All dogs except for the control group had the visceral leishmaniasis confirmed by serology and parasitological exam. Detection of dogs with VL in endemic areas is difficult because clinical signs are very variable and frequently similar to manifestations consistent with other diseases [8, 9]. Our results revealed that more frequent manifestations were onicogriphosis (83.3%), skin lesions (83.3%), conjunctivitis (75%), local or generalized lymphadenopathy (66.6%), and weight loss (58.3%) (Additional file 1). Since symptomatic dogs independent of number of signs had the diagnosis of visceral leishmaniasis confirmed, we suggest these five clinical manifestations of dogs to be considered for the diagnosis of canine VL. However, in a group of 35 dogs previously studied by us two from 16 animals presenting one or two symptoms were parasitologically negative (data not shown). Therefore we suggest that only one or two of these symptoms are not sufficient, but three of them allow us to consider the animal as suspect, and five as strongly suggestive of VL. Such a clinical criteria may contribute to quickly identify dogs with visceral leishmaniasis in endemic areas.
To analyze the transmission potential of these dogs, xenodiagnosis was performed on the ear, where the parasite load was observed to be higher. From the 12 clinically affected dogs, xenodiagnosis was performed in nine. Among them, six resulted in transmission to Lutzomyia longipalpis, while none of the five asymptomatic dogs were infective to the sandfly. These data suggest that dogs that may represent a real threat for Leishmania transmission to the sandfly are those symptomatic ones (p = 0.0310, Fisher's exact test) data that are corroborated by similar results from other studies performed with Lutzomyia longipalpis, Lutzomyia youngi and Phlebotomus perniciosus [4, 11]. A previous study has shown that dogs with the potential to infect the sandfly are those with at least one of the four main clinical manifestations [13]. Furthermore, five asymptomatic dogs with no parasites, as assessed by PCR of the skin in one study, and dogs with no parasites in the skin as assessed by immunohistochemistry in another study were also unable to transmit Leishmania to the sandfly [4, 10]. On the other hand asymptomatic dogs of an endemic area in Spain have been shown to be effective to transmit Leishmania infantum to Phlebotomus perniciosus [12]. These data showing differences between studies carried out in Mediterranean region and South America may be due to Leishmania strain and vector species differences since it is known that Phlebotomus perniciosus is more effective than Lutzomyia longipalpis as vector to transmit viscerotropic Leishmania [4].
The parasite burden in the skin was analyzed from 22 different regions of the body of dogs with VL. Higher parasite load was observed in the ear and ungueal region, and lower parasite load was observed in the abdomen using material stained by different methods. The median value is presented in Additional file 2 in tissue sections stained by immunoperoxidase. Clinically affected dogs exhibited amastigotes in at least one of the regions examined, and positive regions were always associated with inflammatory processes. In the asymptomatic (Figure 1A) and control dogs (Figure 1B), no amastigotes were present in any of the regions examined. The parasite load was higher in clinically affected dogs when compared with the asymptomatic animals (P < 0.05, Kruskal Wallis and Student-Newman-Keuls tests) (Figure 2A).
From 12 symptomatic dogs, skin lesions were observed in 10 animals. However, an inflammatory infiltrate in the skin was present in all animals, even in those without symptoms, and was more intense in the clinically affected dogs than in asymptomatic and control dogs (p = 0.006, Kruskal Wallis and Student-Newman-Keuls tests) (Figure 2B and Additional file 1). Similar results have been previously observed in Leishmania infantum-infected dogs [10]. Although skin inflammation is a common finding in dogs because of sandfly bites, traumas or other parasitic infections, our data showing minimal intensity of inflammatory infiltrate in the absence of amastigotes in the skin of asymptomatic and control dogs, and a correlation between the number of amastigotes and the intensity of the inflammatory infiltrate (R = 0.64; P < 0.05, Spearman test) (Figure 2C) suggest that, in clinically affected dogs, the lesions were likely related to VL [17–19].
In clinically affected dogs, when few or none Leishmania amastigotes were observed (Figure 1C), the inflammatory infiltrate was constituted mainly by lymphocytes and macrophages (Figure 1D). When many parasites were present (Figure 1E), the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs) (Figure 1F). In two of these dogs (animals 5 and 7, Additional file 2), the inflammatory pattern constituted by the monomorphic macrophage infiltrate was associated with high parasite load (Fig 1E) as observed in a study by dos Santos et al. [17]. The intense inflammatory infiltrate constituted by lymphocytes, macrophages and PMN associated with high parasite load in clinically affected dogs differs from the histopathological pattern seen in tegumentar leishmaniasis, which is illustrated by defective cell-mediated immunity in which Leishmania-loaded macrophages are predominant and lymphocytes are scarce [20]. In asymptomatic dogs, the inflammatory infiltrate was constituted predominantly by macrophages and lymphocytes, similar to that observed in control dogs. The characteristics of the inflammatory infiltrate in clinically affected dogs with low parasite burden and the absence of amastigotes in asymptomatic dogs suggest that these animals are immune competent and therefore able to control the infection for long time.
A granulomatous inflammatory infiltrate was also observed in at least one of the regions analyzed in ten symptomatic and four asymptomatic dogs. Since the presence of a granulomatous lesion was associated with low or no parasites in the skin in four asymptomatic dogs, it suggests protective role in the tissue. Granulomatous reaction in the control of Leishmania infection has been studied thoroughly in Leishmania donovani-infected BALB/c mouse strains and also in L. donovani-infected hamster [21–23]. The control of the infection by granulomatous reaction depends on the cytokines secreted by inflammatory cells, mainly interferon-γ and IL 2 [24].
A parameter from the inflammatory process in the skin, the presence of PMN, seems relevant for the presence of higher parasite load. This parameter may be analyzed in smears obtained by scarification of the ear for a quick diagnostic approach.
Although the presence of Leishmania in the skin of the VL dog is considered to be important for the transmission of the parasite to the sandfly vector, it is not clear whether the parasites in the skin themselves are transmitted to the insect or if they are transmitted from the capillary blood to the sandfly during blood meal. If the latter is the case, the presence of parasites in the skin and the more intense inflammatory infiltrate observed are just an indication that the parasites are reaching the skin in great amounts through capillary flow. The results from the present study do not clarify this point, but suggest that the latter hypothesis is likely since, among symptomatic dogs, xenodiagnosis was positive when lymphadenopathy and/or splenomegaly were present, conditions probably related to higher parasitaemia and allowing for easy transmission to the insect. A study showing no correlation of the presence of Leishmania product in the skin and transmission potential to the vector also reinforces the latter hypothesis [4].
