Gaetano Oliva

A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum

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.

Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis

Canine leishmaniosis (CanL) due to Leishmania infantum is a life threatening zoonotic disease with a wide distribution in four continents and importance also in non-endemic regions. The purpose of this report is to present a consensus of opinions on the diagnosis, treatment, prognosis and prevention of CanL in order to standardize the management of this infection. CanL is a disease in which infection does not equal clinical illness due to the high prevalence of subclinical infection among endemic canine populations. The most useful diagnostic approaches include serology by quantitative techniques and PCR. High antibody levels are associated with severe parasitism and disease and are diagnostic of clinical leishmaniosis. However, the presence of lower antibody levels is not necessarily indicative of disease and further work-up is necessary to confirm CanL by other diagnostic methods such as cytology, histopathology and PCR. We propose a system of four clinical stages, based on clinical signs, clinicopathological abnormalities and serological status. Suitable therapy and expected prognosis are presented for each of the stages. The combination of meglumine antimoniate and allopurinol constitutes the first line pharmaceutical protocol. However, although most dogs recover clinically after therapy, complete elimination of the parasite is usually not achieved and infected dogs may eventually relapse. Follow-up of treated dogs with blood counts, serum biochemistry, urinalysis, serology and PCR is essential for prevention of relapses. Protection against sand fly bites by topical insecticides is effective in reducing infection, and recent development of vaccines has indicated that prevention by vaccination is feasible.

LeishVet guidelines for the practical management of canine leishmaniosis.

The LeishVet group has formed recommendations designed primarily to help the veterinary clinician in the management of canine leishmaniosis. The complexity of this zoonotic infection and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of canine leishmaniosis challenging. The recommendations were constructed by combining a comprehensive review of evidence-based studies, extensive clinical experience and critical consensus opinion discussions. The guidelines presented here in a short version with graphical topic displays suggest standardized and rational approaches to the diagnosis, treatment, follow-up, control and prevention of canine leishmaniosis. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient.

Incidence and Time Course of Leishmania infantum Infections Examined by Parasitological, Serologic, and Nested-PCR Techniques in a Cohort of Naïve Dogs Exposed to Three Consecutive Transmission Seasons

ABSTRACT Most experience in the comparison of diagnostic tools for canine leishmaniasis comes from cross-sectional surveys of dogs of different ages and breeds and in cases with unknown onset and duration of leishmaniasis. A longitudinal study was performed on 43 beagle dogs exposed to three transmission seasons (2002 to 2004) of Mediterranean leishmaniasis and examined periodically over 32 months through bone marrow microscopy and nested PCR (n-PCR), lymph node culture, serology (immunofluorescent-antibody test), and evaluation of clinical parameters. Starting from January 2003, the highest rate of positives was detected by n-PCR at all assessments (from 23.3% to 97.3%). Sensitivities of serologic and parasitological techniques were lower but increased with time, from 15.8% to 75.0 to 77.8%. Some dogs that tested positive by n-PCR but negative by other tests (“subpatent infection”) remained so until the end of the study or converted to negative in subsequent assessments, whereas all dogs with positive serology and/or microscopy/culture (“asymptomatic patent infection”) exhibited progressive leishmaniasis; 68% of them developed clinical disease (“symptomatic patent infection”) during the study, at 7 (range, 3 to 14) months after being positive to all tests. Postexposure infection incidences were high and were significantly different between 2002 and 2003 exposures (39.5% and 91.7%, respectively). The time course of infection was highly variable in each dog, with three patterns being identified: (i) rapid establishment of a patent condition (0 to 2 months from detection of infection); (ii) a prolonged subpatent condition (4 to 22 months) before progression; and (iii) a transient subpatent condition followed by 10 to 21 months of apparent Leishmania-negative status before progression.

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.

Guidelines for treatment of leishmaniasis in dogs

rug treatment of leishmaniasis in dogs is a chal-lenge for veterinary practitioners. Because of its complex pathogenesis, leishmaniasis may manifest with various clinical signs, ranging from mild and nonspe-cific to those reflecting severe involvement of several organs. The immune response plays an important role in the development, outcome, and response to treat-ment of

Multicentric, controlled clinical study to evaluate effectiveness and safety of miltefosine and allopurinol for canine leishmaniosis.

The aim of this trial was to evaluate the effectiveness and safety of miltefosine-allopurinol combination therapy vs. the current reference combination therapy, meglumine antimoniate-allopurinol, for canine leishmaniosis. Dogs included in the study exhibited clinical signs of the disease, were positive by PCR and serologically positive by immunofluorescent antibody test for leishmaniosis, and negative for ehrlichiosis. Dogs were divided into two groups: Group 1 was treated with 2 mg/kg of miltefosine orally once daily for 28 days and 10 mg/kg of allopurinol orally twice daily for 7 months; Group 2 was treated with 50 mg/kg of meglumine antimoniate sub-cutaneously twice daily for 28 days and allopurinol (same dose as Group 1) for 7 months. Dogs were examined according to the following schedule: pre-inclusion, Day 0 (D0), D14, D28, D84, D140 and D196. At each visit, blood, urine and bone marrow samples were collected. Parameters monitored included haematology, biochemistry, protein electrophoresis, serology, urinary protein/creatinine ratio and RTQ-PCR performed on bone marrow aspirates. A significant reduction in total clinical score and parasite load was observed in both groups over the 7-month study period (P < 0.0001), with no significant difference between groups (P = 0.3). The safety of miltefosine-allopurinol combination therapy was confirmed by lack of effect on renal and hepatic parameters and adverse reactions. Miltefosine, in combination with allopurinol, offers a safe, convenient and effective alternative treatment option for canine leishmaniosis compared to the reference therapy.

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.

A Randomised, Double-Blind, Controlled Efficacy Trial of the LiESP/QA-21 Vaccine in Naive 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.

A New Model of Progressive Visceral Leishmaniasis in Hamsters by Natural Transmission via Bites of Vector Sand Flies

BACKGROUND Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. METHODS We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. RESULTS Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)-10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%-82%) rather than total GMPL (2.0 × 10(4)-8.0 × 10(4)) per midgut. CONCLUSIONS This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL.