Use of Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of Leishmaniasis in Cats
T.A.C. Costa; C.N. Rossi; M.D. Laurenti; A.A.D. Gomes; J.P. Vides; L.S.V. Sobrinho; D.C. Costa; M. Marcondes
The natural domestic cat infection with Leishmania sp. was first described in 1912 in Argelia, in a four-month old animal that lived with a dog and a child with visceral leishmaniasis. The diagnosis was based on amastigote form findings in the bone marrow, without the identification of the species which caused the disease (Sergent et al. 1912). From the first clinical case description up to these days the literature worldwide has records of about 50 positive cases identified using parasitological diagnosis. From these cases, 24 (53.33%) occurred in the New World and 21 (46.67%) occurred in the Old World. The data compiled from literature show records of feline leishmaniasis cases in several countries, such as in the United States, France, Spain, Italy, Portugal, Switzerland and Brazil. There are divergences in the literature concerning the susceptibility of domestic felines to Leishmania sp. infection. While Pennisi et al. (1998) verified a seroprevalence of 62% in cats from Sicília, Poli et al. (2002) observed a positivity of 0.9% in Toscana, Martín-Sánchez et al. (2007) and Solano-Gallego et al. (2007) observed a prevalence of 60% and 5.25% respectively in Spain, and Maia et al. (2008) observed 30.4% in Portugal. The purpose of the present study was the determination of the seroprevalence of Leishmania sp. infection in 200 cats originated from an endemic visceral leishmaniasis area, using the ELISA technique.
Materials and Methods
Samples from two groups of cats were collected. The first group, composed of 200 adult cats from Araçatuba, São Paulo-Brazil, an endemic area for visceral leishmaniasis; and the second group composed of 53 cats from a non-endemic area for the referred disease. In all cats an anti-Leishmania sp. investigation was performed using the ELISA technique. Parasitological diagnosis was made by needle aspiration biopsy of lymph nodes, bone marrow, spleen and liver. For the execution of the ELISA technique the serum was diluted in 1:400 and IgG in 1:40000. The chromogen used in the test was tetrametilbenzidine dihidroclide (TMB). The cutoff determination (0.365) was performed with the serum of cats from a nonendemic area.
From the 200 cats assessed, 23 were serologically positive (11.5%) and 8 (4%) had amastigote forms of Leishmania sp.; out of these, two were positive for both exams. Four cats showed amastigote forms of Leishmania sp. only in lymph node cytology and the parasite was observed in the liver of one animal only, being negative in the remaining tissues; in one feline, Leishmania sp. was observed both in lymph nodes and bone marrow; and finally, in two other animals amastigote forms were identified in lymph nodes, spleen and bone marrow. From the positive animals, only two (25%) displayed alterations upon physical examination, characterized by dermatological crusty lesions on the dorsal cervical region, followed by hepatomegaly and splenomegaly.
Discussion and Conclusions
The crescent increase in the prevalence of visceral leishmaniasis in Brazil has caused concern in professionals who work alongside public health organizations, in small animals veterinarian clinics and also in the population dwelling in the risk area. In such context, it is necessary to identify possible domestic sources, which include the felines, as highlighted by Simıes-Mattos (2005). Despite the existence of some research on the infection seroprevalence in feline populations dwelling in endemic areas, it is not yet clear whether low prevalences of the infection and disease in cats coming from endemic areas are due to errors in the antibody detection or due to the fact that cats have a natural leishmaniasis resistance. The presence of infected asymptomatic cats with low antibody titers, as found in the present study, corroborate the findings of Maia et al. (2008). According to Solano-Gallego et al. (2007), the anti-Leishmania sp. antibody serum concentrations in cats are inferior to the concentrations found in dogs, consequently requiring a lower serum and conjugated dilution. From the eight cats which were detected positive in the parasitological exam, only two were serologically positive. This observation is in accordance with the reports of Martín-Sánchez et al (2007) who verified that animals with the highest antibody titers presented the lowest positivity proportion in the PCR technique, when evaluating serum samples of 183 cats. On the other hand, the highest PCR positive proportions occurred in cats which had low antibody titers. These results suggest that the immune response to Leishmania sp. infection in cats is different to the response observed in dogs, which should explain the small number of infected and symptomatic animals. This fact can underestimate the real figure of infected cats, thus facilitating the parasite's transmission. That said, serological techniques routinely employed in dogs do not appear to be an efficient tool for infection determination in cats.
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