Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis

The dog is the main reservoir of Leishmania infantum, the parasite responsible for visceral leishmaniasis in Mediterranean countries. The infection in dogs shows different clinical presentations, from subclinical/asymptomatic to a fully developed disease, depending on the host's immune responses. The Th1/Th2 dichotomy is not clear in the different forms of canine leishmaniasis, since the data available from studies of immunity response in canine leishmaniasis are scarce and fragmented. The present work describes the cytokine expression in peripheral blood mononuclear cells (PBMC) obtained from asymptomatic dogs experimentally infected with L. infantum that present a cellular protective immune response. The results obtained from freshly isolated PBMC showed expressions of TNF-alpha, IL-2, IFN-gamma, IL-10 and IL-18 mRNA, similar to those from non-infected dogs. However, there was almost no expression of IL-4 mRNA detected in the asymptomatic infected dogs compared to the control dogs. Unspecific stimulation with ConA promoted the expression in a greater or lower degree of all the cytokines studied. In vitro stimulation of PBMC with soluble leishmanial antigen (SLA) promoted the expression of IL-2, IFN-gamma, TNF-alpha, IL-18, IL-4, IL-6 and IL-10 mRNA, with the two first being specifically induced. Although both Th1 and Th2 cytokines are produced, cell mediated immunity observed in these L. infantum-infected asymptomatic dogs depended on the preferential expression of Th1 cytokines.     

Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis

While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.     

The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.     

Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi

The role of anti-leishmanial immune response underlying the susceptibility/resistance during canine visceral leishmaniasis (CVL) has been recognized throughout ex vivo and in vitro investigations. Recently, we demonstrated that immunoglobulin levels (Igs), as well as the parasite load are relevant hallmarks of distinct clinical status of CVL. To further characterize and upgrade the background on this issue, herein, we have evaluated, in Leishmania (Leishmania) chagasi naturally infected dogs, the relationship between tissue parasitism (skin, bone marrow, spleen, liver and lymph node), the CVL clinical status (asymptomatic (AD), with no suggestive signs of the disease; oligosymptomatic (OD), with maximum three clinical signs-opaque bristles; localized alopecia and moderate loss of weight; symptomatic (SD), serologically positive with severe clinical signs of visceral leishmaniasis), and the humoral immunological profile of anti-Leishmania immunoglobulins (IgG, IgG1, IgG2, IgM, IgA and IgE). Our major statistically significant findings revealed distinct patterns of tissue parasite density within L. chagasi-infected dogs despite their clinical status, pointing out the spleen and skin as the most relevant sites of high parasitism during ongoing CVL. Parasite density of bone marrow and spleen were the most reliable parasitological markers to decode the clinical status of CVL. Moreover, the parasite density of bone marrow better correlates with most anti-Leishmania Igs reactivity. Additionally, a prognostic hallmark for canine visceral leishmaniasis was found, highlighting strong correlation between IgG1 and asymptomatic disease, but with IgA, IgE and IgG2 displaying better association with symptomatic disease. The new aspects of this study highlighted pioneer findings that correlated the degree of tissue parasite density (low (LP), medium (MP) and high (HP) parasitism) with distinct patterns of anti-Leishmania Igs reactivity. In this scope, our data re-enforce the anti-Leishmania IgG but with IgA reactivity as the better marker for overall tissue parasitism. The association between clinical status, Ig profile and the tissue parasitism support a novel investigation on the impact of humoral immune response and susceptibility/resistance mechanism during ongoing CVL.     

Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi

Recent studies suggest that asymptomatic dogs infected with canine visceral leishmaniasis (CVL) develop a Th1 immunological profile whilst oligosymptomatic and symptomatic CVL-infected animals present a Th2 profile. In the present study, an RT-PCR method has been standardised and employed to evaluate the frequency and the semi-quantitative level of expression of the cytokines IL-4, IL-10, IL-12, INF-gamma and TNF-alpha in splenocytes of 30 dogs naturally infected with Leishmania chagasi and of 7 non-infected dogs (NID). An increase in the level of expression of IL-12 (p=0.059) was detected in all CVL-infected dogs compared with NID. In dogs exhibiting high parasitism, the frequency of expression of IL-10 was higher (p=0.011) than in animals presenting low parasitism or medium parasitism (MP) and in NID animals, whilst the level of expression of IL-10 was higher (p=0.0094) than in animals exhibiting MP and in the NID group. Positive correlations between the levels of expression of IL-10 with respect to the progression of the disease (IL-10: r=0.3510; p=0.0337) and the levels of expression of IL-10 and INF-gamma increase in parasitism (IL-10: r=0.3428; p=0.0438 and INF-gamma: r=0.4690; p=0.0045) were observed. Such data suggest that CVL is marked by a balanced production of Th1 and Th2 cytokines, with a predominant accumulation of IL-10 as a consequence of an increase in parasitic load and progression of the disease, and INF-gamma was related with the increase in parasitic load.     

Pretreatment of macrophages with the combination of IFN-gamma and IL-12 induces resistance to Leishmania major at the early phase of infection

Interferon (IFN)-gamma is essential but not sufficient to control leishmaniasis. It is known that IFN-gamma is one of the major macrophage-activating cytokines, and the activated macrophages are a principal source of interleukin (IL)-12, which induces autocrine macrophage activation. In this study, the combined effect of IFN-gamma and IL-12 on the susceptibility of macrophages to Leishmania major infection was evaluated. Macrophages pretreated with IFN-gamma and/or IL-12 were infected with the parasites. Four hr post-infection (p.i.), the levels of infection and parasite load in the macrophages treated with the combination of IFN-gamma and IL-12 (IFN-gamma/IL-12) were significantly lower than those in the nontreated cells. However, the macrophages treated with either IFN-gamma or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-gamma/IL-12-treated and IFN-gamma-treated macrophages showed significantly lower levels of infection and parasite load than the nontreated cells, and higher levels of resistance was observed in the IFN-gamma/IL-12-treated macrophages than in the IFN-gamma-treated macrophages. Although IFN-gamma/IL-12 treatment of macrophages prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to the macrophage culture. These results suggest that IFN-gamma/IL-12 treatment prior to the infection restricts the early phase of the infection.     

Peripheral blood mononuclear cell supernatants from asymptomatic dogs immunized and experimentally challenged with Leishmania chagasi can stimulate canine macrophages to reduce infection in vitro

Leishmania chagasi is the causative agent of visceral leishmaniasis in both humans and dogs in the New World. The dog is the main domestic reservoir and its infection displays different clinical presentations, from asymptomatic to severe disease. Macrophages play an important role in the control of Leishmania infection. Although it is not an area of intense study, some data suggest a role for canine macrophages in parasite killing by a NO-dependent mechanism. It has been proposed that control of human disease could be possible with the development of an effective vaccine against canine visceral leishmaniasis. Development of a rapid in vitro test to predict animal responses to Leishmania infection or vaccination should be helpful. In this study, an in vitro model was established to test whether peripheral blood mononuclear cell (PBMC) supernatants from dogs immunized with promastigote lysates and infected with L. chagasi promastigotes could stimulate macrophages from healthy dogs in order to control parasite infection. PBMC from a majority of the immunized and experimentally infected dogs expressed IFN-gamma mRNA and secreted IFN-gamma when stimulated with soluble L. chagasi antigen (SLA) in vitro. Additionally, the supernatants from stimulated PBMC were able to reduce the percentage of infected donor macrophages. The results also indicate that parasite killing in this system is dependent on NO, since aminoguanidine (AMG) reversed this effect. This in vitro test appears to be useful for screening animal responses to parasite inoculation as well as studying the lymphocyte effector mechanisms involved in pathogen killing by canine macrophages.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies

Visceral leishmaniasis (VL) is a growing zoonosis with an increasing number of new cases and a rapid geographical spreading of the disease. In the present study, a canine survey was carried out in the city of Montes Claros (320,000 inhabitants), an endemic area of American visceral leishmaniasis in the state of Minas Gerais, Brazil. A total number of 4795 dogs were examined by serology, which showed a rate of seropositivity of 5%. Isoenzymatic analysis confirmed Leishmania infantum chagasi as the local aetiological agent of CVL. Canine tissues were assayed for the presence of Leishmania parasite DNA using different techniques. The infectivity of asymptomatic, oligosymptomatic and symptomatic seropositive dogs was tested by xenodiagnosis using laboratory reared Lutzomyia longipalpis. Rates of infection of 5.4%, 5.1% and 28.4% were found for the phlebotomine sand flies that fed in asymptomatic, oligosymptomatic and symptomatic dogs, respectively. Our results indicate that, under experimental conditions, symptomatic dogs are about four times more infective to VL vectors than oligosymptomatic or asymptomatic animals. The lower infectivity rates of dogs displaying any of the last two clinical forms of leishmaniasis, however, must be taken into account in the epidemiology of CVL.     

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.     

Clinical and serological aspects of visceral leishmaniasis in northeast Brazilian dogs naturally infected with Leishmania chagasi

Human visceral leishmaniasis is endemic in the northeast of Brazil, where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. In this study, we evaluated the clinical signs of canine visceral leishmaniasis (CVL), serum protein profile and the antileishmanial IgG antibody production in 86 dogs living in northeast endemic areas of leishmaniasis. Thirty dogs from a leishmaniasis-free area were used as a control group. The major clinical signs of CVL seen were emaciation and skin ulcers (80%), followed by onychogryphosis and conjunctivitis (73%). Depilation was observed in 60% of animals while lymphadenomegaly, splenomegaly, liver enlargement or kidney involvement was less frequent (< or =20%). VL seropositive dogs presented with serum hyperproteinemia, hypoalbuminemia, hypergammaglobulinemia and decreased albumin/globulin ratio. A lower sensitivity and higher specificity was observed for promastigote indirect fluorescent antibody test (IFAT) (83 and 100%, respectively) compared with enzyme-linked immunosorbent assay (ELISA) (94 and 90%), which uses a crude extract of Leishmania. There was a positive correlation between IFAT and ELISA titers of antileishmanial IgG antibodies (Spearman test, P < 0.05), which was augmented in CVL dogs. This study found that the determination of serum protein, A/G ratio and the use of two different leishmanial serological tests like IFAT and ELISA are essential in CVL screening.     

Diagnostic and prognostic potential of antibodies against O-acetylated sialic acids in canine visceral leishmaniasis.

Employing bovine submaxillary mucin (BSM) as the coating agent, an enzyme-linked immunosorbent assay (BSM-ELISA) was developed to detect antibodies directed against O-acetylated sialic acids (O-AcSA) in canine visceral leishmaniasis (CVL). Serum samples were collected from 50 dogs previously screened by a parasite-ELISA to detect anti-leishmanial antibodies and designated as seropositive (n = 30) and seronegative (n = 20). The BSM-ELISA detected anti-O-AcSA antibodies in 29 out of 30 seropositive dogs and was negative in 15 out of 20 seronegative dogs; the sensitivity and specificity of the assay being 96.6% and 75%, respectively. Seven dogs from an endemic area in central Israel were longitudinally monitored for 15 months clinically, serologically and cultured for parasite. The levels of antibodies directed against O-AcSA increased with the appearance of clinical symptoms and/or seropositivity, disappeared when the disease was self-limiting as also with chemotherapeutic response and reappeared with relapse. The BSM-ELISA, therefore, represents a valuable tool for assessment of disease progression.     

Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.     

Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells

Oligodeoxynucleotides containing the cytosine-phosphate-guanine (CpG) motif (CpG-ODNs) have been shown to induce T(H)1 immune responses in animals. Since the sequences of CpG-ODNs that induce T(H)1 responses are considered to vary among animal species, it is necessary to identify effective CpG-ODNs in each animal. In order to identify the sequences of CpG-ODNs that induce T(H)1 responses in dogs, mRNA expression and protein production of IFN-gamma were examined in peripheral blood mononuclear cells (PBMCs) from healthy dogs treated with 11 kinds of synthetic CpG-ODNs. One of the 11 CpG-ODNs (No. 2 CpG-ODN, 5'-GGTGCATCGATGCAGGGGGG-3') was shown to significantly increase mRNA expression and protein production of IFN-gamma in canine PBMCs in a manner dependent on the sequence of the CpG motif. This CpG-ODN also enhanced the expression of IL-12 p40 mRNA in canine PBMCs, whereas expression of IL-12 p35, IL-18, and IL-4 mRNAs was not induced by this CpG-ODN. These results indicate that this CpG-ODN was able to produce IFN-gamma by induction of T(H)1-skewed immune response in dogs. CpG-ODNs may be useful for inducing prophylactic and therapeutic immunity against allergic diseases, viral infection, and tumors in dogs.