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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.     

Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs

Leishmune vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixed L. (L.) chagasi promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania (Leishmania) chagasi infected dogs and Leishmune vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128-1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP=20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasi infected dogs from Leishmune vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP 相似文献   

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

Serological, clinical and histopathological changes in naturally infected dogs with Leishmania infantum in the Khemisset province, Morocco

Canine leishmaniasis (canL) is widespread in the north of Morocco and the Leishmania infantum local strains are highly virulent. An epidemiological survey was carried out in 1993-1995 in the Khemisset province. In this region, the severity of the disease was assessed during regular visits to the identified foci by clinical examination of 323 dogs. Clinical signs were protean and occurred in various combinations. Biopsies were made on available sick dogs; the main histological changes were severe infiltration of the spleen, lymph nodes and bone marrow by mononuclear cells and hyperplasia of macrophage cells with amastigotes in their cytoplasm. The seroprevalence among 323 dog sera tested by ELISA showed a rate of 16.71%. The highest prevalence of the disease was 23.6% in the Sid El Ghandour hamlet. A comparison of the results of this study with those from the year following the first examination on the same site (Sid El Ghandour) of 67 dogs showed that the disease prevalence had not increased significantly (23.6% to 25.33%).     

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.     

Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT), including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania.

Methods

Twenty mongrel dogs naturally infected with Leishmania (Leishmania) infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG) state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry) evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania). The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs.

Results

The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal) irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT.

Conclusion

The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance).     

Regional parasite density in the skin of dogs with symptomatic canine leishmaniosis

In canine leishmaniosis, the parasitic density of the skin may be important for the infection of sandflies, and increased accumulation of inflammatory cells infected with Leishmania is believed to occur in dermal areas subjected to mechanical trauma. Parasite density and inflammatory responses in the upper and lower dermis of three body sites: flank (control site), dorsal muzzle (sandfly feeding site), and footpads (mechanical stress sites) were thus investigated in 15 dogs with symptomatic leishmaniosis. Parasite density did not differ between the control and tested sites or between the upper and lower dermis, apart from the footpads where it was higher in the upper dermis, and there was no correlation with severity of the macroscopic lesions or inflammatory infiltrate, except for the lower footpad dermis. No selective accumulation of the parasite in the muzzle that would favour its transmission to sandflies occurred, and the mechanical stress imposed on the footpads was not associated with increased parasitic density, or with inflammatory infiltrate.