Risk factors for canine leishmaniasis in an endemic Mediterranean region

Human visceral leishmaniasis is an emergent/re-emergent parasitic zoonotic disease in Europe caused by Leishmania infantum, with domestic dog as its main reservoir host. This study presents the results of a canine epidemiological survey in a mediterranean region where human and canine leishmaniasis (CanL) are endemic - Portugal. The main goal was to identify risk factors, which can be relevant for Leishmania infection control. The national survey was carried out in January 2009 with a screening of 3974 dogs from all 18 districts of mainland Portugal. Direct Agglutination Test was used for the detection of anti-Leishmania antibodies in canine blood. An overall CanL true prevalence of 6.31% was observed. Apparent prevalence at district level ranged from 0.88% to 16.16%, with the highest prevalence in the interior regions. Identified risk factors for positivity were: dogs of 2 years and older (adjusted odds ratio OR=5.39); spending exclusively/most of the time outdoors (OR=2.51); origin from the interior of Portugal in comparison to littoral/coast districts (OR=2.51); not having long fur (OR=2.03); and being pure exotic (OR=1.67). The results confirm the leishmaniasis endemicity in Portugal and the dynamic character of prevalence as new foci emerged and old foci lost their importance. The dog's age, fur size, district and living outdoors as opposed to indoors were more important than dog breeds and insecticide treatment in the transmission of Leishmania infection. The future of CanL prevention and control rely on an integrated approach involving veterinarians, dog owners and health authorities in order to reduce the canine infection risk and consequently, the human zoonotic visceral leishmaniasis.     

Recombinant K39 dipstick immunochromatographic test: a new tool for the serodiagnosis of canine leishmaniasis.

The spread of human leishmaniasis has prompted the scientific community to study dogs as reservoirs for Leishmania infantum. Canine leishmaniasis (CanL) is widespread in the Mediterranean area with a prevalence of up to 50%. The first step toward controlling the disease is to monitor its distribution, mainly in stray dogs. The validity of a recombinant K39 (rK39) dipstick test, commercially available for the serodiagnosis of human leishmaniasis, was evaluated using sera from 165 dogs selected on the basis of positive or negative lymph node smears at parasitological examination. The results were compared with the indirect fluorescent antibody test (IFAT) (cutoff 1:80). Sera from a group of dogs with other diagnosed diseases but negative for leishmaniasis were also tested to evaluate any cross-reactivity. Various procedures were used for testing whole blood samples. The relative specificity of the rK39 dipstick and IFAT was 100% (97 of 97) and 98.97% (96 of 97), whereas the relative sensitivity was 97.06% (66 of 68) and 98.53% (67 of 68), respectively. The results of the dipstick and IFAT corresponded except for 2 sera (k = 0.987). This data confirm the usefulness of rK39 antigen for diagnosing CanL both in symptomatic and asymptomatic dogs. The rK39 dipstick proved to be a rapid, sensitive, and specific test that may be very useful in the field for large-scale screening and also in veterinary practice, requiring minimal equipment and operator expertise.     

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of S?o Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical signs of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Ara?atuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick-stained for optical microscopy. Direct immunofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific.     

Clinical and serological aspects of visceral leishmaniasis in northeast Brazilian dogs naturally infected with Leishmania chagasi

Human visceral leishmaniasis is endemic in the northeast of Brazil, where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. In this study, we evaluated the clinical signs of canine visceral leishmaniasis (CVL), serum protein profile and the antileishmanial IgG antibody production in 86 dogs living in northeast endemic areas of leishmaniasis. Thirty dogs from a leishmaniasis-free area were used as a control group. The major clinical signs of CVL seen were emaciation and skin ulcers (80%), followed by onychogryphosis and conjunctivitis (73%). Depilation was observed in 60% of animals while lymphadenomegaly, splenomegaly, liver enlargement or kidney involvement was less frequent (< or =20%). VL seropositive dogs presented with serum hyperproteinemia, hypoalbuminemia, hypergammaglobulinemia and decreased albumin/globulin ratio. A lower sensitivity and higher specificity was observed for promastigote indirect fluorescent antibody test (IFAT) (83 and 100%, respectively) compared with enzyme-linked immunosorbent assay (ELISA) (94 and 90%), which uses a crude extract of Leishmania. There was a positive correlation between IFAT and ELISA titers of antileishmanial IgG antibodies (Spearman test, P < 0.05), which was augmented in CVL dogs. This study found that the determination of serum protein, A/G ratio and the use of two different leishmanial serological tests like IFAT and ELISA are essential in CVL screening.     

Diagnosis of Canine Leishmaniasis in the Endemic Area of Belo Horizonte, Minas Gerais, Brazil by Parasite, Antibody and DNA Detection Assays

Canine leishmaniasis caused by Leishmania chagasi (L. infantum) is found throughout the South American continent, including Brazil, and dogs are considered to be the main reservoir host for this parasite. To support the implementation of a diagnostic protocol for surveillance of the disease in the region of Belo Horizonte (Minas Gerais, Brazil) we have compared the sensitivity and specificity of two serological tests, indirect immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), with the combination of direct microscopy–culture–PCR as the gold standard, using samples obtained from 103 dogs in the city of Belo Horizonte, Minas Gerais. The currently used standard serodiagnostic test, IFAT, had a sensitivity of 100% and its specificity was 74% compared to the gold standard of the study. The sensitivity and specificity of the DAT were 100% and 91%, respectively. On the basis of this study it is recommended to change from the IFAT to DAT for the serodiagnosis of canine leishmaniasis because of the superior specificity of the test combined with its user-friendliness.     

Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).     

Phenotype evaluation of human and canine isolates of Leishmania infantum

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.     

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.     

Questionnaire-based survey on the clinical management of canine leishmaniosis in the Madrid region (central Spain)

This paper describes a questionnaire designed to gain information on how veterinarians clinically manage canine leishmaniosis (CanL) in the Madrid region (central Spain). The present survey is one of the several similar questionnaire-based surveys conducted within the framework of the project EDEN (Emerging Diseases in a changing European eNvironment). The questionnaire sought to obtain data regarding the main clinical manifestations observed, the diagnostic methods used and the preventive measures recommended. Its Spanish version was sent by post to veterinary practitioners within the study area in two lots, one sent out in December 2006 and the other in March 2007. Only 174 of the 760 questionnaires sent were completed and returned (reply rate of 23%). Among the completed questionnaires, clinics differed widely in terms of features such as the habitats of the dogs (urban, peri-urban or rural) and patient volumes. Clinics attending dogs from peri-urban/rural habitats reported more suspected (p<0.001), confirmed (p=0.001) and newly diagnosed (p=0.001) cases/year than clinics providing service to a city clientele alone. According to the veterinary practitioners, skin lesions, lymphadenomegaly and weight loss were commonly observed, although these signs are not specific to CanL. Signs described to be of high diagnostic value were epistaxis and kidney disease. All the veterinarians polled reported that a suspicion of Leishmania infantum infection was confirmed by at least a serological method; the immunofluorescence antibody test (IFAT) being the technique most used. To prevent the disease, most vets recommended topical synthetic pyrethroids applied as impregnated collars or spot-ons. It is observed that despite considerable progress is being made in clinical management and controlling the disease, in Madrid Region its incidence continues to increase.     

One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis

The purpose of this international, multicentric, and non-comparative field trial was to obtain complementary data on long-term clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis (CanL). Seventy-four dogs with clinical signs of CanL and without severe renal failure were recruited in France, Spain and Italy, and 61 of them were part of the analysis. Each dog was treated with palatable tablets of marbofloxacin at 2 mg/kg once a day for 28 days. A clinical and parasitological follow-up was performed regularly up to 12 months. Efficacy was demonstrated in 42 dogs (68.9%), within 51 days (mean value), 10 of them (23.8%) being clinically cured after 3 months. A decrease of 61% in the sum of clinical scores was observed after 3 months. Haemato-biochemical parameters improved in general, supporting the observed clinical efficacy. Relapse was observed in 20/38 dogs (52.6%) approximately 5.5 months after treatment completion. The blood parasite load generally developed in conformity with the clinical outcome, even if exceptions were not rare. Lymph nodes remained positive by culture or PCR for a long time, even in dogs for which a good clinical response was observed. Despite the incomplete parasite clearance, as is also the case with other anti-leishmanial drugs, these results nevertheless confirm the relevance of marbofloxacin as a CanL treatment.     

Efficacy of a combination of 10% imidacloprid/50% permethrin for the prevention of leishmaniasis in kennelled dogs in an endemic area

The efficacy of imidacloprid 10%and permethrin 50% (Advantix; Bayer AG, Germany) in a spot-on formulation was evaluated in the field as a control measure to prevent canine leishmaniasis (CanL) in dogs in an endemic area of southern Italy. In February 2005, out of 845 dogs initially tested for CanL, 631 dogs which tested negative (315 from a kennel in Bari (KB) and 316 from a kennel in Ginosa (KG)) in a serological and a parasitological examination were allocated to one of three groups: Group A-treated with imidacloprid 10% and permethrin 50% once a month; Group B-treated every 2 weeks; and Group C-untreated control animals. All the dogs were examined serologically and parasitologically for CanL prior to the start of the study, in November 2005 (end of the sandfly season) and in March 2006 (end of the study). An initial CanL seroprevalence of 24.7% (209 dogs) was detected in KB and KG. In KB Leishmania infection, inferred by positivity in at least one of the three tests performed at the interim or final follow-up, was found in one animal from Group A and in nine from Group C. No positive animals were detected in Group B, thus giving a final protection efficacy of 88.9% in Group A and 100% in Group B. In KG Leishmania infection was identified in one animal from Groups A and B, respectively, and 11 from Group C (protection efficacy of 90.36% in Group A and 90.73% in Group B). The incidence density rates (IDRs) of infection in both Groups A and B at each kennel were statistically significantly lower than that registered in Group C (KB p<0.05 and KG p<0.01). The results clearly show that a combination of imidacloprid 10% and permethrin 50%, by virtue of its repellent activity against sandflies, is effective under both application regimes in preventing CanL in the field in endemic areas.     

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.     

Comparison of two immunochromatographic assays and the indirect immunofluorscence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana

Two rapid tests evaluated in dogs considered to be of high risk of infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays: Trypanosoma Detect™ for canine, InBios, Seattle, WA and CHAGAS STAT-PAK™ assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test. From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT. The serological survey using IFAT showed a prevalence of T. cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays, 13 and 11 animals were positive on rapid assay: Trypanosoma Detect™ for canine, InBios and CHAGAS STAT-PAK™, Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T. cruzi infection. Early detection of T. cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk, clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana.     

Importance of Lutzomyia longipalpis in the dynamics of transmission of canine visceral leishmaniasis in the endemic area of Porteirinha Municipality, Minas Gerais, Brazil

A study of Lutzomyia longipalpis (Lutz and Neiva, 1912) (Diptera: Psychodidae), the primary vector of American visceral leishmaniasis (AVL), and the canine form of the disease, was carried out in Porteirinha. The city is situated in the northern part of the Brazilian State of Minas Gerais and is an endemic area of AVL. Systematic phlebotomine captures were performed in seven districts with previously reported cases of canine visceral leishmaniasis, during 2 years (January 2000--December 2001). A total of 2328 specimens of L. longipalpis were captured. The association between the local climate variables and the population density of L. longipalpis was evaluated and rainfall was determined to be a major factor, with increased populations during the rainy season (October--March). At the same time period, blood samples from every dog domiciled in the same seven districts, in total 14,077 animals, were analyzed for infection by viscerotropic Leishmania using indirect immunofluorescence assay (IFA). Accumulated incidence rates of canine VL per district varied from 3.40 to 14.34 for the 2-year period. A positive correlation between the population density of L. longipalpis and the canine cases of visceral leishmaniasis in Porteirinha was observed.     

Laboratory tests for diagnosing and monitoring canine leishmaniasis

Although several reviews on canine leishmaniasis have been published, none thoroughly described clinicopathologic abnormalities and their clinical usefulness. The aim of this review was to provide information concerning current diagnostic tests relevant for clinical pathologists and from a practical perspective. Specifically, in canine leishmaniasis, nonregenerative normocytic normochromic anemia, thrombocytopenia, or leukogram changes may be present. Clinical chemistry and urinalysis may indicate renal dysfunction (azotemia, decreased urine specific gravity, proteinuria) and an inflammatory/immune response (increased acute phase proteins [APP] or α₂‐ and/or γ‐globulins). Although a potential gammopathy is usually polyclonal, it may also appear oligo‐ or monoclonal, especially in dogs coinfected by other vector‐borne pathogens. When lesions are accessible to fine‐needle aspiration (lymphoadenomegaly, nodular lesions, joint swelling), cytology is strongly advised, as the presence of Leishmania amastigotes in a pattern of pyogranulomatous inflammation or lymphoplasmacytic hyperplasia is diagnostic. If the cytologic pattern is inconclusive, the parasite should be identified by histology/immunohistochemistry or PCR on surgical biopsies. Alternatively, cytology and PCR may be performed on bone marrow samples where amastigotes, along with erythroid hypoplasia, myeloid hyperplasia, plasmacytosis, or secondary dysmyelopoiesis can be observed. Dogs with overt leishmaniasis generally have high antibody titers, while low titers predominate in immunologically resistant infected dogs or in exposed dogs with no parasite confirmation. Quantitative serology is recommended in clinically suspect dogs as high‐titer antibodies titers may confirm the clinical diagnosis. In confirmed and treated dogs, renal function and inflammatory/immune response variables should be periodically monitored.     

Seroprevalence of Leishmania sp. Infection in Healthy Horses Housed in Endemic Areas in Tuscany

This study carried out an epidemiological survey of seroprevalence of positive immunofluorescence antibody test (IFAT) results for Leishmania infantum in horses living in Tuscany, where the disease in the dog is endemic and no cases of equine leishmaniasis were observed. Inclusion criteria were (1) horses housed for more than 2 years in endemic areas; (2) horses grazing 24 hours a day outside; and (3) horses living on farms where affected dogs were housed. Two blood samples each were collected from 277 horses from June to October 2011 (T1) and from December 2011 to February 2012 (T2), and IFAT was performed for L. infantum. A dermatologic examination was performed to detect the presence of skin lesions. No animals had skin abnormalities. At T1, 18 of 277 horses had positive results for IFAT, while at the second sampling (T2) 277 of 277 samples were negative. In conclusion, our seroprevalence is lower than that in Spain but higher than that in Greece. Our results suggest the presence of a transient humoral response to L. infantum in horses.     

Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis.After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role.The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis.Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Incidences of canine leishmaniasis in an endemic area of southern Italy

Canine leishmaniasis (CanL), caused by Leishmania infantum, is widely distributed in many Mediterranean countries and is considered endemic in southern and central Italy with prevalence reaching up to 48.4%. Determination of the incidence would be useful as a measure of the risk of infection, then to evaluate the usefulness of control measures and to estimate whether a new focus is autochthonous or imported. This study was performed on two sites in the Apulia region of southern Italy, namely sites A and B. A total of 262 dogs were included in the evaluation of incidence, 94 farm dogs from site A and 168 dogs (92 farm and 76 kennel dogs) from site B. The incidence of infection was determined by using two different approaches: in site A by means of incidence density rate (IDR); in site B by the yearly seroconversion rate. In site A, the IDR was calculated at 4.25% dog-years; in site B the yearly incidence rate was of 9.52% (6.5% and 13.1% in farm and kennel dogs, respectively). The strength and weakness of the two different approaches (i.e. annual monitoring or monthly interval monitoring) for calculating the incidence of CanL in an endemic area have been discussed.     

Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.