Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.     

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.     

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.     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Profile of anti-Leishmania antibodies related to clinical picture in canine visceral leishmaniasis

This research investigated the profile of anti-Leishmania antibodies in different clinical forms of canine visceral leishmaniasis (CVL). Naturally infected dogs were divided into two groups: subclinical dogs (SD, n=10) and clinical dogs (CD, n=68). Non-infected dogs (ND, n=7) comprised the negative control group. The humoral response was evaluated by the profile of total IgG, IgG1, IgG2, IgM, IgA and IgE, determined by ELISA. Infected animals showed increased levels of total IgG, IgA and IgE in addition to IgG1 and IgG2 in groups SD and CD, when compared with group ND. Furthermore, it was observed that IgG2 and IgM were correlated with symptomatology, while total IgG, IgG1 and IgA were negatively correlated and IgE showed no correlation. It follows that serum levels of IgG2 anti-Leishmania are correlated with typical clinical signs of disease. Furthermore the determination of specific anti-Leishmania antibodies could be an important tool in monitoring CVL clinical picture.     

Immunoglobulin G subclass distribution in canine leishmaniosis: a review and analysis of pitfalls in interpretation

Infection with Leishmania may have different outcomes in genetically distinct individuals and the course of infection is determined by the nature of the host innate and adaptive immune response. Thus in experimentally infected mice, and in naturally infected dogs or humans, the protective (self-healing or asymptomatic) phenotype is associated with the induction of Th1-regulated cell-mediated immunity. By contrast, a Th2-regulated humoral immune response is associated with severe symptomatic disease. In the murine model system there is strong correlation between clinicopathological phenotype and the nature of the antigen-specific humoral immune response. Symptomatic infection and Th2-regulation is associated with elevation in antigen-specific IgG1 and IgE, whereas asymptomatic infection with Th1-regulation is linked with IgG2a production. IgG subclass restriction is less clear in human disease with only some clinical forms being correlated to a specific serological profile. Although numerous studies have questioned whether infected dogs develop skewed IgG subclass usage, the results of these have been conflicting-suggesting bias towards IgG1 or IgG2 or neither subclass in different investigations. This confusion could relate to the specificity of the commercially available polyclonal antisera used to detect the canine IgG1 and IgG2 subclasses. More meaningful results might be obtained by the use of the panel of monoclonal antibodies with well-validated specificity for all four canine IgG subclasses.     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

Periodic administration of allopurinol is not effective for the prevention of canine leishmaniosis (Leishmania infantum) in the endemic areas

A total of 95 clinically healthy and seronegative for Leishmania infantum dogs, residing an area highly endemic for canine leishmaniosis (CL) and living an outdoor life-style, were split into positive and negative groups, and then were randomly assigned to receive allopurinol (n = 51; 20 mg/kg once daily), or placebo (n = 44) for 1 week per month, from April to November. Forty per cent (38/95) of these dogs were not reexamined and retested at the end of the trial for reasons unrelated to CL. None of the remaining 57 dogs exhibited the symptomatic form of the disease at the end of the 1-year follow-up period. Of the 15 allopurinol-treated dogs that were non-infected (negative PCR and tissue smear microscopy) at the beginning of the trial, 6 (40% P = 0.03) became PCR-positive, of which 3 became also seropositive, at the end of the observation period. In contrast, only 1 of 7 (14.3%) placebo-treated non-infected dogs became PCR positive at the same time point. Of the 19 allopurinol-treated dogs that were infected (PCR-positive) at the beginning of the trial, 18 (94.7%) remained PCR-positive and one (5.3%) seroconverted, at the end of the observation period. Of the 16 initially infected and placebo-treated dogs, 14 (87.5%) remained PCR positive, of which one (6.7%) also turned positive by tissue smear microscopy. Therefore, it is concluded that the use of allopurinol, at the daily dose of 20 mg/kg, for 1 week per month, during the period of sandfly activity, does not prevent the infection of non-infected dogs by L. infantum, and, also, does not help in the elimination of the parasite from dogs with asymptomatic infections.     

Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum

BACKGROUND: Lymphadenopathy in canine leishmaniosis has been reported as reactive lymphoid hyperplasia or granulomatous (histiocytic) lymphadenitis. However, we are unaware of information on the effect of latent Leishmania infection on lymph node cytology compared with clinically affected dogs. OBJECTIVES: The aim of the present study was to investigate cytologic patterns of lymphadenopathy in dogs with clinical and subclinical forms of leishmaniosis and to correlate cytologic findings with the density of Leishmania amastigotes in fine needle aspiration (FNA) smears. METHODS: FNA cytology of prescapular or popliteal lymph nodes was evaluated on 32 dogs with clinical evidence of leishmaniosis (group A), 24 subclinically infected dogs (group B), and 17 clinically healthy noninfected dogs (group C); groups were based on the results of serologic and PCR tests for Leishmania sp. Differential nucleated cell counts (based on 300 cells) and amastigote density were determined microscopically. Cytologic findings were categorized and compared among groups. RESULTS: Cytologic abnormalities were found in 19 of 32 (59.4%) dogs in group A, 1 of 24 (4.2%) dogs in group B, and 2 of 17 (11.8%) dogs in group C and were significantly more frequent in group A than group B (P <.001) or C (P = .001). In group A, 68.7% of the dogs had lymphoid hyperplasia, 12.5% had lymphoid hyperplasia and histiocytic lymphadenitis, 6.3% had histiocytic lymphadenitis, and 3.1% had lymphoid hyperplasia and neutrophilic lymphadenitis. Lymphoid hyperplasia was also noted in 1 dog in group B, and lymphoid hyperplasia and eosinophilic lymphadenitis were each found in 1 dog in group C. Lymph node smears from 31 (96.9%) dogs in group A and 6 (25%) dogs in group B were positive for Leishmania amastigotes; however, no correlation was found between the density of amastigotes and cytopathologic patterns of lymphadenopathy. CONCLUSION: Abnormal lymph node cytology is much more common in dogs with clinical leishmaniosis than in dogs with subclinical infection, and primarily involves lymphoid hyperplasia. Despite finding no association between the density of amastigotes and type of lymphadenopathy, lymph node cytology still is a valuable diagnostic tool for diagnosing canine leishmaniosis.     

Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis

To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seronegative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in comparison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clinical status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pronounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.     

Serum protein response and renal failure in canine Babesia annae infection

Babesia annae piroplasms have recently been recognised as a cause of infection and disease among dogs in Europe. The pathogenesis and clinical implications of this emerging disease remain poorly understood. We conducted this study to describe the electrophoretic profiles associated with the infection and to determine if B. annae associated azotaemia is caused by renal failure. We examined by microscopy 2,979 canine blood samples submitted to a diagnostic laboratory in NW Spain between September 2001 and April 2002. Small ring-shaped piroplasms were detected in blood smears of 87 samples and the identity of 58 of these presumptive cases were confirmed by PCR. This group of 58 infected dogs and a reference group of 15 healthy non-infected dogs were our study population. For all the dogs, serum protein response to -albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin- was measured by capillary electrophoresis. The response of infected and non-infected dogs was compared and within infected dogs, the response of those with azotaemia (19) was compared with that of non-azotaemic dogs (39). Infected dogs presented a significant elevation of total proteins and all the different globulin fractions, and significantly lower levels of albumin compared to non-infected dogs. Among infected dogs, those presenting azotaemia had significantly lower concentrations of total proteins, albumin, beta and gamma globulins, and significantly higher values of alpha-2 globulin. Specific gravity was below the threshold of 1,025 for all dogs with azotaemia for which a urine sample was available (7) suggesting that azotaemia, in these dogs was of renal origin. Azotaemic dogs had higher concentrations of cholesterol and triglycerides, probably as a result of a liver compensatory response to the loss of proteins. We conclude that serum protein response in B. annae infected dogs corresponds to the pattern of a haemolytic syndrome with intense inflammatory reaction and that the azotaemia associated to the infection is very likely of renal origin.     

Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤ 0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.     

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.     

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.     

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.     

The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection

Veterinarians working in the Balearic Islands (Mallorca), an endemic region of canine leishmaniosis, have reported very few cases of leishmaniosis in Ibizian hounds while concurrently observing that dogs of other breeds had a high incidence of clinical canine leishmaniosis. To further investigate this observation, two populations of dogs from the Balearic Islands were examined for the presence of Leishmania-specific cellular immunity using a delayed type hypersensitivity test (DTH) to leishmanin and for the presence of Leishmania-specific humoral immunity using an ELISA. Fifty-six asymptomatic dogs, 31 Ibizian hounds and 25 dogs belonging to other breeds were examined. Seventy-seven percent of the dogs demonstrated a specific immune response against Leishmania, either humoral or cellular. This finding suggests that the infection rate (77%) was higher than previously considered. For Ibizian hounds 81% were DTH positive while only 48% of the other dogs were DTH positive. A statistical association between Ibizian hounds and positive DTH response was found. A specific humoral response was found in 48% of Ibizian hounds and in 56% of the other dogs. No statistical association relative to the Leishmania-specific IgG1 and IgG2 levels were found between the two groups. The Ibizian hound has been reported to be more resistant to Leishmania infection and we found that the Ibizian hound mounts a significant cellular response to infection. Thus, the Ibizian hound may be an interesting canine model for the investigation of protective anti-Leishmania immune response.     

Enzyme immunoassay of Dirofilaria immitis-specific IgE in infected dogs

An ELISA procedure was developed for monitoring the specific IgE response in dogs to Dirofilaria immitis infection. The results of this assay correlated well with, and appeared to be more sensitive than, the passive cutaneous anaphylaxis test. The IgE ELISA values of the positive reference serum and the passive cutaneous anaphylaxis test results showed that a serum to negative absorbance ration of 1.45 was statistically significant for discrimination and was used to evaluate the specific IgE response in the sera from 90 clinically diagnosed heartworm cases. This ELISA procedure was more sensitive, as it detected 78% of the 90 cases as compared to a detection rate of only 43-47% by IgG ELISA or IFA. Sera obtained from 23 experimentally infected dogs at 4-week intervals for 20 weeks post-infection, were assayed for D. immitis-specific IgE by ELISA. A group of the infected dogs was also treated with diethylcarbamazine during the course of infection. All the experimentally infected dogs developed a specific IgE response, with treated dogs generally responding earlier.     

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.     

Serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term treatment

OBJECTIVE: To evaluate changes in serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term therapy in accordance with 2 treatment protocols and determine whether concentrations of acute-phase proteins could be used to monitor the initial response of dogs to treatment. ANIMALS: 12 dogs naturally infected with Leishmania infantum. PROCEDURE: Dogs were allocated into 2 groups. Dogs of group 1 were treated by use of meglumine antimonate (100 mg/kg, SC, q 24 h) administered concurrently with allopurinol (15 mg/kg, PO, q 12 h) for 20 days and then with allopurinol alone at the same dosage for the subsequent 30 days. Dogs of group 2 were treated by administration of allopurinol alone (15 mg/kg, PO, q 12 h) for 60 days). Blood samples were obtained before and during treatment for measurement of serum concentrations of acute-phase proteins and determination of CBC counts, serum biochemical analyses, and electropherograms. RESULTS: All dogs evaluated in the study had increased concentrations of C-reactive protein, haptoglobin, and ceruloplasmin at the time of diagnosis of leishmaniosis. Mean concentration of serum amyloid A before treatment was also increased, but some of the dogs had concentrations of serum amyloid A that were within the reference range. Concentrations of C-reactive protein and ceruloplasmin decreased significantly in all dogs at the end of the study period. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of concentrations of selected acute-phase proteins, such as C-reactive protein or ceruloplasmin, could be used to evaluate the initial response of dogs with leishmaniosis to treatment.     

Histopathological and immunohistochemical investigations of the hepatic compartment associated with parasitism and serum biochemical changes in canine visceral leishmaniasis

The immunopathological evaluation of the hepatic compartment associated with parasitism and biochemical findings are essential for understanding the genesis of hepatomegaly in canine visceral leishmaniasis (CVL). Three clinical groups of dogs naturally infected with Leishmania chagasi [i.e., asymptomatic (AD, n=12), oligosymptomatic (OD, n=12) and symptomatic (SD, n=17)] were assessed and compared with a group of non-infected dogs (NID, n=11). Intense reaction of the Kupffer cells, capsule and portal inflammation, and the presence of intralobular granulomas, were observed in the different clinical groups. Dogs in the SD group presented a higher frequency of parasitism compared with the AD group. Inflammatory alterations were more intense in the SD group and were associated with parasitism. Our results indicated an association between histological liver changes and the progression of biochemical alterations according to progression of clinical forms of CVL, and the direct relationship between clinical symptoms and frequency of hepatic parasitism.