The clinical and laboratory findings observed in 150 dogs naturally infected by Leishmania infantum, from a large endemic area of southern Italy, are described. There was a gradual onset of clinical signs and the course of the disease was progressive in almost all the cases. The majority of the dogs were mongrels (43.3 per cent), male (64.7 per cent), of medium size (50.6 per cent), three to seven years old (64.7 per cent), and living outdoors (60 per cent). They showed generalised (56.7 per cent) or symmetrical (32 per cent) lymphadenomegaly; the mucous membranes of 87 of the dogs (58 per cent) were pale and moderate or severe splenomegaly was diagnosed in 80 dogs (53.3 per cent); weight loss was observed in 32 per cent of the animals. Skin abnormalities were very common, and included dry exfoliative dermatitis (56 per cent), ulcers (40 per cent) periorbital alopecia ('lunettes') (18 per cent), diffuse alopecia (14 per cent) and onychogryphosis (24 per cent). Ocular signs were observed in 24 dogs (16 per cent) including 16 cases of keratoconjunctivitis (three with keratoconjunctivitis sicca), six cases of moderate uveitis and two cases of panophthalmitis. The acute form of the disease was diagnosed in only six dogs and was characterised by fever and generalised lymphadenomegaly, and by the absence of skin lesions. Another six dogs had severe renal failure without systemic clinical signs of leishmaniasis. The most important laboratory findings were a severe or moderate increase in gammaglobulins, hypoalbuminaemia, hyperproteinemia and anaemia. Cultures or cytology tests for L infantum parasites were positive in 134 of the dogs. Following the standard procedures developed for human lymph node and bone marrow cytology tests, the leishmania density in the dogs varied from 1+ to 2+. Leishmania antibody titres were high (> 1:160) in almost all the dogs. Immunological tests for autoantibodies were positive in 25 of 53 dogs tested in the antinuclear antibody (ANA) test, in 15 of 43 dogs tested in the latex test and in five of 24 dogs tested in the Coombs test.

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BackgroundPhlebotomine 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 FindingsSera 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.ConclusionsThese 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.

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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.

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Serologic assays using crude antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus type 1 (HIV)-seropositive patients have been shown to lack sensitivity and specificity, particularly in AIDS patients. Antibodies to a cloned antigen, recombinant (r) K39, of Leishmania chagasi are specific for members of the Leishmania donovani complex and have been shown to indicate active disease in immunocompetent persons. This study demonstrated that antibodies to rK39 were also detectable in HIV-seropositive patients coinfected with Leishmania infantum. Furthermore, the rK39 ELISA was more sensitive than an IFA for detecting L. infantum infections in patients with AIDS. In addition, antibody titers to rK39 in HIV-negative patients infected with L. infantum or L. chagasi declined during treatment with meglumine antimoniate or liposomal amphotericin B. In contrast, most patients who clinically relapsed showed increased antibody titers to rK39. These data demonstrate the diagnostic and prognostic utility of rK39 in detecting active visceral leishmaniasis.