i Eur Veterinary Conference

Symposium "Advanced management of Canine Leishmaniosis" September 30, 201 I

Barcelona, Spain

Canine Leishmaniosis prevention: the challenge of a vaccine for Europe Maite Verde Can ine Leishmaniosis (CL ) is a high prevalence disease in dogs o f the Med i te r ranean Basin countr ies. A n y d o g is susceptible t o the disease w h e n it is bite by infested Ph lebo tominae . A n individual can present the disease, clinically, pr imari ly depend ing on the immune response, concomi tan t parasitic diseases, possible re-infections and the pathogenici ty o f the Leishmania strain.

In the last th i r ty years significant research has been achieved resulting in a disease mode l and a weal th o f informat ion about the clinical forms, the mos t sensitive and specific laboratory tests, prognost ic considerat ions based on the patient biopathological situation and the o the r concomi tan t diseases, wh i ch has made us to bet m o r e confident, making bet ter decisions on h o w t o t reat and obtain successful ou tcomes .

T h e therapeut ic opt ions are based on the c o m b i n e d use o f glucant ime o r mil tefosine, wi th al lopur inol . But in any case, the appl icat ion o f prevent ive measures to aver t disease deve lopmen t is essential. W e are attending the presentat ion o f the latest progress in prevent ive medic ine against C L in Europe: C a n i L e i s h ® (the first vaccine against C L registered in the European Medic ines A g e n c y in 201 I, by V i rbac Laborator ies) .

A t this sympos ium w e are h o n o r e d t o at tend a C L state-of- the-art discussion that may interest animal clinicians hos ted by Drs . Guada lupe Mi ro , Fernando Farinas and Liu is F e r r e r T h e s e expe r t s c o m b i n e exper t i se and exper ience o f the highest level in the field o f immunology, pathology, parasitology, diagnosis and t rea tment o f C L . In addi t ion, M r Dav id M c G a h i e and M r Ch r i s tophe R e m e will prov ide data on the safety and efficacy o f C a n i L e i s h ® vaccine and o n h o w t o use it f r o m a practical perspect ive.

W e are looking fo rward to take this oppor tun i t y t o the next level in o u r C L fund o f knowledge and the mos t expec ted prevent ive

opt ion .

Update on the epidemiology of Canine Leishmaniosis in Southern Europe jj Guadalupe Miro Can ine Leishmaniosis ex tends mainly in th ree geographical areas: Brazil, C h i n a and the Med i te r ranean basin. In t he Med i te r ranean basin, the d o g is the main reservo i r o f L infantum infection domes t i c cycle.

A l s o exists a sylvan cycle o f the L. infantum infection affecting wi ld canidae such as the fox, the wo l f o r the jackal.The prevalence found in foxes using the P C R techn ique in investigations pe r f o rmed in Spain and Southern Italy was 40 -75%; therefore, the fox cou ld be cons idered a secondary reservo i r wh ich is thought t o be the link be tween bo th cycles due t o its preference for living near domest ic populat ions. Leishmania infantum has also been de tec ted in wolves, rodents, horses and cats (although there are many issues t o solve abou t the epidemiological role o f these species).

In endemic areas the re is a high percentage (50%) o f infected but clinically healthy dogs. In contrast, the percentage o f dogs wi th

leishmaniosis is l o w e r (3-5%) because Can ine Leishmaniosis is a disease in wh ich infection is no t always equal t o clinical disease.

There fo re , Can ine Leishmaniosis prevalences registered in Spain and Southern Europe are highly variable depend ing o n w h e t h e r the data obta ined by mo lecu la r techn iques refers t o seropreva lence o r infection.

In the ep idemio logy o f this impor tan t zoonos is the a r t h r o p o d vec to r Phiebotomus spp plays an essential role, and the presence o f L infantum will be inf luenced activity and incidence rates.

Current ly, w e should be consider ing o the r non-vector ia l t ransmission routes o f this infection, wh ich have been proven: vert ical t ransmission (although the cases inc luded in the l i terature are anecdotal ) , b l o o d transfusion (of great impor tance in endemic areas w h e r e b lood d o n o r s may often be sub-clinically infected), venereal t ransmission (the presence o f semen in infected males has been detected, w h o infected healthy females after in tercourse) ,Thei r impor tance is still barely known and w e will have t o take into account in the near future.

O t h e r hypothesis cons idered wi th respect t o potential t ransmission routes, but that have not been proven yet, are t ransmission through o the r vectors, such as ticks and fleas (though exper imenta l t ransmission t o dogs is still t o be proven) and transmission through direct dog - to -dog contact (a hypothesis cons idered t o explain t he presence o f native cases in non -endemic areas in wh ich the presence o f any effective a r t h r o p o d vec to r cou ld be proven, as in the case o f the Un i ted States).To define the infectious ability o f dogs wi th parasites is ve ry impor tant , wh ich can only be exper imental ly ev idenced by direct xenodiagnosis. In general, sick seroposi t ive dogs s h o w greater infectivity for Ph lebo tominae than sub-clinical seroposi t ive dogs and remain 'sterile' fo r Ph lebo tominae be tween 4 and 6 months after t reatment .

Finally, it has been p r o p o s e d to cons ider humans as a reservo i r o f Leishmaniosis in L infantum/HN co- infect ion, e i ther by sharing used syringes o r by the bite o f Ph lebo tominae wi th parasites after being fed by sick peop le , in both cases w e w o u l d be facing a real anthroponos is as the dog, and no o the r ver tebra te than the human, take par t in the disease cycle.

A s for the characterist ics o f the host, it is w o r t h ment ion ing that it is thought that certa in d o g breeds are m o r e p rone t o having the disease such as, Boxen C o c k e r Spaniel, Rot twe i le r and G e r m a n Shepherd dog; and that the deve lopmen t o f the disease has a age-related b imodal curve, showing m o r e cases in animals less than 2-3 years o ld and in dogs m o r e than 8 years o ld.

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and canine leishmaniasis fbcus.Trans R S o c T r o p M e d H y g 74(1): 77 -83 • Ca rdoso L, Rodrigues M, Santos H, Schoone GJ, Carre ta RVarejao E, van Benthem B, A fonso M O , Alves-Pires C , Semiao-Santos SJ,

Rodr igues J, Schallig H D , 2004, Sero-epidemiological study of canine Leishmania spp. infection in the municipality ofAlijo (Alto Douro,

Portugal).Vet Parasitol 121 ,21-32 • C o u t i n h o M T Bueno LL, S te rz ikA , Fujiwara R T Bote lho JR, D e Mar ia M, G e n a r o O , Linardi P M , 2005,Participation ofRhipicephalus

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mammals?Vet Parasitol 147, 320-325

• C r iado -Forne l i o A , Gut ier rez-Garc i 'a L, Rodr iguez -Caabe i ro F, Reus -Garc ia E, Ro ldan-Sor iano M A , D iaz-Sanchez M A , 2000, A parasitological survey of wild red foxes (Vulpes vulpes) from the province of Guadalajara, Spain.Vet Parasitol 92 ,245-251

• da Silva S M , Ribeiro V M , Ribeiro RRTafur i W L , M e l o M N , Michal ick MS, 2009, First report of vertical transmission of Leishmania

(Leishmania) infantum in a naturally infected bitch from Brazil.Vet Parasitol 166, 159-162 • Dantas-Torres R 201 I, Ticks as vectors of Leishmania paras/tes.Trends Parasitol • Dantas-Torres R Lorusso VTest in i G , de Paiva-Cavalcanti M, F igueredo L A , Stanneck D, M e n c k e N , Brandao-Fi lho SRAIves L C ,

O t ran to D, 2010, Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 106,857-860 • de Freitas E, M e l o M N , da Cos ta -Va l A R Michal ick MS, 2006, Transmission of Leishmania infantum via blood transfusion in dogs:

potential for infection and importance of clinical factors.Vet Parasitol 137, 159-167 • D ip ine to L, Manna L, Baiano A , G a l a M , Fioretti A , Grav ino A E , M e n n a LR 2007, Presence of Leishmania infantum in red foxes

(Vulpes vulpes) in southern Italy.J W i l d l Dis 4 3 , 5 18-520 • D u p r e y Z H , Steurer RJ, R o o n e y JA, Kirchhoff LV, Jackson JE, R o w t o n ED, Schantz P M , 2006, Canine visceral leishmaniasis, United

States and Canada, 2000-2003. Emerg Infect Dis 12 ,440-446 • Pisa R, Gal lego M, Casti l lejo S, A i s a MJ, S e r r a T Riera C , C a r r i o j , Ga l lego J, Por tus M, 1999, Epidemiology of canine leishmaniosis

in Catalonia (Spain) the example of the Priorat focus. Ve t Parasitol 8 3 , 8 7 - 9 7 • Gal lego M, Prat long R Fisa R, Riera C , R ioux JA, D e d e t JR Por tus M, 2001 , The life-cycle of Leishmania infantum MON-77 in the

Priorat (Catalonia, Spain) involves humans, dogs and sandflies; also literature review of distribution and hosts of L. infantum zymodemes

in the Old Wor/d.Trans R S o c T r o p M e d H y g 95 ,269 -271

• Ga lvez R, M i ro G , Desca lzo M A , N i e t o j , D a d o D, Mar t in O , C u b e r o E, Mo l ina R, 2009, Emerging trends in the seroprevalence of

canine leishmaniosis in the Madrid region (central Spain).Vet Parasitol. • Kil l ick-Kendrick, R, 1999, The biology and control of phlebotomine sandflies. C l in . Dermato l . , 17 279 -289 • Ma ia C , C a m p i n o L, 201 I, Can domestic cats be considered reservoir hosts of zoonotic leishmaniasis?Trends Parasitol 27,341 -344 • Mar t in -Sanchez J, Mora les-Yuste M, A c e d o - S a n c h e z C , Baron S, D iazV , Mor i l l as -Marquez F, 2009, Canine leishmaniasis in

southeastern Spain. Emerg Infect D is 15 ,795-798 • Michalsky E M , R o c h a MF, da R o c h a L ima A C , Franca-Silva J C , Pires M Q , Ol ive i ra FS, Pacheco RS, dos Santos SL, Barata R A ,

R o m a n h a AJ, For tes -D ias C L , Dias ES, 2007, Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to

Lutzomyia longipalpis phlebotomine sand f//es.Vet Parasitol 147 ,67-76 • M i ro G , C a r d o s o L, Pennisi M G , O l i v a G , Baneth G , 2008, Canine leishmaniosis - new concepts and insights on an expanding

zoonosis: part two.Trends Parasitol 24, 371 -377 • M i r o G , Mo l ina R, 2006, Leishmaniosis canina: manejo clinico y situacion actual en Espaha. Edic iones Bayer H e a l t h C a r e • M i r o G , Ga lvez R, Fraile C , Desca lzo M A , Mo l ina R, 201 I, Infectivity to Phlebotomus perniciosus of dogs naturally parasitized with

Leishmania infantum after different treatments. Parasites & V e c t o r s , 4 : 5 2

• Mo l ina R, A m e l a C , N i e t o J, San-Andres , M, G o n z a l e z F, Cast i l lo JA, Lucientes J, A l v a r J, 1994, Infectivity of dogs naturally infected

with Leishmania infantum to colonized Phlebotomus perniciosus.Trans R S o c T r o p M e d H y g Jul-Aug; 88(4): 491 -493 • Mori l las F, Benavides I, G o n z a l e z J, Magana A ,Va le ro A , 1985, Decouverte de Leishmania sp. dans des Rattus rattus de la province

de Grenade (Espagne). A n n Parasitol H u m C o m p 6 0 , 7 6 8 - 7 7 0 • O w e n s SD, O a k l e y D A , Mar ryo t t K, Hatchet t W W a l t o n R, No lanTJ , N e w t o n A , Steurer F, Schantz R G ige r U, 2001 , Transmission

of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs.] A m Vet M e d A s s o c 219, 1076-1083 • Pangrazio KK, C o s t a EA , Amar i l l a S R C i n o A G , S i l v a T M , Pa i xaoTA , C o s t a LR Dengues E G , D iaz A A , Santos RL, 2009, Tissue

distribution of Leishmania chagasi and lesions in transplacental'^ infected fetuses from symptomatic and asymptomatic naturally

infected bitches.Wet Parasitol 165,327-331 • Papadogiannakis E, Spanakos G , Kon tos V, M e n o u n o s PG,Tegos N.Vakalis N , 2009 , Molecular Detection of Leishmania infantum

in Wild Rodents (Rattus norvegicus) in Greece. Z o o n o s e s and public health. • Petersen C A , Barr S C , 2009, Canine leishmaniasis in North America: emerging or newly recogn/zed?Vet C l in N o r t h A m Small A n i m

Pract 39, 1065-1074, vi. • Quinnel l RJ, Cour tenay 0 , 2 0 0 9 , Transmission, reservoir hosts and control of zoonotic viscergl leishmaniasis. Parasitology 136, 1915-1934 • Ro lao N , Mar t ins MJ, Joao A , C a m p i n o L, 2005, Equine infection with Leishmania in Portugal. Parasite 12, 183-186 • Rosypal A C T r o y G C . Z a j a c A M , Prank G , Lindsay D S , 2005 , Transplacental transmission of a North American isolate of Leishmania

infantum in an experimentally infected beagle. J Parasitol 9 1 , 9 7 0 - 9 7 2 • Sastre N , Francino O , Rami rez O , Ensenat C , Sanchez A , A l t e t L, 2008, Detection of Leishmania infantum in captive wolves from

Southwestern Europe.Vet Parasitol 158, I 17-120 • So lano-Gal lego L, Baneth G , 2008 , Canine leishmaniosis - a challenging zoonosis. European Journal o f C o m p a n i o n A n i m a l Pract ice

18,232-241 • So lano-Ga l lego L, Fernandez-Be l lon H , Ser ra R, Gal lego M, Ramis A , Fondevi la D, Fer rer L, 2003 , Cutaneous leishmaniosis in three

horses in Spain. Equine Ve t J 3 5 , 3 2 0 - 3 2 3 • So lano-Ga l lego L, Koutinas A , M i r o G , C a r d o s o L, Pennisi M G , Fer rer L, Bourdeau R O l i v a G , Baneth G , 2009, Directions for the

diagnosis, clinical staging, treatment and prevention of canine leishmaniosis.Vet Parasitol 165, 1-18 • So lano-Ga l lego L, M i ro G , Koutinas A , C a r d o s o L, Pennisi M G , Ferrer L, Bourdeau R O l i va G , Baneth G , 201 I, LeishVet guidelines

for the practical management of canine leishmaniosis. Parasit Vec to rs 4 , 8 6 • So lano-Gal lego L, More l l R A r b o i x M , A l b e r o l a J, Fer rer L, 2001 a, Prevalence of Leishmania infantum infection in dogs living in an

area of canine leishmaniasis endemicity using PGR on several tissues and serology, j C l in M ic rob io l 3 9 , 5 6 0 - 5 6 3 • Solano-Gal lego L, Riera C , Rou ra X , Iniesta L, Gal lego M.Valladares JE, Fisa R, Castil lejo S, A lbe ro la J, Ferrer L, A r b o i x M, Portus M,

2001 b, Leishmania infantum-specific IgG, IgG I and lgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in

the course of infection and after treatment.Vet Parasitol 9 6 , 2 6 5 - 2 7 6

• Tabar M D , R o u r a X , Francino O , A l t e t L, Ruiz de G o p e g u i R, 2008, Detection of Leishmania infantum by real-time PGR in a canine

blood bank.] Small A n i m Pract 49, 325-328

Immunology of Canine Leishmaniosis. What do I have to know? Fernando Farinas Introduction to the immune response Infectious diseases in any individual are not only caused by the presence o f a pathogen but by its interact ion wi th the host immune sys tem.The immune response can be classified in t w o types: the initial response that tr ies t o contain rapidly the pathogen, in a non-specif ic, s tereotyped way and unable t o create any m e m o r y (innate immune response), and that able t o set up a n u m b e r o f part icular and highly specific strategies in o r d e r t o el iminate the invading agent and to create a m e m o r y (adaptive immune response).To simplify, this adaptive response may consist o f a p r e d o m i n a n t T h I cellular response, p roduc ing high levels o f cytokines IFN-y, IL-12 a n d T N F -a and the p roduc t ion o f lgG2a, a p redominan t T h 2 humora l response wi th the p roduc t ion o f cytokines IL-4, IL-10 and IL-5 and immunoglobul ins A , E and IgG I, o r a m ixed T h 1 ATh2 response.

T h e immunity o r innate response is an immedia te response activated via receptors codif ied in a germl ine and known as pattern recognit ion receptors (PRRs) that can recognize molecu lar patterns that have been conse rved through evolut ion in many pathogens.These molecules, k n o w n as pathogen-associated molecu lar patterns (PAMPs) , st imulate cell signalling, gene express ion and, therefore, the activation o f in f lammatory and antimicrobial funct ions.The innate response, apar t f r om being a rapid line o f defence against infection, initiates the process that enables the eventual deve lopmen t o f the adaptive immune response and sets the immune memory .

Mammals can be infected by a huge n u m b e r o f microorganisms (viral, bacterial, p ro tozoa l , etc.) that fundamental ly have different structures, biology and spread mechanisms. However ; the innate immune system has a l imited n u m b e r o f available PRRs that enable the recognit ion o f s o m e c o m m o n parts in very different infectious agents.

P A M P s are microbial molecu lar structures that have been conse rved through evolut ion and, therefore, are shared by different microbial species. M o r e o v e r P A M P s are essential fo r the g rowth o f m ic robes and rarely modi f ied by the organisms, enabl ing the recognit ion by the innate immune sys tem.The second principle in the innate immune recognit ion is the detec t ion o f molecules wi th aberrant localization, mainly nucleot ide structures.

Microbia l infections are associated wi th the insert ion o f nucleot ides ( D N A and R N A ) both in the e n d o s o m e and cytoplasm,

localizations cons idered as abnormal for these structures.

T h e innate immune system detects nucleot ides in these aberrant localizations thanks to the express ion o f PRRs in the cellular

e n d o s o m e and cytop lasm in such a way similar t o microbial recogni t ion.

A l l v iruses and a subset o f bacter ia and parasites spread in the infected host cell. A m o n g parasites, s o m e p r o t o z o a enter the cells and create a single m e m b r a n e sur rounding the cytoplasmic compar tmen t , the paras i tophorous vacuole, w h e r e repl icat ion takes p lace.The m e m b r a n e glycophosphat idyl inosi to l (GPI) is the main P A M P for the recogni t ion o f p r o t o z o a in the extracel lular space, whereas in the e n d o s o m e , receptors for D N A and d s R N A have been repo r t ed t o contr ibute t o the innate immune response against t h e m .

Immunology of Canine Leishmaniosis T h e resistance o f dogs to Leishmania infection depends on the deve lopmen t o f a strong cellular immune response, in wh ich active C D 4 + T h I lymphocytes take par t by means o f the synthesis and release o f cytokines such as interleukin-2, inter leukin-12 and in ter feron-gamma (IFN-y), each o f t h e m requi red fo r macrophage activation, the effector response o f C D 8 + cytotoxicT-cel ls and the cytotox ic activity o f natural killer cells (NK) , tha t lead t o the destruct ion o f the parasi te.The leishmanicidal activity is due t o the increased capacity t o p roduce tox ic oxygen and nitrogen radicals ( N O ) by macrophages in response t o IFN-y. However , if the response is leaded by C D 4 + T h 2 cells that p roduce interleukins 4 and 10, it may pred ispose t o a m o r e ser ious clinical picture.

S o m e publ ished studies suggest that canine leishmaniasis cou ld be a ve ry useful mode l in the investigation o f human visceral leishmaniasis. Certainly, one cou ld argue that there are s o m e differences be tween canine and human leishmaniasis clinical and pathological aspects, such as the greater human resistance to infection and the presence o f clinical manifestations in dogs (e.g. ulcerative dermati t is associated w i th infection) that have never been r e p o r t e d in humans.

Desp i te this, bo th species have many immune similarit ies.Thus, as already ment ioned , the resistance t o infection is related t o the type and quality o f the immune response both in human and canine leishmaniasis and w h e t h e r it is cutaneous, mucocu taneous o r visceral leishmaniasis. In human cutaneous leishmaniasis, the local p roduc t ion o f IFN-y med ia t ingTh I responses plays a critical role in the resistance by means o f the activation o f infected macrophages. Likewise, dur ing the active clinical phase o f visceral leishmaniasis, the re is a marked depress ion o f bo th the Le/shmanio-specif ic lymphoprol i ferat ive responses and the p roduc t ion o f IFN-y a n d the delayed hypersensit ivity responses against different antigens o f the parasite,This anergy seems t o be media ted, at least partially, by a suppressive effect o f IL-10 and low levels o f IL-12. A l l these mechanisms are essentially identical t o those descr ibed in dogs excep t fo r the role o f IL-10 in dogs, w h e r e it wou ldn ' t seem t o play such an impor tan t o r decisive role in the pathology.

Paying attent ion t o humora l immuni ty against leishmaniasis in sick humans and dogs, the re have been polyclonal, and somet imes monoc lona l , increases in immunoglobu l in p roduc t ion by hyperact ivat ion o f B cel ls.These ant ibodies are non-pro tec t ive and the i r level is even positively cor re la ted to the presence and severity o f the disease.Thus, resistant humans and dogs tend t o p roduce all kind o f ant ibodies in a l ow f requency and concent ra t ion whereas the critically-ill ones do the contrary. A m o n g s t these immunoglobul ins, IgG p redomina te whereas IgM, IgE and IgA are p r o d u c e d in much l ower amounts. Even, it's been found that s o m e o f these ant ibodies created dur ing infection have characterist ics o f auto-ant ibodies against erythrocytes, myocytes and nuclear componen ts , mainly in the dog, wh ich cou ld explain in par t the n u m b e r o f clinical manifestations o f the disease in them.

O n the o t h e r hand, Leishmania parasites are not inert beings but have a number o f c o m p l e x strategies to attack, infect and survive within macrophages .The host may 'fail' t o cont ro l t he disease d u e t o s o m e strains that have the ability t o resist the microbicidal act ion o f the activated macrophages and t o p roduce a state o f severe immunosuppress ion .

A s w e said, Leishmania has several surface molecules that act as v i ru lence factors as wel l as t o evade the host immune system. These mechanisms include a dense glycocalyx f o r m e d by l ipophosphoglycans and glycophosphat idyl inosi to l as wel l as proteins secreted by the parasite, including an arsenal o f glycoconjugates, sulfated proteoglycanes, acid phosphatases and meta l loprote inase gp63, that abounds in the surface o f promast igotes.

T h e r e is no doub t that the knowledge o f t hese immune mechanisms has cont r ibu ted and will fu r ther cont r ibute t o the deve lopmen t o f immunoprophy lac t ic and immunotherapaut ic strategies that will a l low a bet ter con t ro l and t reatment o f the disease,

Introduction T h e basis for establishing a co r rec t diagnosis is the understanding the dif ference be tween infection and disease. Studies s h o w that the percentage o f infected dogs in areas w h e r e the disease is endemic is very high (probably ove r 5096), but only s o m e are seroposi t ive and an even smal ler percentage are deve lop ing the disease. It is known that o n c e the promast igote is inoculated into the dog's skin, the disease progression fo l lows different paths. Maybe (not shown in a reliable way), in a small percentage o f animals, innate immune mechanisms abo r t the infection locally. In mos t cases, h o w e v e r the infection spreads locally and triggers a specific immune response. D e p e n d i n g on the type o f response, the infection progresses t o clinical disease o r remains under cont ro l . In those animals that deve lop a T h I -domina ted cel l -mediated immune response (probably most) , there is activation o f macrophages and destruct ion o f parasites by mechanisms med ia ted by N O . In contrast, in t hose animals w i th a T h 2 - d o m i n a t e d immune response, wi th p redominance o f humora l ant ibody product ion (IgGI, lgG2), infection cannot be cont ro l led and progresses to severe clinical disease.

T h e factors that make a certa in animal progress towards the immune cont ro l o f the disease o r t o the clinical disease are unknown. Genet ics is probably the mos t impor tan t .There are dog breeds in wh ich clinical disease is rare (Ibizan hound) and o thers in wh ich clinical disease is very c o m m o n (Rottweiler, G e r m a n Shepherd , etc.). A recent study in t w o breeds (Boxer, G e r m a n Shepherd) attr ibuted t o genetics a 6096 o f impor tance in the progression o f the infection. A n impor tan t aspect is that the situation o f 'resistant' (non-effective response) or 'suscept ib le ' (ineffective humora l response) is not definitive. A n immunosuppress ive disease, d rug t rea tment o r o t h e r factors may make that an animal that has kept the infection under cont ro l fo r years deve lops clinical signs

Liu is Ferrer

o f leishmaniasis, Similarly, the current therapy (antimonials, miltefosine, al lopurinol) gets the immune response d i rected towards the effective cel l -mediated response and contro ls the disease.

In animals w h e r e the infection progresses, several pathogenic mechanisms are tr iggered. First, the infection spreads to many organs and systems (spleen, lymph nodes, skin and mucous membranes , liver pancreas, testes, bowel...), in wh ich granulomatous inf lammatory processes o c c u r Fu r the rmore , circulating immune comp lexes (ICs) are p r o d u c e d and depos i ted in renal glomerul i , uvea, b l ood vessels and joint synovium. Depos i t i on o f ICs is a major cause o f clinical sign. Moreover ; o the r pathogenic mechanisms such as the fo rmat ion o f autoant ibodies o r chron ic anaemia are p r o d u c e d in the cou rse o f the disease.

Diagnosis of the disease T h e diagnosis o f leishmaniosis can be ve ry easy o r very difficult.The difficulties arise f rom:

1. T h e different clinical presentat ions o f disease 2. T h e confusion be tween infection and disease 3. T h e presence o f secondary infections / parasitic diseases

It is wel l known that the clinical leishmaniasis is a very p l eomorph i c disease. It is expressed differently in each animal because o f the genetic background and associated infections, amongst o the r factors. Briefly, the main clinical signs and clinical presentat ions o f the disease are:

1. Skin lesions: exfoliative dermatit is, skin ulcerat ions and mucocu taneous junct ion ulcerat ion, skin nodules, etc. 2. Lymphadenopa thy (reactive lymphatic hyperplasia) 3. Weakness , anorex ia, weight loss, muscle atrophy, mild hyper thermia 4. Renal failure (proteinuria, azotemia) 5. O c u l a r lesions (keratitis, uveitis, panophthalmit is, g laucoma) 6. Lameness (arthritis, myositis)

7. Epistaxis 8. Large bowe l chron ic d ia r rhoea (colitis)

T h e disease has an insidious onset and a chronic course and deve lops ove r weeks o r months. Somet imes, it is useful t o k n o w that the process has not responded t o antibiotics o r steroids.

Clinical tests are also highly variable, reflecting the variety o f pathogenic mechanisms as wel l as the deve lopmen t and severity o f the disease. Hype rp ro te i nem ia due t o hyper-gamma-globul inemia, mode ra te non-regenerat ive anaemia and prote inur ia o f varying severity are c o m m o n findings, but there are ve ry different presentat ions.

In summary, w e cou ld say that the diagnosis is established in a patient wi th consistent clinical signs or clinicopathological alterations and a positive quantitative serology (IFAT, ELISA).

It must be r e m e m b e r e d that the diagnosis is a clinical op in ion, issued by the surgeon after evaluating a series o f findings and results (history, exams, analysis...). T h e r e are no ' D I A G N O S T I C tests. A c c o r d i n g t o the so-cal led ev idence-based medic ine, a g o o d diagnostic test is that o n e that increases the probabi l i ty o f a specific diagnosis.

Quant i tat ive serology is the best way to conf i rm a diagnosis o f leishmaniosis in clinically suspected cases because:

i) It is known that a high ant ibody t i tre is cor re la ted wi th clinical disease (i.e., it does not only indicate infection) ii) It's s imple, fast and inexpensive and is readily available iii) It can de used to assess patient response to therapy

M u c h o f t he confus ion stems f r o m the use o f P C R techniques, m o r e adapted to the identification o f the infection than to the regular d iagnosis.The diagnosis o f infection is only interesting in specific epidemiological o r research works . It can be d o n e by means of:

- P C R (different techniques)

- Sero logy - Cu l tu re ( low sensitivity and high cost)

Cy to logy and histopathology ( low sensitivity) Intradermal Test

T h e p rob lem is that the use o f P C R techn iques (simple, nested o r quantitative) has been ex tended on the conv ic t ion that it a l lowed a highly sensitive diagnosis o f the disease. H o w e v e r o n e must bear in mind that these techniques only r e p o r t the presence o f the parasite's D N A in the sample .There fore , a negative result is compat ib le wi th:

1. A dog that is not infected, that does not suffer leishmaniosis 2. A dog infected w i th the parasite in o the r parts o f the b o d y but no t in the sample ( c o m m o n situation)

3. A d o g infected and wi th leishmaniosis, wi th the parasite in o the r parts o f the body but not in the sample (rare but c o m m o n situation)

Likewise, a posit ive result is consistent wi th an infected animal that has no leishmaniosis (a situation m o r e c o m m o n in endemic areas) and wi th a sick animal.The p rob lem is that P C R is a qualitative techn ique (posit ive/negative) and does no t help t o distinguish an infected-healthy animal f r o m an infected-sick one . In endemic areas, this is a ser ious p rob lem. Quant i ta t ive P C R partially resolves this p rob lem. However ; the corre lat ion be tween clinical picture and test results is much greater wi th serology, as shown by var ious studies.

T h e figure shows a possible p ro toco l fo r a definitive diagnosis [So lano-Gal lego et al, 201 I]:

F igu re I I D i a g n o s t i c a p p r o a c h t o d o g s w i t h s u s p e c t e d c l in ica l s igns a n d / o r c l i n i c o p a t h o l o g i c a l a b n o r m a l i t i e s c o n s i s t e n t w i t h C a n i n e L e i s h m a n i o s i s

Confirmed CanL

POSITIVE

LOW

POSITIVE

NEGATIVE -̂1 s High

suspicion H of CanL

I—>i NEGATIVE —'f ComUer other

References • Baneth G . Ganine leishmaniosis - new concepts and insights on an expanding zoonosis: part one.Trends in Parasitology (2008); 24:

324-30.

• Ga lvez R. Emerging trends in the seroprevalence of canine leishmaniasis in the Madrid region (central SpoinJ.Veterinary Parasitology (2010); 169: 327-34.

• So lano-Ga l lego L et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veter inary Parasitology (2009); 165: 1-18.

• So lano-Ga l lego L et al. Leishvet guidelines for the practical management of canine leishmaniosis. Parasites and Vec to rs (201 I); 4:86 (ht tp: / /www.parasi tesandvectors.eom/content /4/1 /86) .

CaniLeish®: Development of a. new -preventive tool, How do you produce* a vaccine which really works? David McGahie History of the development T h e h istory o f C a n i L e i s h ® research dates back t o the early 1980s. A t this t ime a ca tegory o f proteins, the ESPs, standing for Excre ted Secre ted Proteins, had been identified in numerous species o f unicellular parasites such as Leishmania, Babesia and s o m e others .Th is category o f proteins was thought t o be involved in macrophage infection and there fore held potential fo r use as antigens, e i ther in diagnostic kits o r in immunotherapeut ic o r immunoprophy lac t i c vaccine applications. H o w e v e r these ESPs w e r e p roduced only in ve ry l imited quantity and purif ication f rom the o the r c o m p o n e n t s o f the culture m e d i u m was difficult as Leishmania requi red ve ry rich culture med ia and inoculat ion in animal hosts t o comp le te its life cycle.

T h e first breakthrough was an invention f r o m the t eam o f Jean-Loup Lemesre in the Institut de Recherche p o u r le D e v e l o p p e m e n t (IRD) o f a comple te ly def ined in vi t ro m e d i u m containing only precise quantit ies o f small molecu les such as sugars, amino acids and vitamins that was able t o sustain the deve lopmen t and mult ipl ication o f the parasite, I This m e d i u m is also se rum free and cell free, wh ich is impor tan t because the only proteins that will be found in the culture m e d i u m supernatant are o f Leishmania origin - the Leishmania infantum ESP (L iESP).The IRD t e a m then began a co l laborat ion w i th Bio V e t o Test (BVT) , a diagnostics company that is n o w a who l l y -owned subsidiary o f V i rbac, t o exp lo re the potential o f the L iESP antigens, initially in the con tex t o f some diagnostic tests they w e r e deve lop ing and then later in the con tex t o f bo th therapeut ic and prophylact ic canine vaccinat ion.The IRD granted an exclusive l icence to B V T for the use o f the invented m e t h o d o f p roduc ing L iESP for animal health applications.

Be tween 1997 and 2003 , in conjunct ion w i th the French Nat iona l Veter inary Schoo l o f Lyon, trials w e r e pe r fo rmed wi th a p ro to type L iESP vaccine adjuvanted wi th muramyl d ipept ide ( M D P ) wh ich p r o d u c e d encouraging results w h e n used as immunotherapy and also for disease prevent ion in bo th challenge mode l and field cond i t i ons . 2 3 , 4 In the field trial the infection rate found in the contro l g roup was l o w (less than 796 after t w o years), but the potential fo r use o f ESP in a prophylact ic vaccine was conf i rmed. H o w e v e r the M D P adjuvant was only used in exper imenta l settings and it is known that its derivatives can also induce a T h 2 response in aqueous condi t ions. 5 Fu r t he rmore ESP p roduc t ion at industrial levels had not been def ined.Therefore significant fu r ther w o r k was requi red t o deve lop the concep t o f ESP use t o a vaccine wh ich cou ld be p r o d u c e d commerc ia l ly .The V i rbac R & D team jo ined the co l laborat ion t o add the i r exper t i se in vaccine formulat ion and deve lopmen t in o r d e r t o br ing the vaccine to its cur rent fo rm.

Developmental steps Var ious deve lopmenta l steps w e r e required. It was necessary t o define the parameters required fo r the industrial scale-up o f the ESP p roduc t ion f rom the small vo lumes o f the pi lot batches t o batch sizes wh ich w o u l d enable the p roduc t t o be made available widely t o general pract i t ioners. It was also necessary t o fu r ther define the character o f the ESP A s par t o f this process the presence o f the major antigens in the vaccine - parasite surface antigen (PSA) was con f i rmed .These proteins are present in a m e m b r a n e - b o u n d f o rm (on bo th promast igotes and amastigotes) and also excre ted /secre ted in free f o rm and are involved in macrophage-paras i te interactions making t h e m suitable vaccine ta rge ts . 6 The unique produc t ion system used fo r C a n i L e i s h ® , using the parasites themselves t o p r o d u c e the proteins, means that the proteins retain the i r native con fo rmat ion .The character izat ion o f the ESP also a l lowed the fu r ther d e v e l o p m e n t o f specific qual i ty-control assays wh ich prov ide assurance o f the consistency o f the prote in profi le f r o m batch to batch.

In addi t ion t o character iz ing and defining p roduc t ion me thods for the ESP it was also necessary t o select a suitable adjuvant fo r the vacc ine .The main requ i rement is the safe induct ion o f a T h I profi le response, Var ious opt ions w e r e cons idered and finally Q A - 2 1 was selected as it has an o p t i m u m benef i t r isk profi le and is an excel lent inducer o f the in ter feron-gamma (IFN-y) med ia ted T h I response.

Proving the concept - the action on the immune system C h o o s i n g an antigen and adjuvant based on the l i terature ev idence and pi lot studies is not sufficient t o prov ide conf idence in the ability o f the vaccine t o pe r fo rm wel l in full efficacy testing. It is also necessary t o fo l low the impact the vaccine has on the immune system o f the dog.This can also a l low s o m e cal ibrat ion o f the co r rec t levels o f antigen and adjuvant.

Dur ing the deve lopmen t o f C a n i L e i s h ® several assays w e r e used to assess the way in wh ich it modu la ted the immune response. In conjunct ion wi th the laboratory o f D r J M o r e n o in the W H O Col labora t ing C e n t r e for Leishmaniasis, Instituto de Salud Ca r l os III in Madr id the ability o f the vaccine to induce m e m o r y cells wh ich w o u l d then prol i ferate specifically in response to exposure t o Leishmania infantum was conf i rmed. In the same laboratory this was then taken a step fu r ther and by means o f the ELISpot test, 7 wh ich measures the p r o p o r t i o n o f the replicating cells wh ich are produc ing IFN-y, t h e T h I polarity o f these m e m o r y cells was con f i rmed.The c o m b i n e d use o f these assays demons t ra ted that w h e n exper imenta l dogs w e r e vaccinated wi th C a n i L e i s h ® they w e r e able t o p roduce the expec ted T h I m e m o r y cells t o L infantum.

H o w e v e r it is also possible t o take this o n e step fur ther In conjunct ion wi th the labora tory o f Professor IVouldoukis at I N S E R M , U P M C - U M R S 945, Immunite et Infections, Paris, it was possible t o utilize the Can ine Macrophage Leishmanicidal Assay ( C M L A ) 8

which con f i rmed that not only did the vaccine induce m e m o r y cells o f a T h I polarity, but that these m e m o r y T cells w e r e capable o f stimulating auto logous macrophages to kill L infantum promast igotes even I year after the vaccinat ion. Fur ther assays also con f i rmed that the level o f parasite killing was cor re la ted w i th induct ion in the macrophages o f inducible nitric ox ide synthase and produc t ion o f a nitric ox ide-based oxidat ive burst .This is consistent w i th t he k n o w n mechan ism o f act ion o f the desired T h I-domina ted cel l -mediated immune response. 9

Fur ther use o f these assays dur ing an exper imenta l challenge trial con f i rmed that even after a vaccinated dog is chal lenged wi th massive levels o f living L. infantum parasites the cor rec t T h I -domina ted cel l -mediated immune profi le is retained, C o m b i n i n g this knowledge o f the m o d e o f act ion o f the vaccine wi th the posit ive results o f an exper imenta l challenge study provides the conf idence to progress to a natural challenge study wh ich is the only suitable way to prov ide a final assessment o f the efficacy o f any vaccine against Can ine Leishmaniosis.

Conclusion T h e successful p roduc t ion o f the first vaccine in Eu rope against Can ine Leishmaniosis began wi th the select ion o f an appropr ia te antigen and adjuvant combina t ion wi th each c o m p o n e n t being known for the i r potential in this regard. This was fo l lowed by significant investment t o ensure that consistent p roduc t ion o f the antigens was possible and by fu r ther w o r k t o understand the mechan ism o f act ion o f the vaccine in the immune response o f dogs.This cu lminated in the decis ion to progress to a natural challenge study o v e r t w o years (which will be discussed in ano ther session o f this sympos ium) and the p roduc t ion o f the relevant data requi red for the successful registration through the E M E A in the first half o f 201 I.

References 1. Lemesre JL, 1993. Patent Application number FR 2 705 358 - Al. Procede de culture in vitro de differents stades de parasites

tissulaires obtenus et applications biologiques.

2. Bourdo iseau G , Hugne t C , Papierok G , Lemesre j . Canine leishmaniosis due to L. infantum: immunotherapy trials. Bull A c a d Ve t France, 2004; 157: 63-67.

3. Lemesre JL, Ho l zmu l l e r R Cava leyra M , et al. Protection against experimental visceral leishmaniasis infection in dogs immunized with

purified excreted secreted antigens of L. infantum promastigotes.Vaccine, 2005 ; 2 3 : 2 8 2 5 - 2 8 4 0 . 4. Lemesre JL, Ho lzmu l le r R Gonca lves RB, et al. Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP

vaccine in endemic areas of France: double-blind randomised efficacy field trial.Vaccine, 2007; 2 5 : 4 2 2 3 - 4 2 3 4 . 5. C o x J C , C o u l t e r AR. Adjuvants - a classification and review of their modes of action.Vaccine, 1997; 15 :248-256 6. Kedzierski L, M o n t g o m e r y J, Bullen D, et al.A Leucine-Rich Repeat Motif of Leishmania Parasite Surface Antigen 2 Binds to

Macrophages through the Complement Receptor 3.J. Immunol., 2004; 172 : 4 9 0 2 - 4 9 0 6 7. M o r e n o J, N i e t o J, C h a m i z o C , et al.The immune response and PBMC subsets in canine visceral leishmaniasis before, and after,

chemotherapy.Wet. Immunol . Immunopathol . , 1999; 71: 181 -195 8. Vou ldouk is I, D r a p i e r J C , N u s s l e r A K , et al. Canine visceral leishmaniasis: successful chemotherapy induces macrophage antileishmanial

activity via the L-arginlne nitric oxide pathway. An t im ic rob . Agen ts C h e m o t h e r , 1996; 4 0 : 2 5 3 - 2 5 6

9. W a n a s e n N , S o o n g L. L-arginine metabolism and its impact on host immunity against Leishmania infection. Immunol Res., 2008; 41:15-25,

Results of safety trials with CaniLeish®. What can be expected? ^^^^ David McGahie Introduction T h e safety profi le o f any vaccine is a very impor tan t factor fo r pract i t ioners consider ing its use. Vaccines are normal ly used t o prevent disease and there fore are used in previously healthy animals w h e r e administer ing the vaccine is a cho ice for the o w n e r This means that adverse react ions are perce ived as being less acceptable than wi th medicat ions used to t reat ser ious illnesses. Addit ionally, the populat ion wh ich is vaccinated is likely t o be much larger than the populat ion wh ich w o u l d receive a therapeut ic medicat ion and so even rare adverse responses may be m o r e apparent.

A d v e r s e reactions t o vaccines are generally cons idered using t w o different approaches: 1) React ions are classified as e i the r 'no rma l ' or ' inappropr ia te ' .Trans ient fever, malaise, local inf lammation and mild local pain have

been classified as 'normal ' react ions t o vaccines because they are the result o f the norma l immune response t o vaccinat ion. 1

React ions persisting over a long durat ion o r o f greater intensity w o u l d be classified as ' inappropriate' .

2) React ions are d iv ided into loca l ' and 'general ' . Local react ions occur r ing at the site o f injection s o o n after the vaccinat ion is adminis tered are relatively easy t o link wi th the act o f vaccinat ion. Gene ra l reactions such as gastrointestinal upsets are less easy to link to the administrat ion o f the vaccine wi th conf idence, as they can also be caused by o the r unrelated events.

Significant w o r k has been d o n e th roughout the deve lopmen t o f C a n i L e i s h ® t o ensure that the high level o f p ro tec t ion it prov ides is also associated wi th a g o o d safety profi le and to assess the expec ted react ions that may be encoun te red dur ing its use.

T h e r e are th ree main sets o f data available t o be discussed dur ing this session. A ) Specific laboratory studies t o assess the safety o f ex t ra doses and overdoses B) Field trials t o assess the safety o f the vaccine under m o r e normal condi t ions o f use in a w ide range o f breeds and ages C ) Post- launching pharmacovigi lance and informal feedback f r o m Portugal w h e r e the vaccine is current ly marke ted

Safety data

A) Laboratory studies assessing the safety of an extra dose or an overdose T w o studies w e r e pe r fo rmed . Firstly the safety o f four doses - the normal p ro toco l is th ree doses at three w e e k intervals, and secondly the safety o f an over -dose w h e r e a doub le dose is given. For bo th o f these studies w e have a similar study design: • T h e vaccine doses w e r e formula ted t o contain 10% m o r e antigen than normal commerc ia l doses. • Ten dogs w e r e vaccinated, and five dogs w e r e kept as controls. • T h e dogs w e r e all four months o ld beagles ( two months younger than the recommenda t i on o n the l icense, younger dogs

being m o r e likely t o suffer adverse react ions t o vaccinat ion). • T h e injection site was shaved t o a l low easy and accurate assessment o f any react ions occur r ing in the local area. • A f te r each vaccinat ion a detai led examinat ion was pe r fo rmed o f the clinical status and the local injection site daily fo r 14 days. • W e e k l y haematological analysis was pe r fo rmed .

In the first study, ten ou t o f ten puppies had local swellings o n at least one occas ion .The m a x i m u m size was ten cent imeters, but mos t o f the swellings w e r e only not iceable by palpat ion, despite the site being shaved. A l l resolved spontaneously wi th in 12 days at the most. A single puppy had mi ld hyper the rmia but only after the first injection. Overa l l the fou r injections w e r e wel l to lera ted.

In the second study, w h e r e a doub le dose was administered, the re was a slightly higher inc idence o f mild hyper the rmia (three o f the ten puppies, each fo r only I day). Seven o f the ten demons t ra ted a local swelling: m a x i m u m size seven cent imeters, m a x i m u m durat ion five days. Aga in the vaccine was ve ry wel l to le ra ted.

B) Field trials: Again t w o studies w e r e pe r fo rmed. Both involved c l ien t -owned animals o f var ious breeds and ages given the normal pr imary course o f 3 injections at 3 w e e k intervals.The injection sites w e r e no t shaved for these studies.

T h e first study involved 15 I dogs. 61 w e r e puppies aged 4 o r 5 months, and 9 0 w e r e 6 months o f age o r o l de r (the l icensed age range). A l l animals received veter inary examinat ions pre-vaccinat ion, 4 hours after vaccinat ion and days 2, 7, 14 and 21 post vaccinat ion as wel l as daily checks by the o w n e r s to specifically include examinat ion o f the injection s i te .The puppies received addit ional veter inary examinat ions on days 1, 3 and 4. A r o u n d 2 5 % o f the animals aged 6 months o r o lde r had detectable local reactions at the injection site w h e n assessed by the veter inary surgeons.The m a x i m u m durat ion was 15 days in a single animal after the first in ject ion.The m a x i m u m durat ion after the second and th i rd injections was I week. In this age g roup the incidence o f animals showing at least o n e general sign such as lethargy, hyper thermia, gastrointestinal upsets etc was in the region o f 15 t o 2096 after each injection. H o w e v e r this includes events such as apathy and d ia r rhoea beginning as late as 14 days after the administrat ion o f the vaccine wh ich cou ld there fore be unrelated. T h e incidence o f local reactions and general signs was, unsurprisingly, higher in the underage puppies. W i t h regards t o the general signs, in mos t cases it was not the same animals wh ich reacted after each vaccinat ion. F o r example , wi th the animals displaying hyper thermia, o n e puppy had hyper the rmia after the first and third injections but not the second. O n e adult had hyper thermia after each o f the th ree injections, but this animal also reco rded episodes o f hyper thermia unrelated to vaccinat ion. A l l o the r cases o f hyper the rmia w e r e isolated and not repeated.

T h e second field study obta ined the percept ion o f the owners , w h o w e r e aware that this was a vaccine in deve lopmen t and there fore had t o be assessed fo r safety. It involved 23 I dogs aged 6 months t o 12 years. A r o u n d o n e quar te r o f these w e r e young dogs close to 6 months o ld .The dogs w e r e assessed by a veter inar ian on the days o f the vaccinations, and 2 w e e k s after the third vacc ine.The o w n e r s w e r e asked to check the i r animal each day and specifically t o check the injection site for swell ing o r pain. A n y abnormal i ty was to be repo r t ed to the veterinarian. In this study, 8 dogs (3.596) had local react ions repo r ted , and in only one o f these dogs was it r epo r t ed after each o f the injections. In this dog it was o f sho r te r durat ion and mi lder intensity at each subsequent occas ion.The mos t c o m m o n general sign no ted was lethargy, no ted in 5 dogs (2.296) lasting fo r I day in 4 o f the dogs, and 3 days in the o ther .Therefore w h e n used in a study under normal field condi t ions w e saw a slightly higher rate o f r epo r t ed reactions than is usual fo r a convent ional vaccine such as D H P P i L , but still found the vaccine t o be ve ry wel l to le ra ted in the majority o f animals.

C) Post-launching pharmacovigilance and informal feedback Clear ly this is a dynamic situation, and at the t ime o f wr i t ing insufficient data are available t o d raw any meaningful conclusions as the vaccine has been marke ted for only a ve ry shor t t ime. In the coming months and years this will be an impor tan t source o f m o r e detai led informat ion.

Conclusion: Veterinarians can expec t s o m e dogs t o s h o w shor t - te rm lethargy, mode ra te hyper the rmia and local reactions. In mos t cases these should be self-limiting o r respond rapidly t o symptomat ic t rea tment if requ i red.Very occasional ly this may be p ro longed and rare cases o f allergic react ions have been repo r ted .

Not i fy ing the o w n e r that s o m e shor t - te rm lethargy o r local swell ing may be expec ted in a minor i ty o f animals is sufficient in most cases t o ensure that the i r expectat ions are met and t o reduce any d isappoin tment if this occurs . O n l y in very rare cases is it d e e m e d necessary t o not cont inue wi th the remainder o f the pr imary vaccinat ion schedule due t o an adverse response after assessment o f the r iskbenef i t in the individual situation by the veterinarian. Indeed there appears t o be a t rend for local reactions to be o f a smal ler size and reduced intensity at subsequent vaccinations.

Overa l l this vaccine is well to le ra ted and the data conf i rms that C a n i L e i s h ® has a g o o d safety profi le fo r an effective anti-Leishmania

vaccine.

Reference: I. T izard IR. Veterinary Immunology: an introduction. 8th Edition, 2009. Saunders Elsevier, St Louis, Missouri. ISBN: 978-1 -4160-4989-0

Results of efficacy testing with CaniLeish®. How does it perform against high level natural challenge? Christophe Reme Tools and classification To understand h o w the efficacy o f C a n i L e i s h ® was assessed, it is necessary t o first appreciate the different too ls used to m o n i t o r

infection wi th Leishmania and h o w they w e r e used t o classify the progression o f the disease.

T h e first set o f too ls looks at the effects o f the parasite on the dog.This includes, fo r example , mon i to r ing clinical signs such as apathy, anorex ia and dermato logica l signs, and measurement o f laboratory abnormal i t ies such as reversed albumin-globul in ratio o r increased total p ro te ins .The measurement o f the level o f ant ibodies p r o d u c e d by the dog in response t o the parasite is also d o n e using IFAT

T h e second set o f techn iques is fo r detect ion o f the parasites themselves in the dog. Live Leishmania may be directly obse rved in aspirates f r o m the b o n e m a r r o w o r the lymph nodes, and these aspirates can also be cul tured by inoculat ion in specific med ium. Cu l tu re is the m o r e sensitive m e t h o d o f the two .

A n even m o r e sensitive m e t h o d is the use o f P C R t o detec t Leishmania D N A . T h i s techn ique al lows us to detec t even t races o f the parasites, and quantitative P C R has the addit ional advantage o f al lowing us t o measure the parasite load.

T h e s e too ls can be c o m b i n e d t o evaluate the status o f the dog towards infection and t o classify this into several categories:

• Stage I: Dogs that have never met the parasite are Leishmania free .They will be negative in all tests. • Stage 2: D o g s that ha rbo r the parasite, but in wh ich the parasite is not actively multiplying o r is present at only very l ow levels

will normal ly be P C R positive but negative on all o the r tests.This is t e r m e d subpatent infection.

In these first t w o categories, the re is no ev idence o f active infection.

• Stage 3: W h e n the parasite starts t o multiply the d o g is not only P C R posit ive but will also b e c o m e culture posit ive. Initially, there are still no clinical signs, and so this active infection is t e r m e d asymptomatic active infection. H o w e v e r it is only a mat ter o f t ime before the disease progresses and b e c o m e s clinically apparent.

• Stage 4: In this final stage the IgG titre rises and clinical signs start t o develop.This is there fore symptomatic active infection.

In these t w o final stages the re is conf i rmat ion o f active infection, and already the d o g is losing the battle wi th the parasi te.The goal o f any vaccine against leishmaniosis is t o stimulate and direct the immune response o f the dog such that it responds appropr iate ly t o parasite infection before it progresses to this active stage, prevent ing dogs f rom switching t o the disease state,

Experimental challenge model C a n i L e i s h ® was first evaluated using an exper imenta l challenge mode l . I 0 a 5 Leishmania infantum taken f r o m an infected dog's spleen w e r e injected by IV route t o 20 dogs. Hal f o f these dogs had been vaccinated o n e year before wi th C a n i L e i s h ® , w i th th ree injections as a pr imary course , whi le the o the r ten dogs w e r e kept unvaccinated.The dogs w e r e o b s e r v e d ove r a for ty-seven w e e k per iod a f te r the challenge wi th regular b l ood sampling t o assess haematology, b iochemis t ry and IFAT parameters, and regular bone m a r r o w aspirates t o detect the parasite using bo th culture and quantitative P C R methods.

Seven o f the ten cont ro l dogs deve loped a persistent infection th roughout the study w i th posit ive P C R and posit ive culture results conf i rming active infect ion.This ex t remely high n u m b e r emphasizes the severity o f the challenge as it is normal ly expec ted that much less than 7096 o f dogs are naturally susceptible t o develop ing the disease. O n e contro l dog became posit ive wi th P C R by the end o f the study but remained negative on culture and so this was classified as a subpatent infection. In the o the r t w o dogs the re was a t ransient posit ive P C R result, but these dogs re turned to the negative state by the end o f the study per iod . T h e y b locked the infection and these dogs are naturally highly resistant t o the disease.

By contrast seven o f the ten vaccinated dogs b locked the infection. Four o f these w e r e posit ive fo r P C R a n d / o r culture at s o m e point but they rever ted t o the Leishmania f ree state by the end o f the study and the o t h e r th ree w e r e Leishmania f ree at all test points.

In th ree dogs, h o w e v e r persistent active infection deve loped dur ing the study per iod . This is a small n u m b e r consider ing the severity o f challenge and the results in the cont ro l group, and the fact that the challenge t o o k place o n e year after the vaccinat ion course and no boos te r injections w e r e given.

This study conf i rmed that C a n i L e i s h ® induces a long-lasting immune response that was proven to be effective ove r o n e year after the last vaccinat ion

High level natural challenge - the pivotal efficacy assessment. In o r d e r t o truly assess the efficacy o f a vaccine against Can ine Leishmaniosis, it is necessary t o see h o w it per forms against high level natural challenge w h e r e there is a constant high infection pressure prov ided by wild-l iving sandfly vec to rs .The pivotal efficacy study wi th C a n i L e i s h ® used such a natural challenge under field condi t ions.This study was conduc ted in t w o parts. In the first part, 80 Leishmania free beagles o f approx imate ly 6 months age w e r e housed in p ro tec ted labora tory condi t ions and randomized into t w o groups. O n e group was given a pr imary course wi th C a n i L e i s h ® (three injections at th ree weeks intervals), whi le the o the r half o f the dogs remained unvaccinated.The dogs w e r e fo l lowed for nearly o n e m o n t h after vaccinat ion t o m o n i t o r the serological and cellular response in vaccinated dogs.

T h e dogs w e r e then m o v e d t o o p e n kennels in t w o ve ry contaminated areas; o n e in Spain (near Barcelona) and the o the r in Italy (near Naples) wi th equal numbers o f vaccinated and cont ro l dogs at each s i te .The dogs w e r e t ransferred dur ing the sand fly season to ensure rapid strong exposu re and they w e r e e x p o s e d to this natural challenge ove r t w o consecut ive t ransmission seasons in 2007 and 2008. N o repel lent was a l lowed for these dogs, o r any t rea tment against le ishmaniosis.The vaccinated dogs received an annual boos te r wi th C a n i L e i s h ® .

T h e dogs w e r e assessed every th ree months using clinical and paraclinical examinat ions, as wel l as culture o f b o n e m a r r o w o r lymph n o d e aspirates and quantitative and qualitative P C R to detec t parasites.These parameters w e r e used as expla ined previously t o classify the dogs into the fou r infection categor ies and specifically t o evaluate w h e t h e r an active infection was deve lop ing in these dogs o r not.

T h e severity o f the natural challenge in these condi t ions was conf i rmed because 7296 o f the cont ro l dogs became posit ive by P C R at least o n c e ove r the t w o year per iod o f the study, clearly indicating a ve ry strong parasitic pressure.This is fu r ther emphas ized by the fact that 2396 o f con t ro l dogs deve loped clinical symp toms o f the disease wi th in the t w o years o f the study, and in a fu r ther 1096 an asymptomat ic active infection was obse rved . Mean ing that in total about one- th i rd o f the cont ro l dogs deve loped active infection.

In the vaccinated g roup only 796 deve loped symptomat ic infection and 596 deve loped an asymptomat ic active infection, meaning that nearly nine ou t o f 10 vaccinated dogs did not deve lop active infection ove r the study despi te the intense challenge received, This reduct ion in the number o f dogs progressing t o active infection by the end o f the trial is statistically significant (p=0.025). Examining the results o v e r t i m e by means o f a survival analysis gives a similarly significant result (p=0.027)

W h e n only the deve lopmen t o f symptomat ic disease is c o m p a r e d be tween groups the differences are also significant. A t the end o f the trial, 9396 o f dogs in the vaccinated g roup remained free o f symp toms (p=0.046) and survival analysis o f the difference ove r t ime also conf i rms that the vaccine lowers the probabi l i ty o f deve lop ing symptomat ic disease (p=0.047).

T h e impact o f a vaccinat ion at popula t ion level is interesting, but o f course it is the individual benefit o f vaccinat ion for the individual patient wh ich is o f greatest interest in small animal medic ine. In o r d e r t o establish this, risk calculations are used and this can prov ide a measure o f efficacy applicable t o an individual dog .The odds are a classical measure o f relative risk used in clinical studies.They are calculated f r o m the probabi l i ty o f an event happening div ided by the probabi l i ty o f that event not happening and groups are c o m p a r e d by calculating odds ratios. In this study the odds ratio for active infection is I t o 3.6, and for symptomat ic disease the odds ratio is I t o 3,8, wh ich in practical t e rms means that the vaccine reduces the risk o f disease approx imate ly four-fold.This four-fold reduct ion o f risk is there fore the official claim on the app roved S P C o f the product .

Conclusion U n d e r severe challenge condi t ions, in highly endemic areas, C a n i L e i s h ® has demons t ra ted a significant ability t o reduce the risk o f a d o g develop ing active infection o r symptomat ic disease four-fold.The results obta ined wi th the vaccine are consistent w i th the m o d e o f act ion. In c o m m o n wi th the vast majori ty o f vaccines it does not prevent the pathogen f r o m enter ing the body, but rather stimulates and directs the immune response in o r d e r t o c o p e wi th the challenge, thus prevent ing progression t o active infection.

Practical use of C-^aniLeish * What do I need to know to get the best from this new tool? David McGahie Introduction T h e pu rpose o f this session is t o cons ider s o m e o f the data sheet recommenda t ions and requ i rements as wel l as s o m e practical suggestions t o enable pract i t ioners t o understand the opt imal use o f the vaccine w h e n it b e c o m e s available in the i r countr ies,

The basic protocol W i t h C a n i L e i s h ® , the basic initial starting p ro toco l is th ree injections (subcutaneously) at th ree w e e k intervals.This is slightly different t o a convent ional parvovi rus o r d i s temper vaccinat ion and is because the object ive is t o induce a T h I -domina ted cel l-med ia ted immunity, Simply inducing ant ibodies is not sufficient fo r p ro tec t ion against Leishmania, Studies pe r fo rmed during deve lopmen t have con f i rmed that this is the opt imal schedule for the pr imary c o u r s e . T h e declared onset o f immunity is four weeks after the third injection.

T h e start ing age f r o m wh ich puppies can be vaccinated is six mon ths o f age and oider.The efficacy test ing in all o f the deve lopmenta l studies was d o n e f rom the age o f six months and it is bel ieved that the maturat ion o f the cel l -mediated immune response capability in young dogs is s lower than that o f the humora l response.

Cur ren t l y there is no data o n the s imultaneous administrat ion o f C a n i L e i s h ® wi th o the r vaccines such as D H P P i L . T h e r e f o r e the cur rent r ecommenda t i on is t o separate the administrat ion o f C a n i L e i s h ® f r o m all o t h e r vaccinat ions by at least 2 weeks, wh ich is a fairly c o m m o n recommenda t i on in situations w h e r e specific test ing has not been pe r fo rmed t o conf i rm compatibil ity. T h e r e is a requ i rement for an annual boos te r wi th a single dose o f the vaccine t o maintain immunity.

The vaccine T h e vaccine itself is suppl ied in a very convent ional manner : a lyophil ized pellet (freeze dr ied pellet) and the solvent wh ich is isotonic saline solut ion. It is interesting to no te that the lyophil ized pellet looks significantly larger than for o the r convent ional dog vacc ines.The solvent is a normal vo lume (I ml), and the lyophil ized pellet dissolves into the di luent t o p roduce a normal I ml vo lume o f a reddish solut ion for injection.

A s the vaccine contains a high prote in content , v igorous shaking dur ing reconst i tut ion will result in the fo rmat ion o f s o m e foam, and it is r e c o m m e n d e d that the solut ion is simply m ixed gently t o ensure comp le te dissolut ion, wh ich occurs very easily.

In c o m m o n wi th mos t o the r vaccines it should be s tored in the fridge, and it should be taken f r o m the fridge and reconst i tuted immediate ly before use. A s wi th any refr igerated vaccinat ion it is always appropr ia te t o w a r m it slightly in the hand to bring it c loser t o body tempera tu re for the c o m f o r t o f the animal w h e n injected, and as norma l it is r e c o m m e n d e d t o lightly massage the injection site.

Pre-vaccination screening T h e market ing author izat ion is fo r the active immunizat ion o f Leishmania negative dogs . 2 The object ive o f vaccinat ion is t o prevent disease, not t o treat disease, and therefore, a d o g wh ich is already wel l on the way t o deve lop ing the disease is not a candidate for prevent ion but rather requires t reatment, W e k n o w wi th Leishmania that active infection, w h e n the d o g has progressed t o having a high parasite load o f actively replicating parasites, rarely if ever regresses back t o the negative state w i thou t assistance.

Var ious opt ions are possible t o detec t infection: • Sero logy is a convent ional and rout ine me thod , and high t i ters in sero logy tests such as IFAT are closely associated wi th

advanced and progressive disease. It is s imple, rapid and inexpensive t o per fo rm. • D i rec t visualization o f amastigotes in smears f rom the lymph n o d e o r the b o n e m a r r o w conf i rms that the d o g is infected.

H o w e v e r this techn ique is not sensitive enough for rout ine pre-vaccinat ion screening and dogs may still test negative despite having the disease.

• Immunohis tochemis t ry o f such smears can increase the sensitivity, but is not routinely used by many pract ices and is not adapted to the screening situation.

• Parasite culture is even m o r e sensitive again, but is not available for rout ine use in general pract ice. • Finally, P C R is the mos t sensitive techn ique o f all, but a posit ive P C R result gives no indication as t o w h e t h e r the parasite is

actually still present in a living state. Du r i ng the active t ransmission season, dogs may transiently test P C R posit ive despite not develop ing the disease. It is not adapted to the screening situation.

It is c lear that serological test ing is the mos t appropr ia te m e t h o d o f screening before vaccinat ion in the general pract ice situation

T h e data sheet o f C a n i L e i s h ® states' the detec t ion o f Leishmania infection using a rapid serological diagnostic test is r e c o m m e n d e d pr ior t o vaccination'. Rapid tests a l low a lmost instantaneous real-t ime results, al lowing vaccinat ion t o take place on the same day as testing w h e r e required wh ich is m o r e convenient fo r the o w n e r H o w e v e r it is c lear that o the r serological tests such as IFAT are also wel l adapted fo r pre-vaccinat ion screening in cases w h e r e there is no urgency fo r the result, and in the event o f a posit ive test have the addit ional advantage o f providing a t i tre wh ich is useful w h e n assessing future courses o f act ion.

W h e n using a rapid test in the con tex t o f pre-vaccinat ion screening, it is impor tan t that a test w i th excel lent conco rdance w i th IFAT is used, and ideally wi th a known thresho ld wh ich is not excessively high to avoid situations w h e r e dogs wh ich are already develop ing the disease are vaccinated. O n e such test is Speed Leish K ™ w h e r e recent ly presented data s h o w e d an excel lent conco rdance wi th IFAT and an appropr ia te cut-off th resho ld o f be tween I / 8 0 and I / 1 0 0 on the IFAT scale. 3 If a d o g tests negative wi th an appropr ia te rapid test such as Speed Leish l ^ M o r by IFAT.the d o g can be vaccinated.

Other tips A s wi th any vaccinat ion, it is r e c o m m e n d e d that animals should be d e w o r m e d pr io r t o vaccinat ion, It is wel l k n o w n that heavy w o r m burdens p r o m o t e a T h 2 - d o m i n a t e d immune response, and the requ i rement fo r prevent ion o f leishmaniosis is t o stimulate a T h I -domina ted response. O t h e r parasites may d o the same; there are publ ished suggestions that Ehrl ichia may wel l p r o m o t e the establ ishment and deve lopmen t o f leishmaniosis in dogs.There fore g o o d general parasite contro l , including internal parasites such as w o r m s and external parasites such as ticks, is no t only beneficial fo r the general health o f the dog, but also reduces o t h e r factors wh ich may act con t ra ry t o the beneficial effects o f C a n i L e i s h ® ,

U s e o f the vaccine should not prevent measures to reduce exposu re t o sandf l ies.The reason being that they are work ing in entirely different ways. Reduct ion o f exposu re to sandflies will minimise the challenge the dog receives. H o w e v e r C a n i L e i s h ® is the only op t ion a imed at stimulating immune responses t o the challenge that will still inevitably be encoun te red .

References 1. Day MJ. Immune System Development in the Dog and Cat. Journal o f Compara t i ve Pathology, 2007; i 37(Suppl I): S10-S15

2. h t tp : / /www,emaeurc>paeu/doc^en_GB/do

3. C h e n e J, Bourdo iseau G , C h a b a n n e L e t al. Comparison of a rapid immunochromatographic test with immunofluorescence assay

for the detection of anti-L. infantum antibodies in dogs. 2nd Cong resso Internazionale S C I V A C - Leishmaniosi canina. Pisa, Italy

Apr i l 2010,

How can I best treat clinical cases of Canine Leisnmaniosis! Current guidelines Guadalupe Miro Can ine Leishmaniosis can present wi th a b road range o f clinical manifestat ions.The basic principle o f the t rea tment should include a g o o d definit ion o f clinical cases o f leishmaniosis fo r the assessment and accurate diagnosis.

Current ly, there are not 100% effective drugs against this disease; but in general, the clinical cond i t ion improves after t rea tment in

most dogs. 'Parasitological cure ' doesn ' t happen. In s o m e cases, relapses may be possible that requi red reconsider ing diagnosis and

t reatment.

Even though t rea tment p ro toco ls and clinical fo l low-up has changed considerably in the last years, the drugs used are unchanged. Fu r the rmore , thanks to the improvemen t in veter inary assistance, the expectat ions o f clinical - no t parasitological- cure are much higher than in the past. Occas iona l ly it is not possible t o prevent relapse f r o m immunosuppress ion cases (stress, debil itating diseases, etc). A m o n g all available drugs, the mos t used leishmanicides are pentavalent ant imonials ( G l u c a n t i m e ® ) , fo l lowed by miltefosine ( M i l t e f o r a n ® ) in combina t ion w i th al lopur inol ( Z y l o r i c ® ) the leishmaniostatic agent par excel lence, due t o innocuity and efficacy.

Current ly, the t rend is t o w a r d general consensus on ant imonial administrat ion pro toco ls , r e c o m m e n d i n g cycles o f 7 5 - 1 0 0 mg/kg/ day fo r 4-8 weeks (with total doses div ided tw ice a day). Pharmacokinet ic studies have shown that pentavalent antimonials subcutaneous administrat ion is the mos t adequate f o r m o f administrat ion. An t imon ia l toxic i ty may lead t o sti bo into lerance that appears after the first doses, o r t o st ibotoxici ty that may o c c u r at the end o f t rea tment by cumulat ive effect. Both condi t ions present wi th asthenia, exerc ise intolerance, vomi t ing and diarrhea, appet i te decrease and in s o m e cases fever Even though somet imes it is difficult t o de te rm ine if the d isorders that occurs in a sick pe rson are side effects o f chemothe rapy o r a consequence o f the disease itself; the toxic i ty presents in patients w i th nephrotox ic i ty is greater in bo th dogs and humans.The combina t ion o f pentavalent ant imonials and al lopur inol is the mos t d o c u m e n t e d therapy. Both drugs have a synergistic effect that boosts efficacy, prolongs clinical remission and delays the relapse.

A n o t h e r op t ion that cou ld be appl ied is an alkyl phosphol ip id , miltefosine ( M i l t e f o r a n ® ) , at an oral dose o f 2 mg/kg for 4 weeks, always adminis tered wi th f o d d e r It is indicated in animals that have nei ther lost appet i te n o r have digestive prob lems, It is the first op t ion in dogs wi th prote inur ia and /o r renal disease.

Both leishmanicides are always used in combina t ion wi th al lopur inol at a dose o f 10-20 mg/kg BID orally adminis tered for long per iods o f t ime (mean 12-18 months) in the cases o f classic clinical leishmaniasis. Pro longed t rea tment w i th al lopur inol is effective t o prevent remission o f the clinical condi t ion depend ing on the animal's ability t o cont ro l the disease; this will have be a t ime variability. Somet imes , it is necessary t o m o n i t o r the possible adverse reactions present in the long t e r m in s o m e patients, e.g. xanthine induced urolithiasis and /o r nephrolithiasis. Clinical improvemen t o f sick dogs will vary depend ing on thei r previous cl inical-pathological condi t ion, and it is normal ly seen be tween the first and th i rd mon th o f t reatment . It is wel l known that dogs w i th severe renal damage will generally have a w o r s e clinical recovery. Discont inuat ion o f al lopur inol is r e c o m m e n d e d w h e n the patient is comple te ly recovered f r o m the clinical perspect ive (history, physical e x a m and labora tory tests) and w h e n the ant ibody levels are negative o r slightly posit ive.

Recommended references • Baneth G , Shaw SE, 2002 , Chemotherapy of canine leishmaniosis.Vet Parasitol 106, 315 -324

• Bianciardi R Brov ida C.Va len te M, A r e s u L, Cavicchio l i l_Vischer C , G i r o u d L, Castagnard M, 2009, Administration of miltefosine

and meglumine antimoniate in health dogs: Clinicopathological evaluation of the impact on the k/'dneys.Toxicol Pathol 3 7 , 7 7 0 - 7 7 5 • C iaramel la R O l i va G , Luna R D , G r a d o n i L, A m b r o s i o R, C o r t e s e L, Scalone A , Persechino A , 1997, A retrospective clinical study

of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum.Vet Rec 141 ,539-543 • Denero l l e R Bourdo iseau G , 1999, Combination allopurinol and antimony treatment versus antimony alone and allopurinol alone in

the treatment of canine leishmaniasis (96 coses). J Ve t Intern M e d 13 ,413-415 • Koutinas AR, Po l i zopou lou Z S , Sar idomichelakis M N , Argyr iadis D, Rytianou A , Plevraki K G , 1999, Clinical considerations on canine

visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989-1996). J A m A n i m H o s p A s s o c 35, 376-383 • Koutinas A R Saridomichelakis M N , Mylonakis M E , Leont ides L, Pol izopoulou Z , Billinis C , Argyriadis D, D iakou N , Papadopou los O,

2001 , A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis.\/et Parasitol 98 ,247-261 • M a n n a L.Vitale F, Reale S, Picil lo E, Neg l ia G.Vesc io R Grav ino A E , 2009, Study of efficacy of miltefosine and allopurinol in dogs with

leishmaniosis.Vet J 182,441 -445 • M a t e o M, 2007, Estudios sobre la eficacia comparada y la tolerancia de la miltefosina y el antimoniato de n-metilglucamma, y la

monitorizacion post-tratamiento con alopurinol en la infeccion natural porLeishmania infantum en el perro.Tesis doctoral. Un ivers idad C o m p l u t e n s e de Madr id

• M a t e o M, Maynard L V i s c h e r C , Bianciardi R M i ro G , 2009, Comparative study on the short term efficacy and adverse effects of

miltefosine and meglumine antimoniate in dogs with natural leishmaniosis. Parasitol Res 105, 155-162 • M i r d G , C a r d o s o L, Pennisi M G , O l i va G , Baneth G , 2008, Canine leishmaniosis - new concepts and insights on an expanding

zoonosis: part two.Trends Parasitol 24,371 -377 • M i r o G , Mo l i na R, 2006, Leishmaniosis canina: manejo di'nico y situation actual en Espaha. Edic iones Bayer Hea l thCare . • M i ro G , O l i va G , C r u z I, Canavate C , M o r t a r i n o M.V ischer C , Bianciardi P, 2009,Multicentric, controlled clinical study to evaluate

effectiveness and safety of miltefosine and allopurinol for canine leishmaniosis.Vet D e r m a t o l 2 0 , 3 9 7 - 4 0 4 • O l ive i ra A L , B r u s t o l o n i Y M , F e r n a n d e s T D , D o r v a l M E , C u n h a RV, Boia M N , 2009a,Severe adverse reactions to meglumine

antimoniate in the treatment of visceral leishmaniasis: a report of 13 cases in the southwestern region ofBrazil.Trop D o c t 39, 180-182 • Pennisi M G , D e Majo M, Masucci M, Britti D.Vitale R De l M a s o R, 2005 , Efficacy of the treatment of dogs with leishmaniosis with

a combination of metronidazole and spiramycin.Vet Rec 156(1 I) 346 -349

Ribeiro RR, M o u r a ER P i m e n t e l V M , Sampaio W M , Silva S M , Schettini D A , A lves C R M e l o RA.Tafuri W L , Demiche l i C , Me lo , M N , Frezard F, Michal ick MS, 2008, Reduced tissue parasitic load and infectivity to sand flies in dogs naturally infected by Leishmania (Leishmania) chagasi following treatment with a liposome formulation of meglumine antimoniate. Ant imicrob ia l agents and chemothe rapy 5 2 , 2 5 6 4 - 2 5 7 2

So lano-Gal lego L, Kout inas A , M i r d G , C a r d o s o L, Pennisi M G , Fer rer L, Bourdeau R O l i v a G , Baneth G , 2009, Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis.Vet Parasitol 165, 1-18

So lano-Ga l lego L, M i r d G , Koutinas A , C a r d o s o L, Pennisi M G , Rerrer L, Bourdeau R O l i va G , Baneth G , 201 I, LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vec to rs 4 , 8 6

Valladares JE, A l b e r o l a J, Esteban M, A r b o i x M, 1996, Disposition of antimony after the administration of N-methylglucamine antimoniate to dogs. Ve t Rec 138, 181-183

W o e r l y V , Maynard L, Sanquer A , Eun H M , 2009, Clinical efficacy and tolerance of miltefosine in the treatment of canine leishmaniosis. Parasitol Res 105 ,463-469

Zagh lou l IY Al - jasser M, 2004, Effect of renal impairment on the pharmacokinetics of antimony in hamsters. Annals o f t ropical medic ine and parasitology 9 8 , 7 9 3 - 8 0 0

Treatment was successful... What next? Liu is Ferrer Introduction Can ine Leishmaniosis, as stated before, is an extraordinar i ly c o m p l e x and diverse disease.Thus, it cannot have a sole adequate therapeut ic p ro toco l fo r all cases and requires a clinical classification that al lows the implementat ion o f different therapeut ic approaches that are m o r e appropr ia te and a m o r e accurate prognosis.

A s w e saw before, in the mos t c o m m o n stages (II and III), the mos t effective and safe t rea tment is the combina t ion o f a parasiticide drug such as ant imony salts (meglumine ant imoniate - G l u c a n t i m e ® ) o r mil tefosine, w i th al lopur inol , a parasitostatic drug. Cur ren t l y publ ished papers repo r t a similar efficacy for the combina t ion o f miltefosine wi th al lopur inol and for the classical t rea tment (meglumine ant imoniate + al lopurinol) (Table I) [M i rd et al, 2009],

T a b l e I i T r e a t m e n t o f C a n i n e L e i s h m a n i o s i s

Duration Side effects

vVeakness, anorexia, pain

' 1 • i i n« diarrhoea, anore*

Urolithiasis (xanthine)

This t rea tment p roduces very g o o d o u t c o m e s in dogs w i thou t a severe renal d isorder (stages II and III). M o r e than 8096 o f animals exhibi t an ev ident clinical improvemen t within the first t w o months, a 9096 after 3 months. General ly, clinical and pathological parameters normal ise within 3 months (including the prote in e lectrophoresis) . An t i bod ies titres are ano ther mat ter W h i l e the mean titre tends to fall s lowly in t reated animals, seronegativity cannot be expec ted wi th in the first six months o f t reatment. In addit ion, ant ibody t i tre remains high for years in a significant percentage o f cases. [Torres et al, 2010] ,

Patient fo l low-up dur ing and after the t rea tment is as impor tan t as the t reatment itself.The goals o f patient fo l low-up are:

1. D e t e c t if the response is the expec ted o n e and if the patient shows an adequate clinical progress. D e t e c t t rea tment failures. 2. Identify potential adverse effects o f t rea tment and cor rec t t h e m 3. Identify late-onset lesions associated w i th the disease (caused by immune complexes , associated t o immunodef ic iency,

coinfections, etc.) 4. De tec t leishmaniosis relapses as s o o n as possible

Thus, the fo l low-up p ro toco l must be a imed t o achieve all these goals. Table 2 summar ises a standard fo l low-up p ro toco l once the t rea tment has begun. General ly, clinical examinat ion, b l ood tests (particularly protein e lectrophoresis) and serology a l low the assessment o f the progression o f the disease in a pat ient T h e biggest difficulty might be the co r rec t evaluat ion o f an animal being t reated for months that has responded to medicat ion and presents clinical signs consistent wi th leishmaniosis (weight loss, weakness, lameness, skin o r ocu lar lesions), ^Must these be simply at tr ibuted t o the previously d iagnosed leishmaniosis? ^ H o w t o make a decision?

T a b l e 2 ) M o n i t o r i n g o f p a t i e n t s w i t h l e i s h m a n i o s i s f r o m t h e t r e a t m e n t o n

I . Check-up at I, 2, 3, 6 and 12 months

II. Then, twice a year

III. Every check-up must include:

Total protein - protein electrophoresis. Serology (won't be needed for 3 mon

stry and

IV. Occasionally, it can also include:

• CD3+, CD4+ and CD8+ count. • Leishmanin skin test. • Assessment of proteinuria. • Quantitative bllod and/or bone marrow PCR (identification of n • Acute Phase Protein (PCR, Haptoglobin

Normal ly , if t he clinical signs are associated wi th a change in prote in e lect rophores is and serology, w e are in f ront o f a relapse. If not (e.g. the dog has always maintained a high titre), it may be m o r e difficult. In these cases, it is best t o use C-react ive prote in and, even bet ter quantitative P C R (Rea lT ime) [Mar t inez et al, 201 I].

Fu r the rmore , it is ve ry impor tan t that the animals that have undergone clinical leishmaniasis and have been t reated will receive

special cares, including:

1. Highest quality nutr i t ion accord ing to the kind o f dog and its lifestyle. C o n s i d e r using a diet fo r the prevent ion o f urolithiasis, 2. Prov ide pe rmanen t access t o fresh w a t e r (prevent ion o f xanthine uroliths associated w i th al lopurinol). 3. Regular parasit icide t reatments (both for e n d o and ectoparasi tes). Annua l faecal exams. 4. U s e sandfly repel lents (del tamethr in, permethr in) since sick dogs have the highest parasite burden. 5. A v o i d , w h e n e v e r possible, any immunosuppress ive t rea tment (steroids). 6. A v o i d drugs that cou ld interact, part icularly w i th al lopur inol (azathioprine).

References 1. Mar t inez V. Canine leishmaniasis: the key points for qPCR result interpretation. Parasites and Vec to rs (201 I); 4 : 5 7

ht tp: / /www.parasi tesandvectors.eom/content /4/1 / 5 7 2. M i ro G et al. Multicentric, controlled clinical study to evaluate effectiveness and safety of miltefosine and allopurinol for canine

leishmaniosis.Veterinary D e r m a t o l o g y (2009); 2 0 : 3 9 7 - 4 0 4 3. So lano-Gal lego L et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veter inary

Parasitology (2009); 165: 1-18. 4. So lano-Ga l lego L et al. Leishvet guidelines for the practical management of canine leishmaniosis. Parasites and Vec to rs (201 I); 4:86

ht tp: / /www.parasi tesandvectors.eom/content /4/1 / 86 5. Torres et al. Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and treated with meglumine antimoniate

and allopurinol Met Journal (2010) (in press, e lectronic access)