Part 7 WORKSHOP REPORTS

Transcription

1 Part 7 WORKSHOP REPORTS Co-editors: Dr Karen L. Campbell, University of Illinois, Urbana, USA and University of Missouri Veterinary Health Center, Wentzville, USA Dr Kinga Gortel, Lake Country Veterinary Specialist Hospital, Lake Country, BC, Canada

2 Vaccines for canine leishmaniasis 7.1 C. Noli 1 (Chairperson) and A. Fondati 2 (Secretary) 1 Servizi Dermatologici Veterinari, Peveragno, Italy 2 Ambulatorio Veterinario Trastevere-Veterinaria Cetego, Rome; Clinica Veterinaria Colombo, Lido di Camaiore, Italy Introduction on vaccines in leishmaniasis (C. Noli) Chiara Noli (Italy) welcomed the attendants to the workshop and opened it by briefly explaining that canine leishmaniasis (CanL) is a severe, chronic, vector-borne, protozoan disease which cannot be easily cured with current therapies. In endemic areas it is estimated that approximately 30% of dogs are seropositive and some will eventually become clinically ill. A vaccine capable of preventing the development of the disease after the animal has been infected would be highly desirable. It would protect vaccinated dogs and, through reducing disease incidence, also decrease infection rates in non-vaccinated dogs and humans. To be effective the vaccine should induce a strong and long-lasting Th1-mediated immune response. Different antigens and adjuvants, as well as different combinations of antigens and adjuvants, have been investigated during the past 15 years. They have led to the development of three generations of vaccines, based on antigen type: 1 attenuated or killed Leishmania parasites, 2 purified or recombinant Leishmania proteins, 3 DNA-encoding Leishmania or salivary sandfly proteins. Different types of antigen-delivery system have also been investigated, including liposomes, nanomaterials and electroporation. However, due to difficulties in the production of Leishmania vaccines, only the following products have reached the market thus far: Leish-Tec (Hertape Calier Saude Animal S.A., Juatuba, Brazil) and Leishmune (Fort Dodge Saude Animal Ltd, Campinas, Brazil) in Latin America and CaniLeish (Virbac S.A., Carros, France) in Europe. a Leishmune has been removed from the market and only Leish-Tec is available in Latin America. Unfortunately, none of these vaccines offers 100% protection against the disease. Based on published data, the overall absolute risk reduction in progression to symptomatic active infection for Leishmania vaccines ranges from 0.2 to 0.54 and the clinical efficacy ranges from 57 to 80%. CaniLeish was released in Europe approximately 5 years ago and still there are many questions about its use. Hopefully, invited speakers will help to answer some of these questions, including, for example, the following. 1 When should we advise the owner to vaccinate his or her dog in endemic and non-endemic areas? (P. Bourdeau) 2 It is recommended by the CaniLeish manufacturer that only healthy dogs, serologically negative for leishmaniasis should be vaccinated. How do we define a healthy dog? (M. Saridomichelakis) 3 Which cellular and humoral immune responses should we expect in a vaccinated dog? (L. Solano Gallego) 4 Is this vaccinated dog infected or ill? How can we confirm or rule out leishmaniasis in seropositive vaccinated dogs? (L. Ordeix) 5 Leishmania vaccine side effects: facts or urban legends? (L. Ferrer) Chiara Noli thanked the attendees for participating and the Secretary (A. Fondati). She then introduced the next speaker. Should my dog be vaccinated? Necessity of vaccination in endemic and non-endemic areas (P. Bourdeau) Patrick Bourdeau (France) explained that the rationale for vaccination in CanL is based on the elicitation of a specific and protective Th1-mediated immune response. If infection occurs, the vaccine should protect against the disease or reduce its severity. In fact, the aim of the currently available vaccines is protection not against infection but against its consequences. For this reason, vaccination should be considered only in dogs already protected by insecticides having a strong, clearly demonstrated repellent activity against sandflies. Vaccinating such dogs would integrate the protective, albeit incomplete, effect of insecticides. As for any vaccine, only Leishmania vaccines with efficacy recognized by the European Medicines Agency (EMA) should be used, manufacturer recommendations must be followed and the balance between benefit (efficacy) and risk (side effects) must be evaluated. When considering whether or not to vaccinate dogs, several factors have to be taken into account, including the duration of exposure to sandflies and the endemicity of the zone where the patient resides. In some endemic (syn. enzootic) areas, exposure of dogs to infecting sandfly bites may occur up to 8 months per year. In these areas, aside from a few exceptions, promoting vaccination in the dog population is justified. On the other hand, non-endemic areas are characterized by the absence or a reduced number of vectors and a 2017 The Authors. Compilation 2017 ESVD and ACVD 187

3 Workshops low number of infected sick dogs. In these areas the risk of vectorial transmission of CanL is negligible and vaccination does not provide significant benefits. Special attention has to be paid to fringe (syn. periendemic) areas such as northern Italy, northern Spain or southwestern France, which are at the periphery of wellidentified endemic zones. Here the disease is rapidly expanding through the increase of both sandfly populations and of dogs travelling to and from enzootic zones. In addition, Leishmania parasites are likely transferred from enzootic to fringe areas by the movements of infected wild animals. Therefore, these areas should be considered equal to enzootic zones and vaccination should be recommended. In a collective prophylactic approach, if a high percentage of susceptible dogs were vaccinated, CanL expansion could be reduced. However, the global prophylactic benefit would be limited if wild animals play an important role in CanL expansion. For dogs travelling from non-endemic to endemic areas the choice to vaccinate will depend on duration and/or frequency of travels during the sandfly season. For short visits (e.g. 1 month), adequate protection can be afforded by insecticides alone and vaccination might be optional. On the other hand, vaccination should be considered with repeated or longer visits. It has been estimated by a mathematical model 1 that the combination of vaccination and insecticides in dogs imported from a non-endemic to an endemic area is more effective than either measure alone for preventing the development of active CanL infection. Chiara Noli thanked Patrick Bourdeau for his contribution, opened the discussion and asked Patrick Bourdeau s advice about vaccination for those dogs living in nonendemic areas, such as Germany, spending, for instance, 1 week during the owner s vacation in a highly endemic area, such as Spain. Patrick Bourdeau remarked that vaccination of dogs that are going to spend up to 2 months in endemic areas could be done but is probably not very useful. Two more aspects have to be taken into account before recommending vaccination: namely, financial aspects and the delay of vaccination effects. To be effective the vaccination protocol should be started many weeks before travelling. When financial constraints are a concern, insecticides should be the first choice. Nevertheless, vaccination should be promoted in endemic areas because, despite not being protective against infection, it is protective against disease. In addition, Leishmania vaccine reduces dogs infectivity and, as a result, the risk of infection to other dogs living in the area. Chiara Noli summarized the messages from the previous presentation. The use of insecticides with repellent activity should be mandatory for all dogs living in or travelling to endemic areas. Vaccination is recommended for dogs living in endemic regions and is also suggested for dogs that move from non-endemic to endemic regions for a long period of time. Provided that vaccination does not represent a financial problem, dogs spending short periods of time in endemic areas might also be vaccinated. Patrick Bourdeau remarked that the combination of repellents and vaccine should also be promoted in dogs living in periendemic areas, in order to reduce the rapidity of infection and disease expansion. Alessandra Fondati (Italy) asked if pyrethroids can be considered true repellents; in other words, whether or not pyrethroids prevent sandfly contact with the skin of treated dogs. Patrick Bourdeau explained that pyrethroids do not possess a true repellent activity; that is, they do not keep insects at a distance. Sandflies do contact the dogs skin but they do not bite because they die rapidly or are repelled by contact with pyrethroids. Pure repellents have no residual effect and, so far, no marketed veterinary product has true repellent activity. While companies state that pyrethroids have an anti-feeding effect on sandflies, this lack of feeding is the result of a rapid killing effect combined with a repellent effect that prevents bites. Laura Ordeix (Spain) asked for information about the length of the interval between the beginning of the vaccination protocol and travel to endemic regions. Patrick Bourdeau replied that vaccine protection is complete 1 month after the last injection, when a Th1 cell response has developed, according to experimental studies. Therefore, the vaccination schedule should be completed 1 month before exposure. Alessandra Fondati asked whether it would be appropriate to personalize vaccine recommendations depending, for example, on a dog s breed, age or lifestyle. Patrick Bourdeau commented that data on the variability of vaccination efficacy with dog breeds are lacking. However, there is likely to be individual variation of vaccination responses. Guadalupe Miro (Spain) pointed out that tailoring the vaccination to different factors, for example a dog s lifestyle, time of exposure to sandflies and other factors, is very important. Recommendations for a hunting dog should differ from those for a Chihuahua living in a carrying bag. Chiara Noli introduced and welcomed the next speaker and highlighted that given the high prevalence of infection in endemic areas, deciding which dogs are truly healthy that is, suitable for vaccination can be challenging. Can I vaccinate this dog? Is this dog healthy enough to be vaccinated? Criteria to decide if a dog is really healthy (M. Saridomichelakis) Manolis Saridomichelakis (Greece) began by stating that at the time of vaccination against an infectious agent, it is usually necessary to ascertain that the dog is not infected by that agent. However, in endemic areas, many seemingly healthy dogs that are candidates for vaccination against CanL are actually infected by Leishmania 188

4 Vaccines for canine leishmaniasis infantum. Subsequently, even if the vaccination schedule starts early in life, it is probable that a large number of infected dogs will be vaccinated against L. infantum. To vaccinate dogs, clinicians should confirm that they are clinically healthy (do not currently present mild CanL) and they are not progressing to develop the disease in the near future (i.e. they are not in the prepatent period of CanL). Careful physical examination is the first step to decide if a dog is really healthy and thus whether it can be vaccinated. It is advisable to pay special attention to detecting subtle clinical signs of CanL, including mild peripheral lymphadenomegaly, papular dermatitis or early exfoliative dermatitis. Documentation of seronegativity that is, lack of Leishmania-specific circulating IgG antibodies is also necessary before vaccination. Many infected, clinically healthy seropositive dogs are in the process of developing clinically overt CanL. It is known that in seropositive dogs histologic lesions of various tissues and organs, including skin, masticatory muscles, intestine, liver, joints and kidneys, develop before the ensuing clinical signs and laboratory abnormalities. It is worth highlighting that the results of serological tests, especially in dogs with low antibody levels, depend on the accuracy, sensitivity and specificity of the test. Consequently, some dogs in the process of developing the disease might test seronegative despite having low levels of circulating antibodies and be accidentally vaccinated. There are few data on the effect of vaccination in these dogs. The use of Leishmune as immunotherapy in seropositive dogs with CanL was associated with some benefits without obvious side effects. Similar data are not available for CaniLeish. Chiara Noli thanked Manolis Saridomichelakis and commented that this presentation raised some questions. It is not known whether vaccination of an infected dog might help to clear the infection or, instead, might be harmful. Alessandra Fondati asked for advice about vaccination in non-infected dogs suffering from concurrent cutaneous or internal organ disease. Manolis Saridomichelakis replied that normally only healthy animals should be vaccinated, similar to other canine and feline vaccines. It is not known what will happen if sick dogs are vaccinated against CanL; additionally, the efficacy of the vaccination might be compromised. Side effects are not expected but no data are available. Chiara Noli introduced the next speaker. Which antibody and cellular immunity responses should be expected in a dog vaccinated against leishmaniasis? (L. Solano Gallego) Laia Solano Gallego (Spain) explained that vaccines against canine L. infantum infection should elicit protective humoral and cellular immune responses. Limited knowledge is available about the complex dog parasite interactions; however, it is well known that a predominant Th1-like response is capable of controlling L. infantum infection in dogs. Therefore, a vaccine should elicit a Th1- like response. Considerations regarding the immune response induced by the vaccination include antigen recognition, kinetics of antibody- and cell-mediated responses, differences in assay diagnostic performance and individual variability among dogs. Limited studies are available regarding the humoral and cellular immune responses after vaccination in dogs living in endemic areas. Antibodies reactive with Leishmania antigen have been reported in experimental healthy dogs to peak 2 weeks after completion of the primary vaccination course with CaniLeish, which consists of three injections 3 weeks apart in dogs at least 6 months of age, and to persist for at least 4 6 months. Detection of antibodies persisting after vaccination likely depends on the sensitivity of the serological technique. The sensitivity of rapid serological tests is lower compared to that reported for quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT). These latter techniques are capable of detecting antibodies elicited by vaccination, whereas Speed Leish K (Virbac BVT, La Seyne-sur-Mer, France), the test also used to screen dogs prior to vaccination, does not commonly detect these antibodies. No data are currently available regarding the antibody peak and persistence after the annual booster with CaniLeish. The specific L. infantum T-cell response assessed by lymphocyte proliferation and/or interferon-γ production develops 3 weeks after the completion of the primary course and has at least 1 year s duration. There are limited studies on the Th1-cellmediated immune response to vaccination. However, it might be hypothesized that in a large-scale study on dogs of various breeds, the T-cell-mediated immune response to vaccination would not be as homogenous as in experimental beagle dogs and that it might include non- or lowresponders. Future research should be directed at the development of new techniques for the discrimination of humoral and cell-mediated immune responses due to vaccination from those resulting from natural infection. These tests would be essential from a diagnostic point of view and would allow for a better understanding of the effect of vaccinating dogs against leishmaniasis in endemic areas. Chiara Noli thanked Laia Solano Gallego and asked the audience for comments or questions. Manolis Saridomichelakis commented that our current knowledge on antibody kinetics after the primary course of vaccination is based on data from healthy laboratory animals living in a sandfly-free environment. It is likely that the situation will be different in dogs living in endemic areas and exposed to bites from infected and noninfected sandflies before and after the vaccination. Laia Solano Gallego agreed and added that there are only limited studies on antibody kinetics after either a booster or the primary course of vaccination. Personal studies showed that in the majority of dogs the maximum peak of antibodies is reached 2 weeks after the third dose of a primary course of CaniLeish and is followed by a decrease. Nevertheless, there are exceptions. 189

5 Workshops Chiara Noli asked if Leishmania parasites continuously inoculated by infecting sandflies in vaccinated dogs living in endemic areas might eventually act as a booster, keeping the antibody levels high. Laia Solano Gallego responded that this is not known and might depend on the susceptibility of the dog. A very resistant dog might produce antibodies only after vaccination. Chiara Noli introduced the next speaker. Problems with confirming or ruling out leishmaniasis in vaccinated dogs (L. Ordeix) Laura Ordeix (Spain) presented the preliminary results of a descriptive study on 17 dogs from an endemic area that developed leishmaniasis after CaniLeish vaccination. Clinical and clinico-pathological alterations were similar to those observed in non-vaccinated dogs and included ulcerative and nodular cutaneous lesions, lymphadenomegaly, mild-to-moderate non-regenerative anaemia, hypergammaglobulinaemia, proteinuria and renal azotemia. Most dogs were diagnosed several months after vaccination, suggesting that they were previously infected. Diagnosing CanL in vaccinated dogs can be challenging as serological tests that is, IFAT and ELISA are not normally helpful because they detect vaccine-elicited antibodies. Demonstration of Leishmania in diseased tissues through cytology, histopathology, immunohistochemistry or molecular techniques is essential to confirm the disease in these patients. However, the detection of parasites in lymph nodes, bone marrow or blood does not allow one to directly correlate the infection with the cutaneous disease. Only a favourable response to antileishmanial treatment allows a causal role to be attributed to the parasite. After the presentation of two clinical cases, Laura Ordeix concluded that a history of Leishmania vaccination does not allow CanL to be ruled out, and that the disease should be included in the list of differential diagnoses for vaccinated dogs. Parasite detection in typical skin lesions through cytology, histology or immunohistochemistry confirms the disease. If the results are negative, RT-PCR should be performed on skin samples. Detection of the parasite or its DNA in organs other than the skin (e.g. blood, bone marrow or lymph node) demonstrates the infection but not the causal relationship between the parasite and skin lesions. Chiara Noli asked about the possibility of a different clinical presentation of CanL in vaccinated versus unvaccinated dogs. Laura Ordeix recalled that while no differences were observed in the preliminary study, this was an uncontrolled study. Gaetano Oliva (Italy) stated that, according to the study he authored, 2 less severe renal damage was observed in vaccinated experimental beagle dogs compared to untreated controls. Laia Solano Gallego added that in the preliminary study shown by Laura Ordeix, most dogs presented clinical signs of leishmaniasis very early after vaccination. This suggests that at least some dogs were vaccinated when already infected. Therefore it is more likely a failure to identify infected dogs, possibly related to the low sensitivity of diagnostic tests (e.g. rapid qualitative serological tests) rather than a failure of vaccine protection. Chiara Noli remarked that this would also help to explain why no clinical differences between vaccinated dogs and dogs with natural infection were noticed in this preliminary study. On the other hand, Gaetano Oliva s study showed a difference because dogs were infected after vaccination. Chiara Noli then asked Laura Ordeix whether the results of the study she presented had been stratified according to the time span between vaccination and development of CanL. Laura Ordeix responded that the number of dogs was too small to analyse results in this way. Manolis Saridomichelakis asked if the vaccinated dogs that developed CanL had received immunosuppressive treatment for other diseases. Laura Ordeix replied that no dogs were treated with immunosuppressive drugs. Patrick Bourdeau commented that any infected dog can have Leishmania in the skin; thus the presence of parasites in the skin by itself does not demonstrate their causal role. In infected dogs Leishmania parasites circulate inside macrophages and they can concentrate in lesions primarily unrelated with leishmaniasis. The only way to attribute a causal role to parasites is the response to specific antileishmanial treatment. Laura Ordeix partly disagreed with the comment from Patrick Bourdeau and explained that there might be a difference depending on the clinical picture of the dog. If Leishmania is demonstrated in the skin of a dog living in an endemic area and showing typical clinical signs and suggestive/compatible cytological and/or histopathological alterations, a causal role of parasites is likely. However, when dermatological signs are atypical and many other causes of skin disease have to be considered, response to antileishmanial treatment is more important for the diagnosis. Guadalupe Miro shared that she saw two cases of lymphoma in dogs 2 3 years after vaccination and asked whether anyone had a similar experience, or if other diseases, apart from leishmaniasis, had been observed in vaccinated dogs. Chiara Noli and the attendants did not report a similar experience. Nevertheless, Chiara Noli added that since vaccination for CanL is more widespread in Spain than in Italy, the odds that vaccinated dogs develop concurrent diseases is high. 190

6 Vaccines for canine leishmaniasis Alessandra Fondati asked for suggestions on the most suitable tests to rule out Leishmania infection in clinically healthy recently vaccinated dogs, for example those that have to be treated with immunosuppressive therapy. Laura Ordeix recommended blood work, looking for clinico-pathological alterations and both quantitative and qualitative serological tests if vaccination dates back to less than 6 months. Depending on the results, a very sensitive test to diagnose infection, for example RT-PCR on bone marrow, could be also performed. Manolis Saridomichelakis stated that if Leishmania infection had to be excluded in every dog living in an endemic region and needing immunosuppressive therapy, then, considering the high prevalence of infected dogs and the chance that non-infected dogs will become infected during the course of the treatment, immunosuppressive drugs should not be used at all in these areas. Fortunately, it seems that only a minority of infected dogs under long-term immunosuppressive treatment develop CanL. Chiara Noli thanked Laura Ordeix and introduced the next speaker. Adverse reactions of the vaccine against CanL: facts or urban legends? (L. Ferrer) Lluis Ferrer (USA) reported that his sources of information on adverse reactions to CaniLeish were represented by pharmacovigilance data from the EMA and the French agency Agence Nationale de Securite du Medicament et des Produits de Sante (ASMPS), by a survey performed in seven large veterinary hospitals in highly endemic areas in Spain (including files from more than 4000 vaccinated dogs) and by data from the Virbac Pharmacovigilance Department. Information from social networking sites was also consulted. Data referred only to short-term adverse effects. Side effects reported in more than 10% of cases included local pain and injection-site inflammation, and fever. According to data from veterinary hospitals, the frequency of these side effects ranged from 5 to 25% of cases, whereas according to the EMA local reactions accounted for 0.079% of cases. They appeared to be more common in small dogs, were probably associated with the adjuvants and resolved spontaneously in 2 15 days. They could easily be prevented by administering non-steroidal anti-inflammatory drugs. Based on data from pharmacovigilance, uncommon side effects reported in 0.1 1% of dogs included anorexia, emesis, lethargy and localized-to-generalized transient urticarial reactions (which can be prevented by antihistamine administration). Very rare side effects described in fewer than 0.01% of dogs were ulcerative necrotizing dermatitis, panniculitis and vasculitis at the injection site. Severe and fatal anaphylactic reactions have thus far only been reported in social networks. In conclusion, side effects are similar to those reported for other vaccines. The only difference is the greater frequency of injection-site reactions, likely associated with the type of adjuvants included in the vaccines. Owners and veterinarians must be informed and the preventative use of anti-inflammatory drugs might be recommended. In any case, the reported short-term side effects should not be a reason to avoid vaccinating dogs. Long-term side effects, if any, are currently unknown. Chiara Noli thanked Lluis Ferrer for reassurances about the lack of serious side effects of CaniLeish and asked for questions or comments. Guadalupe Miro pointed out that data from Virbac pharmacovigilance were valuable; however, they were lower than data obtained directly from practitioners and clinics because of the reluctance of veterinarians to officially report adverse reactions to drugs. The importance of pharmacovigilance should be stressed among veterinarians. Chiara Noli thanked all the speakers, Alessandra Fondati and the audience for their attendance and contributions, and closed the workshop. Note a After the workshop took place (June 2016), a new vaccine called Letifend (Laboratorios LETI, Madrid, Spain) was authorized in Europe. References 1. EFSA Panel Animal Health and Welfare. Scientific opinion on canine leishmaniasis. EFSA J 2015; 13: Oliva G, Nieto J, Foglia Manzillo V et al. A randomised, doubleblind, controlled efficacy trial of the LiESP/QA-21 vaccine in naıve dogs exposed to two Leishmania infantum transmission seasons. PLoS Negl Trop Dis 2014; 8: e