Evaluation of serum cystatin-C in dogs with visceral leishmaniasis

Serum Cystatin C (sCys-C) is one of the most important serum markers of renal function assessment in dogs. The purpose of this study was to determine the sCys-C concentration in dogs with visceral leishmaniasis (VL). In the study, 16 dogs with VL and 10 clinical healty dogs (control) were used. Mean sCys-C concentration of the infected dogs was significantly higher than that of the control group (p < 0.05). Mean serum creatinine concentration was lower and mean blood urea nitrogen, albumin and globulin concentrations were higher in dogs with VL; however, these changes were not statistically significant. Mean total protein and phosphorus concentrations were found to be higher in dogs with VL than healthy dogs (p < 0.05). No significant correlation had been determined between sCys-C and other variables. Visceral leishmaniasis in dogs has increased sCys-C concentration indicating a possible renal impairment; however, further studies are needed to be performed together with renal biopsies in the investigation sCys-C in dogs with VL.     

Factors associated with the occurrence of epistaxis in natural canine leishmaniasis (Leishmania infantum)

BACKGROUND: Canine leishmaniasis (CanL) is a common cause of epistaxis in dogs residing in endemic areas. The pathogenesis of CanL-associated epistaxis has not been fully explored because of the limited number of cases reported so far. HYPOTHESIS: Epistaxis in CanL could be attributed to more than 1 pathomechanism such as hemostatic dysfunction, biochemical abnormalities, chronic rhinitis, and coinfections occurring in various combinations. ANIMALS: Fifty-one dogs with natural CanL. METHODS: The allocation of 51 dogs in this cross-sectional study was based on the presence (n = 24) or absence (n = 27) of epistaxis. The potential associations among epistaxis and concurrent infections (Ehrlichia canis, Bartonella spp., and Aspergillus spp.), biochemical and hemostatic abnormalities, and nasal histopathology were investigated. RESULTS: Hypergammaglobulinemia (P= .044), increased serum viscosity (P= .038), decreased platelet aggregation response to collagen (P= .042), and nasal mucosa ulceration (P= .039) were more common in the dogs with epistaxis than in those without epistaxis. The other significant differences between the 2 groups involved total serum protein (P= .029) and gamma-globulin (P= .013) concentrations, which were higher, and the percentage platelet aggregation to collagen, which was lower (P= .012) in the epistaxis dogs. CLINICAL IMPORTANCE: CanL-associated epistaxis appears to be the result of multiple and variable pathogenetic factors such as thrombocytopathy, hyperglobulinemia-induced serum hyperviscosity, and nasal mucosa ulceration.     

Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum

In order to evaluate if the presence of Trypanosoma caninum can lead to a confuse diagnosis of canine visceral leishmaniasis (CVL), we investigated the serological status of dogs infected by T. caninum and assessed the serological cross-reactivity with CVL. A set of 117 serum samples from dogs infected by T. caninum, Leishmania chagasi and not infected dogs (n = 39 in each group) was tested using commercial kits – indirect immunofluorescence (IFI-LVC), ELISA (EIE-LVC) and immunochromatographic test (DPP) – and in house tests with T. caninum (IIF-Tc and ELISA-Tc) and L. chagasi antigens (IIF-Lc and ELISA-Lc). IIF-Tc and ELISA-Tc presented sensitivity of 64.1% and 94.9% and specificity of 23.1% and 35.9%, respectively. The sensitivity of the IFI-LVC, EIE-LVC and DPP tests was 100% and the specificity was 70.5%, 68% and 97.5% respectively. The concordance between the tests was considered as satisfactory. The specificities of IFI-LVC, EIE-LVC and DPP were higher when the group Tc was excluded, with significant values for IFI-LVC (χ² = 4.36, P-value = 0.036), thus suggesting that the infection by T. caninum can confuse the diagnosis of CVL.     

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.     

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease.In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen.Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61–0.80) very good (0.81–0.99) or perfect (1.00), for most of the parameters analyzed.Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.     

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.     

Analysis of immune responses in dogs with canine visceral leishmaniasis before, and after, drug treatment.

The incidence of canine visceral leishmaniasis (CVL) is increasing in the Mediterranean region. Many drugs have been tested for treatment of CVL, but little is known regarding their effect on test immune responses. In our study, three dogs naturally infected with Leishmania infantum and five dogs experimentally infected with the same strain, were treated with dimethasulfonate pentamidine (Lomidine) and the immune response evaluated before, and after, treatment. After the last injection, animals began to gain weight and the major clinical signs disappeared. Antibody titers gradually decreased to low levels, six months after treatment. At the same time, antigen specific lymphoproliferation reappeared in the sampled animals. This study shows that, after treatment, immune cellular responses to leishmanial antigens, involved in protection against Leishmania infection, were established.     

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.     

Pancytopenia in a cat with visceral leishmaniasis

Abstract: A 4‐year‐old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune‐mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.     

Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs.

OBJECTIVE: To conduct serologic surveillance for Leishmania spp in English foxhounds from a kennel, as well as recipients of blood from these dogs, and determine whether L infantum organisms could be transmitted via blood transfusion. DESIGN: Serologic prevalence survey. ANIMALS: 120 English foxhounds and 51 dogs of various breeds receiving blood from these donors. PROCEDURE: Foxhound blood donors, foxhound nondonors, and nonfoxhound blood recipient dogs were evaluated serologically for Leishmania spp by indirect fluorescent antibody testing. Dogs that received packed RBC (PRBC) transfusions from foxhound donors from mid-1996 through mid-2000 were identified. Furthermore, dogs were serologically evaluated if they had received fresh frozen plasma (FFP) transfusions in 1999 and 2000 from seropositive foxhound blood donors. RESULTS: Thirty percent of the English Foxhounds were seropositive for Leishmania spp (titer > or = 1:16), although the degree of seropositivity varied considerably during the period. Furthermore, 57 foxhounds had been used as donors from 1996 to 2000, and 342 units of PRBC had been transfused to at least 227 patients. All 25 dogs screened that received PRBC from seronegative foxhound donors tested negative, whereas 3 of 7 dogs that received PRBC from seropositive donors tested positive. All 9 dogs that received FFP from seropositive foxhound donors remained seronegative. CONCLUSIONS AND CLINICAL RELEVANCE: To our knowledge, this report documents the first transmission of Leishmania spp by blood transfusion. The use of foxhounds as blood donors may not be advisable in North America.     

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.     

Experimental visceral leishmaniasis in the opossum

Adult wild-trapped opossums were infected with Leishmania donovani (Khartoum strain, WR 378) and evaluated as an animal model of visceral leishmaniasis. All infected opossums died within 32 days. Loss of body fat, hepatomegaly, and petechiae of skin and abdominal musculature were seen at necropsy. Microscopically, numerous amastigote-laden macrophages were seen in histologic sections of liver, spleen, and lymph nodes; fewer parasite-laden macrophages were in the bronchial-associated lymphoid tissues and renal glomeruli. Hematological findings included thrombocytopenia (terminal), neutropenia, and lymphopenia. Blood lymphocyte blastogenesis in response to concanavalin A and phytohemagglutinin was decreased markedly at day 24 post-infection (PI). Serum antibodies (1:40 dilution) to promastigotes of L. donovani were detected in five of eight infected opossums tested on days 10 and 24 PI. Total bilirubin concentrations and alanine aminotransferase and aspartate aminotransferase activities were increased after day 25 PI. Activated partial thromboplastin times and one-stage prothrombin times were prolonged before death. Concurrently, factors V, VIII, and XII activities were decreased.     

Evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis

The efficacy of specific immunochemotherapy against Leishmania infantum infection in dog was studied. The effects on transmission of the disease, as well as the cellular and humoral immune response were examined. The treated animals showed a significant reduction in the infection rates that were detected in Phlebotomus perniciosus females fed on the dog. The humoral immune response, assayed with an indirect immunofluorescence antibody test (IFAT), did not show significant variations under the influence of the therapy. The characterisation of the peripheral blood mononuclear cells (PBMC) using flow cytometry indicated a significant increase in the proportion of T lymphocytes, especially of CD4/TcR(alpha)(beta)(+) and CD4/CD45RA(+) cells, without showing evidence for modifications in the other leukocyte subsets. Cellular lymphoproliferation studies indicated a lack of a specific response to soluble leishmanial antigen (SLA), but the non-specific lymphoproliferative capacity assayed with phytohemagglutinin (PHA) was maintained.     

Experimental visceral leishmaniasis in the owl monkey

Visceral leishmaniasis developed in eight owl monkeys (Aotus trivirgatus) after intravenous inoculation with a Khartoum strain (WR378) of Leishmania donovani. Six monkeys died within 93 days, and two monkeys recovered from the disease. Clinically, signs were weight loss, anemia, and hepatosplenomegaly. Hematologic findings included anemia, granulocytopenia, thrombocytopenia, and lymphocytosis. Analysis of serum or plasma revealed hyperbilirubinemia, azotemia, hyperglobulinemia, hypoalbuminemia, and altered hemostasis. All monkeys developed positive antibody titers to promastigotes of L. donovani and had increases in immunoglobulins M and subsequently G. Liver, spleen, bone marrow, and lymph nodes were the principal organs containing numerous parasitized macrophages. The owl monkey was highly susceptible to L. donovani infection and should be a useful animal model for the study of visceral leishmaniasis.     

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.     

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

Canine visceral leishmaniasis and its emergence in the United States.

Leishmaniasis is an important zoonotic disease that accounts for approximately 57,000 deaths among people each year. Dogs are reservoirs for the disease, and dog ownership is a risk factor in endemic areas. This article discusses insect vectors for this disease, as well as diagnosis and treatment for dogs.