Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi

Visceral leishmaniasis (VL) presents vigorous Th2 immune response, which is mainly characterized in human by augmented expression of Il-4, polyclonal B cell activation, intense hypergammaglobulinemia and production of antileishmanial IgE antibodies. However, few aspects of this type of immune response have been demonstrated in studies of canine visceral leishmaniasis (CVL). This work investigated by ELISA and western immunoblotting the production of antileishmanial IgE antibodies (IgE Ab) in symptomatic and asymptomatic dogs naturally infected by Leishmania chagasi, and also compared this IgE immune response with those of IgG, IgG1 and IgG2 antibodies. Three groups of dogs were evaluated: 12 VL dogs with positive Leishmania biopsies (GI), 44 dogs with a positive leishmanial indirect fluorescent antibody test (IFAT), 30 of them presenting clinical signs of VL and 14 asymptomatic (GII) and 21 healthy dogs living in kennels located in leishmaniasis endemic areas (GIII), which were seronegative in the IFAT. Eighteen dogs from an area free of CVL were used as controls (GIV). Antileishmanial IgE antibodies were detected in 4 of 12 VL dogs from group I (33%) and 14 of 30 symptomatic dogs from group II (47%). While all asymptomatic dogs from group II (100%) were seronegative for antileishmanial IgE Ab, 7 of 21 healthy animals from group III (33%) had these immunoglobulins. A strong correlation was verified between antileishmanial IgG and IgG2 antibody titers in all symptomatic dogs, but only 15 of these 42 animals (36%) produced simultaneously IgE, IgG, IgG1 and IgG2 antibodies to Leishmania. IgE antibodies recognized leishmanial antigens of 12, 36, 61, 81 and 118 KDD, while a more complex pattern of immunoblotting was verified mainly for IgG and IgG2 antibodies from symptomatic animals. IgG1 and IgG2 antibodies shared the recognition of L. chagasi polypeptides of 118, 81, 61, 36, 18, 14 and 12 KDD, being more intense the immune reactions between IgG1 Ab and the leishmanial polypeptides of 61 and 36 KDD, and also between IgG2 antibodies and the antigens of 26, 21, 18, 14 and 12 KDD. Our results suggest that the polyclonal production of antileishmanial antibodies that includes IgE Ab could characterize a Th2 immune response in CVL and can help the laboratory diagnosis of this disease.     

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver "imprints" and in vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely.     

Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs

Although visceral leishmaniasis is primarily transmitted by a biological invertebrate vector, transmission in the absence of the vector has been reported, including venereal transmission in humans. Considering the possibility of venereal transmission, we studied genital lesions in dogs naturally infected with visceral leishmaniasis and shedding of Leishmania sp. in the semen. Approximately 200 dogs were serologically tested for anti-Leishmania antibodies and divided into three groups: 1) serologically negative dogs (n = 20), 2) asymptomatic serologically positive dogs (n = 20), and 3) symptomatic serologically positive dogs (n = 20). Samples from both testes, all segments of both epididymes, prostate gland, glans penis, and prepuce were histologically evaluated and processed for immunodetection of Leishmania sp. Semen samples were obtained from 22 symptomatic serologically positive dogs and processed for detecting Leishmania DNA by polymerase chain reaction. A significantly higher frequency of inflammation was observed in the epididymes, glans penis, and prepuce of dogs with visceral leishmaniasis, which was associated with a high frequency of immunohistochemically positive tissues (up to 95% of tissues from symptomatic dogs were positive by immunohistochemistry). Leishmania DNA was detected in eight of 22 semen samples from symptomatic dogs. Together these findings indicate that genital lesions and shedding of Leishmania sp. (donovani complex) in the semen are associated with visceral leishmaniasis. Additional studies should address the possibility of venereal transmission of the disease in the dog.     

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.     

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

Apoptosis, inflammatory response and parasite load in skin of Leishmania (Leishmania) chagasi naturally infected dogs: A histomorphometric analysis

The skin has an important role in infection by Leishmania chagasi. Apoptosis modulates the inflammatory response acting distinctively either on the progression or regression of the lesions. The parasites interact with multiple regulatory systems inducing apoptosis in host cells, during cell invasion, stabilization and multiplication of pathogens. In this context, the aim of this study was to evaluate cell death within the inflammatory infiltrates, and to correlate these results with parasite load and clinical features of dogs naturally infected with L. chagasi. Fragments of skin pinnas (8 symptomatic+8 asymptomatic+6 negative controls) were used to characterize and measure the inflammatory response, parasite load and apoptosis. Diagnosis of canine leishmaniasis was confirmed by the detection of anti-Leishmania antibodies by IFA and ELISA in serum, direct visualization of the parasite and culture in spleen, liver, pinna, bone marrow and lymph nodes, and PCR (pinna). Histomorphometry was performed with images obtained from 20 representative histological fields in a light microscope. Ultra-thin sections were mounted over a 300 mesh grids, contrasted with 2% uranyl acetate and lead citrate and examined under a Transmission Electronic Microscopy. Amastigotes were only found in the skin of symptomatic animals (31.94±18.81). The number of foci and cellularity of the inflammatory infiltrates in symptomatic dogs were higher than in other groups and in asymptomatics were higher than in controls (p<0.05; Tukey). The average area, perimeter and extreme diameters of the inflammatory infiltrates obtained in symptomatic dogs were higher than in controls (p<0.05; Tukey). The apoptotic index was higher in symptomatic than in other groups and there was no difference between asymptomatics and controls (p<0.05; Tukey). Ultrastructurally, apoptotic cells were shrunken, with condensed nuclear chromatin and cytoplasm. Condensed nuclei were frequently fragmented. Internucleosomal DNA fragmentation occurred only in symptomatic cases. Amastigotes were observed within neutrophils and macrophages. Apoptosis is directly related to parasite load, intensity of inflammatory response and clinical manifestations in L. chagasi naturally infected dogs.     

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.     

Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum

Cell-mediated and humoral immune response in naturally and experimentally infected dogs was studied using crude and pure antigens. Both types of infections induced severe signs of visceral disease, but the symptoms observed in natural infections were more pronounced than in experimental infections. In addition, asymptomatic infections were not observed in experimentally infected animals. Disease evolution in laboratory infections was rapid and an increase in antibody titer to crude parasite antigen was correlated with the appearance and aggravation of clinical symptoms. Peripheral blood lymphocyte proliferation to crude antigen and pure gp63 was observed early following experimental infection, but was abolished once the infected dogs began to exhibit clinical signs. A similar pattern was observed in naturally infected dogs. Serum from all patent dogs showed high antibody titers to rK39 in enzyme-linked immunosorbent assays (ELISA), and reacted by western blotting with several antigens, 12 to 120 KDa, including gp63 and gp70. In the case of asymptomatic dogs. antibody titers to crude antigen were low and only a few antigens were identified by western blotting. None of the pure proteins examined, gp63, gp70, and rK39 were recognized by western blotting or ELISA. However, asymptomatic dogs exhibited specific lymphocyte proliferation to both crude antigen and the potential vaccine candidate gp63.     

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs.     

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-β₁, IL-10, and INF-γ in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-β₁, IL-10, and INF-γ. Naturally active in vitro produced TGF-β₁ was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-β₁ levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-γ concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-γ is being produced in canine infection, mean levels of TGF-β₁ and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.     

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.     

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.