Effect of Lutzomyia whitmani (Diptera: Psychodidae) Salivary Gland Lysates on Leishmania (Viannia) braziliensis Infection in BALB/c Mice

Leishmaniases are parasitic diseases caused by protozoan parasites of the genus Leishmania that produce either tegumentary (cutaneous, mucocutaneous, diffuse) or visceral clinical presentations in man and are responsible for significant morbidity and mortality in many areas throughout the world (Pearson et al. 1999). The mammalian host becomes infected with Leishmania when the sand fly vector, Phlebotomus species in the Old World and Lutzomyiaspecies in the New World, probes the skin of parasitized host for a blood meal and injects the parasite admixed with its saliva (Titus et al. 1994).

Previous studies have shown that salivary gland lysates of Lu. longipalpis and P. papatasi markedly enhance the infectivity of L. major in mice (Titus & Ribeiro 1988, Theodos et al. 1991). This exacerbating effect on the course of cutaneous leishmaniasis applies not only to infection with L. major, causative agent of Old World cutaneous leishmaniasis, but also to infection with other parasites causing cutaneous leishmaniasis in the New World, such as L. mexicana (Theodos et al. 1991) and L. braziliensis (Samuelson et al. 1991, Lima & Titus 1996). Additionally, it has been shown that the enhancement of infectivity is not due to a direct effect of the saliva on the parasite but rather to an effect on the host. Sand fly saliva has several pharmacological activities, including vasodilation, inhibition of platelet aggregation, and inhibition of coagulation (Ribeiro 1987). Besides, sand fly saliva may also have immunomodulatory effects related to the pathogenesis of leishmanial infections (Hall & Titus 1995, Qureshi et al. 1996, Mbow et al. 1998).

We report here the effect of salivary gland lysate of Lu. whitmani on the course of L. (V.) braziliensis infection in BALB/c mice. The rationale for extending these observations to a model using L. (V.) braziliensis plus salivary gland lysate fromLu. whitmani is that this sand fly is the most important natural vector for L. (V.) braziliensis in Ceará, Brazil (Queiroz et al. 1994). Moreover, L. (V.) braziliensis is transmitted in nature neither by Lu. longipalpis nor P. papatasi, the sand fly species targeted on saliva studies up to now.

The L. (V.) braziliensis strain (MCAN-BR-92-19914) used in this study was maintained in Schneider's insect medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Sigma), 2% sterile normal human urine, 100 U/ml penicillin (Sigma), 100 µg/ml streptomycin (Sigma) and 2 mM L-glutamine (Gibco BRL, Grand Island, NY) at 26oC. Parasites were used at no later than the fourth passage. Salivary gland lysates were obtained from sugar-fed 3-5-day-old adult laboratory-reared, female Lu. whitmani from a closed laboratory colony maintained at Núcleo de Medicina Tropical/UFC and used as described previously (Titus & Ribeiro 1988).

When L. (V.) braziliensis promastigotes were inoculated with salivary gland lysate, the lesion appeared around seven days post-infection. They peaked in size at 28 days of evolution and persisted for up to 42 days, then regressed in a slower way than the lesions of the controls without saliva (Figure). Also, in the animals coinjected with L. (V.) braziliensisplus Lu. whitmani saliva, the lesions developed always larger than those of the controls did, which it was statistically significant (p < 0.05) at virtually all time point examined (Figure).

In the BALB/c model of infection, L. braziliensis induces only transient and non-severe cutaneous lesion. Nonetheless, when L. braziliensis parasites are injected into BALB/c mice with sand fly saliva, they cause progressive skin lesions and infection is significantly enhanced as measured by lesion size, parasitic burden, and outcome of infection (Samuelson et al. 1991, Lima & Titus 1996, Donnelly et al. 1998). In this work we found that coinjection of L. (V.) braziliensis plus Lu. whitmani salivary gland lysate also increases the size of cutaneous lesions in BALB/c mice. However, while in the works using coinjection of Lu. longipalpis saliva plus L. (V.) braziliesies the cutaneous lesions persisted for the lifetime of the mice (Samuelson et al. 1991, Lima & Titus 1996), we showed that coinjecting saliva ofLu. whitmani plus L. (V.) braziliensis it formed spontaneously resolving cutaneous lesions. The mechanism(s) by whichLu. whitmani saliva exacerbates L. (V.) braziliensis infection in BALB/c mice may only be speculated, since most of the salivary factor(s) responsible for the enhancement of infectivity of Leishmania have not yet been identified. In the salivaof Lu. longipalpis one key molecule is the peptide maxadilan, which besides being a potent vasodilator may also have immunomodulatory properties (Lerner et al. 1991, Qureshi et al. 1996, Soares et al. 1998). P. papatasi lacks maxadilan, but its saliva contains large amounts of adenosine and 5'-AMP (Ribeiro et al. 1999). It has been demonstrated that adenosine is probably the factor in P. papatasi that interferes with the ability of activated macrophages to kill parasites (Katz et al. 2000).

So far as we are aware, the present report is the first to show that salivary gland lysate of Lu. whitmani enhance the infectivity of L. (V.) braziliensis in mice. Our data support other studies that have demonstrated that the vectors' saliva somehow modulates the long-term pathology of the disease. We hope with this work to contribute for a better understanding of the role of Lu. whitmani saliva in the natural transmission of L. (V.) braziliensis cutaneous leishmaniasis in this region of the world.

ACKNOWLEDGMENTS

To Antônio Wilson Vasconcelos and Eddie WP Santana for revising the manuscript.

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