Despite the possible importance of the rodent´s role as reservoir hosts for canine and human leishmaniosis, to our knowledge, no previous studies on Leishmania infection in any rodent species had been published in Portugal.
Our results revealed the presence of Leishmania amastigotes in eight Mus musculus and in one Rattus norvegicus visceral sample. Despite Leishmania amastigote detection in the liver and spleen, no positive results were obtained by qPCR from these samples. Regarding nucleic acid detection, positive samples were obtained from the skin but not from the liver and spleen suggesting a cutaneous Leishmania infection. Considering the high sensitivity of the qPCR assay visceral negative results may be due to random selected fragments for amplification and microscopic observation.
In experimental studies, early dissemination of Leishmania parasites occurs in the spleen . In susceptible mice, the spleen constitutes the organ where the adaptive immune response to the parasite mainly takes place. In this study, in spite of systemic dissemination being present on 6 rodent’s livers (on parasitological analysis) and 8 rodent´s spleens (3 on parasitological analysis and 5 on histopathological analysis), no positive results were obtained by qPCR on spleen samples. Similar PCR results were also described by other authors working with specimens of Mus musculus infected with Leishmania major.
In the molecular assay, 9 ear lobe skin samples from Mus musculus (n=27) were positive to Leishmania DNA. Although no DNA sequencing was performed, the use of a specific Taqman® probe to Leishmania infantum in the qPCR strongly suggests the detection of this species in our samples. Furthermore, Leishmania infantum is known to be the only Leishmania species causing zoonotic visceral leishmaniosis in Portugal and in the Mediterranean countries .
In our study, an infection rate of 33.3% (9/27) was found in ear lobe skin of Mus musculus. This result is higher than the ones obtained in Brazil with 13.7% (3/22)  and Iran with 19% (4/21) . This occurrence may have an important epidemiological role on the transmission of the parasite.
The identification of positive ear lobe skin samples in our Mus musculus rodents indicates the parasite cutaneous availability to Phlebotomus species, allowing the infection to spread during sandfly feeding, not only between rodents but also to other hosts. In fact, in an experimental study, using specimens of Rattus rattus and Leishmania tropica, the ear lobe was suggested as a preferential feeding spot for sandflies, probably due to thinner skin, easy access and abundant blood flow. In addition, the parasite DNA persisted in the ear lobe for 24 months post-inoculation and the ability to transmit the infection to sandflies from month 1 to month 24 post-inoculation was observed . Although the parasite number in the blood was not assessed in this work, considering that the parasite load ranged between 50 to 393 copies in 5 animals, it is safe to conclude that the parasites were present in the skin in sufficient numbers to infect sandflies. Unfortunately, in this work, it was not possible to perform specific antibody titers to correlate data with the obtained molecular results.
This work reports for the first time in Portugal, the natural infection of Leishmania infantum in Mus musculus and Rattus norvegicus, suggesting these animals probably have a role as reservoirs in the Leishmania infantum life cycle. Mus musculus and Rattus norvegicus are extremely prolific animals, with a 12 and 24 months life expectancy respectively, maintaining the parasite availability at least for one year, even during low sandfly activity season. The infection rate obtained by qPCR on Mus musculus rodents (33.3%) strongly suggests the occurrence of contact with infected sandflies, especially considering the preferential nightfall activity period overlap. Also, the absence of skin lesions strongly suggests a non-pathogenic infection course, as already described in Brazil [10, 11] and in Iran [22, 23].
This study was performed in a peridomestic environment in two regions, with a canine leishmaniosis prevalence of 2.3% (5/215) in Sintra and 5.2% (12/230) in Sesimbra. Mus musculus and Rattus norvegicus were found to be naturally infected by Leishmania infantum with an overall prevalence of 43.3% (13/30). These species are known to inhabit in close proximity to human housing, especially where shelter and food are provided like those found in Sesimbra and Sintra. This process of domiciliation may be important in the urban and periurban cycle of Leishmania infantum. Further studies are necessary to evaluate the ability of these rodents to effectively infect vector sandflies in order to establish Mus musculus and Rattus norvegicus as reservoir hosts for Leishmania infantum.
As susceptible hosts with a potential role in the epidemiology of this zoonotic disease, infected rodents may increase the risk for dog and human infection in households and surrounding areas, enhancing the need for an efficient rodent control measure in shelters and risk zones to prevent the transmission.