Canine leishmaniasis: epidemiological risk and the experimental model

TRENDS in Parasitology Vol.18 No.9 September 2002 399Review

http://parasites.trends.com 1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)02347-4

Javier Moreno

WHO CollaboratingCentre for Leishmaniasis,Servicio de Parasitología,Centro Nacional deMicrobiología, Institutode Salud Carlos III, Ctra. Majadahonda-Pozuelo km 2, 28220 Majadahonda,Spain.

Jorge Alvar*

WHO CollaboratingCentre for Leishmaniasis,Centro Nacional deMedicina Tropical,Instituto de Salud CarlosIII, Sinesio Delgado 6,28029 Madrid, Spain.*e-mail: [email protected]

Canids represent reservoirs for Leishmania infantum,the causative agent of visceral leishmaniasis (VL) inChina, the Mediterranean basin and the Americas.The domestic cycle takes place in pet dogs, and aperidomestic cycle is maintained in stray dogs andwild canids, which has progressive synanthropy(association with humans or their dwellings). Wild canids appear to spread the disease [1], but theexistence of a sylvatic cycle, independent of infectiousdogs, is unlikely*. The parasite is transmitted froman infected dog to a non-infected dog by the bite of asandfly, although direct (dog-to-dog) transmission [2]and blood transfusion routes [3] have been reported.Humans become infected accidentally and do not actas reservoir hosts for L. infantum, except in caseswhere contaminated syringes are shared amongintravenous drug addicts [4]. The seroprevalence ofL. infantum is similar in all western Mediterraneancountries, varying from region to region, dependingon ecological aspects. For example, in Italy, the Apulia region has 14.5% prevalence and Tuscany has 24%; in France, the Alpes Maritimes has 3% to17% prevalence; in Spain, Madrid has 5% and thePriorato region has 18%, and in the area surroundingLisbon, Portugal, there is an 8.5% prevalence [5].These serological data suggest that out of 15 milliondogs in these countries, a conservative estimate of 2.5 million dogs (16.7%) are infected. Higherseroprevalences have been reported in SouthAmerica: 33% in Margarita Island, Venezuela [6], and

36% in Jacobina, Brazil [7]. The annual incidence ofcanine leishmaniasis has been studied in only a fewareas; for example, in Madrid, we found 30 infecteddogs in every 1000 dogs per season [8]. If one infecteddog has a life expectancy of 2–3 years (with 20% ofthem harbouring amastigotes in the skin [9]), it is notsurprising that a range of 6–9% seroprevalence isfound. The overall incidence of Leishmania infectionin Jequié, northeast of Brazil, was 6.55 cases for every100 dogs per year, although the incidence variedmarkedly between town clusters [10]. The suspicionthat the seroprevalence underestimates the realnumber of infected dogs in endemic areas [11] hasbeen confirmed by figures recently obtained fromPCR-based screening of seronegative dogs, whichshow prevalences of 80% (24 out of 30) in Marseille,France [12], or 67% in Majorca, Spain [13]. Theepidemiological implications (i.e. infectivity tosandflies) of these high percentages of infected dogs,most of them asymptomatic carriers, have yet to beestablished. However, we know, by direct xenodiagnosis,that asymptomatic dogs (50–60% of all seropositiveand 80% of all infected dogs [9,13]) are highlyinfective to both Phlebotomus perniciosus(54%, 10 out of 19) [14] and Lutzomyia longipalpis†,although a recent study did not find asymptomaticdogs to be infectious to L. longipalpis [15].Symptomatic dogs are even more infective to insectvectors (70%, 31 out of 44) (R. Molina, unpublished)and a strong positive correlation between infectivityand serological response has been evidenced [14].These are key figures for leishmaniasis controlprogrammes to ascertain the level of protection when a vaccine is available, to avoid clinical outcomeof the disease and to determine infectivity of dogs to sandflies.

Epidemiological factors

Studies on the risk factors associated with canineleishmaniasis have shown that, in endemic areas,gender does not appear to be a factor. However, age is agood indicator of the degree of infection transmission –prevalence rises until dogs reach three years of age,declining thereafter until 7–8 years of age when

Increasing risk factors are making zoonotic visceral leishmaniasis a growing

public health concern in many countries. Domestic dogs constitute the main

reservoir of Leishmania infantum and Leishmania chagasi, and play a key role in

the transmission to humans. New reagents and tools allow the detailed

investigation of canine leishmaniasis, permitting the monitoring of the

immunological status of dogs in both natural and experimental infections.

Such studies are essential to determine the basis of the canine protective

immune response and to establish a laboratory model, a significant aspect for

the development of vaccines against canine leishmaniasis.

Published online: 6 August 2002

Canine leishmaniasis:

epidemiological risk and the

experimental model

Javier Moreno and Jorge Alvar

*Courtenay, O. et al. (2001) The role of foxes (Carnivora: canidae) inthe maintenance and transmission of Leishmania infantum:implications for peridomestic control. Summaries of Presentations at the International Canine Leishmaniasis Forum, Crete, Greece,20–24 May 2001, Intervet, pp. 17–23

†Miles, M.A. et al. (1999) Canine leishmaniasis in Latin America:control strategies for visceral leishmaniasis. Proc. Intl. CanineLeishmaniasis Forum, Barcelona, Spain, pp. 46–53

TRENDS in Parasitology Vol.18 No.9 September 2002

http://parasites.trends.com

400 Review

another peak is observed [8]. In general, all breeds are similarly susceptible, with the risk dependent on the dog’s activities: feral, rural and working dogsare more exposed to sandfly bites than are thoseliving in the cities.

More important is the role that is played by aparticular immune response in each dog and, possibly,the virulence of the L. infantum clone transmitted in each bite. The dog has been considered to be themost susceptible part of the transmission cycle ofL. infantum, but infection does not necessarily meanactive disease, and infected dogs from the sameendemic area can develop different cell responses, asassessed by specific delayed-type hypersensitivity(DTH) reaction in a leishmanin skin test [16], andin vitro stimulation assays using leishmanialantigens [17]. After infection, some dogs can controlthe parasite and do not develop the disease in the short term, sometimes for years or for theirlifetime, whereas other infected dogs presentprogressive disease. After an incubation period of 2–4 months, dogs with progressive disease developlymphoadenopathies (93%), dermatitis (90%),onychogryphosis (75%), weight loss (26%), cachexia(24%), locomotion problems (23%), conjunctivitis(18%), epistaxis (9%), high antibody response and an impaired cell-mediated immune response [18].However, a long-term study following several cohortsof dogs in the Priorato region (Tarragona, Spain)revealed that 15% of all infected dogs were able tocircumvent establishment of disease and even resolvedisease spontaneously [19] (Fig. 1).

Immunity

The mechanisms underlying protection andsusceptibility to disease are not known. Cell-mediatedimmunity (CMI), when present, is able to control theinfection and the animal remains asymptomatic [20],whereas the lack of such responses allows progressionof the disease, as seen in humans [21]. Interestingly, aspecific inbred dog from an endemic area – the Ibiziangreyhound – seems to be more resistant to L. infantumthan other breeds due to a long-lasting CMI [22],although in this case the relevance of the geneticbackground on natural resistance to the parasiteremains to be determined.

The study of lymphocyte subsets in the peripheralblood of naturally infected symptomatic dogsindicated a significantly decreased level of CD4+ T cellscompared with those in non-infected dogs. Thisdecrease is responsible for the lack of a specific CMI ininfected dogs, confirmed by a positive relationshipbetween CD4+ levels and in vitro lymphoproliferativeresponse to leishmanial soluble antigens [23].However, infectivity to sandflies, measured by directxenodiagnosis, has an inverse relationship withCD4+ levels [24], as previously demonstrated inhumans [25].

Dogs with clinical signs of canine leishmaniasisare treated or sacrificed. Treatment in canine

leishmaniasis is of low efficacy, independent ofwhether antimonials or amphotericin B are given.Although the majority of treated dogs improve orappear to resolve disease clinically; there is no sterilecure. Relapses occur in ~80% of treated dogs after oneyear. More relevant is that a significant number oftreated dogs (~30%), even when they do not exhibitany clinical signs or symptoms, are able to regaininfectivity to sandflies 3–5 months after the last drugtreatment [26]. Indeed, the drugs led to a clearance ofcirculating parasites and consequent restoration of CD4+ levels, thus reducing the risk of sandflytransmission. Once drug treatment is stopped, theparasites have a chance to multiply and CMI willreturn to its previous low levels, permitting thepropagation of amastigotes and, therefore, sandflyinfectivity [27]. Two epidemiological implications areconsequent: the development of drug resistance andrecurrence of transmission risk.

To sacrifice infected dogs is generally not acceptedfor ethical and social reasons; however, epidemiologicalstudies also suggest that alternative approachesshould be considered. Excluding the successfulcontrol of VL in west China by eliminating infecteddogs, together with intradomiciliary insecticidespreading [28], other VL control programmessacrificing dogs have given contradictory results [29].One study in Brazil compared an intervention area(five years of sacrificing seropositive dogs) with anon-intervention area (not killing and not treatinginfected dogs) [7]. The drop in incidence of canineleishmaniasis (seroconversion from 36% to 6% in oneyear) and a decrease in prevalence (from 35% to 8% intwo years) was observed in the area where interventionoccurred. However, both the incidence and prevalenceincreased after two years (incidence 14% andprevalence 13%) despite continued elimination of

TRENDS in Parasitology

Naive host

Not exposed Exposed to parasite

Non-infectedInfected

Asymptomatic Clinically apparent

Spontaneous clinical healing Chronic infection

Healed infection Persistent infection

Cured and parasite-free Clinical reactivation Asymptomatic

Treatment

Fig. 1. Natural history of canine leishmaniasis. Different phenotypes ofdogs which could occur following a Leishmania-infected sandfly biteare indicated: healthy non-infected (green), symptomatic (red) andasymptomatic (blue) dogs. The sandfly symbol indicates situations inwhich the infected dog is capable of transmitting Leishmania to theinsect vector, as proven by xenodiagnosis. The factors affecting eachstep are not clear; hence, it is not possible to determine why infecteddogs remain asymptomatic, why ill dogs can heal spontaneously orsuffer clinical reactivation after treatment.



401Review

infected dogs during this period. There are threereasons which could explain these results: (1) thepresence of sylvatic reservoirs keeping the cycleactive; (2) the fact that control measures eliminatedonly seropositive dogs, but not all infected dogs(indicated by PCR [12,13]); and/or (3) the incompletecoverage of the intervention area because not allseropositive dogs were culled. Nevertheless, thehuman incidence dropped in the intervention areafrom 12 cases per 1000 inhabitants to 2 per 1000, even years after ending the control measures. In apilot programme set in the island of Elba, Italy, by combining sacrifice and treatment, the canineincidence fell from 11.6% to 4.5% in two years [30].

In a case-control study with the goal of estimatingthe relative risk for VL in houses with a recentVL patient, and in relation to the presence or absenceof pet dogs, it was not possible, statistically, to correlatethe acquisition of human leishmaniasis and presenceof canids; nevertheless, the risk for VL was twice ashigh if there were dogs in the domestic setting than ifthey were absent [31]. There is strong evidence forhuman cases occurring in areas where the prevalencerates in dogs are high [32].

Useful or not, removal of infected dogs is not ameasure followed in most of the endemic countries. To convince owners or veterinarians to sacrifice dogsis difficult for emotional and/or economical reasons.In addition, such control measures are thwartedbecause of the high number of stray dogs (one millionout of 4.5 million in Spain) that will be not covered bythese interventions, and the high proportion ofasymptomatically infected dogs, half of which wouldbe capable of infecting sandflies. In many countries,to screen all dogs every year is not economicallyfeasible at a local, regional or national scale becauseof the low incidence of human leishmaniasis.

The use of deltamethrin-impregnated collars (and, in a lower proportion, lotions or shampoos) toprotect dogs from a significant number of sandfliesbites [33] represents a new control strategy for canineleishmaniasis. The first field-evaluation of theefficacy of these collars against canine leishmaniasisshowed that their impact on the incidence could benegligible during low transmission seasons, but couldbe strong when the level of transmission is high [34].Nevertheless, more fieldwork and evaluation of theimpact on human incidences are necessary to confirmthe efficacy of deltamethrin-impregnated collars as acontrol strategy for canine leishmaniasis.

In summary, the situation for canine leishmaniasisrepresents an epidemiological situation whereinfection does not always mean active disease;symptomatic disease and subclinical cases overlap inendemic areas and, currently, no effective controlstrategies exist. The introduction of new diagnostictechniques has revealed the real level of infection;however, further work is required (immunologicaland epidemiological) to understand the basis forprogression or resistance to the disease.

The increasing awareness that the dog represents amajor key point to control parasite transmission tohumans has recently promoted interest to develop acanine experimental model. If reached, it will beuseful not only for a better understanding of thedisease, but also for developing control measures ofinfection. This also has economical implications forthe pharmaceutical industry.

Experimental model of canine leishmaniasis

Dogs constitute an excellent model to studyleishmaniasis. This species acts as a patient, a targetfor control and a good model for human leishmaniasisbecause the symptoms in dogs are similar to thosedeveloped in humans, with the exception ofdepilation, onychogryphosis and emaciation [35].Hence, progress in the knowledge of canineleishmaniasis will help to prevent the disease inhumans [36]. Experimental infections in dogs have been carried out since the beginning of the20th century, when the role of the dog as a reservoirhost was confirmed. One major problem in the study of canine leishmaniasis has been the lack ofimmunological markers and reagents for dogs. Thispaucity of specific research tools has significantlydelayed the progress of canine immunology.Therefore, little information is available on theimmunological basis of canine leishmaniasis, bycontrast to the abundant data on human andexperimental murine leishmaniasis [37]. Researchduring the past few years has allowed standardizationof laboratory work with dogs, and basic specific toolsare now available for the required experimentalassays. The production of monoclonal antibodies(mAb) against canine homologues of the humancluster of differentiation (CD) antigens [38] providesthe opportunity to define canine lymphoidpopulations [39], and to monitor these cell subsetsunder normal [40,41] and pathological conditions[42,43]. The recent cloning, sequencing andexpression of many canine cytokines [44–47] haveallowed development of diverse reverse transcriptasePCR (RT-PCR) protocols to analyze messenger RNA(mRNA) expression of cytokines and the type of T cellproduced in response to the parasite [48–50].

All these newly available reagents and tools allowdetailed investigation of canine leishmaniasis, thuspermitting the monitoring of the immunologicalstatus of dogs in a natural as well as an experimentalinfection. Such studies will be essential to determinethe basis of the canine protective immune responseand whether those mechanisms are similar to theaspects observed in rodents and humans.

A review of experimental infections in the caninemodel concludes that one main problem is theunpredictable nature of the response (i.e. the sameinoculation procedure induces different clinicalpatterns of the disease). Although most researchersconsider this variability as a reflection of the clinicalresponse observed in natural infections, it deprives



402 Review

these models of the homogeneity and reproducibilitynecessary to obtain definitive conclusions. Instead ofa single natural model, it would be better to establishdifferent experimental models of infection to induce a particular stage of the immune response or disease,such as the long prepatency period, CMI, high level of humoral response and fast appearance of thesymptoms. In this way, it would be possible to obtain a particular clinical response suitable for thedesignated purpose, such as challenge in vaccinetrials, clinical assays to test new drugs or treatmentprotocols, establishment and standardization of earlydiagnostic methods, and selection of antigens withimmunogenic properties.

The different attempts carried out in the pastdecade for establishing an experimental model ofinfection are shown in Table 1. The factors that seemto affect success and progression of experimentalinfections are included: route of inoculation; size ofinoculum; and stage of parasite. The low number of dogs used in these assays (an inherent aspect to all

dog experiments) makes it difficult to obtain definitiveconclusions. Nevertheless, the grouped analysis ofthese factors helps to raise at least some indicationsfor attaining a homogeneous and repetitive clinicalresponse, and how the combination of these factorscan guide the response in one way or another. Fromthe data published and our own experience, it can be inferred that intravenous (IV) inoculation is thebest way to obtain symptomatic disease in infectedanimals. All assays that induced early symptomaticdisease used IV inoculation ([51–54], J. Moreno et al.,unpublished) and, only in one case, intradermal (ID)infection induced clinical disease, although much latercompared with IV infection, and in only 50% of thedogs [55]. However, ID inoculation of the parasitestends to induce long prepatency periods, generating a patent cell-mediated response in some cases.Amastigotes seem to be more effective at inducinginfection and, when compared with promastigotesunder the same conditions, more effective in producingclinical symptoms [54], although this could be due to

Table 1. Experimental infections of dogs with Leishmania infantum in the past decade

Dog

strain

Route of

inoculationa

Parasite stage

(no. of dogs)

Size of inoculum Evidence for infection Clinical

symptoms

Cell mediated

immune response

Refs

Mixed IV Amastigotes (3) 1010–1011 parasites kg–1 dog Seroconversion 3 months p.i. in all cases No Positive [66] breed Promastigotes (3) 107–108 parasites kg–1 dog Seroconversion 2 month p.i. in one case No Negative

ID Amastigotes (1) 1011 parasites kg–1 dog Seroconversion 3–6 months p.i. No NegativeIP Amastigotes and Variable: 106–1010 parasites Seroconversion in all cases No NK [67]

promastigotes (2) in total BM culture positive (six out of eight cases)IV Amastigotes (6) Variable: 5 × 107–1 × 109 Increase in spleen weight, granulomas in No NK

parasites in total spleen and/or liver in all casesIV Amastigotes (3) 108 parasites in total Seroconversion 1 month p.i. in all cases No Negative [68]IV Promastigotes (8) 5 × 107 parasites in total Seroconversion 2 months p.i. in all cases Yes Negative [69]

Parasite isolation 75 days p.i.; and seroconversion in all cases

IV Promastigotes (4) 2 doses of 5 × 105 parasites Yes(3 monthsp.i.)

NK [51]

IV Amastigotes (4) 105 parasites kg–1 dog Clinical signs 9 weeks p.i. in all cases Yes(3 monthsp.i.)

Negative [52]

ID Promastigotes (5) 108 parasites kg–1 dog Seroconversion 1–3 months p.i. in all cases No Negative [70]IV Promastigotes (4) 109 parasites kg–1 dog Parasite isolation at necropsy in 2 out of 4 No Negative [54]

casesIV Amastigotes (3) 109 parasites kg–1 dog Seroconversion 3 months p.i. in all cases Yes

(3 monthsp.i.)

Negativeb

Beagle ID Promastigotes (25) 75 000–180 000 parasites No signs of infection in 13 out of 25 cases No Positive [55]Seroconversion 1–2 years p.i. and parasite Yes Negative isolation in 12 out of 25 cases

IV Amastigotes (5) 4.8 × 108 parasites Seroconversion 2 months p.i. in all cases No Negative [71]IV Promastigotes (6) 5 × 107 parasites Parasite isolation 8–20 weeks p.i. in all Yes (7–11 NK [53]

cases monthsp.i.)

IV Amastigotes (3) 106 parasites kg–1 dog Parasite isolation in all cases No 1 positive [56] out of 3

ID Promastigotes (4) 107 parasites kg–1 dog No signs of infection in all cases No PositiveSeroconversion and parasite isolation 2–3 months p.i. in all cases

IV Promastigotes (5) 108 parasites Yes(2 monthsp.i.)

Negative –c

ID Promastigotes (7) 5 × 108 parasites Seroconversion; asymptomatic for 3 years No Positive in all cases

aBM, bone marrow; ID, intradermal; IP, intraperitoneal; IV, intravenous; NK, not known; p.i., post-infection.bTwo of these dogs showed transitory cell responses 12 months p.i.cJ. Moreno et al., unpublished.



403Review

the fact that amastigotes are usually inoculated IVand are also freshly isolated. Promastigotes tend tolose their virulence after subculturing. The size of theinoculum may play an important role in experimentalinfection. In Nature, infection is due to a low numberof parasites (~1000), although the number of bitesneeded to induce the infection is not known. In theexperimental model, a high number of parasites(108–109) tends to induce a homogeneous response inall individuals, whereas a small number (105–106)tends to lead to a more heterogeneous clinicalresponse. Variability of the response could depend onall these different aspects, but also on host genetics.The use of inbred dogs can reduce variation, but in theassays that used beagle dogs, differences in immuneresponse were observed [55,56].

The data obtained from experimental infection ofL. infantum are also helpful for understanding thenatural history of canine leishmaniasis (Fig. 1,Table 1). The distinct response induced by IV orID route of infection could correspond to differentstages of the disease. ID inoculation of parasites, by natural or artificial procedures, seems to elicit aspecific response in the skin, with the selection ofspecific T-cell clones in the draining lymph node andthe development of a specific CMI to keep the parasiteunder control for a long time. The dog will be thenasymptomatic. On the contrary, if the dog is not ableto mount an efficient immune response and/or theparasite escapes from this control, the parasite willmultiply and spread, invade new organs, leading tospecific immunosuppression, and appearance ofclinical symptoms and open disease. This last stage ofthe disease could be induced directly by IV inoculationof parasites thus allowing quick spread of live parasitesto different organs, in particular, the spleen and liver,where they multiply. In this way, the natural processis accelerated and avoids a long prepatency period,confirming that IV inoculation is sufficient forinducing symptomatic disease (Table 1).

Vaccine development

The laboratory model of canine leishmaniasisrepresents a key aspect for vaccine development. It is clear that vaccines represent the main tool for controlling leishmaniasis, in that successfulimmunization of dogs could significantly reduce theincidence of human visceral leishmaniasis and it isthe most cost-effective control strategy [11]. Severalantigens and approaches for immunization againstLeishmania have been tested: killed parasites; liveattenuated parasites; recombinant and syntheticproteins; synthetic peptides; non-protein antigens;immunogens expressed in bacteria and viruses; andnaked DNA, most of them in the murine model and alimited number in humans [57]. By comparison,

few vaccine trials have been performed in dogs. The first vaccination trial in dogs was done by Dunan et al. in 1989 [58], using a partially purifiedL. infantum-derived preparation. Although thispromastigote fraction was shown to confer resistancein experimentally infected mice [59], the vaccinateddogs presented a higher susceptibility to infection anddisease than the control group. This indicates thatconclusions obtained in the murine model studiesmight not be applicable to dogs.

An effective vaccine against canine leishmaniasishas to induce strong and long-lasting CMI. Such cellresponse can be found in dogs naturally infected withLeishmania, but different laboratory assays haveshown that CMI can be experimentally induced. Dogs vaccinated with merthiolated, sonicatedpromastigotes of L. braziliensis plus BCG [60],autoclaved L. infantum, or L. major plus BCG [61,62]presented a specific and long-lasting CMI assessed byin vitro PBMC proliferation or DTH reaction in skin.Furthermore, vaccination with killed promastigotesof L. infantum is able to induce high levels of IFN-γproduction by PBMC, in addition to a significantincrease of NO production, and phagocytosis andkilling by infected macrophages [63]. The protectivepower of these vaccines after challenge resulted in alower rate of infection in vaccinated dogs whencompared with control animals, although fieldapplication showed poor results [64]. More detailedstudies and long-term monitoring of an effectivechallenge after vaccination are needed to establishthe real protection induced by these vaccines.

In 2001, the first vaccine was reported for canineleishmaniasis, the fucose mannose ligand (FML)vaccine of L. donovani, which elicits a protective effectin the field [65]. This vaccine induced a significant,long-lasting and strong protective effect againstcanine leishmaniasis: all dogs vaccinated showedintradermal reaction to L. donovani lysate sevenmonths after vaccination, indicative of a CMIresponse. After two years, 33% of animals in thecontrol group developed either clinical or fataldisease, whereas only 8% of vaccinated dogs showedmild signs of leishmaniasis and there were no deaths.

Perspective

Many new vaccines and protocols have been developedfor canine leishmaniasis, but they have not been testedin dogs; consequently, much work remains to be done.Owing to the differences between animal species, thevaccine candidates should be tested specifically in dogs.Vaccination against canine leishmaniasis must notonly elicit a protective response and prevent the clinicaloutcome of disease, but also avoid the risk oftransmission. Only under these conditions will thetransmission of the parasite be prevented [64].

Acknowledgements

This work was supportedby a FIS grant ref. 96/0302,an INCO-DC researchproject (no.IC18*CT970213) from theEuropean Commissionand a research project(no. MPY-1031) from theInstituto de Salud Carlos III. We thank Diane McMahon-Pratt andPaul A. Bates for criticalreading and comments onthe article. We wish todedicate this article to thedear memory of ourco-worker Rafael Ortiz.

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TRENDS in Parasitology Vol.18 No.9 September 2002 405Review

Michel Tibayrenc*

UR Génétique desMaladies Infectieuses,UMR Centre National dela RechercheScientifique/Institut deRecherche pour leDéveloppement 9926,IRD, BP 64501, 34393 Montpellier cedex 5, France.*e-mail:[email protected]

Francisco J. Ayala

Dept Ecology andEvolution, University ofCalifornia Irvine, Irvine,CA 92697, USA.

Population structure and mating system of pathogensare tightly linked biological phenomena with crucialconsequences on the epidemiology of transmissiblediseases. For this reason, they have received muchattention from researchers since the availability ofsuitable genetic markers. Pathogens that cause major public health problems have been the subject of more studies and discussions. This is the case forPlasmodium falciparum, the agent of the most severeform of malaria.

The initial debate

The proposal in 1990 of a general clonal theory ofparasitic protozoa [1] followed the same methodologydeveloped for Trypanosoma cruzi, the agent of Chagas disease [2,3]. The tests take, as nullhypothesis, a PANMICTIC (see Glossary) situation.Strong statistical deviations from panmicticexpectations are taken as circumstantial evidencethat the pathogen under investigation undergoespredominant clonal propagation. Particular attentionis paid to the phenomenon of linkage disequilibrium,or non-random association between genotypes atdifferent loci, by means of a set of complementarytests (Table 1). Most of these tests rely on the analysisby MONTE CARLO SIMULATIONS of a substantial sample

(>10) of loci, or of independent components of thegenome. Recent contributions to this issue rely onvery similar approaches, even when using differentlinkage indices [4]. Biases as a result of geographicalor time isolation (WAHLUND EFFECT) have beenconsidered, together with the means to detect thesebiases [5]. The need for analyzing extensive anddiversified natural samples of the species under studyto avoid the so-called ‘iceberg effect’, has been developedat length elsewhere [6]. Unwillingly sampling limitedsubsets of a given species leads to building artifactualextinctions and linkage disequilibrium [6]. Since our initial proposal [1], many articles have beenpublished on the topic of parasitic clonality. However,it seems that a ‘state of the art’ review is useful for tworeasons. First, most articles have focused on only onespecies and no one gives a general view of the problemfor all parasitic protozoa. Second, although manyauthors have considered the clonality versussexuality debate since 1990, the practicalimplications of the debate in terms of molecularepidemiology, strain typing and definition of thespecies have been insufficiently discussed.

The main parasites considered in our initialproposal included T. cruzi, Trypanosoma brucei,Trypanosoma congolense, various Leishmania andNaegleria spp., P. falciparum, Giardia intestinalisand Entamoeba histolytica [1]. In a subsequent study[5], Toxoplasma gondii and fungal organisms(Candida albicans, Cryptococcus neoformans andSaccharomyces cerevisiae) were also included, and the strength of the available evidence forclonality has been ranked for each species considered(Table 2), from i (no evidence for clonality) to iv(CLONAL POPULATION STRUCTURE well ascertained).

The question of population structure in parasitic protozoa has recently gained a

renewed topicality with significant contributions on medically important

pathogens, such as Plasmodium falciparum, Toxoplasma gondii and

Cryptosporidium parvum. The proposals that initiated this debate are reviewed

here and the subsequent developments of the clonal theory, in light of recent

contributions, are examined.

Published online: 6 August 2002

The clonal theory of parasitic

protozoa: 12 years on

Michel Tibayrenc and Francisco J. Ayala

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Leishmania infantum in hamster and dogs. Am. J. Vet. Res. 54, 113–121

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http://parasites.trends.com 1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)02357-7

Canine leishmaniasis: epidemiological risk and the experimental model

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