Canine leishmaniasis

Leishmaniasis is a protozoan disease transmitted by insect vectors (sandflies) and is endemic in 88 countries in the world, including those of Mediterranean Europe. Various Leishmania spp. can cause the disease both in humans and dogs (Tab. 1). In Europe the most diagnosed human cases are zoonotic visceral leishmaniasis and cutaneous leishmaniasis caused by Leishmania infantum  (also called Leishmania chagasi), whose natural reservoir is the domestic dog. The disease that develops in dogs, caused by the same aetiological agent, is called canine leishmaniasis.

AETIOLOGICAL AGENT

Leishmania infantum, a protozoon of the family Tripanosomatidae.

VECTORS

The vectors are Phlebotomus perniciosus, P. perfiliewi, P. ariasi andP. neglectus (in Italy). Sandflies are small (about 2 mm) haematophagous insects with predominantly nocturnal habits (Fig. 1). The males feed on vegetal juices, the females feed on blood. The females bite exclusively in the evening and night-time (from dusk to dawn) in the period between halfway through May to the end of October (the transmission season; the duration of this season varies depending on the latitude). In Italy the areas in which the disease is traditionally considered endemic are the coastal zones of the centre and south of the country and its islands, but in recent years leishmaniasis has also been reported in numerous areas in the north of Italy, previously considered unaffected. The prevalence of the infection in the foci of canine leishmaniasis in the Mediterranean varies between 2 and 40%.

METHODS OF TRANSMISSION

In order to complete its biological cycle, the Leishmania needs an intermediate host, which is a hematophagous  vector (sandfly), and a definitive host, which is a vertebrate. When an uninfected sandfly takes its blood meal from an infected host, it ingests amastigotes (non-infective forms of the protozoan parasite) (Fig. 2). The amastigotes undergo a series of changes within the vector, until transforming into the flagellated promastigote (which is very mobile and infective) (Fig. 3). The haematophagous female sandfly carries the promastigotes in her digestive system and transmits the parasite during the next blood meal to domestic and wild animals and to humans, in which the amastigote form is developed again.

As already mentioned, the natural reservoir is the DOG.  Although foxes and wolves are also natural hosts for L. infantum, they seem to have a limited epidemiological role (wolf) or, at any rate, a role still under discussion (fox). Cats can be infected and, OCCASIONALLY, manifest the disease. Their epidemiological role is unclear. In the light of current knowledge, mice and rats are not considered either certain reservoirs or epidemiologically important.

Other routes of transmission: it has been proven that blood transfusions can be a potential hazard, so donors must be carefully tested for this pathogen. Sexual and uterine transmission are not real sources of contagion, even though some scientific research seems not to exclude these routes absolutely definitively.

Numerous studies carried out in the past and repeated in recent years using biomolecular techniques have tried to shed light on the role of other insects, in particular fleas and ticks, in the transmission of the parasite. So far, there is no sufficiently valid proof demonstrating the possibility of transmission of leishmaniasis by insects other than sandflies.

PATHOGENESIS

The parasite is deposited in the skin of the host almost immediately, partly because the blood meal lasts only a few seconds. The spread of the parasite within the body and the possible development of the disease depend on the type and efficiency of the immune response of the infected dog. Leishmania infection has three main pathogenic characteristics: (i) the targets of the parasite are macrophages, within which the parasite can replicate; (ii) the onset and evolution of the disease depend on the immune or inflammatory response of the host; (iii) the persistence of the infection in tissues. Leishmania tend to localise in all tissues rich in components of the monocyte-macrophage system, where they can be found using direct methods already within a few weeks after the infection.

In the dog, L. infantum causes an infection which is usually chronic. In some cases the infection can be asymptomatic, whereas in other cases an evident symptomatic disease develops. The immune response plays a very important role in this dichotomy (infection or disease): CD4+ T helper lymphocytes can direct the immune system towards a humoural response (Th2) or towards a cell-mediated response (Th1). The two extremes of the clinical expression are represented by infected, clinically healthy dogs and infected, severely sick dogs.  In both dogs and humans, resistance to the disease seems to be associated with a mixed Th1-Th2 type of immune response, while susceptibility to the disease seems to be associated with continuous antigenic stimulation and an exaggerated antibody response which induce hypergammaglobulinaemia, deposition of immune complexes – which can cause glomerulonephritis, vasculitis, polyarthritis, uveitis and meningitis – and production of autoantibodies against platelets and red blood cells.

CLINICAL SIGNS

The period of incubation varies from a few months to years. The state of infection (demonstration of the presence of the parasite) is not always followed by a state of disease (presence of clinical signs and/or abnormal laboratory findings). The disease may start with severe signs (Figs. 4 and 5) but may also be only just apparent (Fig. 6). The most common clinical signs are enlarged lymph nodes, anorexia, sensorial depression, weight loss, muscle atrophy, skin lesions (nodules, ulcers, periorbital alopecia, exfoliative dermatitis), ocular signs (kerato-conjunctivitis, uveitis), lameness and polyuria-polydipsia (Table 2,  Castagnaro et al., 2008, Canine Leishmaniasis Working Group).

Table 2. (Castagnaro et al., 2008, Canine Leishmaniasis Working Group)

It is essential to differentiate infected dogs from dogs with disease. No pathognomonic clinical signs are present.

LABORATORY TESTS

The main findings of laboratory tests include a poorly regenerative anaemia, an increase in total serum proteins, hypoalbuminaemia,  hypergammaglobulinaemia,  increased values of urea and creatinine and proteinuria (Table 3). There are no laboratory alterations pathognomonic for this disease.

Table 3. (Castagnaro et al., 2008, Canine Leishmaniasis Working Group)
(FDP = fibrin/fibrinogen degradation products, AT = antithrombin, CRP= C-reactive protein, Hp= haptoglobin, SAA= serum amyloid A, SG=specific gravity, UP/UC = urinary protein/creatinine ratio, SDS-AGE =s odium dodecylsulphate agarose gel electrophoresis, PT =p rothrombin, aPTT = activated partial thromboplastin time).

DIAGNOSIS

The diagnosis of leishmaniasis can be made from detecting the parasite itself or the anitbodies produced by the body against the parasite. It is, however, useful to  clarify that, in the absence of clinical signs and/or abnormal laboratory test results, it is more accurate to talk of INFECTION, differentiating this from DISEASE, a condition in which the clinical signs found are clearly attributable to the presence of the parasite. The differentiation between infection and disease is fundamental, particularly in endemic regions, because a dog could, for example, be infected (as demonstrated by the detection of the parasite or the presence of antibodies) and present signs of a disease due to other another cause, infectious or otherwise.

Diagnostic tests

Indirect immunofluorescence antibody test (IFAT) for the detection and assay of specific anti-L. infantum antibodies in the serum. Titres more than four times higher the reference laboratory’s threshold in the presence of clinical signs or abnormal laboratory results confirm the state of DISEASE.  For this reason it is always advisable to titre the level of antibodies.

Microscopy: the amastigotes of L. infantum can be seen free or within the cytoplasm of macrophages on smears of material obtained by fine needle aspirate of lymph nodes/bone marrow (video) or skin lesions.

Polymerase chain reaction (PCR): this is used to identify or quantify (RT-PCR) L. infantum-specific DNA in biological samples. This test is most sensitive when carried out on lymph node material or bone marrow. In endemic areas, positivity for the PCR investigation alone, in the absence of progressively increasing antibody titres (seroconversion), is not necessarily an indication of an “active” infection. 

Cultures to isolate the promastigotes of L. infantum from lymph node material or bone marrow (these studies are only available in a few laboratories)

DIFFERENTIAL DIAGNOSIS

The differential diagnosis includes ehrlichiosis, babesiosis, hepatozoonosis, lymphoma and demodectic mange.

TREATMENT

Canine leishmaniasis is a very complex pathology and its treatment is still a difficult problem to resolve. For this reason, only some of the issues regarding the treatment of this much feared zoonosis are dealt with, drawing the reader’s attention to the importance of obtaining a thorough and precise clinical assessment of infected dogs before making any therapeutic decisions.

Which dogs to treat?  Before deciding whether and, if so, how to treat a dog infected with L. infantum, the state of infection must be clearly differentiated from a state of disease. As already mentioned, an infected dog is one in which the presence of the parasite is demonstrated by either direct methods (microscopy, culture, PCR) or indirect methods (evidence from specific antibodies). In order to facilitate the diagnostic categorisation of infected dogs, the  Canine Leishmaniasis Working Group (CLWG) recently produced and published a classification (Paltrinieri et al., 2009). Besides guidelines on the diagnosis and classification of infected subjects, the CLWG has also produced guidelines on the treatment of canine leishmaniasis (Oliva et al., 2008). An infected dog can be defined as ill when it manifests one or more of the clinical signs of leishmaniasis, including changes in haematological, blood-chemistry and urinary test results. Furthermore, a dog infected by L. infantum may remain in a state of infection for months to years before showing clinical signs of disease. This state may be fairly easily diagnosed (patent infection) or be at the limit of detection, even when using various diagnostic techniques, such as the demonstration of anti-Leishmania antibodies, microscopic vision of the parasite and qualitative/quantitative PCR (sub-patent infection). Once the infection has given rise to the disease, this can be expressed although the clinical severity of the manifestations depends on the genetic constitution of the individuals affected, their immune response, any concomitant disorders and the organs involved.

Staging of dogs with leishmaniasis, for therapeutic purposes (Oliva et al., 2008)

Dogs categorised in stage A do not need to be given treatment with anti-Leishmania drugs. These dogs definitely do not have either clinical signs or laboratory abnormalities and the infection is in a stage of unpredictable evolution. The same therapeutic reasoning could also be applied to dogs in stage B; however, in this case the easy identification of the parasite, together with a rising antibody titre (seroconversion) could indicate the need to anticipate treatment, with the aim of preventing the appearance of clinical signs and also of limiting the potential danger of the dog as an active reservoir of the parasite. Since the subjects in stages C and D have overt disease, they must always be treated with specific anti-Leishmania therapy. It is worth remembering that although antiparasite therapy almost always produces a good clinical improvement, it must not be considered the only treatment for repairing organ damage induced by the protozoon. For example, an animal with proteinuric nephropathy should undoubtedly be given other drugs together with the specific anti-parasite treatment. For all dogs in stage E, before considering alternative protocols or modifying the one already being used, the diagnosis should be questioned, the correct application of the protocol confirmed and the presence of any concomitant diseases, infectious or otherwise, be determined. 

How to treat? The first point that must be made is that the main drugs active against Leishmania were designed and developed through the pre-clinical and clinical phases exclusively for the treatment of human leishmaniasis and only subsequently were studied and used in dogs. The drugs currently used by veterinarians in Italy, and in general in the rest of Europe, are described succinctly below; it should be remembered that before being used in clinical practice, an anti-Leishmania drug should be tested in randomised, controlled clinical trials that are adequately powered to detect any statistically and clinically significant differences between the group treated with the drug under investigation and the control group.

As described in the previously mentioned guidelines and as already emphasized by other authors (Noli, 2005), almost all the studies reported in the veterinary literature have numerous methodological weaknesses which sometimes generate serious doubts about the reasons for using a given therapeutic protocol. The most frequent weaknesses are: the study was not blinded; there was no control group; the study population was numerically inadequate for the aims of the study; the groups of dogs were not homogeneous and almost never comparable to those in similar studies; the extreme variability in diagnostic and clinical criteria; the extreme variability in the clinical and parasitological criteria defining a “cure”; and the extreme variability in doses and treatment times even for the same drug. The articles identified through a review of the literature enable an evaluation of the following drugs in the treatment of canine leishmaniasis, listed in order of decreasing number of publications:

• Antimonials (34 studies)
• Allopurinol (19 studies)
• Miltefosine (8 studies)
• Aminosidine (4 studies)
• Amphotericin B (3 studies)
• Pentamidine (1 study)
• Spiramycin/Metronidazole (1 study)
• Enrofloxacin (1 study)
• Marbofloxacin (1 study)
• Domperidone (1 study)

It is clear from the above that the use of some of the anti-Leishmania drugs is often not supported by valid scientific evidence. The dosing regimens of the anti-Leishmania drugs for which there are at least three or four international references are reported below. 

Therapeutic protocols used for canine leishmaniasis, extracted from a review of the literature (Oliva et al., 2008).

In daily clinical practice, protocols derived from associations of several drugs are often used, with the intention of potentiating their action and limiting phenomena of drug-resistance which are possible with all the drugs considered. It is worth emphasizing that also in these cases, the only combinations that should be used are those for which there is scientific support that has been adequately validated at an international level. The association between N-methylglucamine antimoniate and allopurinol is the most widely used protocol in the treatment of canine leishmaniasis and undoubtedly the one for which there is greatest agreement and published evidence. As demonstrated by Denerolle and Bordoiseau (1999), subjects treated with these two drugs have longer remissions than those treated with either drug alone. Another interesting point is the good tolerability of this combination of drugs.

In practice, the most frequently used protocol is N-methylglucamine antimoniate, at a dose of 100 mg/kg s.i.d. s.c. (or 50 mg/kg b.i.d. s.c.) for 1 or 2 months, together with allopurinol, at a dose of 10 mg/kg b.i.d. p.o. continued for many months after clinical remission has been achieved. This protocol, used correctly, almost always leads to a clinical cure in dogs in stage B and C and almost always maintains the remission for more than 1 year. Treatment with the combination of these two drugs also lowers the parasite load drastically for some months and, consequently, the rate of infectivity for sandflies. However, for subjects with stage D disease, although the protocol described above ensures moderate to good improvement, it may not be sufficient to obtain a clinical cure of the animal. Obviously, in dogs with a “severe clinical picture”, in particular in those already with renal impairment, the prognosis is strongly related to the clinical conditions of the animals at the start of treatment and to the indispensable requirement for collateral therapies. As for all the drugs tested so far, the combination of N-methylglucamine antimoniate and allopurinol does not cause a parasitological cure, even when used for long periods. Recurrences are possible, despite continuation of the treatment.

Miltefosine (hexadecyl-phosphocholine) is a recently registered drug. The anti-Leishmania activity of this phospholipid analogue, composed of esters with various different long chains saturated and unsaturated with alkyl groups, is caused by alterations induced in the metabolism of phospholipids in the parasite. In a recent, multicentre, clinical study (Mirò et al., 2008), the efficacy and safety of combined treatment with miltefosine and allopurinol were compared with those of treatment with N-methylglucamine antimoniate and allopurinol in dogs with natural L. infantum infection. It was found that both treatments significantly lowered the clinical score, normalised laboratory data and reduced the parasite load, without there being statistically significant differences between the results achieved with the two combinations.

The clinical classification of dogs for which anti-leishmania treatment is being considered is, therefore, of paramount importance for prognostic purposes. Another classification proposed recently in the international literature (Solano Gallego et al., 2009) is based essentially on a measurement of the antibody titre and assessment of the clinical signs and haematological, biochemical and urinary parameters. According to this classification, the disease is defined as:

• Mild (Stage I): when the clinical signs are mild (e.g. lymphadenopathy or papular dermatitis), the haematological, biochemical and urinary parameters are normal and antibodies are not detectable or give only weak reactions.
• Moderate (Stage II): when the dog, besides having the signs reported for Stage I, has signs such as exfoliative dermatitis, onychogryphosis, ulcers, anorexia, weight loss, fever and epistaxis, has laboratory abnormalities such as a mild, non-regenerative anaemia, hypergammaglobulinaemia, hypoalbuminaemia, serum hyperviscosity syndrome, a still normal renal function and a low to high titre of antibodies.
• Severe (Stage III): when the subject, besides the clinical signs of Stages I and II, has signs attributable to damage caused by immune complexes (vasculitis, arthritis, uveitis, glomerulonephritis), when chronic renal failure (IRIS I or II) is present in addition to the laboratory abnormalities reported for Stage II and the antibody titre is medium or high.
• Very severe (Stage IV): when the dog with severe disease has pulmonary thromboembolism, nephrotic syndrome and end-stage renal disease, and when, in addition to the laboratory abnormalities reported in stage III, it has IRIS III or IV renal failure or nephrotic syndrome. The serum levels of antibodies are moderate or high.

How to monitor a dog during and after treatment? There are still no sufficiently standardised protocols, so the monitoring of dogs during and after treatment must be adapted to each, individual clinical case. The following, deliberately simplified, scheme can be proposed for dogs in groups B and C which, on the basis of a clinical examination and blood tests, do not need support therapy:

• full physical examination and monitoring of common haematological, blood biochemistry and urinary parameters at the end of the treatment course;
• if all findings are within the norm, administer allopurinol at the previously indicated doses for a period of many months (or life-long, according to some authors);
• periodic controls every 6 months, with determination of the antibody titre and quantification of the parasite load by real-time, quantitative PCR analysis of bone marrow or lymph node tissue;
• in the case of early recurrence or if the clinical assessment and/or laboratory parameters are abnormal or do not tend to normalise, the dog is classified in group Ea or Eb and the case must be re-evaluated as described previously.

The treatment of a relapse must always include alternation of several protocols, with the aim of minimising the risk of selection of drug-resistant strains.

It is worth concluding by emphasizing some concepts that can be useful in the therapeutic management of dogs with leishmaniasis:

• definite diagnosis and differentiation between infection and disease;
• exclude/identify other concomitant disorders;
• categorise the patient’s clinical picture correctly, performing the most precise additional examinations possible;
• choose the therapeutic protocol to use, giving priority to drugs for which there is good scientific support in the international literature; 
• avoid combining drugs with anti-Leishmania properties if the combination has not been tested;
• when necessary (certainly for dogs in stage D) administer the appropriate collateral treatment;
• follow the therapeutic protocol carefully, respecting the times and methods of administration suggested by the international literature;
• monitor the patient correctly during and after treatment.

PROPHYLAXIS AGAINST THE VECTOR

Action against the vector is a crucial part of the strategy to control L. infantum infections. Where tried, environmental interventions with DDT, carbamates, malathion, permethrin and pyrethroids to decrease the population of sandflies have been unsuccessful. The reasons for the failure to control sandflies in the environment include the difficulty of dosing the insecticides, the impossibility of reaching the sites where these insects rest and reproduce, the risk of environmental pollution, residual activity against other organisms and, lastly, the high costs.

Not allowing the sandfly to feed on dogs means preventing inoculation of the protozoon and, therefore, infection of a receptive host by Leishmania. This simple strategy is the basis of what is currently considered the best solution for controlling canine leishmaniasis. Pyrethroids (Table 4) are not only insecticides, but also have an “anti-feeding” effect (the ectoparasites do not take a blood meal from the host), which is commonly called a “repellent effect”.

Table 4. (Maroli et al., 2009, Canine Leishmaniasis Working Group).

There are various different commercially available pyrethroid-based preparations which should be applied to both healthy dogs as well as infected/diseased dogs (Table 5). 

Table 5. (Maroli et al., 2009, Canine Leishmaniasis Working Group). 

PROPHYLACTIC VACCINATION

Anti-Leishmania treatments do not clear parasites from infected dogs, but the elimination of infected dogs does not seem to be a feasible solution for ethical reasons and because of the social role played by dogs in western countries. In any case, it should be noted that in countries in which this strategy has been used, the results in terms of decreasing the incidence of leishmaniasis in humans have appeared contradictory and not adequate for supporting the adoption of this strategy. Prevention of bites by the sandfly vector is a form of prophylaxis that should always be promoted, independently of the use of drugs or vaccines, which can never be considered to resolve the problem completely. Despite abundant research on the use of immunostimulatory drugs, prophylactic vaccination is still a challenge it its earliest stage. Among the various types of vaccines investigated over the last decades (Table 6), the ones that have produced the best results are those based on purified Leishmania antigens.

Table 6. Vaccines investigated for the prophylaxis of leishmaniasis.

Numerous proteins and lipophosphoglycans with immunogenic power have been selected from various Leishmania spp. for use in vaccines. These include two antigens that have been experimented in phase III trials with very good results in the dog. One of these antigens is called fucose-mannose-ligand (FML), a glycoprotein fraction which, enriched with a saponin (Quillaja saponaria (QS)21; an aldehyde containing deacylated saponins of QS) as an adjuvant, was the first vaccine registered for canine leishmaniasis (Leishmune®) and is currently marketed in Brazil. Numerous studies, starting in 2002, have demonstrated the protective effect of this vaccine in phase III studies, with an efficacy ranging between 92% and 95%. According to the researchers, the vaccine is particularly effective at preventing severe clinical disease, even though there are other studies aimed at demonstrating that the vaccine can render vaccinated dogs “not infectious”. Finally, recent studies have demonstrated that the marked efficacy of the vaccine is, in part, due to the adjuvant used, since this is capable of evoking a Th1 response at the site of the inoculation, following the inflammation produced.

The second antigen raising considerable interest is LiESAp, an excretion/secretion product obtained from cultures of L. infantum: it is a protein of about 54 kDa. This product, to which muramyl dipeptide (MDP) is added as an adjuvant, has been tested in two separate phase III studies, one of which was a preliminary investigation (6 dogs) while the other was much larger (404 dogs enrolled in the south of France). The results of this latter study, which were undoubtedly encouraging (vaccine efficacy of 92%), suffered strongly from the very low incidence of recorded deaths in the 2-year follow-up, reflecting the low infective pressure of the endemic area, and preventing definitive conclusions from being drawn on the real efficacy of the vaccine.

Vaccines based on the use of purified antigens do, therefore, represent one of the possible solutions for vaccines in the very near future. One definite limitation to their large scale use derives from the problems of industrial production and the difficulties in obtaining a standardised product.

The first anti-Leishmania vaccine registered in the Old Continent (Canileish®-Virbac) has been available on the European market since about one year. The vaccine consists of Leishmania infantum “secreted-excreted” antigens cultured in a special, “pure” growth media, that is without the addition of proteins or matrices other than those generated by the same parasite. The main antigens responsible for the immunostimulating activity of the vaccine belong to the PSA (promastigote surface antigens) complex, the constitutive proteins of cell membranes in both promastigote and amastigote morphological stages. The adjuvant chosen for the Canileish®  formulation is a purified extract of Quillaja saponaria  (QA-21), a saponin which has shown in various trials an excellent capacity to stimulate cell-mediated immunity. Data on the efficacy of the vaccine are derived from pre-registration clinical trials (Phases I, II and III). In particular, in vitro studies have shown that Canileish® can stimulate a significant Th1 type response, with the consequent production of interferon-gamma (IFN-g), activation of the killing capacity of macrophages and the consequent reduction of the parasitic load._^a The immune response is highest at the end of the entire vaccination schedule, which requires three administrations of the product, each three weeks apart, in dogs which are seronegative and in good physical conditions. Booster immunization requires instead a single, yearly administration of the vaccine. With regard to clinical efficacy the experimental data derive from a field trial carried out in two different highly endemic areas (Italy and Spain), in which the protection rate was tested on a group of 90 six-month-old naïve beagles, followed for two years (two consecutive transmission seasons). In the above described conditions, in spite of not assuring an absolute protection rate, Canileish® reduced by about 4 times the risk of developing the disease in the group of vaccinated animals versus the control group._^b A preliminary study carried out on a selected group of dogs taken from the clinical efficacy trial has furthermore reported some results which seem to show that the sandfly infection rate in vaccinated dogs is reduced compared to the rate found in non-vaccinated dogs._^c With regard to public health, the role of vaccinated dogs as a possible reservoir of infection is without doubt the most important aspect to be considered and which requires further and more thorough assessments. The vaccine is currently sold in 11 European countries, mostly in Portugal, France, Spain, Greece and Italy. Existing data report sales of around 1,000,000 doses, which correspond to more than 300,000 vaccinated subjects. In view of the clear demonstration that vaccinated dogs may still result infected, the use of repellents of proven efficacy against sandflies is to be always recommended, also in dogs undergoing vaccination.

RISKS TO HUMANS

The transmission of lesihmaniasis from an animal to a human occurs exclusively via the bites of infected sandflies. Predisposing factors include infancy, malnutrition, human immunodeficiency virus and severe immunosuppression. Humans can develop a visceral form or a cutaneous form. The visceral form is characterized by an irregular fever, weakness, anorexia, pallor, splenomegaly, hepatomegaly and enlarged lymph nodes. The cutaneous form is manifested by single lesions, ulcerated or not, usually located at the site of the sandfly bite. The drug of choice for the treatment is liposomal amphotericin B. This drug usually provides a clinical and parasitological cure.

References

1. Guidelines for prevention of leishmaniasis in dogs. Maroli M, Gradoni L, Oliva G, Castagnaro M, Crotti A, Lubas G, Paltrinieri S, Roura X, Zini E, Zatelli A. J Am Vet Med Assoc. 2010 Jun 1;236(11):1200-6. Review
2. Guidelines for treatment of leishmaniasis in dogs. Oliva G, Dvm XR, Crotti A, Maroli M, Castagnaro M, Gradoni L, Lubas G, Paltrinieri S, Zatelli A, Zini E. J Am Vet Med Assoc. 2010 Jun 1;236(11):1192-8. Review
3. Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Paltrinieri S, Solano-Gallego L, Fondati A, Lubas G, Gradoni L, Castagnaro M, Crotti A, Maroli M, Oliva G, Roura X, Zatelli A, Zini E; Canine Leishmaniasis Working Group, Italian Society of Veterinarians of Companion Animals. J Am Vet Med Assoc. 2010 Jun 1;236(11):1184-91.
4. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, Baneth G. Vet Parasitol. 2009 Oct 28;165(1-2):1-18. Epub 2009 Jun 6. Review
5. Canine leishmaniosis--new concepts and insights on an expanding zoonosis: part two. Miró G, Cardoso L, Pennisi MG, Oliva G, Baneth G. Trends Parasitol. 2008 Aug;24(8):371-7. Epub 2008 Jul 4. Review.
6. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Trends Parasitol. 2008 Jul;24(7):324-30. Epub 2008 May 29. Review
7. Vaccines for leishmaniasis in the fore coming 25 years. Palatnik-de-Sousa CB. Vaccine. 2008 Mar 25;26(14):1709-24. Epub 2008 Jan 30. Review

a)   Moreno J, Vouldoukis I, Martin V, McGahie D, Cuisinier AM, Gueguen S. Use of a LiESP/QA-21 vaccine (CaniLeish) stimulates an appropriate Th1-dominated cell-mediated immune response in dogs. PLoS Negl Trop Dis.2012;6(6):1683. doi:10.1371/journal.pntd.0001683. Epub 2012 Jun 19.

b) Oliva  G,  Nieto J,  Foglia Manzillo V, Cappiello S, Fiorentino E, Di MuccioT, Scalone A, Moreno J, Chicharro  C, Butaud T, Guegand L, Martin V, Cuisinier AM, Gueguen S, Cañavate C, Gradoni  L. Evidence for protection against active infection and disease progression in naïve dogs vaccinated with LiESP/QA-21 (CaniLeish_^®) exposed to two consecutive Leishmania infantum transmission seasons. WSAVA Proceedings, Birmingham, April 2012.

c) Bongiorno G, Paparcone R, Foglia Manzillo V, Oliva G,  Cuisinier AM, Gradoni L. Vaccination with LiESP/QA-21 (CaniLeish_^®) reduces the intensity of infection in Phlebotomus perniciosus fed on Leishmania infantum infected dogs – results of a pilot xenodiagnosis study. WSAVA Proceedings, Birmingham, April 2012.