Research Article Epidemiological Investigation of Canine ...downloads.hindawi.com/archive/2014/104697.pdf · Research Article Epidemiological Investigation of Canine Leishmaniasis

Research ArticleEpidemiological Investigation of Canine Leishmaniasis inSouthern Morocco

Samia Boussaa12 Mohamed Kasbari3 Amal El Mzabi4 and Ali Boumezzough2

1 Institut Superieur des Professions Infirmieres et des Techniques de Sante (ISPITS) Ministere de Sante 40 000 Marrakech Morocco2 Equipe Ecologie Animale et Environnement-Lab L2E (URAC 32) Faculte des Sciences Semlalia Universite Cadi Ayyad40 000 Marrakech Morocco

3 ANSES French Agency for Health and Safety Animal Health Laboratory Leishmaniasis and Sand Flies Team94 700 Maisons-Alfort France

4 Equipe Modelisation Economique-Lab PEL Faculte des Sciences Juridiques Economiques et SocialesUniversite Hassan 2 20 650 Mohammedia Morocco

Correspondence should be addressed to Samia Boussaa samiaboussaayahoofr

Received 20 April 2014 Revised 21 August 2014 Accepted 9 September 2014 Published 24 September 2014

Academic Editor Xu-Sheng Zhang

Copyright copy 2014 Samia Boussaa et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dogs are the major reservoir of Leishmania infantum the causative agent of human and canine visceral leishmaniasis in theMediterranean basin In Morocco canine leishmaniasis (CanL) is usually believed to be widespread mainly if not only in thenorthern regions and few data are available about the situation in southern parts of the country Here we report the results of apreliminary clinical and serological study carried out in 2004ndash2007 in four provinces of southern Morocco Serological analyseswere processed using two different Elisa techniques a homemade Elisa test and IDVET commercial kit and confirmed by twodifferent western blot (WB) tests homemade and LDBIO commercial kits We highlighted the presence of CanL infection insouthern regions known until then as free of the disease 198 (48243) of examined dogs displayed clinical signs compatiblewith CanL and the seroprevalence was particularly high respectively 818 and 878 by Elisa and western blot tests Our currentdeveloped and validated homemade (Elisa and WB) tools will be cost-effective and useful for next large-scale epidemiologicalstudies on Moroccan leishmaniasis animal reservoir

1 Introduction

Canine leishmaniasis (CanL) is a zoonotic disease causedby Leishmania infantum a trypanosomatid protozoan trans-mitted through infected sand fly (Diptera Psychodidae)bites CanL is endemic in the Mediterranean basin whereseroprevalence ranges between ten and 37 [1 2] andsymptoms in dogs occur in various combinations [3] Bothsymptomatic and asymptomatic dogs are sources of theparasite and phlebotomine sand flies play an active role in thetransmission of Leishmania to humans [4]

In northern Morocco natural CanL was first reportedin the region of Tangier [5] Further subsequent cases havebeen reported in Taounate Al Hoceima Chefchaouen andOuezzane provinces [6] in Sefrou and Zouagha MoulayYacoub [7] in Khemisset province [8] and in the province

of Nador [9] Leishmania infantum zymodeme MON-1 is theunique causative agent of CanL in the northern slopes of theRif mountains [6] and L infantumMON-24 was identified indogs from the Pre-Rif area [10] In the Rif region specificallydog seroprevalence ranges between ten and 41 [9] and Linfantum strains are highly virulent [8 9]

In northern slope of High Atlas Moroccan mountainsDereure et al [11] reported the presence of dogs infectedwith L tropica (MON-102 and MON-113) AdditionallyGuessous-Idrissi et al [12] reported a case of dogwith visceralleishmaniasis caused byL tropica in Taounate provinceMorerecently in Al Hoceima province L tropica MON-279 wasidentified in dogs with clinical signs of CanL [13]

To date only sporadic cases of human visceral leish-maniasis are reported in southern Morocco [14 15] In thepresent study we report the results of clinical and serological

Hindawi Publishing CorporationAdvances in EpidemiologyVolume 2014 Article ID 104697 8 pageshttpdxdoiorg1011552014104697

2 Advances in Epidemiology

Table 1 Species composition of the sand fly fauna in study area

Province Species composition References

Al Haouz

Phlebotomus papatasi

[33 36 37 45]

P sergentiP perniciosuslowast

P longicuspisP alexandri

Sergentomyia fallaxS minutaS dreyfussi

Azilal


[36 37 46]


P longicuspisSergentomyia fallax

S minuta

Chichaoua

Phlebotomus perniciosuslowast

[34 36 37]

P sergentiP longicuspis

P ariasiP papatasiP alexandriP mariae


Marrakech


[36 37 47 48]P sergenti


S minutalowastAs atypical form [36ndash39]

surveys on CanL in southern Morocco an area traditionallyconsidered to be free from CanL in spite of the omnipresenceof dogs and phlebotomine sand flies Our investigations werecarried out in four provinces of Morocco Al Haouz AzilalChichaoua andMarrakech where species composition of thesand fly fauna was also established (Table 1)

2 Materials and Methods

21 Sampling An epidemiological survey was carried outin 27 localities (Figure 1) in four provinces of southernMorocco Al Haouz (31∘221015840N 7∘511015840W) Azilal (31∘581015840N6∘341015840W) Chichaoua (31∘321015840N 8∘451015840W) and Marrakech(31∘361015840N 8∘021015840W)

Two hundred and ten dogs in 2004ndash2006 and 33 dogsin 2007 were studied An individual form was completedfor each dog date locality name and environment dog agesex type (domestic or feral) and Leishmania specific clinicalsigns The detail of sampling is shown in Table 2

Because of practical economic and security constraintsit was not possible in 2004ndash2006 to take blood samples from

1

2

3

4

AtlanticOcean

Algeria

Rabat

Spain

Figure 1 Moroccan study area provinces (1) Chichaoua (2)Marrakech (3) Al Haouz (4) Azilal

stray and feral dogs with unknown rabies infection statusIn 2007 after clinical examination blood was collected byjugular vein puncture and 33 sera were stored at minus80∘C forserological tests

22 Serological Assays For serological analyses the 33 serawere processed using two ELISA techniques a homemadeELISA test and IDVET commercial kit and the results wereconfirmed by two western blot (homemade and LDBIOcommercial kit) techniques

221 Preparation of the L Infantum Antigen Antigens wereproduced from Leishmania infantum promastigotes grown inRPMI 1640 medium supplemented with 10 fetal calf serum(Sigma) L-glutamine (Gibco) and penicillin-streptomycin(Gibco) Log phase promastigotes were harvested by cen-trifugation (14000 g for 15min at 4∘C) and washed threetimes in sterile phosphate buffered saline (PBS) Cells werecounted and adjusted to a concentration of 35 times 108promastigotesmL and as previously described [16] andlysed by boiling for 5min in sample buffer (05M Tris-HClpH 68 001M EDTA 5 sodium dodecyl sulphate (SDS)5 2-mercaptoethanol and 00125 bromophenol blue)Protein concentration (15mgmL) was determined by thebicinchoninic acid method (Pierce) and antigen was storedat minus80∘C

222 WB Analysis

Homemade Western Blot Kit SDS-polyacrylamide gel elec-trophoresis was performed on a 15 polyacrylamide gel witha Mini-Protean II apparatus (Biorad) and 200 120583g of antigenwas used per gel width with molecular mass proteins stan-dards (standard low range BioRad) as previously described[17] Gels were run at 50V for 3 hours at room temperature

Advances in Epidemiology 3

Table2Orig

inandnature

ofstu

died

dogpo

pulations

Date

Provinces

Localities

Male

Female

Total

Age

rangelowast

Ferald

ogs

Dom

estic

dogs

Veterin

ary

follo

w-up

Suspected

dogs

Dogsw

ithou

tclinicalsigns

2004ndash2006

Marrakech

Semlalia

42

62ndash4y

60

00

6Land

fillsite

4317

6018mndash4

y60

00

357

Tadchirt

20

23ndash5y

02

00

2Ao

udad

60

61ndash6y

06

00

6Be

lbakkar

10

18y

10

01

0Ch

ouflidi

10

11y

01

00

1Amerchich

20

22-3y

20

00

2Dou

arLaasker

11

220

mndash3

y2

00

02

Sansou

20

220

mndash2

y0

20

20

Guenn

oun

20

225ndash3y

11

01

1

AlH

aouz

Ghm

ate

225

274m

ndash6y

1017

210

17Ourika

23

52m

ndash6y

32

12

3

Chichaou

a

Zinit

30

33ndash8y

03

00

3AitMou

ssa

40

42-3y

40

00

4Timsal

10

13y

01

00

1AitIshaq

12

33ndash7y

03

01

2Ikhriben

61

72ndash16y

16

02

5

Azilal

Tanant

71

88m

ndash2y

26

11

7Fo

umJemaa

63

918mndash4

y8

10

90

AitTagalla

52

78m

ndash2y

16

00

7Aittkhajet

30

36m

ndash5y

03

10

3AitCh

warit

10

12m

01

10

1AitMalek

40

46m

ndash2y

04

00

4Tigit

32

54m

ndash35y

23

12

3Im

adahen

113

142m

ndash11y

68

04

10Makou

ssa

132

155m

ndash10y

312

05

10Ze

lguen

64

108m

ndash2y

19

01

9

2007

Marrakech

Land

fillsite

55

106m

ndash5y

82

11

9

AlH

aouz

Ghm

ate

94

136m

ndash3y

85

41

12Ourika

64

105m

ndash7y

82

22

8To

tal()

182(749)

61(251)

243(100)

mdash137(564)

106(436)

14(58)

48(19

8)

195(802)

lowast

mm

onthyyear


After electrophoresis the proteins were transferred onto anitrocellulose sheet (Hybond ECL Amersham Biosciences)The sheets were blocked in PBS containing 5 skimmedmilkand 02 Tween 20 overnight at 4∘C and then washed fivetimes The dry membranes were cut and kept refrigerated

For immunoblotting two dilutions of sera in PBS 05skimmed milk and 02 Tween 20 were used with totalantigen 125 and 1100 The nitrocellulose individual stripswere incubated for 3 hours with sera washed five timesin PBS 02 Tween 20 and incubated in a 1300 dilutionof alkaline phosphatase-conjugated rabbit anti-canine IgG(PARIS Anticorps compiegne France) for 2 hours Afterfive washes the bound proteins were revealed with nitroblue tetrazolium (NBT) andbromo-chloro-indolyl phosphate(BCIP) as substrates The reaction was stopped with distilledwater Positive and negative controls were included in eachprocedure The interpretation was based on the presence ofthe antibodies to 14 and 16 kDa antigens which are commonto all Leishmania species [18]

Commercial Western Blot Kit Leishmania LDBIO kit (refLES-WB12G Lyon France) was used according to the manu-facturerrsquos specifications

223 Enzyme-Linked Immunosorbent Assay (ELISA)

Homemade ELISA Kit Quantification of antibodies by ELISAwas performed according to following procedure adaptedfromMary et al and Correia da Costa et al [17 19]

96-well microtiter plates were coated with the previouslydescribed antigen diluted at four different concentrations (510 20 and 50 120583gmL) in PBS pH 72 in a volume of 100 120583LPlates were then incubated for 2 hours at 37∘C and overnightat 4∘C

Sera were serially diluted from 140 to 11280 in PBS 05skimmed milk and 02 Tween 20 (PBS-T) and incubatedon microplates previously coated for 1 hour at 37∘C Afterthree washes a labelled anti-dog IgG alkaline phosphataseconjugate (PARIS Anticorps compiegne France) diluted to11000 in PBS-T was added per well and incubated for 1hour at 37∘C Enzymatic activity was revealed by hydrolysisof para-nitrophenyl phosphate for 15min at 37∘C and thereaction stopped by adding K

2HPO42M Plates were read

at 405 nm in an ELISA reader (MultiscanThermoFischer)

Commercial ELISA KitThe ID Screen Leishmaniasis Indirect(ref LEISHS-2P IDVET Montpellier France) kit was usedaccording to the manufacturerrsquos specifications

23 Statistical Analysis Data was analysed by crosstab andlogistic regression

3 Results

31 Characterisation of the Dog Population A total of 243dogs were clinically examined 749 males and 436domestic type (pets) versus 564 stray dogs Only 58 dogs

91

62

04

7482

5849

25

0012

00

16

0123456789

10

a b c d e f g h i j k l

Freq

uenc

y (

)

Figure 2 Frequency of each clinical sign displayed by all symp-tomatic and suspected dogs (a) looking old (b) cachexia (c)anorexia (d) weakening (e) dry exfoliative dermatitis andorperiorbital alopecia (f) ulcers (g) onychogryphosis (h) noise atwalk (i) epistaxis (j) ocular signs (keratoconjunctivitis and uveitis)(k) lymphadenopathy (l) splenomegaly

1813 11

3 2 102468

1012141618

1 2 3 4 5 6

Nbr

of d

ogs

Nbr of signs

Figure 3 Number (Nbr) of clinical signs displayed by all symp-tomatic and suspected dogs

were under veterinary follow-up and 198 were clinicallysuspected (Table 2)

In this study we investigated twelve commonly describedclinical cutaneous and visceral CanL signs as reported in thescientific literature Gallego [20] confirmed that the most fre-quent signs are skin abnormalities (dry exfoliative dermati-tis ulcers periorbital alopecia and onychogryphosis) butocular signs (keratoconjunctivitis and uveitis) and lymphade-nomegaly are also common According to Solano-Gallegoand Baneth [21] the main clinical signs associated with CanLare skin lesions lymphadenomegaly splenomegaly ocularabnormalities onychogryphosis weight loss and generalpoor body condition Carre et al [22] classified these symp-toms to general visceral and mucocutaneous signs

In our results alteration of general aspect (91) andskin abnormalities (82) were the most common clinicalsigns while ocular (12) and visceral (16) signs were verydiscrete (Figure 2)

Figure 3 shows the association of clinical signs Most ofthe infected dogs were oligosymptomatic 375 of symp-tomatic dogs presented only one sign 271 two signs 229three signs and 125 four or more clinical signs

32 Serological Results Firstly concerning the validation ofthe homemade western blot protocol better intensity and


Table 3 Seroprevalence of canine leishmaniasis infection in southern Morocco comparison of commercial and homemade ELISA andwestern blot tests

Localities Dogs clinicallysuspected ()

Seropositive dogs ()ELISA Western blot

Homemade kit IDVET kit Homemade kit LDBIO kitLandfill site 110 (10) 70 (710) (510) 50 80 (810) 80 (810)Ghmate 113 (77) 923 (1213) (713) 538 923 (1213) 100 (1313)Ourika 210 (20) 80 (810) (210) 20 60 (610) 80 (810)Total 433 (121) 818 (2733) 424 (1433) 788 (2633) 878 (2933)95 confidence interval [097 232] [686 949] [255 592] [648 927] [821 935]

Table 4 Concordance percentage between the different serologicalkits

ELISAHomemade IDVET

WB Homemade 88 606LDBIO 94 546

ELISA Homemade versus IDVET 606WB Homemade versus LDBIO 88

readability of antigenic bands were found with sera dilutedto 1100 in PBS rather than dilution of 125

Besides homemade ELISAprotocol showed better resultswhen antigen coating concentration was 20120583gmL in avolume of 100 120583L per well (2120583gwell) For the next broaderepidemiological studies on prevalence of canine leishma-niasis in all these preprospected areas we recommend theuse of a sera dilution of 1100 A threshold at 024 OD wasestablished after testing 30 negative control sera (mean + 2SD)

The highest seroprevalence of leishmaniasis infection wasfound to be respectively 818 (95 confidence interval(CI) 686ndash949) and 878 (95 CI 821ndash935) with thehomemade ELISA test and LDBIO commercial western blotkit (Table 3)

With the exception of IDVET commercial ELISA kit alltests revealed a very high prevalence of infection around80 No significant difference (Fisherrsquos exact test 119875 gt05) was found between western blot and ELISA resultsprobably because of high titer of anti-Leishmania antibodieswithin this population of dogs Among the 29 seropositivedogs only four were symptomatic (138) As expected allclinically suspected dogs were both ELISA and western blotseropositive

The best concordance percentage (94) was foundbetween LDBIO commercial western blot kit and homemadeELISA test (Table 4) Only two samples out of 33 werenegative with both ELISA tests but positive with the westernblot kits The concordance percent between the commercialIDVET and homemade ELISA tests was low (606) wherethe homemade ELISA test was much more sensitive How-ever the significant difference (Fisherrsquos exact test 119875 = 0002)in sensitivity between the two ELISA techniques may bedue to the conservation of sera samples The IDVET ELISAkit was the last test used when sera had already undergone

several cycles of freeze-thawing We also cannot exclude aproblem of batch or inappropriate conservation of IDVET kitreagents themselves

Western blot was the most sensitive test and there was nosignification difference (Fisherrsquos exact test 119875 gt 05) betweenthe sensitivity of the commercial versus homemade westernblot kits

Using a crosstab analysis the results of theChi-square testshowed no significant correlation between clinical signs ofdisease and seropositivity (119875 gt 005 and Cramerrsquos 119881 lt 070)Logistic regression was applied to study further possible cor-relations between the disease on the one hand and the local-ity age sex and type of dogs (domestic or feral) on the otherhand Our results based on 119875 value and Cox and Snellrsquos andNagelkerkersquos 119877-square measures showed no significant corre-lation between disease and locality (Sig = 0205) age (Sig =0058) sex (Sig = 0109) or type of dogs (Sig = 0999)

4 Discussion

CanL constitutes a considerable veterinary challenge as wellas important public health problem because infected dogs illor asymptomatic act as reservoir hosts for the humandiseasesand flies infected by a blood meal taken from infected dogsmay during a later blood meal infect humans

With the exception of sporadic cases of human visceralleishmaniasis [14 15] southern Morocco is currently consid-ered L infantum- and CanL-free area To provide a betterunderstanding of L infantum epidemiology in southernregions of Morocco clinical and serological surveys werecarried out to establish the presence of L infantum in caninepopulations

The western blot analysis is a valuable tool for epidemi-ological studies on visceral leishmaniasis especially when itis associated for confirmation with Elisa test It providesa significant increase in the sensitivity because the 14-kDandor 16-kD antigens are recognized bymost part of infectedanimals or patients (if not all) and show no cross-reactivity[17 23]

We showed the presence of L infantum in our studyarea with a high serological prevalence in dogs around80 Therefore southern Morocco might be highly endemiccompared to the lower range seroprevalence (10 to 40)usually reported in northern Morocco [8 9] and in theMediterranean area [24ndash26]


These unusual high seroprevalence results could beexplained by different factors Firstly the previous multipleand successive stray dog culling campaigns have been morefrequent and regular in the northern populated provinceswell known for leishmaniasis endemicity rather than in southareas Secondly the previous leishmaniasis seroprevalencestudies carried out in the northern Morocco or in Europeancountries around Mediterranean basin concerned rather petdogs with veterinary follow-up while 564 (137243) ofanimals in our study were stray dogs (Table 2) and amongthe 436 of domestic dogs with owners (pets) only 58have been subjected to veterinary follow-up and possibletreatment Lastly considering the small sampling size ofthis preliminary study an unknown selection bias cannot becompletely excluded

Our results corroborate with Dereure et al [27] whodemonstrated the presence of an autochthonous caninevisceral leishmaniasis cycle in the High and Anti-Atlasmountains of Morocco not far from our study area

Clinical diagnosis of leishmaniasis can be difficult dueto the great variety of symptoms and should thereforebe confirmed by parasitological serological or molecularmethods [20] We showed that all clinically suspected dogs(433) were Elisa and western blot seropositive HoweverCanL usually shows a high prevalence of subclinical infection[21 22] commonly explained by the long incubation time ofthis disease [28] and here supported by the high frequency ofasymptomatic dogs (862) we found

Besides few human visceral leishmaniasis cases reportedin our study area suggest that the dynamic of these southernMoroccan foci of CanL is different from northern ones Thisunusual pattern has sometimes been observed in Khemissetprovince (northern Morocco) where CanL does not sustainhuman visceral leishmaniasis [29] CanL and human visceralleishmaniasis distribution can geographically overlap wherethe incidence of the former is much higher [30]

On the other hand the distributions of L infantumand L tropica overlap in Morocco The causative agentsof cutaneous leishmaniasis in Sefrou province (northernMorocco) are attributed to both L tropica and L infantumby species-specific ITS1-PCR-RFLP assay [31] Furthermoreseveral cases of canine visceral leishmaniasis caused by Ltropica have been reported in regions where canine visceralleishmaniasis is caused by L infantum [32]

In our study area Azilal Chichaoua and El Haouzprovinces are simultaneously anthroponotic foci of Ltropica cutaneous leishmaniasis [33ndash35] Previously somezymodemes of L tropica have been several times found indogs in anthroponotic foci of L tropica cutaneous leishmani-asis in Taounate and Al Hoceima northern provinces [12 13]but also in south part (Azilal province) [11] Consequently inour study area we cannot exclude the possibility of visceralleishmaniasis in dogs that could be due to some specificstrains of L tropicaTherefore this study should be continuedby the isolation and typing of the strains circulating in allsouthern CanL foci

Moreover concerning the vector L infantum inMoroccois known to be transmitted by two species of the subgenus

Larroussius Phlebotomus perniciosus and P ariasi [15] How-ever our previous entomological investigations in this area(Table 1) showed the presence of three species of subgenusLarroussiusP ariasi atypicalmorph ofP perniciosus [36ndash39]and P longicuspis

The vectorial role of P ariasi has been shown in northernMorocco [40] while the epidemiological roles of atypicalmorph of P perniciosus and P longicuspis are not yet elu-cidated Furthermore P papatasi and some species of thegenus Sergentomyia largely spread throughout Morocco andour study area might also be able to transmit L infantum[41 42] Hence it is essential to identify the still unknownsand fly species involved in the CanL transmission in thesesouthern regions It could help to understand the reasonof low prevalence of human visceral leishmaniasis in thesesouthern CanL foci

In southern Algeria human visceral leishmaniasis isknown to be sporadic but during the last decade the numberof cases has increased significantly whereas entomologicalinvestigations showed the absence of sand flies of subgenusLarroussius [43]

Regarding canine leishmaniasis control it remains adifficult task inMorocco because of important stray and feraldog populations low attendance level to veterinary clinicsby the owners of domestic dogs rare use of insecticide-impregnated dog collars an absence of proven effectivevaccines at affordable cost and chemotherapeutic agentshaving a limited efficacy for carrier dogs and a high cost

For control the World Health Organization [44] rec-ommends the treatment of human cases insecticide vectorcontrol and infected dog sacrifice for the control of caninevisceral leishmaniasis So the elimination of infected orat least symptomatic stray and feral dogs is justified inMorocco It is an operation integrated into a global strategy ofcontrol of canine zoonosis with high impact on public health(especially rabies echinococcosis and leishmaniasis) but itis an activity difficult to realize This suggests the need fora strong collaboration between the veterinary and medicalservices the authorities and the local populations

To conclude we reported a high seroprevalence of CanLin southern Morocco although it is considered until now tobe free of this infection Both of our homemade serologicalkits were validated are cost-effective and will be useful forfurther large-scale epidemiological studies on the animalreservoir of visceral leishmaniasis

These preliminary results could be a helpful basis forfurther investigations in these CanL high prevalent foci ofsouthern regions in order to identify the still unknownsand fly species involved in the transmission to isolateand characterize the circulating Leishmania strains and tounderstand better the reason of lower prevalence of humanvisceral leishmaniasis in the southern Morocco

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper


Acknowledgments

This modest work is dedicated to the memory of KhalilRamaoui the authorsrsquo laboratory colleaguewho died during afieldmissionThe authors are very grateful to Bernard Pessonand Shazia Mahamdallie for the critical review of the paperand kind assistance

References

[1] R Fisa M Gallego S Castillejo et al ldquoEpidemiology of canineleishmaniosis in Catalonia (Spain) the example of the Prioratfocusrdquo Veterinary Parasitology vol 83 no 2 pp 87ndash97 1999

[2] V Sideris G Papadopoulou E Dotsika and E KaragounildquoAsymptomatic canine leishmaniasis in Greater Athens areaGreecerdquo European Journal of Epidemiology vol 15 no 3 pp271ndash276 1999

[3] V J Kontos and A E Koutinas ldquoOld world canine leishmani-asisrdquo Compendium on Continuing Education for the Practisingvol 15 pp 949ndash960 1993

[4] RMolina C Amela J Nieto et al ldquoInfectivity of dogs naturallyinfected with Leishmania infantum to colonized Phleboto-mus perniciosusrdquo Transactions of the Royal Society of TropicalMedicine and Hygiene vol 88 no 4 pp 491ndash493 1994

[5] G Jeaume ldquoUn cas de leishmaniose naturelle generalisee chezle chien auMarocrdquo Bulletin de la Societe de Pathologie Exotiquevol 25 pp 225ndash227 1932

[6] R Nejjar M Lemrani A Malki S Ibrahimy H Amarouchand A Benslimane ldquoCanine leishmaniasis due to Leishmaniainfantum MON-1 in northern Moroccordquo Parasite vol 5 no 4pp 325ndash330 1998

[7] R Nejjar M Lemrani L Boucedda H Amarouch and ABenslimane ldquoVariation in antibody titres against Leishmaniainfantum in naturally infected dogs in northern MoroccordquoRevue de Medecine Veterinaire vol 151 no 8-9 pp 841ndash8462000

[8] A Natami H Sahibi S Lasri M Boudouma N Guessouss-Idrrissi and A Rhalem ldquoSerological clinical and histopatho-logical changes in naturally infected dogs with Leishmaniainfantum in the khemisset province Moroccordquo VeterinaryResearch vol 31 no 3 pp 355ndash363 2000

[9] M Rami T Atarhouch M Sabri M Cadi Soussi T Benazzouand A Dakkak ldquoCanine leishmaniasis in the Rif mountains(Moroccan Mediterranean Coast) sero-epidemiological sur-veyrdquo Parasite vol 10 no 1 pp 79ndash85 2003

[10] C Haralambous A Dakkak F Pratlong J-P Dedet and KSoteriadou ldquoFirst detection and genetic typing of LeishmaniainfantumMON-24 in a dog from the Moroccan Mediterraneancoast genetic diversity of MON-24rdquo Acta Tropica vol 103 no1 pp 69ndash79 2007

[11] J Dereure J-A RiouxM Gallego J Perieres F Pratlong and JHMahjour Saddiki ldquoLeishmania tropica inMorocco Infectionin dogsrdquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 85 no 5 p 595 1991

[12] NGuessous-Idrissi B BerragM RiyadH SahibiM Bichichiand A Rhalem ldquoShort report Leishmania tropica etiologicagent of a case of canine visceral leishmaniasis in NorthernMoroccordquo The American Journal of Tropical Medicine andHygiene vol 57 no 2 pp 172ndash173 1997

[13] M Lemrani R Nejjar and F Pratlong ldquoA new Leishmaniatropica zymodememdashcausative agent of canine visceral leishma-niasis in Northern Moroccordquo Annals of Tropical Medicine andParasitology vol 96 no 6 pp 637ndash638 2002

[14] MoroccanMinistry of Health ldquoLutte Contre les LeishmaniosesDirection de Lrsquoepidemiologie et de Lutte Contre les MaladiesService des Maladies Parasitairesrdquo in Guide des Activites 2010

[15] M Rhajaoui ldquoHuman leishmaniases in Morocco a nosogeo-graphical diversityrdquo Pathologie Biologie vol 59 no 4 pp 226ndash229 2011

[16] M J Aisa S Castillejo M Gallego et al ldquoDiagnostic potentialof Western blot analysis of sera from dogs with leishmaniasisin endemic areas and significance of the patternrdquoTheAmericanJournal of Tropical Medicine and Hygiene vol 58 no 2 pp 154ndash159 1998

[17] C Mary D Lamouroux S Dunan and M Quilici ldquoWesternblot analysis of antibodies to Leishmania infantum antigenspotential of the 14-kD and 16-kD antigens for diagnosis andepidemiologic purposesrdquo The American Journal of TropicalMedicine and Hygiene vol 47 no 6 pp 764ndash771 1992

[18] O Aoun C Mary C Roqueplo et al ldquoCanine leishmaniasisin South-East of France screening of Leishmania infantumantibodies (Western blotting ELISA) and parasitaemia levelsby PCR quantificationrdquoVeterinary Parasitology vol 166 no 1-2pp 27ndash31 2009

[19] J M Correia da Costa A B Neogy I Vouldoukis M LSampaio Silva M Gentilini and L Monjour ldquoAntigenic com-ponents of partially purified antigens of Leishmania donovaniinfantum recognized by sera from dogs with asymptomatic oractive visceral leishmaniasisrdquoThe American Journal of TropicalMedicine and Hygiene vol 55 no 5 pp 511ndash515 1996

[20] M Gallego ldquoZoonosis emergentes por patogenos parasitos lasleishmaniosisrdquo Revue Scientifique et Technique (InternationalOffice of Epizootics) vol 23 pp 661ndash676 2004

[21] L Solano-Gallego and G Baneth ldquoCanine leishmaniosismdashachallenging zoonosisrdquo European Journal of Companion AnimalPractice vol 18 pp 232ndash241 2008

[22] N Carre M Collot P Guillard M Horellou and J-PGangneux ldquoVisceral leishmaniasis epidemiology diagnosistreatment and prophylaxisrdquo Journal de Pharmacie Clinique vol29 no 3 pp 121ndash148 2010

[23] P Marty A Lelievre J F Quaranta A Rahal M Gari-Toussaint and Y le Fichoux ldquoUse of the leishmanin skin testandWestern blot analysis for epidemiological studies in visceralleishmaniasis areas experience in a highly endemic focus inAlpes-Maritimes (France)rdquo Transactions of the Royal Society ofTropical Medicine and Hygiene vol 88 no 6 pp 658ndash659 1994

[24] J Dedet F B Osman A Chadli H Croset and J A RiouxldquoLeishmaniasis in Tunisia Sero immunological survey of thefrequency of infestationrdquo Annales de Parasitologie Humaine etComparee vol 48 no 5 pp 653ndash660 1973

[25] S Mansueto M D Miceli and P Quartararo ldquoCounterim-munoelectrophoresis (CIEP) and ELISA tests in the diagnosisof canine leishmaniasisrdquo Annals of Tropical Medicine andParasitology vol 76 no 2 pp 229ndash231 1982

[26] I Amusategui A Sainz E Aguirre and M A Tesouro ldquoSero-prevalence of Leishmania infantum in Northwestern Spain anarea traditionally considered free of leishmaniasisrdquoAnnals of theNew York Academy of Sciences vol 1026 pp 154ndash157 2004

[27] J Dereure I D Velez F Pratlong et al ldquoLa leishman-iose viscerale autochtone au Maroc meridional Presence


de Leishmania infantum MON-1 chez le Chien en zonepresahariennerdquo in Leishmania Taxonomie et PhylogeneseApplications eco-epidemiologiques pp 421ndash425 Colloque Inter-national CNRSINSERM Montpellier France 1986

[28] R W Ashford and S Bettini ldquoIn the leishmaniasis in Biologyand Medicinerdquo in Ecology and Epidemiology W Peters and RKillick-Kendrick Eds New World Academic Press LondonUK 1987

[29] H Sahibi A Natami A Moufaid S Lasri and A RhalemldquoClinical and seroepidemiological study of Leishmaniasisin Northern Moroccordquo Revue Marocaine des SciencesAgronomiques et Veterinaires vol 21 no 4 pp 227ndash2322001

[30] M Gramiccia ldquoRecent advances in leishmaniosis in pet ani-mals epidemiology diagnostics and anti-vectorial prophylaxisrdquoVeterinary Parasitology vol 181 no 1 pp 23ndash30 2011

[31] H Asmae A Fatima F Hajiba et al ldquoCoexistence of Leish-mania tropica and Leishmania infantum in Sefrou provinceMoroccordquo Acta Tropica vol 130 no 1 pp 94ndash99 2014

[32] M Rhajaoui A Nasereddin H Fellah et al ldquoNew clini-coepidemiologic profile of cutaneous leishmaniasis MoroccordquoEmerging Infectious Diseases vol 13 no 9 pp 1358ndash1360 2007

[33] K Ramaoui S Guernaoui and A Boumezzough ldquoEntomo-logical and epidemiological study of a new focus of cutaneousleishmaniasis inMoroccordquo Parasitology Research vol 103 no 4pp 859ndash863 2008

[34] S Guernaoui A Boumezzough B Pesson and G PichonldquoEntomological investigations in Chichaoua an emerging epi-demic focus of cutaneous leishmaniasis in Moroccordquo Journal ofMedical Entomology vol 42 no 4 pp 697ndash701 2005

[35] F Pratlong J A Rioux and J Dereure ldquoLeishmania tropicaau Maroc IV Diversite isozymique intrafocalerdquo Ann ParasitolHum Comp journal vol 66 pp 100ndash104 1991

[36] S Guernaoui B Pesson A Boumezzough and G PichonldquoDistribution of phlebotomine sandflies of the subgenus Lar-roussius in MoroccordquoMedical and Veterinary Entomology vol19 no 1 pp 111ndash115 2005

[37] S Boussaa A Boumezzough P E Remy N Glasser andB Pesson ldquoMorphological and isoenzymatic differentiation ofPhlebotomus perniciosus and Phlebotomus longicuspis (DipteraPsychodidae) in Southern Moroccordquo Acta Tropica vol 106 no3 pp 184ndash189 2008

[38] I Benabdennbi B Pesson M Cadi-Soussi and F M MarquezldquoMorphological and isoenzymatic differentiation of sympatricpopulations of Phlebotomus perniciosus and Phlebotomus longi-cuspis (Diptera Psychodidae) in northern Moroccordquo Journal ofMedical Entomology vol 36 no 1 pp 116ndash120 1999

[39] B Pesson J S Ready I Benabdennbi et al ldquoSandflies of thePhlebotomus perniciosus complex mitochondrial introgressionand a new sibling species of P longicuspis in the Moroccan RifrdquoMedical and Veterinary Entomology vol 18 no 1 pp 25ndash372004

[40] A Hamdani Etude de la faune phlebotomienne dans trois foyersde leishmanioses au Nord du Maroc especes abondance saison-nalite et incrimination du vecteur These de 3eme cycle [PhDthesis] Faculte des Sciences Semlalia Marrakech Morocco1999

[41] R Yavar K Hadi A M Reza et al ldquoFirst detection ofLeishmania infantumDNA inwild caughtPhlebotomus papatasiin endemic focus of cutaneous leishmaniasis South of IranrdquoAsian Pacific Journal of Tropical Biomedicine vol 3 no 10 pp825ndash829 2013

[42] M W Senghor M N Faye B Faye et al ldquoEcology ofphlebotomine sand flies in the rural community of MontRolland (Thies region Senegal) area of transmission of canineleishmaniasisrdquo PLoS ONE vol 6 no 3 Article ID e14773 2011

[43] K Benallal B Gassen L Bouiba J Depaquit and Z Har-rat ldquoEntomological investigation following the resurgence ofhuman visceral leishmaniasis in southern Algeriardquo Acta Trop-ica vol 128 no 3 pp 518ndash521 2013

[44] WHO ldquoControl of the leishmaniasis report of a meeting of theWHOexpert committee on the control of Leishmaniasesrdquo TechRep no 949 World Health Organization Geneva Switzerland2010

[45] S Boussaa B Pesson and A Boumezzough ldquoFaunistic studyof the sandflies (diptera Psychodidae) in an emerging focusof cutaneous leishmaniasis in Al Haouz province MoroccordquoAnnals of Tropical Medicine and Parasitology vol 103 no 1 pp73ndash83 2009

[46] S Boussaa M Neffa B Pesson and A BoumezzoughldquoPhlebotomine sandflies (Diptera Psychodidae) of south-ern Morocco results of entomological surveys along theMarrakech-Ouarzazat and Marrakech-Azilal roadsrdquo Annals ofTropical Medicine and Parasitology vol 104 no 2 pp 163ndash1702010

[47] S Boussaa S Guernaoui B Pesson and A BoumezzoughldquoSeasonal fluctuations of phlebotomine sand fly populations(Diptera Psychodidae) in the urban area of MarrakechMoroccordquo Acta Tropica vol 95 no 2 pp 86ndash91 2005

[48] S Boussaa B Pesson and A Boumezzough ldquoPhlebotominesandflies (Diptera Psychodidae) of Marrakech city MoroccordquoAnnals of Tropical Medicine and Parasitology vol 101 no 8 pp715ndash724 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Species composition of the sand fly fauna in study area

Province Species composition References

Al Haouz


[33 36 37 45]


P longicuspisP alexandri


Azilal


[36 37 46]



S minuta

Chichaoua

Phlebotomus perniciosuslowast

[34 36 37]

P sergentiP longicuspis

P ariasiP papatasiP alexandriP mariae


Marrakech


[36 37 47 48]P sergenti


S minutalowastAs atypical form [36ndash39]

surveys on CanL in southern Morocco an area traditionallyconsidered to be free from CanL in spite of the omnipresenceof dogs and phlebotomine sand flies Our investigations werecarried out in four provinces of Morocco Al Haouz AzilalChichaoua andMarrakech where species composition of thesand fly fauna was also established (Table 1)

2 Materials and Methods

21 Sampling An epidemiological survey was carried outin 27 localities (Figure 1) in four provinces of southernMorocco Al Haouz (31∘221015840N 7∘511015840W) Azilal (31∘581015840N6∘341015840W) Chichaoua (31∘321015840N 8∘451015840W) and Marrakech(31∘361015840N 8∘021015840W)

Two hundred and ten dogs in 2004ndash2006 and 33 dogsin 2007 were studied An individual form was completedfor each dog date locality name and environment dog agesex type (domestic or feral) and Leishmania specific clinicalsigns The detail of sampling is shown in Table 2

Because of practical economic and security constraintsit was not possible in 2004ndash2006 to take blood samples from

1

2

3

4

AtlanticOcean

Algeria

Rabat

Spain

Figure 1 Moroccan study area provinces (1) Chichaoua (2)Marrakech (3) Al Haouz (4) Azilal

stray and feral dogs with unknown rabies infection statusIn 2007 after clinical examination blood was collected byjugular vein puncture and 33 sera were stored at minus80∘C forserological tests

22 Serological Assays For serological analyses the 33 serawere processed using two ELISA techniques a homemadeELISA test and IDVET commercial kit and the results wereconfirmed by two western blot (homemade and LDBIOcommercial kit) techniques

221 Preparation of the L Infantum Antigen Antigens wereproduced from Leishmania infantum promastigotes grown inRPMI 1640 medium supplemented with 10 fetal calf serum(Sigma) L-glutamine (Gibco) and penicillin-streptomycin(Gibco) Log phase promastigotes were harvested by cen-trifugation (14000 g for 15min at 4∘C) and washed threetimes in sterile phosphate buffered saline (PBS) Cells werecounted and adjusted to a concentration of 35 times 108promastigotesmL and as previously described [16] andlysed by boiling for 5min in sample buffer (05M Tris-HClpH 68 001M EDTA 5 sodium dodecyl sulphate (SDS)5 2-mercaptoethanol and 00125 bromophenol blue)Protein concentration (15mgmL) was determined by thebicinchoninic acid method (Pierce) and antigen was storedat minus80∘C

222 WB Analysis

Homemade Western Blot Kit SDS-polyacrylamide gel elec-trophoresis was performed on a 15 polyacrylamide gel witha Mini-Protean II apparatus (Biorad) and 200 120583g of antigenwas used per gel width with molecular mass proteins stan-dards (standard low range BioRad) as previously described[17] Gels were run at 50V for 3 hours at room temperature


Table2Orig

inandnature

ofstu

died

dogpo

pulations

Date

Provinces

Localities

Male

Female

Total

Age

rangelowast

Ferald

ogs

Dom

estic

dogs

Veterin

ary

follo

w-up

Suspected

dogs

Dogsw

ithou

tclinicalsigns

2004ndash2006

Marrakech

Semlalia

42

62ndash4y

60

00

6Land

fillsite

4317

6018mndash4

y60

00

357

Tadchirt

20

23ndash5y

02

00

2Ao

udad

60

61ndash6y

06

00

6Be

lbakkar

10

18y

10

01

0Ch

ouflidi

10

11y

01

00

1Amerchich

20

22-3y

20

00

2Dou

arLaasker

11

220

mndash3

y2

00

02

Sansou

20

220

mndash2

y0

20

20

Guenn

oun

20

225ndash3y

11

01

1

AlH

aouz

Ghm

ate

225

274m

ndash6y

1017

210

17Ourika

23

52m

ndash6y

32

12

3

Chichaou

a

Zinit

30

33ndash8y

03

00

3AitMou

ssa

40

42-3y

40

00

4Timsal

10

13y

01

00

1AitIshaq

12

33ndash7y

03

01

2Ikhriben

61

72ndash16y

16

02

5

Azilal

Tanant

71

88m

ndash2y

26

11

7Fo

umJemaa

63

918mndash4

y8

10

90

AitTagalla

52

78m

ndash2y

16

00

7Aittkhajet

30

36m

ndash5y

03

10

3AitCh

warit

10

12m

01

10

1AitMalek

40

46m

ndash2y

04

00

4Tigit

32

54m

ndash35y

23

12

3Im

adahen

113

142m

ndash11y

68

04

10Makou

ssa

132

155m

ndash10y

312

05

10Ze

lguen

64

108m

ndash2y

19

01

9

2007

Marrakech

Land

fillsite

55

106m

ndash5y

82

11

9

AlH

aouz

Ghm

ate

94

136m

ndash3y

85

41

12Ourika

64

105m

ndash7y

82

22

8To

tal()

182(749)

61(251)

243(100)

mdash137(564)

106(436)

14(58)

48(19

8)

195(802)

lowast

mm

onthyyear













3 Results


91

62

04

7482

5849

25

0012

00

16

0123456789

10


Freq

uenc

y (

)


1813 11

3 2 102468

1012141618

1 2 3 4 5 6

Nbr

of d

ogs

Nbr of signs













ELISAHomemade IDVET











4 Discussion























Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Orig

inandnature

ofstu

died

dogpo

pulations

Date

Provinces

Localities

Male

Female

Total

Age

rangelowast

Ferald

ogs

Dom

estic

dogs

Veterin

ary

follo

w-up

Suspected

dogs

Dogsw

ithou

tclinicalsigns

2004ndash2006

Marrakech

Semlalia

42

62ndash4y

60

00

6Land

fillsite

4317

6018mndash4

y60

00

357

Tadchirt

20

23ndash5y

02

00

2Ao

udad

60

61ndash6y

06

00

6Be

lbakkar

10

18y

10

01

0Ch

ouflidi

10

11y

01

00

1Amerchich

20

22-3y

20

00

2Dou

arLaasker

11

220

mndash3

y2

00

02

Sansou

20

220

mndash2

y0

20

20

Guenn

oun

20

225ndash3y

11

01

1

AlH

aouz

Ghm

ate

225

274m

ndash6y

1017

210

17Ourika

23

52m

ndash6y

32

12

3

Chichaou

a

Zinit

30

33ndash8y

03

00

3AitMou

ssa

40

42-3y

40

00

4Timsal

10

13y

01

00

1AitIshaq

12

33ndash7y

03

01

2Ikhriben

61

72ndash16y

16

02

5

Azilal

Tanant

71

88m

ndash2y

26

11

7Fo

umJemaa

63

918mndash4

y8

10

90

AitTagalla

52

78m

ndash2y

16

00

7Aittkhajet

30

36m

ndash5y

03

10

3AitCh

warit

10

12m

01

10

1AitMalek

40

46m

ndash2y

04

00

4Tigit

32

54m

ndash35y

23

12

3Im

adahen

113

142m

ndash11y

68

04

10Makou

ssa

132

155m

ndash10y

312

05

10Ze

lguen

64

108m

ndash2y

19

01

9

2007

Marrakech

Land

fillsite

55

106m

ndash5y

82

11

9

AlH

aouz

Ghm

ate

94

136m

ndash3y

85

41

12Ourika

64

105m

ndash7y

82

22

8To

tal()

182(749)

61(251)

243(100)

mdash137(564)

106(436)

14(58)

48(19

8)

195(802)

lowast

mm

onthyyear













3 Results


91

62

04

7482

5849

25

0012

00

16

0123456789

10


Freq

uenc

y (

)


1813 11

3 2 102468

1012141618

1 2 3 4 5 6

Nbr

of d

ogs

Nbr of signs













ELISAHomemade IDVET











4 Discussion























Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3 Results


91

62

04

7482

5849

25

0012

00

16

0123456789

10


Freq

uenc

y (

)


1813 11

3 2 102468

1012141618

1 2 3 4 5 6

Nbr

of d

ogs

Nbr of signs













ELISAHomemade IDVET











4 Discussion























Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















ELISAHomemade IDVET











4 Discussion























Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Epidemiological Investigation of Canine ...downloads.hindawi.com/archive/2014/104697.pdf · Research Article Epidemiological Investigation of Canine Leishmaniasis