Recombinant forms of Leishmania amazonensis excreted/secreted promastigote surface antigen (PSA) induce protective immune responses in dogs

International audiencePreventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombi-nant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21-and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recom-binant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates

Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France : Double-blind randomised efficacy field trial

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. Dogs constitute the major reservoir of Leishmania infantum/chagasi responsible for human visceral leishmaniasis. We have recently demonstrated that the combination of naturally excreted/secreted antigens, easily purified from culture supernatant of Leishmania infantum promastigotes (LiESAp) as vaccine antigen in formulation with muramyl dipeptide (MDP) as adjuvant, conferred 100% protection to dogs experimentally infected with L. infantum by inducing in vaccinees a significant, stable and long-lasting Th1-type cell response [Lemesre JL, Holzmuller P, Cavaleyra M, Bras Goncalves R, Hottin G, Papierok G. Protection against experimental visceral leishmaniasis infection in dogs immunised with purified excreted secreted antigens of L. infantum promastigotes. Vaccine 2005; 23:2825-2840; Holzmuller P, Cavaleyra M, Moreaux J, Kovacic R, Vincendeau P, Papierok G, Lemesre JL. Lymphocytes of dogs immunised with purified excreted secreted antigens of L. infantum co-incubated with Leishmania-infected macrophages produce IFNgamma resulting in nitric oxide-mediated amastigote apoptosis. Vet. Immunol. Immunopathol. 2005, 106:247-257]. In this report, protection against visceral leishmaniasis is investigated in naturally exposed dogs of endemic areas of the South of France vaccinated with LiESAp/MDP vaccine. A double-blind randomised efficacy field trial was developed on a large-scale dog population composed of vaccinees (n = 205) and placebo-treated animals (n = 209), which were prospectively studied for a 2-year period. Of the initial 414 enrolled dogs, 340 (175 controls and 165 vaccinees) were analysed for clinical, serological and parasitological studies at 24 months post-vaccination, after two sand fly seasons. Strong seroconversion disclosed by an L. infantum indirect immunofluorescence antibody test (IFAT) associated with suspicious clinical symptoms, considered an indication that the animals had an established progressive infection, was only observed in the placebo group. The seropositive and/or symptomatic dogs were selected for further examination for possible Leishmania infection by culturing parasites from bone-marrow aspirate. The presence of leishmanial infection was also evaluated by means of the PCR analysis of bone marrow samples in all enrolled dogs prior to vaccination and in all evaluated animals (175 controls and 165 vaccinees) at 24 months post-vaccination. After two transmission cycles completed, the Leishmania infection rate was 0.61% (1/165) in vaccinated dogs and 6.86% (12/175) in the placebo group. The efficacy of the vaccine was calculated to be 92% (P = 0.002). A clear difference between the dogs that received vaccine and those that received placebo was also established by the results of their immune status. Increased anti-LiESAp IgG2 reactivity and significant enhanced NO-mediated anti-leishmanial activity of canine macrophages in response to higher IFN-gamma production by T cells were almost exclusively revealed in vaccinees. The LiESAp-MDP vaccine induced a significant, long-lasting and strong protective effect against canine visceral leishmaniasis in the field

Intoxicações natural e experimental por amitraz em eqüídeos: aspectos clínicos Natural and experimental poisoning by amitraz in horses and donkey: clinical aspects

A administração oral e a aspersão com amitraz reproduziram experimentalmente em 17eqüinos e um asinino um quadro de intoxicação muito similar a outro que vinha ocorrendo em cavalos no Estado do Rio de Janeiro. O início dos sintomas após a administração oral variou entre 15min. e 2h05min., na aplicação por aspersão variou entre 6h28min. e 8h38min. A evolução nos casos de administração oral foi de 4 a 9 dias, nos de aspersão de 5 a 6 dias. Somente morreram animais que receberam a administração oral. Um animal aspergido com o amitraz foi sacrificado. Por via oral foram usadas dosagens de 5,5 mg/kg (uma administração), 5,8 mg/kg (duas administrações) e num terceiro animal, doses que variaram entre 7,2 e 36,4 mg/kg (cinco administrações). Nas aplicações por aspersão, a intoxicação foi reproduzida com soluções nas concentrações de 0,1 e 0,2%. Com relação ao sistema nervoso, os principais sinais observados foram apatia, sonolência, ptoses palpebral e auricular, dificuldade de apreensão, mastigação e deglutição do alimento, arrastar das pinças dos cascos no solo, exposição do pênis, sensibilidade cutânea diminuída/ausente, instabilidade em estação, abdução dos membros, cabeça baixa, incoordenação, bocejos, flacidez labial, exposição da língua, cruzamento dos membros ao caminhar, resposta postural diminuída após cruzar e abduzir os membros, reflexos do lábio superior, palatal, lingual, de deglutição e flexor diminuídos/ausentes, reflexos auricular, palpebral e de ameaça diminuídos e resposta ambulatória diminuída ao teste de girar em círculo de pequeno raio. No que se refere ao sistema digestivo, foram evidenciados, principalmente, hipomotilidade/atonia intestinal, edema dos lábios, distensão abdominal, deitar e levantar com freqüência, rolar no solo, olhar para o flanco, gemer e impactação do intestino grosso. Observaram-se ainda taquicardia, aumento do tempo de preenchimento capilar e mucosas congestas, estridor, taquipnéia, dispnéia, secreção nasal, bradipnéia e respiração abdominal. Todos os três casos naturais ocorreram após aspersão do amitraz. Os primeiros sintomas foram observados 2 e 3 dias após o banho. A evolução foi de 6, 7 e 17 dias. Um animal manifestou a maioria dos sinais referentes ao sistema nervoso observados nos experimentos, com exceção dos sinais de cruzamento dos membros ao caminhar, bocejos, lábios flácidos e exposição do pênis. Outro animal, intoxicado espontaneamente, manifestou somente sintomas digestivos como rolar, ''patear'', hipomotilidade/atonia intestinal e impactação do intestino grosso. Um terceiro animal, inicialmente manifestou sintomas digestivos caracterizados por patear, rolar, atonia intestinal e impactação do intestino grosso, com conseqüente desenvolvimento de laminite; na fase final exibiu acentuada sintomatologia nervosa mostrando compressão da cabeça contra obstáculos, incoordenação motora com cruzamento dos membros ao caminhar e relutância em se movimentar. Baseados no quadro clínico observado, são sugeridos possíveis locais de lesão no sistema nervoso.<br>Poisoning by amitraz was experimentally reproduced in 17 horses and a donkey. First symptoms were observed between 15 min. and 2 h 5 min. after oral administration, and between 6 h 28 min. and 8 h 38 min. after spraying with amitraz. The course of poisoning after oral administration was 4 to 9 days, and after spraying 5 to 6 days. Death of experimental animals occurred only after oral administration. One animal was euthanized after spraying. Doses of 5.5 mg/kg (1 administration), 5.8 mg/kg (2 administrations) and doses which varied between 7.2 and 36.4 mg/kg (5 administrations) were used when amitraz was given by mouth, all causing symptoms of poisoning. When the administration of amitraz was by spraying, poisoning was reproduced with solutions of 0.1 and 0.2 % . Regarding the nervous system, the main signs observed were recumbency, somnolence, palpebral and auricular ptosis, difficulties in apprehension, chewing and swallowing of food, dragging of the hooves, exposure of the penis, diminished or absent cutaneous sensibility, instability, abduction of the legs, lowering of the head, incoordination, jawning, labial flacity, exposure of the tongue, crossing of the legs when walking, diminished postural response after crossing or abducting the legs, diminished/absent reflex of the upper lip, palatal, tongue, flexor and swallowing reflex, diminished auricular, palpebral and menace reflex. The ambulatory response was diminished when tested by walking in circles of small radius. Regarding the digestive system, the signs were mainly intestinal hypomotility/atony, edema of the lips, abdominal distention, frequent lying down and standing up, rolling on the ground, looking at the flancs, groaning and impaction of the large bowel. Regarding the circulatory system, the main clinical signs were tachycardia, increase of the refilling time of capillaries, congested mucosa and splitting of the cardiac sounds. Regarding the respiratory system, stridor, tachypnoea, dyspnoea, nasal discharge, bradypnoea and abdominal respiration was observed. Beside these signs, there were alterations of the general condition, as apathy and hypothermia. All natural cases occurred after spraying with amitraz. First symptoms were seen 2 to 3 days after the application of amitraz. The course was 6, 7 and 17 days. One animal showed mainly the nervous signs as seen in the experiments, with exception of the signs of crossing the legs when walking, yawning and exposure of the penis. Another animal had only digestive symptoms as rolling, pawing, intestinal hypomotility/atony and impaction of the large bowel. A third animal initially showed digestive symptoms characterized by pawing, rolling, intestinal atony and impaction of the large bowel, followed by laminitis; in the final stage this animal showed severe nervous signs as pressing the head against obstacles, incoordination with crossing the legs when walking and reluctance to move. Possible location of lesions in the nervous system according to the clinical signs are suggested