n)-PCR result. A: no.10 healthy dogs before the experimental infection; B: no.10 clinically healthy dogs infected experimentally, IFAT negative (= reciprocal titer <160) and n-PCR positive; C: no.10 clinically healthy dogs naturally infected, IFAT positive at titers 160-320 and n-PCR negative; D: no.10 sick dogs experimentally infected, IFAT positive at titer >320 and n-PCR positive; E: no.12 sick dogs naturally infected, IFAT positive at titer >320 and n-PCR positive. CICs levels were assessed by ELISA method (canine CIC assay-Cloude-Clone Corporation, USA). The two groups characterized by negative IFAT (A and B) had the lowest median level of CICs (16.09 and 12.78 µg/ml, respectively). CICs value increased progressively in the group C and reached the highest levels in the groups D and E, both characterized by high antibodies titer and severe disease, independently from the mode of infection. Significant differences in CICs concentration (p < 0.0001) were demonstrated between A, B, and C groups when compared with D or E groups of dogs. No differences were found inside the first three groups, while differences were recorded between the last two groups of sick dogs. CICs serum concentration increased with the progress of leishmaniasis, being significantly correlated with the increase of specific antibodies over time. High CICs levels detectable by commercial ELISA proved specific to an established Leishmania infection in dogs in the absence of other concomitant infections, as demonstrated by the similar trend assessed in experimentally and naturally infected dogs.

Canine leishmaniosis in the Italian northeastern Alps: A survey to assess serological prevalence in dogs and distribution of phlebotomine sand flies in the Autonomous Province of Bolzano - South Tyrol, Italy.

The Autonomous Province of Bolzano-South Tyrol (APB), located in the northernmost territory of the Italian eastern Alps, is still considered non-endemic for canine leishmaniosis (CanL) despite clinical cases being observed and a competent Leishmania infantum vector (Phlebotomus perniciosus) having been recorded since 2008. A serological survey of leishmaniosis among a randomly-selected subpopulation of registered owned dogs was carried in 2018, followed by entomological investigations performed in 2019 and driven by canine survey results. A total of 457 resident dogs from all over the APB territory were examined through IFAT for antibodies against L.infantum, of which 63 (13.8%) tested positive. Thirty-five seropositive cases (7.7%) were considered autochthonous to APB, i.e. dogs born and lived in the province, or imported dogs with no travel history in the past 5 years. Most of these animals showed an antibody titre at the threshold level of 1:40, suggesting a low degree of parasite transmission/contacts. In 2 autochthonous cases with moderately high IFAT titre, the infection was confirmed by nested-PCR in peripheral blood. Thirty-one georeferenced sites were monitored for sand flies by means of interception (sticky papers) and attraction (CDC miniature light traps) collection devices. Traps were set during summer approximately on monthly basis, and extended up to October for positive sites. Only 2 sites were found positive for a total of 317 phlebotomine specimens collected by sticky traps, which included a previously known P. perniciosus-endemic site near Bolzano town. Sergentomyia minuta was by far the most prevalent (98.1%) and the only recorded sand fly species in the most northerly Italian site ever investigated (Coldrano municipality in Venosta valley). For the first time, Leishmania serology and n-PCR positive dogs autochthonous to APB were identified, however the spread of sand flies competent for L. infantum transmission could not be demonstrated in several places where endemic seropositive cases were recorded. APB can be considered a territory of low CanL endemicity, however awareness and continuous monitoring are needed to detect changes in the epidemiological status of the zoonosis.

Emerging feline vector-borne pathogens in Italy.

BACKGROUND: The epidemiology of feline vector-borne pathogens (FeVBPs) has been less investigated in cats than in dogs. The present study assessed the prevalence of Rickettsia spp., Babesia spp., Cytauxzoon spp. and Leishmania infantum infections in cat populations living in central Italy, by molecular and serological tools. RESULTS: A total of 286 healthy cats were randomly selected from catteries and colonies in central Italy. Peripheral blood and conjunctival swab (CS) samples were collected during surgical procedures for regional neutering projects. Sera were analysed by IFAT to detect anti-Rickettsia felis, R. conorii, Babesia microti and Leishmania IgG antibodies using commercial and home-made antigens. DNA extracted from buffy coats (BCs) was tested for Rickettsia spp., and Piroplasmida species, including Cytauxzoon spp. and Babesia spp. by PCR. Buffy coats and CS samples were assayed by a nested (n)-PCR for Leishmania spp. Sixty-two cats (21.67%) were seropositive to at least one of the tested pathogens. The serological assay revealed 23 (8.04%) and 18 (6.29%) positive cats for R. felis and R. conorii, respectively, with low titers (1/64-1/128). No antibodies against B. microti were detected. Neither Rickettsia nor Piroplasmida DNA were amplified using the specific PCR assays. Thirty-one cats (10.83%) tested positive to anti-Leishmania IgG, with titers ranging from 1:40 to 1:160 and 45 animals (15.73%) tested positive to Leishmania CS n-PCR, whereas none of the animals tested positive to BC n-PCR. Considering the results obtained by IFAT and CS n-PCR, a moderate agreement between the two tests was detected (κ = 0.27). CONCLUSIONS: The results of the serological and molecular surveys showed a moderate exposure to Leishmania in the investigated cats and highlighted the limited molecular diagnostic value of BC versus CS samples for this pathogen. Conversely no evidence supported the circulation of Cytauxzoon spp. in domestic cats, in contrast with previous detections in European wild cats in the same areas monitored. The low positive titres for R. felis in association with no DNA BC amplification prevent speculation on the exposure of feline populations to this FeVBP due to the cross-reactivity existing within spotted fever group rickettsiosis (SFGR).

An investigation of polymorphisms in innate and adaptive immune response genes in canine leishmaniosis.

The outcome of infection with Leishmania infantum in dogs is variable, which is thought to be due to the nature of the immune response mounted by the host. As a consequence, the clinical signs and severity of canine leishmaniosis vary between individual dogs. Host immunogenetic factors might play an important role in determining the outcome of infection. The aim of this study was to examine polymorphisms in innate and adaptive immune response genes, to determine whether any of these were associated with susceptibility or resistance to L. infantum infection. Genomic DNA was obtained from two groups: pet dogs in endemic regions of Europe and a group of Beagles exposed to sand fly infection as part of a vaccine study. Genotyping was performed using a SNP (single nucleotide polymorphism) array for selected immune response genes. The first part of the study compared 62 clinical cases with 101 clinically unaffected dogs that were seronegative for Leishmania antibodies. One SNP in the CIITA gene demonstrated a significantly higher minor allele frequency in the case group, compared with the control group at the individual SNP level after permutation, but was not significant after correction for multiple testing. The second part of the study examined 48 Beagle dogs exposed to L. infantum over two transmission seasons. Twenty-seven dogs with a resistant phenotype (no evidence of clinical disease, seronegative at the end of the study period, negative on lymph node culture and only transiently PCR positive in bone marrow) were compared with 21 dogs demonstrating a susceptible phenotype (clinical disease, seropositive, positive lymph node culture and consistently PCR positive in bone marrow). Three SNPs in TLR3, two SNPs in PTPN22 and one SNP in TLR4 and IL1A were associated with the susceptible phenotype in the Beagle group at the individual SNP level after permutation analysis, but were not significant after correction for multiple testing. Further validation of these SNPs is required in a larger cohort of dogs, ideally with extreme phenotypes to confirm an association with the outcome of L. infantum infection.

Nostril mass caused by Leishmania spp. in a dog.

Canine leishmaniasis (CanL) is a systemic zoonotic disease caused by the protozoan Leishmania, an intracellular macrophage parasite, transmitted by the bite of phlebotomine sandflies. In dogs, the clinical disease is mostly characterised by symptoms associated with viscerocutaneous lesions such as lymphadenopathy, splenomegaly, skin lesions, and renal and ocular disease caused by the deposition of immune complexes. The parasite may provoke mucosal lesions which cause atypical clinical signs. The aim of this study is to describe an atypical nostril mass in a dog infected by Leishmania. Clinical examination did not show any systemic clinical signs, while haematological, biochemical, and urinary parameters demonstrated a mild disease stage. Diagnosis was confirmed through the isolation of cultured live parasites by biopsy. The dog was treated with a combination of miltefosine and allopurinol, showing full remission of clinical symptoms after 2 months. The authors outline the importance of considering CanL in the differential diagnosis of mucous and tumour-like lesions.

Ongoing outbreak of cutaneous leishmaniasis in northwestern Yemen: clinicoepidemiologic, geographic, and taxonomic study.

BACKGROUND: Cutaneous leishmaniasis (CL) is widespread in Yemen but has not been fully documented. OBJECTIVES: This study aimed to investigate the clinicoepidemiologic and geographic aspects of CL in northwest Yemen and the taxonomic profile of the causative Leishmania species. METHODS: All CL cases diagnosed at the Dermatology Clinic of the Saudi Hospital at Hajjah during 1997-2012 were reviewed. Diagnoses were based on clinical, microscopic and, occasionally, histopathologic examinations. Leishmania species identification was carried out in 712 microscopically positive samples by multi-locus enzyme electrophoresis and polymerase chain reaction-restriction fragment length polymorphism. RESULTS: During the surveillance period, 1343 cases of CL were diagnosed. Lesions per patient ranged from one to 71, but most patients had a single facial lesion, classified as representing the "dry type" in 1315 (97.9%) and "wet type" in 28 (2.1%) patients. Leishmania typing in 576 cases identified Leishmania tropica as the main species responsible (n = 529), followed by Leishmania infantum (n = 20), Leishmania donovani (n = 11), and members of the L. donovani complex (n = 8). Atypical molecular patterns were observed in eight CL cases diagnosed in areas in which the three Leishmania species were found sympatrically. CONCLUSIONS: Cutaneous leishmaniasis appears to be endemic in northwest Yemen, where its incidence has recently increased abruptly. The disease presents clinically as the "dry type" and is caused mainly by L. tropica and occasionally by L. infantum, L. donovani, and L. donovani complex species. A sympatric diffusion of the three species is present in some governorates.

Hunting dogs as sentinel animals for monitoring infections with Trichinella spp. in wildlife.

BACKGROUND: Nematode parasites of the genus Trichinella are important foodborne pathogens transmitted by ingestion of striated muscles harbouring infective larvae. Wild carnivorous and omnivorous animals are the most important reservoirs of these parasites. Hunting activities play an important role in Trichinella spp. EPIDEMIOLOGY: The aim of the present work was to assess if serological detection of anti-Trichinella IgG in hunting dogs can be a tool to indirectly monitor Trichinella spp. infections in wildlife. METHODS: An ELISA and a Western blot (Wb) were developed and validated. To validate the assays, serum samples were collected from 598 dogs considered to be Trichinella-free, 15 naturally infected dogs, and six experimentally infected foxes. Sera were tested by ELISA with Trichinella spiralis excretory/secretory antigens. The diagnostic sensitivity and specificity of ELISA were 100 % (95 % CI: 83.89-100 %) and 95.65 % (95 % CI: 93.69-97.14 %), respectively. Sera from Trichinella-infected dogs/foxes tested by Wb showed a three-band pattern ranging from 48 to 72 kDa. Since the prevalence of Toxocara canis is very high in dogs, the specificity of the ELISA and Wb was further assessed by testing sera for anti-T. canis IgG using T. canis excretory/secretory antigens. No cross-reactivity was observed. To evaluate the test's reliability in the field, serum samples were collected from wild boar hunting dogs from Central Italy where Trichinella britovi was circulating among wildlife. RESULTS: Out of 384 hunting dog sera, 189 (49.2 %) tested positive by ELISA and of these, 56 (29.6 %) tested positive by Wb, showing an overall prevalence of 14.6 % (56/384) in the wild boar hunting dog population of the investigated area. The serological prevalence in hunting dogs was significantly (P < 0.001) associated with the hunting district's altitude. This is in agreement with previous investigations, which had shown that the prevalence of T. britovi in wildlife was higher in mountainous areas than in lowland areas of Italy. CONCLUSION: The results suggest that the circulation of Trichinella spp. among wildlife can be monitored by testing sera from hunting dogs, which could act as sentinel animals of Trichinella spp. circulation in wildlife.

Epidemiological survey on Leishmania infection in red foxes (Vulpes vulpes) and hunting dogs sharing the same rural area in Southern Italy.

Southern Italy, particularly Campania region, is an area where canine leishmaniasis (CanL) and zoonotic human visceral leishmaniasis (VL) are endemic. The red fox (Vulpes vulpes) has been hypothesized to play a role in occurrence of CanL in Italy but specific studies are poor. The aim of the present survey was to investigate the prevalence of Leishmania infection in dogs and foxes living in the same rural area (Picentini hills). 123 sera from autochthonous fox-hunting dogs were examined by immunofluorescent-antibody test (IFAT) using a cut-off of 1:160. The seroprevalence of dogs examined was 17.9%. Moreover, 48 foxes were examined after having been shooted by hunters or road accidents. Spleen, liver and lymph node samples were analyzed by specific Leishmania nested PCR (n-PCR). 10 foxes were found infected by L. infantum (20.8%) of which 4 animals in spleen, 2 in lymph nodes and 4 both in spleen and lymph nodes. The overall n-PCR positivity was 17.4% for spleen samples and 13.3% for lymph nodes; all liver samples resulted negative. In positive PCR foxes no signs clearly referable to leishmaniasis were recorded at necropsy. The results confirmed the presence of L. infantum infection in red foxes from Southern Italy, with a moderate level of exposure. Because large proportions of dogs with ascertained progressive leishmaniasis show a prolonged "subpatent condition" during which they are only positive to n-PCR before seroconversion, our results allow to assume that exposure risk in foxes is lower than hunting dogs living in the studied area.

Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. METHODOLOGY/PRINCIPAL FINDINGS: Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. CONCLUSIONS: These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

A randomised, double-blind, controlled efficacy trial of the LiESP/QA-21 vaccine in naïve dogs exposed to two leishmania infantum transmission seasons.

Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.

Sternal Aspiration of Bone Marrow in Dogs: A Practical Approach for Canine Leishmaniasis Diagnosis and Monitoring.

Bone-marrow aspirate material is commonly considered as one of the most sensitive tissues for a reliable diagnosis of leishmaniasis. The procedure herein described may permit less experienced veterinarians to be familiar with a quick and safe assessment method for leishmaniasis diagnosis in their patients. Animals are positioned in right lateral recumbency, and the area corresponding to the second, third, or fourth sternebra is identified and aseptically prepared. A 18-gauge needle connected to a 10 mL syringe is driven through the skin, up to the bone wall, and firmly pushed forward while rotating. Entry into the sternebra's cavity is clearly perceived by the fall of resistance offered by the cortex. Some 2,500 sternal bone-marrow samplings were safely and efficiently performed on 887 dogs of different breeds and aging from 6 months to 14 years, during eight years of clinical activity for routine diagnosis of canine leishmaniasis in pets or for the efficacy evaluation of anti-Leishmania immunobiologicals in dogs naturally exposed to parasite transmission. Most of the samples (1716) were from 387 dogs enrolled for anti-Leishmania vaccine studies. The safety of the method was particularly assessed on these dogs that as per study protocol were submitted to repeated bone-marrow aspirations (2-4 per year) in follow-up examinations.

Prospective study on the incidence and progression of clinical signs in naïve dogs naturally infected by Leishmania infantum.

The incidence of clinical and clinicopathological signs associated with the progression of infection was evaluated prospectively in 329 naïve young dogs exposed to Leishmania infantum transmission and examined periodically during 22 months (M). The dogs were part of Leishmania vaccine investigations performed under natural conditions. Vaccinated groups were considered in the evaluation when the vaccine resulted non-protective and the appearance and progression of signs did not differ statistically from controls at each time point, otherwise only control groups were included. 115 beagles were part of 3 studies (A to C) performed in the same kennel; 214 owned dogs (29 breeds, 2.3% beagles) were included in a study (D) performed in 45 endemic sites. At M22 the prevalence of any Leishmania infection stage classified as subpatent, active asymptomatic, or symptomatic was 59.8% in studies A-C and 29.2% in study D. Despite different breed composition and infection incidence, the relative proportion of active infections and the progression and type of clinical and clinicopathological signs have been similar in both study sets. All asymptomatic active infections recorded have invariably progressed to full-blown disease, resulting in 56 sick dogs at M22. In these dogs, lymph nodes enlargement and weight loss--recorded from M12--were the most common signs. Cutaneous signs were seen late (M18) and less frequently. Ocular signs appeared even later, being sporadically recorded at M22. Most clinicopathological alterations became evident from M12, although a few cases of thrombocytopenia or mild non-regenerative anemia were already observed at M6. Albumin/globulin inversions were recorded from M12 and urea/creatinine increase appeared mostly from M18. Altogether our findings indicate that any susceptible young dogs naturally infected by L. infantum present a common pattern of progression of signs during 2 years post infection, providing clues for medical and epidemiological applied aspects.

First record of autochthonous canine leishmaniasis in Hungary.

Hungary is traditionally regarded as a leishmaniasis-free country, and human or canine cases diagnosed locally have been recorded as imported. However, recent entomological surveys have verified the presence in Hungary of Phlebotomus neglectus and Phlebotomus perfiliewi perfiliewi, which have been incriminated as competent vectors of Leishmania infantum elsewhere in Europe. Following the occurrence in October 2007 of an undisputable clinical case of L. infantum canine leishmaniasis (CanL) in a 4-year-old female pug in a kennel of 20 dogs in Tolna province, an investigation was performed to assess the infection status in that canine population and to search for putative phlebotomine vectors. Another female pug became sick during the study period (May-November 2008) and L. infantum was confirmed as the causative agent. The other animals appeared clinically healthy; however, 4 additional dogs were found positive by indirect fluorescent antibody test (2 dogs), or by buffy-coat PCR (1 dog), or by both methods (1 dog). Hence the overall Leishmania infection prevalence in the kennel was 30% (6/20). All dogs were born in the same place and had been always kept outdoors. They had neither been abroad nor received a blood transfusion. No sand flies were collected with CDC Standard Miniature Light traps, Mosquito Magnet(®) X (MMX) dry ice-baited traps, or sticky traps placed either in or around the kennel and at nearby chicken yards during July and August of 2008 and 2009. Considering the dogs' historical background and the failure to trap any sand fly vectors in the kennel area, the origin of CanL in this site remains unexplained.

Longitudinal study on the detection of canine Leishmania infections by conjunctival swab analysis and correlation with entomological parameters.

A longitudinal study was carried out on kennelled stray dogs in a canine leishmaniasis (CanL) endemic area, to evaluate early and late diagnostic performance of a non-invasive conjunctival swab (CS) nested (n)-PCR analysis for Leishmania detection in 2 cohorts of dogs, respectively. (A) Sixty-five IFAT- and CS n-PCR-negative dogs exposed to, and followed up once or twice a month during a full sand fly season (July-November 2008). In parallel, a sand fly survey was performed on site using standard sticky traps set twice a month, for a cumulative surface of 63 m(2). (B) Seventeen IFAT- and CS n-PCR-negative dogs found positive in July 2008 at the peripheral blood buffy-coat (BC) n-PCR. These dogs were examined again by BC n-PCR in September and November 2008, and before the subsequent transmission season (May 2009) along with CS n-PCR and IFAT. None of the cohort (A) dogs converted to positive CS n-PCR during the transmission season. Although approximately 2500 phlebotomine specimens were collected with peaks of 100-147 specimens/m(2) sticky trap, the cumulative density of the only proven CanL vector in the area (Phlebotomus perniciosus) was found to be very low (0.5/m(2)). All cohort (B) dogs remained substantially seronegative; BC n-PCR showed an intermittent positive trend during the period surveyed, resulting in 82% conversions to negative by the end of the study, in contrast with 71% conversions to positive at the CS n-PCR analysis. In conclusion, while CS n-PCR was not found effective for the early detection of Leishmania contacts in dogs exposed to a low pressure of vectorial transmission, this assay showed to slowly convert to positive in a high rate of dogs, in the absence of seroconversion. CS n-PCR technique can be a suitable marker for assessing Leishmania exposure in dogs as a non-invasive alternative to current serological and molecular tools.