Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis.

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity.

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.

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.

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.

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.

Quadro clínico de cães infectados naturalmente por Leishmania chagasi em uma área endêmica do estado da Bahia, Brasil / Clinical profile of naturally infected dogs from an endemic area for "Leishmania chagasi" (infantum) in Bahia State, Brazil

A leishmaniose visceral canina (LVC) é uma zoonose de importância em Saúde Pública, causada no Novo Mundo pela Leishmania chagasi e é associada a uma doença sistêmica crônica à qual se atribui uma gama variada de sinais clínicos. Foram examinados 61 cães, identificados como soropositivos pelo teste ELISA indireto para anticorpos anti-Leishmania, provenientes do município de Jequié, área endêmica para LVC na Bahia, tendo-se como o objetivo delinear o perfil clínico dos animais infectados. Os caninos foram submetidos ao exame físico e estudo parasitológico por cultivo do parasito em amostras de aspirado esplênico. Todos os cães foram classificados como sintomáticos, sendo 13 positivos ao exame parasitológico. Foi encontrada correlação entre os resultados positivos de cultura esplênica e a densidade óptica obtida no ELISA dos cães estudados (r=0,8555; p=0,00327) e entre os resultados de ELISA e a presença de alterações cutâneas (p=0,0402), conjuntivite (p=0,050), esplenomegalia (p=0,0272) e dificuldade de locomoção (p=0,0256), além de tendência à redução no número de hemácias nos animais. Observaram-se positividade na cultura e alterações das mucosas (p=0,0106), apatia (p=0,0469), aumento de bastonetes (p=0,0224) e leucocitose com desvio à esquerda. O conjunto de sinais clínicos encontrados mostrou-se equivalente aos sinais descritos na literatura em cães infectados por L. infantum e L. donovani nos países do velho mundo.

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.

Perfil clínico, sorológico e parasitológico em 88 cães naturalmente infectados por L. Chagasi

OBJETIVOS: Definir o perfil clínico em cães naturalmente infectados por L. chagasi em uma área endêmica no Brasil. MÉTODOS: Foram estudados 88 cães provenientes de Jequié (Bahia-Brasil), sendo analisados 215 parâmetros clínicos e realizados hemograma, sorologia (ELISA) e exame parasitológico (cultura de material obtido por punção esplênica). RESULTADOS: Os resultados positivos de cultura esplênica correlacionam-se com a densidade óptica obtida no ELISA, em 64 dos cães estudados (r=0,8555; p=0,00327), demonstrando a associação entre positividade à sorologia e à parasitologia. Em todos os animais do grupo estudado, evidenciou-se correlação entre positividade na cultura e ocorrência de palidez nas mucosas (p=0,0106), apatia (p=0,0469), aumento do número de bastonetes (p=0,0224) e tendência ao aumento na contagem global de leucócitos (evidências clínicas de anemia e resposta inflamatória, esta caracterizada por leucocitose com desvio à esquerda, na presença de infecção). Verificou-se correlação entre os resultados de ELISA e presença de lesões cutâneas pruriginosas (p=0,0402), conjuntivite (p=0,050), esplenomegalia (p=0,0272) e claudicação (p=0,0256), além de tendência à redução do hematócrito e no número de hemácias nos animais com valores mais elevados de densidade óptica de ELISA, confirmando dados de literatura. Surpreendentemente, achados clínicos clássicos, como onicogrifose, emaciação e linfoadenomegalia não mostraram correlação com positividade à sorologia ou à parasitologia. CONCLUSÃO: Alguns resultados obtidos discordam da descrição clássica dos sinais clínicos de leishmaniose visceral canina. Os sinais clássicos, possivelmente, refletem apenas o estado avançado da doença, tornando-se necessário um melhor conhecimento de importantes aspectos da evolução da doença.