T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Visceral leishmaniasis (VL) is a vector-borne disease caused by the intracellular protozoan Leishmania chagasi (Syn = L. infantum) and transmitted by phlebotomine sand flies. VL is a disease with great variability of clinical manifestations, and in dogs it ranges from absence of symptoms to generalized disorders that may result in the host death, such as fever, anemia, cachexia, skin lesions, lymphadenopathy, renal alterations and ocular lesions [1–3]. The clinical manifestations vary according to the host immune response and to the parasite pathogenicity, which differs among species [2]. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis (CCL) [4–7]. Toxoplasmosis is known by causing pulmonary and digestive disorders, retinal lesions and neuropathies [8–10], and canine neosporosis is an important cause of neurological disorders [11, 12].

Canine VL is considered an immune-mediated disease due to the parasite ability to modify the host immune system. During the disease, infected cells from the mononuclear phagocyte system act as antigen presenting cells (APCs), stimulating CD4⁺ T helper (Th) lymphocytes. Th1 response, mediated by macrophages and pro-inflammatory cytokines such as TNF-α, IL-2 and IFN-γ is related to the host ability to control the infection. However, as an evasion strategy, components of the parasite preferentially activate Th2 lymphocytes and when the Th2 response predominates, the production of anti-inflammatory cytokines such as IL-4, IL-10 and TGF-β occurs, resulting in B lymphocytes proliferation and subsequent production of antibodies, mainly IgG, which are not effective against the parasite [2, 13–15].

The involvement of the host immune system due to the Leishmania infection [16] may be related to the infection by other pathogens. In humans, the association between VL and AIDS is becoming common in VL endemic areas and HIV/Leishmania co-infection is considered an emerging disease [17, 18]. Concomitant incidence of VL, neosporosis and toxoplasmosis in dogs have already been described in the region of the municipality of Araçatuba, São Paulo State, Brazil, an endemic area for VL [19]. Toxoplasmosis is an opportunistic disease related to immunosuppressive status [9, 10, 19, 20] and association between canine VL and neosporosis has also been detected [19, 21, 22]. Recently, Lindsay et al. [23] confirmed the role of dogs as the definitive host of N. caninum, and even if dogs are not the definitive host of T. gondii, they may collaborate with mechanical dissemination of toxoplasmosis [9].

VL may be associated to an immunosuppressive state [13, 14] and the contact between the host with the opportunist pathogens Toxoplasma gondii and Neospora caninum possibly will contribute to the occurrence of lesions in the central nervous system (CNS). Recently, we demonstrated that dogs with VL presented encephalic lesions compatible with encephalitis or at least, that the nervous milieu presented a pro-inflammatory status [5, 24, 25]. Hence, the aim of this study was to compare the lymphocytes population (T and B cells) in the brains of seropositive dogs to L. chagasi with those observed in dogs with seropositivity to T. gondii and N. caninum.

The data presented herein provide a picture of the lymphocyte population within the brain of dogs with seropositivity to visceral leishmaniasis, toxoplasmosis and neosporosis. Although L. chagasi, T. gondii and N. caninum are important pathogens, a high amount of the dogs presented no alterations at the necroscopic examination and consequently they were considered asymptomatic. An important epidemiological characteristic of VL is that infected dogs may remain asymptomatic for a long period and may never develop the clinical disease [2, 14]. However, the presence or absence of clinical symptoms did not influence the histopathological lesions in the nervous tissue of naturally infected dogs [25]. None of the dogs presented signs of neurological disorder and no characteristic alterations of toxoplasmosis or neosporosis was observed, except for lymphadenopathy, which was the only symptom observed in the dogs that have been experimentally infected by different strains of. T. gondii [8].

Clinical neosporosis in adult dogs is generally related to the reactivation of a previous infection due to an immunosuppressive status [29], and the occurrence of clinical signs is associated with antibody titer greater than 1:800, while antibody titers equal to or greater than 1:50 only indicate exposure to the parasite [28]. Regarding toxoplasmosis, during experimental infection, some dogs keep asymptomatic, few dogs develop clinical signs and deaths are rarely seen [8].

Even if association among these diseases were not detected, 43.6% (24/55) of the dogs with VL presented antibody titers for T. gondii and/or N. caninum, and on the other hand, 61.5% (16/26) of the dogs diagnosed without VL presented antibody titers for T. gondii and/or N. caninum. The presence of positive antibody titer for toxoplasmosis and/or neosporosis in animals with VL did not influence the amount of CD3⁺ cells within the brain, as occurred in the dogs infected only by L. chagasi or in seropositive dogs only for T. gondii and/or N. caninum. All of these three conditions rendered favourable the accumulation of CD3⁺ T lymphocytes in the nervous milieu, since there was evident difference when compared to control group. Further, the great variability observed among the number of CD3⁺ T lymphocytes detected in the dogs within the same group is likely to be due to the natural character of the infection. Regarding the CD79α⁺ B lymphocytes, the presence of these cells was not significant in none of the above mentioned conditions, as the CD79α immunostaining was minimal and rather equivalent among groups. Although these cells play a role in the peripheral immune response against VL, they seem not to take part in the response within the brain.

The concomitance of VL, toxoplasmosis and neosporosis might be a propitious condition to the occurrence of neurological disorders. Alone, VL may promote an influx of a high amount of T lymphocytes, few phagocytic and B cells in the nervous tissue, primarily in the choroid plexus and leptomeninges, as well as high index of astroglial and microglial activation [24, 25]. Further, the severity of the histopathological lesions and the presence of clinical neurological manifestation in dogs with VL seem not to be correlated [4]. Moreover, toxoplasmosis might promotes mononuclear meningitis as well as mononuclear perivascular cuffs [8] and in mice experimentally infected by N. caninum, the main encephalic alteration was encephalitis, glial nodules, malacia and perivascular cuffs [11]. Several of these histopathological alterations were detected in the brains of dogs included in this study, with inflammatory infiltrates being the most prevalent.

The activation of a systemic immune response promotes the peripheral activation of lymphocytes and their migration across the blood–brain-barrier (BBB) into the nervous environment and brain parenchyma, as previously described by some researchers [30–35]. BBB is extremely susceptible to the action of cytokines, which might activate the endothelial cells and increase the permeability of the barrier [36].

The anti-Leishmania immune response also includes the production of several cytokines [2, 37], which might modulate the BBB selective filtration and allow the passage of inflammatory mediators and immunoglobulins [38]. Receptors for the Fc portion of the immunoglobulins are present in immune cells (mast cells, lymphocytes, macrophages) and also in neurons and glial cells. The activation of these receptors starts a succession of biological processes including phagocytosis, degranulation and cytokine genes activation [39, 40]. High titers of anti-Leishmania antibodies in the CSF have already been described in dogs with natural VL [4, 25, 41, 42], and, therefore, the presence of circulating antibodies within the CNS may contribute to the beginning of the inflammatory process, as previously described in immune-mediated and degenerative diseases of CNS [43, 44].

In dogs, VL can be associated with an influx of CD3⁺ T lymphocytes in the brain similarly as occurs during toxoplasmosis and neosporosis, and the concomitant presence of serum antibodies against Leishmania, Toxoplasma and Neospora does not exacerbate the inflammatory brain lesions. In animals presenting with neurological signs, the use of serological tests is an important tool to diagnose toxoplasmosis or neosporosis, and the encephalitis caused by these agents presents a well described pattern [8, 9, 11, 12, 28]. Nevertheless, this and other studies [4, 24, 25, 42, 45] demonstrated that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes and other inflammatory changes within the nervous milieu. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. Finally, VL has to be considered as differential diagnosis for inflammatory lesions in the brain.