A compared histopathological study on kidneys and eye bulbs in distinct clinical presentations of canine leishmaniasis by Leishmania infantum.

Canine leishmaniasis (CanL) caused by Leishmania infantum commonly progresses with renal and ophthalmic lesions associated with active systemic disease. As chronic inflammation related to immune complex deposits is a pathophysiological factor in the development of both glomerulonephritis and uveitis, we aimed to evaluate renal and ocular histopathological lesions and analyze whether they were related to each other and the clinical degree of the disease. For that, we evaluated 15 dogs from CanL-endemic areas. L. infantum PCR-positive dogs were studied according to disease severity into two different groups: Group-1 (G1) had data from seven dogs with mild to moderate CanL and no history of treatment, and G2 was formed with eight dogs with severe to terminal disease that had not responded to CanL treatment. Histopathological analysis of kidneys showed higher frequencies and intensities of glomerular basement membrane thickening (p = 0.026), deposits in glomeruli (p = 0.016), epithelial necrosis (p = 0.020), tubular dilatation (p = 0.003) and interstitial fibrosis (p = 0.04) in G2 dogs than in G1 dogs. Surprisingly, the histopathology of eye bulbs showed a higher frequency and intensity of retinitis (p = 0.019) in G1 dogs than in G2 dogs. The comparative analysis showed that there was no correspondence between histopathological findings in kidneys versus eyes in milder or more severe CanL. Our findings suggested that (1) clinically undetectable eye alterations can be more precocious than those in kidneys in the development of CanL, and (2) the lower frequency of eye lesions and higher frequency of renal lesions in dogs with terminal disease even after treatment indicate that therapy may have been effective in reducing CanL-associated ophthalmic disease but not proportionally in reducing kidney disease.

Nutritional adjuvants with antioxidant properties in the treatment of canine leishmaniasis.

Clinical improvement of dogs treated for canine leishmaniasis (CanL) requires reducing Leishmania infantum loads, which depend on intracellular oxidant compounds to destroy the parasite. However, oxidative species' excess and antioxidants consumption can culminate in oxidative stress, resulting in increased, widespread inflammation. We aimed to evaluate if early or late addition of nutritional adjuvants (NAs) - omega-3 polyunsaturated fatty acids and B vitamins - to anti-Leishmania drugs (ALDs) in the treatment of CanL would be clinically beneficial. For that, serum biomarkers including oxidative stress parameters were analyzed during 12 months in dogs allocated to two treatment groups: (G1) NAs administered from 30 days prior to the beginning of ALDs; and (G2) NAs administered from 61 days after the beginning of ALDs. Both G1 and G2 continued to receive NAs until the 12th month. The ALDs administered were metronidazole associated with ketoconazole (40 days), followed by allopurinol from day 41 until the 12th month. G1 exhibited superior inflammation control, with reduced globulins (p = 0.025), specific anti-Leishmania immunoglobulins (p = 0.016), total protein (p = 0.031), and an increased serum albumin/globulin ratio (p = 0.033), compared to G2. The early use of NAs associated with ALDs is clinically beneficial in treating dogs with CanL.

Vincristine and ivermectin combination chemotherapy in dogs with natural transmissible venereal tumor of different cyto-morphological patterns: A prospective outcome evaluation.

Vincristine is the first-line drug for the chemotherapy of canine transmissible venereal tumor (CTVT). Drug resistance is related to tumor cyto-morphological patterns of CTVT. There are anti-cancer properties of ivermectin, thus, a combination of ivermectin and vincristine could be an effective chemo-therapeutic treatment regimen for CTVT. Study aims, therefore, were to (1) assess the frequency of CTVT cyto-morphologies, and (2) evaluate treatment efficacy and possible adverse reactions to vincristine compared with a combination vincristine and ivermectin. Dogs (n = 41) with CTVT were characterized by tumor cyto-morphology and disease severity and of those, 20 were randomly allocated into two groups. There was a control group (G-Vin; n = 10) in which there was treatment with vincristine; and an experimental group (G-Iv/Vin; n = 10) in which there was treatment with the ivermectin/vincristine combination. Although dogs in the G-Iv/Vin group had more severe disease at the beginning of the study (P = 0.0031), the number of weeks and chemotherapy sessions until tumor remission were similar among dogs of the two groups, indicating both treatments were effective. There was a decrease in the leukocyte counts (P = 0.0020), related to neutropenia (P = 0.0371) in the G-Vin but not the G-Iv/Vin treatment group. There was no tumor resistance that developed during the study regardless of the treatment regimen used or tumor cytomorphology. In summary, the use of the vincristine/ivermectin combination was well tolerated and efficacious for CTVT treatment.

Mathematical Modelling Using Predictive Biomarkers for the Outcome of Canine Leishmaniasis upon Chemotherapy.

Prediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient's prognosis.

Leishmania infantum-derived lipophosphoglycan as an antigen in the accurate serodiagnosis of canine leishmaniasis.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage.

Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species.

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog's body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors.

The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector.

Xenodiagnosis on dogs with visceral leishmaniasis: Canine and sand fly aspects related to the parasite transmission.

One of the main limitations for the effective control of canine leishmaniasis in endemic areas is the difficulty in identifying infectious dogs. The objective of this study was to determine factors, related to dogs and to parasite detection in sand flies, which are associated with the positive xenodiagnosis of Leishmania infantum using the vector Lutzomyia longipalpis. The xenodiagnosis was performed in 50 owned dogs residing in endemic areas, which were divided into three different groups: G1-26 dogs proved to be infected and classified by severity of VL clinical signs on physical examination; G2-15 dogs proved to be infected and classified by severity of clinical signs and intensity of laboratory abnormalities; G3-nine dogs that were seropositive for anti-Leishmania IgG in ELISA tests. Parasite search in the sand flies after having fed on dogs in the xenodiagnosis was performed by both methodologies, PCR and dissection followed by microscopy. In G1, 58% (15/26) of dogs were able to transmit Leishmania to the vector, when parasite detection in sand flies were performed by PCR technique, 5 days after blood meal, whereas in G2, 53% (8/15) transmitted the parasite to the vector, however, confirmation was performed by direct observation of parasite through optical miscroscopy held 10 days after blood meal. Rate of infectiousness of dogs to sand flies was positively associated to severity of disease (p=0.042 and p=0.040), regardless the method used for clinical classification or for parasite detection in sand flies after xenodiagnosis. In G1 30% (3/10) of dogs with subclinical infection were infectious to the vector, while 80% (12/16) of dogs with clinical disease were also infectious. Even more, 17% (1/6) of dogs that had moderate disease were infectious to the sand flies, while 78% (7/9) of dogs with severe disease were infectious in G2. Still in G2, the proportion of sand flies infected (grade of infectiousness) was significantly lower (p=0.0098) when they fed on dogs with moderate disease (1%) in comparison with dogs with severe disease (38%). The dogs from G3 presented a rate of infectiousness of 11% (1/9), demonstrating that the indirect ELISA is not a good indicator of infectiousness and, therefore, should not be used as a confirmatory test for the euthanasia of dogs, as it is currently done in Brazil.

Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis.

Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4-100) in splenic aspirate; 79.2% (CI 68-90.3) in lymph nodes; 77.3% (CI 64.5-90.1) in skin; 75% (CI 63.1-86.9) in blood; 50% (CI 30-70) in bone marrow; 37.5% (CI 24.2-50.8) in left-eye; and 29.2% (CI 16.7-41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7-79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5-96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG, and subclasses, parasitism, and parasite transmission by xenodiagnosis.

BACKGROUND: The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use. PURPOSE: To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL). METHODS: From 2010 to 2012, healthy dogs were vaccinated with Leishmune(®) (50 animals) or Leish-Tec(®) (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis. RESULTS: Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune(®) and Leish-Tec(®) groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune(®) and on the 21st day after the second dose of Leish-Tec(®). The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune(®) and Leish-Tec(®) groups, respectively. The Leishmune(®) group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec(®) group (p<0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p<0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune(®) group, and 7.9% (3/38) of the Leish-Tec(®) group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively. CONCLUSIONS: No significant differences were observed in dogs vaccinated with Leishmune(®) or Leish-Tec(®), with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune(®)-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec(®) exhibited adverse reactions with greater frequency and severity.

Uso do plasma rico em plaquetas no tratamento da doença articular degenerativa em cão: relato de caso / Use of platelet-rich plasma in the treatment of degenerative joint disease in dog: case report / Uso de plasma rico en plaquetas en el tratamiento de la enfermedad degenerativa de las articulaciones en perro: relato de caso

No presente trabalho é descrito o uso do plasma rico em plaquetas (PRP) no tratamento de um caso de osteoartrose naturalmente adquirida em um cão. O PRP foi obtido a partir de sangue venoso autólogo coletado em citrato de sódio. A separação do precipitado celular do sobrenadante foi realizada de forma estéril. Após adição de cloreto de cálcio e liberação dos fatores de crescimento, o PRP foi infiltrado no joelho do animal em estudo. Após o tratamento, os parâmetros de apoio, crepitação e dor articular apresentaram melhora, não havendo intolerância do animal à infiltração. Preliminarmente, pode-se dizer que é esta é uma técnica minimamente invasiva e de alta aplicabilidade. O emprego do PRP para infiltração mostrou ser simples, rápido, barato e seguro para o tratamento da osteoartrose no animal em questão. Estudos com maior número de casos em cães são necessários, a fim de validar este procedimento como prática terapêutica na rotina clínica...

The use of platelet-rich plasma (PRP) in the treatment of a natural case of osteoarthrosis in a dog is reported in the present work. The PRP was obtained through autologous venous blood collected in sodium citrate. The cellular precipitate was separated from the supernatant in a sterile condition. After the addition of calcium chloride and the release of growth factors, the PRP was infiltrated in the knee of the dog under study. After the treatment, weight-bearing, crackle and joint pain parameters presented improvement without intolerance to the infiltration by the animal. Preliminarily, it can be concluded that this is a minimally invasive method with high applicability, as well as being simple, quick, cheap and safe for the treatment of degenerative joint disease to the described animal. The study of a larger number of cases in dogs is necessary in order to validate the procedure for clinical routine...

En este estudio se ha descrito el uso del plasma rico en plaquetas (PRP) en el tratamiento de un caso de osteoartritis, adquirido naturalmente por un perro. El PRP ha sido obtenido a partir de la sangre venosa autóloga recogido en citrato de sodio. La separación del precipitado celular del sobrenadante se ha realizado de forma estéril. Después de la adición de cloruro de calcio y liberación de los factores de crecimiento, el PRP ha sido infiltrado en la rodilla del animal en estudio. Tras el tratamiento, los parámetros de apoyo, crepitación y dolor articular presentaron mejora, no ocurriendo intolerancia del animal a la infiltración. Preliminarmente, se puede decir que esta es una técnica poco invasiva y de alta aplicabilidad. El empleo del PRP para infiltración ha mostrado ser sencillo, rápido, barato y seguro para el tratamiento de osteoartritis en el animal estudiado. Estudios con mayor número de casos en perros son necesarios, para que se pueda validar este procedimiento como práctica terapéutica en la rutina clínica...

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens.

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis.

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.

Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals.

The purpose of this study was to evaluate the safety of spleen aspiration as a sampling technique for the parasitological detection by culture and microscopy of Leishmania (chagasi) infantum. Two hundred and nine domiciled dogs from an endemic area for visceral leishmaniasis in Bahia State, Brazil, were studied. Most dogs (87%) were seropositive for anti-L. chagasi antibodies by ELISA. Clinical signs of disease were recorded and the animals monitored during and after spleen puncture in order to detect possible complications associated with the procedure. From a total of 257 splenic punctures in the 209 animals, only three minor events occurred, with no significant consequence for the animals and no association with risk factors. Leishmania was isolated from 149/180 (83%) seropositive dogs, and from 6/26 (23%) seronegative animals. The procedure did not cause adverse side effects or unnecessary suffering and confirmed the diagnosis in a large percentage of dogs. We conclude that spleen aspiration can be considered an effective and safe procedure for the definitive diagnosis of canine visceral leishmaniosis.

A novel monoclonal antibody against canine monocytes/macrophages.

The production and partial characterization of a monoclonal antibody, the IgG1 IH1, which recognizes an antigen distributed in canine monocytes/macrophages, is reported here. The distribution and apparent molecular weight of the antigen recognized by the IH1 MAb was determined in peripheral blood leukocytes, peripheral blood monocyte-derived macrophages and tissue sections of spleen, liver and skin, using Western blotting, immunocytochemistry, immunohistochemistry and flow cytometry. The IH1 MAb-recognized antigen was detected in Western blotting under non-reducing conditions spread out as a large band covering the position corresponding to the migration of molecules with molecular weights from 55 to 73 kDa. The IH1 MAb labeled blood monocytes, tissue macrophages in lymph nodes, and in the mantle zone of the spleen, and Kupffer cells in the liver. It did not react with human cells. In flow cytometric analysis, the IH1 MAb reacted with a subpopulation of monocytes. The MAb described herein may become a valuable tool for diagnosis and research on canine diseases.

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs.

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Production of monoclonal antibodies against canine leukocytes.

A panel of anti-canine leukocyte monoclonal antibodies (MAbs) was produced by immunizing BALB/c mice with canine peripheral blood mononuclear cells (PBMC), either resting or stimulated with concanavalin A (ConA). Three out of 28 clones-IH1, AB6, and HG6-screened by ELISA and producing antibody with the highest specificity for canine cell immunostaining, were subjected to three subsequent subcloning steps by limiting dilution, and selected for further characterization. These MAbs belonged to IgG1 (HG6 and IH1) and IgG2a (AB6) isotypes. The distribution of cell populations expressing the antigen recognized by the antibodies was identified by indirect immunoflorescence on canine PBMC and on tissue sections of lymph node, spleen, liver and skin. The possible crossreactivity with human PBMC was also examined in immunocytochemistry. One of the antibodies specifically recognized macrophages. The MAbs presented here can be foreseen as possible valuable diagnostic and research tools to study immune functions in dogs.

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9 percent; in lymph node cultures it was 25 percent. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Modulação da infecção em modelo murino por moléculas de amastigota de Leishmania / Modulation of infection in a murine model of molecules of Leishmania

Embora algumas espécies de Leishmania possam determinar várias formas de doença, existe usualmente uma relação entre a espécie infectante e a apresentação clínica da enfermidade e natureza da resposta imune ao parasito. A Leishmania Braziliensis, por exemplo, costuma ocasionar uma forte resposta imune celular e poucas lesões restritas à pele ou/e à mucosa, enquanto que a Leishmania Amazonensis pode causar uma forma muito grave da doença, conhecida como Leishmaniose cutâneo-difusa (LCD), na qual ocorre uma resposta imunológica ao parasito deficiente e lesões disseminadas pelo corpo do hospedeiro. É possível que diferenças moleculares entre estas duas espécies de Leishmania sejam responsáveis pela associação da LCD apenas com a infecção por L. Amazonensis. Neste trabalho, foi investigada a capacidade de moléculas de L. Amazonensis em potenciar a infecção por L. Braziliensis em camundongos BALB/c. A identificação e caracterização de moléculas parasitárias supressoras podem servir de base para o desenvolvimento de futuras estratégias de produção de vacinas e imunoterápicos...