Variation in antibody titres against Leishmania infantum in naturally infected dogs in northern Morocco

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1 ARTICLE ORIGINAL Variation in antibody titres against Leishmania infantum in naturally infected dogs in northern Morocco R. NEJJAR, M. LEMRANI, L. BOUCEDDA, H. AMAROUCH and A. BENSLIMANE Unité de Microbiologie, Laboratoire des leishmanioses, Institut Pasteur du Maroc, 1 rue Abou Kacem Ez-Zahraoui, Casablanca, Maroc Laboratoire de Biochimie, Biologie Cellulaire et Moléculaire, Faculté des Sciences I Ain Chock, Casablanca, Maroc Correspondance to : NEJJAR Rajae, Unité de Microbiologie, Laboratoire des leishmanioses, Institut Pasteur du Maroc, 1 rue Abou Kacem Ez-Zahraoui, Casablanca, Maroc Tel : (212) , Fax : (212) SUMMARY The variation of the anti-leishmania antibodies titres in a cohort of 46 dogs were studied in two areas in northern Morocco during the period (from July 1996 to July1997) by using the immunofluorescence test (IFAT) and counter-immunoelectrophoresis (CIEP). The 46 dogs had titres 100 and showed specific anti-l.infantum bands by CIEP. Dogs were divided in three groups : group 1 included 20 dogs which produced high antibody titres (titre 800) and developed clinical and fatal canine leishmaniasis. Group 2 had 16 dogs with low titres following infection and which remained asymptomatic during the period of follow up. In group 3, the titres 100 of 10 animals decreased to 50 and became negative by the end of the study. The disease was more prevalent in older dogs than in younger ones. KEY-WORDS : Leishmania infantum - titres variation - Morocco - dogs. RÉSUMÉ Variation des titres en anticorps anti-leishmania infantum chez une population canine naturellement infestée. Par R. NEJJAR, M. LEM- RANI, L. BOUCEDDA, H. AMAROUCH et A. BENSLIMANE. Les variations des titres en anticorps anti-leishmania infantum chez une cohorte de 46 chiens naturellement infestés ont été étudiées dans deux localités au nord du Maroc entre juillet 1996 et juillet 1997 en utilisant les techniques d'immunofluorescence indirecte (IFAT) et d'électrosynérèse (CIEP). Au début de l'étude, les 46 chiens ont montré des titres en anticorps 100 en IFAT et des arcs spécifiques en CIEP. Les chiens ont été divisés en 3 groupes : le groupe 1 comprend 20 chiens symptomatiques qui produisent des titres élevés en anticorps(titres 800) et développent une leishmaniose canine fatale. Dans le groupe 2, 16 chiens ont montré de faibles titres en anticorps et sont restés asymptomatiques durant tout le suivi. Pour le groupe 3, les titres de 10 chiens se sont négativés à la fin de l'étude. De plus, les chiens adultes sont plus sensibles à la maladie que les jeunes chiens. MOTS-CLÉS : Leishmania infantum - variation des titres en anticorps - Maroc - chiens. 1. Introduction Leishmaniasis in dogs is a severe, often fatal, chronic systemic disease caused by a protozoan parasite of the genus Leishmania. In the Old World, leishmaniasis in dogs is almost exclusively caused by L. infantum, which causes visceral leishmaniasis in people. Canidae are the main reservoir host of VL [14] while sandflies of the subgenus Larroussius are the incriminated vectors [19]. Leishmania infantum is distributed in many areas of the Mediterranean basin. Although the incidence of visceral leishmaniasis (VL) in humans is relatively low, the prevalence of VL in canids is very high, ranging from 10 % to 37 % [8]. In Morocco, visceral leishmaniasis is primarily located in the North, with some 50 pediatric cases notified each year to the Ministry of Health [22]. Recent studies on seroprevalence of canine leishmaniasis carried out in 5 provinces in northern Morocco to assess seropositivity percen-

2 842 NEJJAR (R.) AND COLLABORATORS tages, have revealed a wide range of values between provinces with an average of 8,6 % [24]. High frequency of asymptomatic dogs was observed (90 %). Several studies have shown a high seroprevalence in Mediterranean canine population without visible clinical signs in the animal [1, 3, 9]. Hence, dogs are considered to be the main veterinary reservoir of L. infantum, particularly during the long asymptomatic incubation phase preceding the appearance of the clinical symptoms associated with the VL form [10] and "asymptomatic" dogs are infective to sand flies as the "symptomatic" [6]. The aim of this research is to investigate the variation in the IFAT titres against L. infantum of a cohort of 46 dogs during the period (from July 1996 to July 1997) in two areas in northern Morocco where the disease has a significant impact on a naturally infected dog population. 2. Material and methods A) DOGS The data come from a coss-sectional serological follow-up of the dog population carried out in two rural areas in northern Morocco : locality of Azzaba (province of Sefrou) and locality of Brarcha (province of Zouagha Moulay Yacoub). In the beginning, 228 dogs were analysed to determine seropositivity of canine leishmaniasis in the two provinces. For the serological follow-up, a maximum of three blood samples were obtained per animal and the seropositive animals (symptomatic and asymptomatics dogs), received a complete physical examination for clinical signs related to leishmaniasis : enlarged lymph nodes, onychogriphosis, depilation, ulceration, weight loss. Interviews with owners provided information about the number of dogs kept, their names, ages, residence time, presence or absence of clinical symptoms and the names and addresses of the owners. The age of dogs varied from 8 months to 144 months. B) PARASITOLOGY Attempts were made to isolate parasites in NNN culture (Novy, McNeal, Nicolle) from symptomatic dogs (at diagnosis) and seropositive animals (second and third follow-up). Punctures of the popliteal lymph node were taken as the principal procedure for parasite isolation. They were cultured in NNN medium and maintained at 25 C for at least 30 days. The culture were checked weekly for the presence of promastigotes. A culture was considered positive when at least one promastigote was observed. Negative culture were examined again at the end of the third and fourth weeks, being then discarded if remaining negative. Punctures of lymphatic nodes were used to make preparations on slides which were stained with Giemsa and examined with standard light microscopy in search of amastigotes. A total of 46 dogs were ponctionated during one year of follow-up. C) SEROLOGY For the serological study, blood was extracted from the cephalic vein, and the serum were separated from the blood samples under standard conditions and used for an immunofluorescence antibody test (IFAT) and counter-immunoelectrophoresis (CIEP). a) IFAT IFAT on serum samples was performed by using whole promastigotes of Leishmania infantum MON-1 (MHOM/TN/80/IPT-1) as antigen.the parasites were expanded in RPMI medium (Aqual) supplemented with 10 % foetal calf serum (Aqual) at 25 C. The IFAT was carried out as described by PINELLI et al. [26], using serial serum dilutions starting at either 1 :50 to 1 :12800 and titres 100 were considered positive. Detection of anti-leishmania antibodies in canine sera was assayed using an FITC-conjugated antidog Ig G (Sigma). For each test, standard L. infantum positive and negative canine sera were included as controls. b) CIEP The CIEP was performed in respect to the method developed by MONJOUR et al. [23]. The same parasite strain used in IFAT was frozen and thawed repeatedly in liquid nitrogen, to lyse the promastigotes, and was examined microscopically to ensure disruption of promastigote membranes. The protein concentration of the lysate was determined by the method of LOWRY [20]. C) STATISTICAL METHODS Ninety-five percent confidence intervals (95% CI) for clinical signs related to leishmaniasis were calculated according to the method proposed by FLEISS [17]. Results Sixty-four (28 %) out of 228 dogs showed IFAT titres of 100 to and specific anti-l. infantum bands by CIEP. Eighteen (28,1 %) out of the 64 dogs disappeared after one year of follow-up : ten of these dogs died of various diseases and the 8 remaining dogs were killed by the health authorities. The remaining canine sub-population (46 dogs) underwent a visual clinical examination and symptoms related to leishmaniasis were noted (weight loss, onychogriphosis, furfuraceus dermatitis, ulceration, depilation, enlarged lymph nodes). Twenty dogs (43,4 %) were symptomatic, and 26 (56,5 %) out of 46 dogs were asymptomatic but positive in serology (IFAT+CIEP). Finally, all dogs' (46) popliteal lymph nodes were aspirated for culture in NNN medium (symptomatic and asymptomatic dogs). Twenty-two (47,8 %) of these animals(16 from symptomatic and 6 from asymptomatic dogs) yielded parasites in culture. The anti-leishmania titres reported in the 46 dogs are shown in table I. The 46 seropositive dogs were divided into three groups :

3 VARIATION IN ANTIBODY TITRES AGAINST LEISHMANIA INFANTUM IN NATURALLY INFECTED DOGS IN NORTHERN MOROCCO 843 symp : symptomatic ; asymp : asymptomatic ; pos : positive ; neg : negative TABLE I. Variation in antibody titres (IFAT) in a cohorte of 46 dogs with canine leishmaniasis.

4 844 NEJJAR (R.) AND COLLABORATORS symp : symptomatic ; asymp : asymptomatic ; pos : positive ; neg : negative TABLE I. (suite) Variation in antibody titres (IFAT) in a cohorte of 46 dogs with canine leishmaniasis. Group 1 : When the diagnosis of leishmaniasis was established, all dogs (1 to 20) had positive titres (1:800 or higher) and displayed canine leishmaniasis symptoms, especially loss of weight and weakness (80 %, 95 % CI = 97,8-63 %), onychogriphosis (90 %, 95 % CI = %), enlarged lymph nodes (8 %, 95 % CI = 20-4 %) and lesions of the skin and coat (40 %, 95 % CI = %) including furfuraceous dermatitis, ulceration and depilation. Sixteen out of the 20 dogs popliteal lymph node were aspirated, and yielded Leishmania in culture(table I). In dogs 4, 6, 13 and 16, it was not possible to demonstrate the parasite by culture. However, all these animals showed signs of leishmaniasis and produced high IFAT titres ranging from 1/800 to 1/6400. Six months later, 8 out of the 20 dogs had died from leishmaniasis, and the 12 remaining ones died a year later. Group 2 : comprised 16 asymptomatic dogs (cases 21 to 36) but positive in serology. On 8/16 occasions, antibody titres increased 6 months and one year later, and in the other half-population, they remained the same. Dogs 34, 35 and 36 remained positives with low titres of 100. In 6 dogs, promastigotes were noticed in cultures of lymph nodes in the sixth month of diagnosis (table I). These dogs did not show any clinical evidence of disease during the whole investigation. Group 3 : In the beginning of the study, 10 dogs (37 to 46) showed IFAT titres of All dogs became seronegative after one year of follow-up (table I). We noticed a statistically difference in the presence or absence of clinical symptoms of canine leishmaniasis according to the age (table I) : dogs aged > 48 months were in general symptomatic whereas dogs aged < 36 months were asymptomatic(χ2 = 9,73, p < 0,01). Discussion To determine the variation in antibody titres against Leishmania infantum in dogs naturally infected in two rural areas in northern Morocco, we have determined firstly the seroprevalence in these two localities for identifying seropositive dogs for the follow-up. The prevalence in these two areas were 28 %. This value is higher than that estimated for other parts in northern Morocco. A previous study of seroprevalence of canine leishmaniasis in 5 provinces in northern Morocco have shown variations between localities of 0 to 33,3% of positive dogs with an average of 8,6 % [24] and GUESSOUS et al. [18] have noted a prevalence of 27,1 % in the locality of Oulad Hcein (province of Taounate). In 4 symptomatic dogs, the culture of the parasite was negative. This finding is probably due to several factors such as the the very low density of parasites in lymph nodes, which were not revealed by standard techniques [12] and problems with microbial contamination of cultures. Cultures remain the "gold standard" for definitive diagnosis. Unfortunately, the culture of specimen isolate organisms in animals with pathologically confirmed disease less than 80 % of the time [27]. This estimated sensitivity is often decreased because of problems with culture contamination. These difficulties have led to the use of several serodiagnostic tests for visceral leishmaniasis (human and canine). Among these tests, the indirect immunofluorescence assay (IFA) exhibits a high level of sensitivity and specificity [21] and is routinely employed for the diagnosis of canine leishmaniasis. It is interesting to note the differences in seropositivity between the beginning and the end of the study since some dogs shows seroconversion to seronegativity. We distinguished between three groups of dogs : Dogs of groupe 1 were symptomatics, produced high antibody titres and developed clinical and fatal leishmaniasis. The correla-

5 VARIATION IN ANTIBODY TITRES AGAINST LEISHMANIA INFANTUM IN NATURALLY INFECTED DOGS IN NORTHERN MOROCCO 845 tion between the development of the disease and the level of antibody titres (IgG1 and IgG2) were shown by NIETO et al. [25]. The asymptomatic dogs were subdivided into two groups. Dogs of group 2 were asymptomatic but serologically positive. These dogs contribute to transmission [7]. The last group included asymptomatic dogs which were serologically positive and became seronegative (after 6 or 12 months) indicating a remission of the infection. In a highly endemic area in southern Spain, ACEDO-SANCHEZ et al. [4] reported 35 % of remission of positive dogs and FISA et al. [16] also reported a similar percentage in a 3 years serological follow-up of canine population. These dogs probably contain fewer parasites than the other dogs but have a longer life span and have to be considered when estimating the rate of transmission [7]. The remission of the infection was explained by the presence of a strong cellular immune response in these dogs and a negative serology observed by CABRAL et al. [11] and PINELLI et al. [26]. In our study, we have found a difference in the presence or absence of clinical symptoms of canine leishmaniasis according to the age. The disease was more prevalent in older dogs. Older dogs also produce high antibody titres, develop clinical and may die of canine leishmaniasis (Group 1). Because the probability of infection is related to the duration of exposure to infected sandflies, older dogs may be expected to be more infected [1]. AMELA et al. [5] argued that the greater risk of infection at older ages may be linked to lower resistance to infection due to an age-related fall-off in the efficacy of the immune response. In contrast to our study, SIDERIS et al. [28] did not find an influence of age on prevalence but COURTENAY et al. [13] described the disease as being more prevalent in younger dogs because older resistant animals are protected from disease by cellular immunity [10, 26] and would clearly form a larger proportion of dogs in older age classes. The dogs in our study had symptoms typical of the disease reported by veterinarians and cited in published works. However, in comparison with a study carried out by SIDERIS et al. [28], which reported adenopathy in 22 % of dogs, onychogriphosis in 4 % and keratitis in 20 %, the corresponding rates in our study were 40 %, 90 % and 10 %. The reason of the dichotomy symptomatic and asymptomatic dogs are unclear but probably reflect the genetic background, nutritional and immunological status of hosts; degree of repeated introduction of promastigotes by vectors, sandfly vector factors and leishmanial parasite virulence [2]. Furthermore, for epidemiological studies, the prevalence of infection is underestimated if only symptomatic dogs are taken into account because sandflies feed on apparently healthy dogs and become infected and some cases of canine infection are missed [15]. Aknowledgements We would like to thank Dr. RIOUCH delegate of the province of Sefrou, Dr. MOUNIM, head doctor of the CIAP (province of Sefrou), Dr KAMAL delegate of the province of Zouagha Moulay Yacoub as well as the local authorities of the two provinces ; and to Pr. J P. DEDET (Laboratoire de Parasitologie, Faculté de médecine, Montpellier, France) and Pr. M. BELKAID (Institut Pasteur d'algérie) for comment, review and careful reading of the manuscript. References 1. ABRANCHES P., SILVA-PEREIRA M.C.D., CONCEIÇAO-SILVA F.M., SANTOS-GOMEZ G.M. and JANZ J.G. : Canine leishmaniasis : pathological and ecological factors influencing transmission of infection. J. Parasitol., 1991a, 77, ABRANCHES P., SANTOS-GOMES G., RACHAMIM N., CAM- PINO L., SCHNUR L.F. and JAFFE L. : An experimental model for canine visceral leishmniasis. Parasite immunol., 1991b, 13, ACEDO SANCHEZ C., MARTIN SANCHEZ J., VELEZ BERNAL I.D., SANCHIZ MARIN M.C., LOUASSINI M., MALDONADO J.A. and MORILLAS MARQUEZ F. : Leishmaniasis Eco-epidemiology in the Alpujarra region (Granada province, southern Spain). Int. J. Parasitol., 1996, 25, ACEDO SANCHEZ C., MORILLAS MARQUEZ F., SANCHIZ MARIN M.C. and.martin SANCHEZ J. : Changes in antibody titres against Leishmania infantum in naturally infected dogs in southern Spain. Vet. Parasitol., 1998, 75, AMELA C., MENDEZ I., TORCAL J.M., MEDINA G., PACHON I., CANAVATE C. and ALVAR J. : Epidemiology of canine leishmaniasis in the Madrid region, Spain. Eur. J. Epidemiol., 1995, 11, ALVAR J., MOLINA R., SAN ANDRÉS M., TESOURA M., NIETO J., VITUTIA M., GONZALEZ F., SAN ANDRÉS M.D., BOGGIO J., RODRIGUEZ F., SAINZ A. and ESCACENA C. : Canine leishmaniasis : clinical, parasitological and entomological follow-up after chemoterapy. Ann. Trop. Med. Parasitol., 1994, 88, BERRAHAL F., MARY C., ROZE M., BERENGER A., ESCOF- FIER K., LAMOUROUX D. and DUNAN S. : Canine leishmaniasis : identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am. J. Trop. Med. Hyg., 1996, 55, BETTINI S. and GRADONI L. : Canine leishmaniasis in the mediterranean area and its implications for human leishmaniasis. Insect. Sci. Applicat., 1986, 7, BRANDONISIO O., CARELLI G., CECI L., CONSENTI B., FASA- NELLE A. and PUCCINI V. : Canine leishmaniasis in the Gargano promontory (Apulia, south Italy). Eur. 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6 846 NEJJAR (R.) AND COLLABORATORS 20. LOWRY O.H., ROSENBERG M.J., FARR L. and RANDALL R.J. : Protein measurement with the folin phenol reagent. J. Biol. Chem., 1951, 193, MANCIANTI F. and MECIANI N. : Specific serodiagnosis of canine leishmaniasis by indirect immunofluorescence, indirect hemagglutination, and couterimmunoelectrophoresis. Am. J. Vet. Res., 1988, 49, Ministère de la Santé Publique du Maroc, Direction de l'epidémiologie et des Programmes Sanitaires. Bulletin Epidémiologique, 1992, 7, MONJOUR L., MILLE C., DRUILHE P. and GENTILINI M. : Application de l'immunoélectrodiffusion, sur membrane d'acétate de cellulose, au diagnostic de la leishmaniose viscérale humaine et canine. Ann. Soc. Belge. Med. Trop., 1978, 58, NEJJAR R., LEMRANI M., MALKI A., IBRAHIMY S., AMA- ROUCH H. and BENSLIMANE A. : Canine leishmaniasis due to Leishmania infantum MON-1 in Northern Morocco. Parasite, 1998, 5, NIETO C.G, GARCIA ALONSO M., REQUENA J.M., MIRON C., SOTO M., ALONSO C. and NAVARRETE I. : Analysis of the humoral immune response against total and recombinant antigens of Leishmania infantum : correlation with disease progression in canine experimental leishmaniasis. Vet. Immunol. Immunopathol., 1999, 67, 2, PINELLI E., KILLICK-KENDRICK R., WAGENAAR J., BERNA- DINA W., DEL REAL G. and RUITENBERG J. : Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum. Infect. Immun.,1994, 62, SCHNUR LF. and JACOBSON RL. : Appendix III : Parasitological techniques. PETERS W, KILLICK-KENDRICK R, eds. The leishmaniases in Biology and Medicine, 1987, 1, New York : Academic Press, SIDERIS V., KARAGOUNI E., PAPADOPOULOU G., GARIFAL- LOU A. and DOTSIKA E. : Canine visceral leishmaniasis in the great Athens area, Greece. Parasite, 1996, 3, Revue Méd. Vét., 2000, 151,8-9,

No.1 May CVBD DIGEST. Asymptomatic Leishmaniosis in Dogs. Cutting-edge information brought to you by the CVBD World Forum

No.1 May CVBD DIGEST. Asymptomatic Leishmaniosis in Dogs. Cutting-edge information brought to you by the CVBD World Forum No.1 May 2008 www.cvbd.org Asymptomatic Leishmaniosis in Dogs Cutting-edge information brought to you by the CVBD World Forum No.1 May 2008 Introduction When Bayer HealthCare, Animal Health Division, called