Angelo Elio Gravino

Evidence for a relationship between Leishmania load and clinical manifestations.

Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmaniainfantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis.

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.

Presence of Leishmania infantum in red foxes (Vulpes vulpes) in Southern Italy

Skin, lymph node (popliteal), and bone marrow samples were collected from 50 red foxes (Vulpes vulpes) from May 2004 to May 2005 in southern Italy. Samples were tested for Leishmania infantum by polymerase chain reaction (PCR). The parasite was detected by PCR from 20 of 50 (40%) fox carcasses. All 20 positive cases were PCR-positive from lymph node and bone marrow samples, whereas 17 of 20 positive cases were PCR-positive from skin samples. Infection status was not related to age or sex. This is the first report of leishmaniasis in red foxes in Italy based on PCR results, and these results reinforce the assumption that this wild canid can serve as a reservoir for Leishmania.

Leishmania DNA Quantification by Real-time PCR in Naturally Infected Dogs Treated with Miltefosine

A new drug that has just become available in India for treatment of human visceral leishmaniasis (VL) is miltefosine, an alkyphospholipid that was originally developed as an oral antineoplastic agent. Miltefosine is not only directly toxic for Leishmania parasite, but it also enhances both T cell and macrophage activation and production of microbicidal reactive nitrogen and oxygen intermediates. It is highly effective in the treatment of Leishmania infection in mice and human beings. However, adverse effects in dogs treated with miltefosine have been reported, but there are no data on the efficacy of this drug for the treatment of canine visceral leishmaniasis (CVL). The aim of this study was to use a real‐time PCR assay to monitor the Leishmania load in the blood samples and lymph node aspirates of 18 naturally infected dogs before and after treatment with miltefosine (2 mg/kg for 30 days). The results of our study showed that the therapy with miltefosine shows a drastic and progressive reduction of parasite load in lymph node aspirates, but does not suppress the parasite in lymph nodes. In all dogs the real‐time PCR assay demonstrated an irregular presence of parasites in blood. Therefore, blood does not seem a suitable substrate for the purpose of quantifying Leishmania DNA.

Interferon-gamma (INF-γ), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol

In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmaniainfantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-gamma and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-gamma and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment.

Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.

Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report

BackgroundTransmission of canine leishmaniasis (CanL), a severe infection caused by L. infantum, usually occurs through the sand fly bite to the vertebrate host. A venereal route of transmission has also been suggested, but this issue is still controversial.FindingsHere, we report a case of a dog affected by orchitis showing a clinical profile of L. infantum infection. By exploiting a real-time PCR assay, we detected a significantly higher DNA load of the parasite in the lymph node and testis than in blood and urine samples collected from the dog.ConclusionsOur results suggest that: 1) L. infantum infection can be associated with testicular lesions in naturally infected dogs; 2) genital involvement could result in shedding of the parasites in the semen, favoring venereal transmission of the disease.

Development and Clinical Trial of a Novel DNA Vaccine as Immunotherapy during Canine Leishmaniasis

Background: Visceral Leishmaniasis (VL) is a zoonotic disease caused by Leishmaniainfantum in the Mediterranean area. Dogs are the main reservoir of Leishmaniainf antum parasites. Disease management represents a serious problem, since anti-Leishmania drugs have limited efficacy in both symptomatic and asymptomatic dogs, which are infective to phlebotomine vectors. In many tropical and sub-tropical countries the development of a safe and easily-available vaccine has high priority. DNA vaccines represent one of the most recent innovations in the field of immunization. Findings: This study aimed to evaluate the effect of a DNA vaccine based on two Leishmania antigens (Cpb1, PO) in leishmaniotic dogs. Twelve leishmaniotic dogs from a Leishmaniasis-endemic area (Naples, Italy) received three consecutive injections of DNA vaccine at 15-days intervals. Another group of five leishmaniotic dogs received the same amount of pVAX-1 without the coding sequences of Leishmania antigens. Leishmania DNA load, INFγ, IL-4 mRNA expression levels and clinical parameters were tested before and after the therapy, every 3 months for a period of 12 months. Analysis of the data in the vaccinated dogs showed: i) a decrease Leishmania DNA load in lymph node samples, ii) an increase of INFγ and IL-4 mRNA expression levels in PBMC samples. All vaccinated dogs also showed an improvement in the clinical symptoms. Conclusion: Our results show that the vaccine developed in this study may represent a useful tool in the treatment of leishmaniotic dogs. However, since the duration of positive effects is limited in time, further trials are needed to evaluate the effectiveness of immunotherapy alone or in association with conventional therapy.

Fluorescence-based Detection of Leishmania infantum DNA in Phlebotomus Vectors

The geographical diffusion of leishmaniasis can depend on the factors limiting the distribution of sandfly vectors. In Sicily, as in all Mediterranean areas, sandflies are present almost all year round because the climate permits perpetuation of this vectors life cycle. Transmission can occurs in rural and domestic habitats through the bite of infected female phlebotomine sandflies. In Italy, the visceral form of leishmaniasis is commonly found, which is due exclusively to L. infantum. Two species of sandfly are considered the main vectors: Phlebotomus perniciosus and P. perfiliewi. Fluorescence resonance energy transfer technology was used to determine the parasite load in phlebotomine vectors, and the test was targeted on a 117‐bp fragment of kinetoplast DNA minicircles. The assay was evaluated, focusing on analytical sensitivity, discriminatory power, and reliability of quantification. During 2005, a total of 1686 sandflies were trapped in various Sicilian provinces and in farms randomly selected using black light traps. We found 20, 30, and 16 sandflies positive for Leishmania for each kind of analyzed phlebotomine sandfly, respectively, corresponding to 6.5% for the gravid, 2.7% for the fed, and 6.3% for other groups. Previously the insects were identified on the basis of morphology and the most prevalent sandfly species were P. Sergentomyia minuta, P. perfiliewi, and P. perniciosus.