Occurrence of Leishmania (Leishmania) chagasi in a domestic cat (Felis catus) in Andradina, São Paulo, Brazil: case report

This work describes natural infection by Leishmania in a domestic cat where amastigote forms of the parasite were observed in the popliteal lymph node imprint. Positive and negative serological reactions were observed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA), respectively. Polymerase chain reaction (PCR) revealed that the nucleotide sequence of the sample was identical to Leishmania (L.) chagasi. This is the first report of the disease in felines of the city of Andradina, SP, an area considered endemic for canine and human visceral leishmaniasis.

Here we describe a case of natural infection of a domestic cat by Leishmania chagasi in the city of Andradina, located in an area endemic for human and canine visceral leishmaniasis.Andradina is in the region of Araçatuba / São Paulo (20.8961°, 51.37944°) at 405 m of altitude.
This study was approved by the Ethics Committee on Animal Experimentation of the Faculty of Dentistry of Araçatuba -UNESP -Protocol No. 2007-003276.
The feline, a male adult mongrel, was given for euthanasia by his owners to the local Center for Animal Control.This animal was from the urban area and had extensive dermatitis with crusts and small ulcers on its face as well as on the palmar and plantar areas of its legs.
Leishmania spp.amastigotes were seen in the feline's lymph node imprints stained with Quick Panoptic kit (Hematocor Biolog®), but not in imprints from bone marrow, spleen, liver, and skin lesions.Analysis of serum sample was negative by IFA (cut-off 1:40) and positive by ELISA performed as described by Lima et al. (2005) with protein A as an antigen, in triplicate.Serology for feline immunodeficiency virus (FIV) and feline leukemia (FeLV) performed with the kit Snap® Combo Plus Test, was negative.
DNA was extracted from lymph nodes and spleen samples using Genomic Prep™ Cells and Tissue DNA GE Healthcare kit.PCR reactions were performed at final volumes of 25 mL containing reaction buffer (10 mM Tris HCl, 50 mM KCl), 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.2 units Platinum Taq DNA polymerase (Invitrogen), 10ng of extracted DNA, and 10mmol of each of the RV1 and RV2 primers (LACHAUD et al., 2002).RV1 primer sequence (5'-CTTTTCTGGTCCCGCGGGTAG-3') and RV2 primer sequence (5'-CCACCTGGCCTATTTTACACCA -3') produce a 145 bp-conserved region present in all Leishmania species.PCR mixtures were incubated in a thermocycler Mastercycler Gradient (Eppendorf ) using the following cycling profile: initial denaturation at 94 °C for 4 minutes, 40 cycles of 94 °C for 30 seconds, 59 °C for 30 seconds, 72 °C for 30 seconds, and 70 °C for 10 minutes.The amplified products were analyzed by electrophoresis on 1.5% agarose gel stained with SYBR® safe (Invitrogen®).
The amplified fragments were visualized with an image analyzer (GelDoc-IT™ Imaging System), using the software Vision Works® LS.Next, amplicons were purified with the enzyme ExoSAP-IT (USB) and sequenced on MegaBace 1000 DNA sequencer (GE Healthcare) using the termination kit DYEnanic ET Dye (GE Healthcare).The sequences were compared with those deposited in the NCBI database using blastn (Basic Local Tool for Nucleotide Alignment).Positive fragments of L. chagasi were detected in tissue samples of spleen and bilateral popliteal lymph nodes (Figure 1).Database alignments of the amplified fragments showed 97% identity with the DNA sequences of L. chagasi in the GenBank: Z35276.
Taken together, these studies indicate that cats may play an important role in the epidemiology of leishmaniasis, acting as a possible source of infection (DA SILVA et al., 2010).More detailed studies will be necessary to assess feline's contribution in the maintenance and spread of leishmaniasis in different environmental conditions According to the data revealed herein it is plausible to think that in an area endemic for canine visceral leishmaniasis, with confirmed reports of human infection, cats may also be affected by the parasite.

Figure 1 .
Figure 1.Electrophoresis of the PCR products obtained from cat spleen, and right and left popliteal lymph node samples using RV1 and RV2 primers.PCR products of 145 bp are shown.