First clinical case of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis in a domestic cat from French Guiana

Veterinary Parasitology 181 (2011) 325–328

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First clinical case of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis in a domestic cat from French Guiana a ˜ V. Rougeron a,∗ , F. Catzeflis b , M. Hide a , T. De Meeûs c,d , A.-L. Banuls a

Laboratoire MIVEGEC, UMR IRD 224-CNRS 5290-Université Montpellier 1, Montpellier, F-34394, France Laboratoire de Paléontologie, Université Montpellier 2, Place E Bataillon, 34095, Montpellier cedex 05, France c Interactions hôtes-vecteurs-parasites dans les infections par des trypanosomatidae (TRYPANOSOM), UMR IRD/CIRAD 177, Centre International de Recherche-Développement sur l’Elevage en zone Subhumide (CIRDES), 01 BP 454, Bobo-Dioulasso 01, Burkina Faso d Centre National pour la Recherche Scientifique (CNRS), Délégation Languedoc-Roussillon, 1919, route de Mende, 34293 Montpellier cedex 5, France b

a r t i c l e

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Article history: Received 8 December 2010 Received in revised form 4 April 2011 Accepted 12 April 2011 Keywords: Leishmania Viannia braziliensis Felis catus French Guiana

a b s t r a c t We report the first case of natural infection of a domestic female cat (Felis catus) by Leishmania (Viannia) braziliensis in French Guiana. The infected animal had a cutaneous ulcer on the nose and nodules of different sizes in the ears. The diagnosis was confirmed by molecular analysis of cutaneous samples that detected the presence of Leishmania parasites and allowed identifying the Leishmania (Viannia) braziliensis species. The discovery of a cat infected by L. (V.) braziliensis suggests the possibility that cats could be potential secondary reservoirs of Leishmania parasites in French Guiana. Thus, it would be important to investigate the possible epidemiological role of domestic cats in domestic foci of Leishmania in this region. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Leishmaniasis is a public health problem in French Guiana as 348 cases were declared in 2004. The main observed clinical forms are cutaneous and mucocutaneous. The parasitic agents are mainly Leishmania guyanensis and occasionally L. braziliensis, L. amazonensis, L. naiffi and L. lainsoni (Rotureau, 2006). Nevertheless, the 2008 Report published by the French National Reference Center for Leishmania shows that the number of cases due to L. braziliensis, which is responsible of the majority of mucosal lesions, is increasing in French Guiana. Leishmaniasis in domestic cats (Felis catus) has been sporadically reported in various parts of the world, but their role as reservoir hosts is not clear as yet. The fol-

∗ Corresponding author at: New York University School of Medicine, Medical Parasitology Department, 341 East 25th street, New York, NY 10010, United States. Tel.: +1 212 263 4360; fax: +1 212 263 8116. E-mail address: [email protected] (V. Rougeron). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.04.028

lowing parasites have been identified in cats’ cutaneous lesions: Leishmania (Leishmania) mexicana in Texas, USA (Barnes et al., 1993), L. (Viannia) sp. in Brazil (Passos et al., 1996), L. (L.) amazonensis in Brazil (Schubach et al., 2004), L. (V.) braziliensis in Brazil (De Souza et al., 2005; Schubach et al., 2004) and L. (L.) infantum in Brazil (Savani et al., 2004), South of France (Ozon et al., 1998), Spain (Martin-Sanchez et al., 2007) and Italy (Poli et al., 2002). In the present study, we describe the first case of Leishmania infection in a semidomestic cat in French Guiana. 2. Materials and methods In October 2007, during a field study for collection of mammal specimens in the framework of the French National Project ANR 06-SEST-20 IAEL, we found a semidomestic female cat, between 3 and 5 years old, with an approximately 8-month-old cutaneous nasal ulcer of 1 cm in diameter and auricular nodules of different size (Fig. 1). This cat lived in a forest part of the time and also with a family, 3 miles away from the village of Cacao, in a man-made

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V. Rougeron et al. / Veterinary Parasitology 181 (2011) 325–328

Fig. 1. Tegumentary feline leishmaniasis caused by L. (V.) braziliensis. (A) single nodular nasal lesion of about 1 cm in diameter, (B) nodules of different sizes in the ear. Photographed by F. Catzeflis on October 18, 2007 near Cacao, French Guiana.

clearing at the end of a path, 04◦ 35 N/52◦ 26 W (168 m altitude). This finding was unexpected and we did not have all the necessary equipment for microscopic examination and parasite culture. Therefore, two samples were taken using cotton swabs from the ear lesions and placed in 95% ethanol for storage and transport. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Courtaboeuf, France), following the manufacturer’s recommendations. The cat sample and six reference DNA stocks from different species [L. guyanenis (MHOM/FG/84/H166), braziliensis (MHOM/BR/75/M2904), L. lainsoni L. (MHOM/BR/81/M6426), L. amazonensis (IFLA/BR/73/PH8), L. mexicana (MNYC/BZ/62/M379) and L. donovani (MHOM/IN/82/Patna1)] were amplified as in Noyes et al. (1998). Eight microliters of the second-round PCR products were loaded onto a 2% agarose gel with EnVISIONTM DNA Dye (Ambresco). This first analysis allowed detecting the presence of Leishmania parasites in the cat samples. However, since the different New World species could not be distinguished, sequencing of the 6-phosphogluconate dehydrogenase (6pgd) gene was performed. This enzyme has been extremely useful to distinguish the New World Leishmania species for both MLEE (Multilocus Enzyme Electrophoresis) studies and diagnostic PCR (see examples in Cupolillo et al., 1995; Saravia et al., 1998; Tsukayama et al., 2009). PCR amplification of the cat sample and the six reference stocks was thus carried out with the primers 6pdg2L (5 -GGCTTCAACAACGATCAGGT-3 ) and 6pgd2R (5 -ACGACTCACGACCGTCCTT-3 ) (Mimori et al., unpublished data). Each reaction mix was composed of 1 ␮L of each primer (10 ␮M), 5 ␮L template DNA, 1 ␮L dNTP mix (5 mM), 3 ␮L 10× buffer and 0.5 ␮L Taq polymerase (Roche Diagnostics, Meylan, France, 5 UI/␮L). PCR amplifications (35 cycles) were carried out in a thermal cycler using the following reaction conditions: 94 ◦ C for 30 s, annealing

temperature of 56 ◦ C for 1 min, 72 ◦ C for 1 min and a final extension step of 72 ◦ C for 7 min. The cat amplicon (800 bp) was gel-purified (QIAEX kit), cloned into pGEM-T Easy TA cloning vector (Promega, Madison, WI, USA) and sequenced by MWG Biotech Inc. (Hedersberg, Germany). Blast analysis of the EMBL database allowed adding 16 6pgd sequences to our analysis. The sequences were aligned using the BioEdit sequence alignment editor (Hall, 1999). The evolutionary distance (Maximum Composite Likelihood method) was calculated and the Neighbor Joining tree was constructed using the MEGA software (version 4.0.2, Tamura et al., 2007). The stability of the tree topology was evaluated by bootstrapping (n = 500). 3. Results and discussion Leishmania DNA was detected in the cat samples using the nested PCR approach previously described in Noyes et al. (1998). Sequencing and phylogenetic analysis of the 6pgd sequences (Fig. 2) showed that this semi-domestic cat was infected by a member of the L. braziliensis complex (i.e., L. braziliensis or L. peruviana). Since the L. peruviana ˜ species is restricted to the Andean regions (Banuls et al., 2000), we reasonably assumed that the cat was infected by L. braziliensis. This cat was raised by a family living near a secondary forest. It appears that cats are common pets in the Cacao village (F. Catzeflis, personal observation), where several epidemics of leishmaniasis have occurred. These observations suggest an emergent risk of peri- and intradomestic transmission of L. braziliensis. Similarly, the group of Schubach discovered two female cats infected by L. (V.) braziliensis in Rio de Janeiro, Brazil (Schubach et al., 2004). Moreover, the group of Maroli et al. (2007) reported evidence of transmissibility of the feline parasite L. infantum to a proven vector, suggesting that cats could potentially be a domestic reservoir. Cats could thus be a secondary

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Fig. 2. The evolutionary tree was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.43,876,766 is shown. The percentages of replicated trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units akin to those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and they were expressed in units that correspond to the number of nucleotide substitutions per site. Included codon positions were 1st + 2nd + 3rd + non-coding. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). There were a total of 785 positions in the final data set. Phylogenetic analyses were carried out using MEGA4 (Tamura et al., 2007).

reservoir and a link between sylvatic cycles and peri- and intra-domestic cycles of leishmaniasis. Finally, the detection of a cat with cutaneous ulcerations indicates that further investigations are required to determine their possible epidemiological role in domestic foci of Leishmaniasis in French Guiana. Indeed, a better understanding of the epidemiological situation in this region by an entomological study and an investigation concerning the domestic mammals (cats, domestic rodents, etc.) as well as the commensal wild mammals, such as Rattus rattus, that are present in this region will contribute to our understanding of the epidemiological situation of French Guiana and consequently will help setting up appropriate prevention and control strategies against this disease. Acknowledgements The authors would like to acknowledge the family of Claude Duffay (owners of the cat), Gilles Peroz for his efficient help in the field as well as C. Arnathau and P. Durand for helpful discussions. We thank also Pr. Tatsuyuki Mimori for the development of the 6pgd PCR analysis. The authors acknowledge the assistance of Dr. Linda Northrup and Elisabetta Andermarcher for correction of the manuscript. We are grateful to the Institut de Recherche pour le Développement (IRD) and the Centre National de la Recherche Scientifique (CNRS) for financial support. This work was also supported by the French National Project ANR 06SEST-20 IAEL.

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