Abstract
Visceral leishmaniasis (VL), caused by Leishmania infantum, is an endemic zoonosis in Iran. In recent years, Leishmania infection in cats has been reported in several countries where leishmaniasis is present. The aim of this study was to survey Leishmania infection in cats and to detect its causative agents in VL endemic areas in Iran. Forty stray cats were captured from two areas where VL is endemic, Fars and East Azerbaijan provinces. Infection with Leishmania was evaluated by parasitological and molecular methods. Leishmania parasites were detected in 4 out of 40 cats (10%). The parasite was isolated from the spleen of three and the liver of one cat and mass cultivated in the culture medium. Molecular and isoenzyme characterization revealed that the parasites isolated from the four cats were all L. infantum. Considering the high rate of feline Leishmania infection in this study, it may be suggested that cats have an epidemiological role in areas where VL is endemic in Iran.
Introduction
Leishmaniasis in cats was first reported in 1912 in Algeria (Sergent et al. 1912). Since then, more than 40 cases have been reported in several countries where the diseases is endemic. Cases of feline Leishmania infection caused by L. infantum have been described in France, Italy, Spain, Switzerland, and Brazil, and more recently in Portugal (Ozon et al. 1998, Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004, Rufenacht et al. 2005, Martin-Sanchez et al. 2007, da Silva et al. 2008, Maia et al. 2008).
Although the cutaneous form has been the common form of feline leishmaniasis, cases reported from France, Italy, and Spain have been VL. Now, cats are considered as a potential reservoir of L. infantum, and it is necessary to evaluate their infection status in areas where VL is endemic.
In Iran, leishmaniasis is widespread in the canine population (Mohebali et al. 2005). In areas where canine leishmaniasis is endemic, up to 25% of the dogs are infected, most of them not presenting clinical signs (Gavgani et al. 2002, Parvizi et al. 2008). Cases of feline Leishmania infection have not yet been documented in Iran; therefore, this study aimed at evaluating Leishmania infection in cats in areas where VL is endemic in Iran.
Materials and Methods
Studied subjects
After getting approval from the ethics committee of Shiraz University of Medical Sciences, 40 domestic cats from two well-known VL endemic areas, Fars and East Azerbaijan provinces, were captured by special traps. Among them, 11 cats were from East Azerbaijan (Kalaybar and Azarshahr districts), and the rest (29 cats) were from Fars province (Norabad, Firozabad, Jahrom, Darab, and Kavar districts). Twenty of the cats were male, and the remaining 20 were female. Clinical examination was performed for each animal. The animals were euthanized, using ketamin plus xylocaine, and samples (at least three samples) of the skin, liver, spleen, and blood were taken from each animal.
Parasitological investigations
Infection with Leishmania in cats was evaluated through touch smear preparation of the liver and spleen samples, thin smear of blood samples, and the skin (biopsies) samples. In animal with skin lesion, samples (three) were taken from the borders of each lesion. Moreover, skin, liver, and spleen samples were cultured, inoculated in NNN (Novy-MacNeal-Nicolle) medium, and incubated at 26°C. The cultures were checked regularly for 2 months before being considered negative. Mass cultivation of positive cultures was made in BHI (Brain Heart Infusion Agar) medium supplemented with 15% fetal calf serum.
Polymerase chain reaction
Polymerase chain reaction (PCR) was performed on isolated parasites and also on tissue samples of all captured cats. DNA was extracted from Leishmania promastigotes obtained from mass cultivation of the parasite in BHI medium. DNA was also extracted from the liver and spleen tissues of the cats.
PCR reactions were done in a final volume of 25 μL, containing 1 × PCR buffer, 2 mM MgCl2, 1.5 μL of each primer LINR4 (5′-GGG GTT GTT GTA AAA TAG GG-3′), reverse LINR17 (5′-TTT GAA CGG GAT TTC TG-3′), and 2 U of Taq DNA polymerase. DNA was first denatured at 94°C for 3 min and then cycled. The cycles were 94°C for 1 min, 65°C for 30 s, and 72°C for 1 min. Amplified products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and viewed by ultraviolet light.
Isoenzyme characterization
Isoenzyme electrophoresis was performed using discontinuous polyacrylamide gel electrophoresis as previously described (Hatam et al. 2005). Briefly, the parasite cultures were harvested at the end of logarithmic phase of the growth, and the number of organisms was adjusted to 1–1.5 × 107 promastigotes/mL. After centrifugation (2000 g for 20 min at 4°C), the pellet of promastigotes was washed three times in cold-proline–balanced salt solution. To the washed pellet of promastigotes, an equal volume of hypotonic aqueous solution of enzyme stabilizers (1 mM ɛ-amino-N-caproic acid, 1 mM dithiothreitol, and 1 mM ethylenediaminetetraacetic acid; Sigma, St. Louis, MO) was added and mixed thoroughly. After freezing and thawing, the extract was centrifuged at 30,000 g for 30 min at 4°C, and the supernatant was aliquoted and stored at −70°C. Each isolate was examined for the activity of the six enzymatic systems—glucose phosphate isomerase, malate dehydrogenase, nucleoside hydrolase 1& 2, phosphoglucomutase, and glucose 6 phosphate dehydrogenase. Electrophoretic band-developing conditions were used for each enzyme system.
Results
In this study, 40 cats were evaluated by parasitological and molecular techniques to detect Leishmania infection. One cat from Kalaybar had two skin lesions (Table 1); all the other cats presented no clinical sign. Leishmania parasites were detected in 4 out of 40 cats (10%) collected from Fars and East Azarbayjan provinces. The parasites were detected in the Giemsa-stained spleen (one cat) and liver (one cat) tissues. Moreover, Leishmania parasites were isolated from the spleen of three and the liver of one cat, and mass cultivated in culture medium. Using molecular method (PCR), Leishmania DNA was detected in the liver samples of three and the spleen samples of two cats. Analysis of PCR products using primer LINR4 and LINR17 resulted in amplification of a 720-bp fragment. Comparison of the obtained bands with those of the WHO references confirmed that the isolated parasites from the four cats are all L. infantum (Fig. 1).
Polymerase chain reaction products obtained with DNA samples from cat tissues. Line 1, negative control; line 2, DNA from a sample of the liver of a cat; line 3, DNA from Leishmania infantum; line 4, DNA from Leishmania major; line 5, DNA from Leishmania tropica; line 6, molecular marker.
PCR, polymerase chain reaction.
Isoenzyme analysis of the isolated parasites revealed that enzymatic profiles of all four isolates are consistent with WHO reference strain of L. infantum, except in glucose 6 phosphate dehydrogenase system, where two isolates (cat number 1 and 4 in Table 1) showed an extra fast running band.
Discussion
The increasing number of cases of Leishmania feline infection in the scientific literature suggests that cat may also have a role in the epidemiology of VL. Most of the reported Leishmania-infected cats show no clinical signs, but they might be infectious to the sand fly vectors of Leishmania, as recently documented (Maroli et al. 2007).
To the best of our knowledge, this is the first report of both Leishmania infection and leishmaniasis in cats in Iran, where we detected the presence of L. infantum in 10% of the studied cats. Further, in two animals we detected amastigotes of the parasite in the tissues, and in three animals we were able to isolate and mass cultivate the parasite.
The studied cats were native to the area where autochthonous human VL and canine leishmaniasis have been frequently reported. The cats lived in two areas where VL is endemic, Fars and East Azarbayjan, which are two important foci of VL in Iran. In East Azarbayjan, Kalaybar is a district where most of the VL cases in children have been reported in the last two decades (Alborzi et al. 2007, Kadivar and Moslehi 2007). In the current study, one of the highly infected cats was collected from this area.
The rates of seropositivity levels in dogs in Fars and Azarbayjan areas stand between 10% and 25%. The main reservoir for VL in these areas is reported to be dogs, although infection with L. infantum in rodents (Meriones persicus and Mesocricetus auratus) and foxes has also been documented (Mohebali et al. 2004). While most cases of Leishmania infection in cats in our study are asymptomatic, dogs, the main reservoir of L. infantum, are susceptible to develop the disease (23.9% of the seropositive domestic dogs had clinical signs in Mohebali et al. 2004 study). Since the cat has been suggested as a reservoir for leishmaniasis in other VL endemic countries (Mancianti 2004, Martin-Sanchez et al. 2007, Maia et al. 2008) and in view of the fact that transmission of Leishmania from feline to sand flies has been confirmed, and considering the high rate of infection in cats, it can be suggested that cats may act as an additional reservoir (an animal in which a parasite lives and multiplies without significant damaging its host), rather than just an incidental one, for VL, in areas where VL is endemic in Iran. However, the presence of amastigotes in liver or spleen of cats without peripheral parasitemia or skin lesion might indicate that cats are not an important reservoir for VL.
Further studies in other areas where VL and CL (Cutaneous Leishmaniasis) are endemic are needed to find out more about the role of cats in the epidemiology of leishmaniasis in endemic settings in Iran.
Footnotes
Acknowledgment
This work received financial support from Fars Management and Planning Organization and office of vice chancellor for research at Shiraz University of Medical Sciences.
Disclosure Statement
No competing financial interests exist.