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Canine leishmaniasis (CanL) is a vector-borne disease caused byLeishmania infantumand is transmitted by female phlebotomine sand flies primarily between animals and secondarily to humans. The course of infection may be different from one individual dog to another, ranging from spontaneous cure to acute evolution that leads to death, if proper management and therapy are not adopted. A parasitological cure is rarely achieved and clinical recurrences in CanL are frequent. Vaccination associated with the use of topical insecticides is undoubtedly the most effective form of prevention and control of the disease. In order to integrate the most important scientific knowledge of the literature in one objective publication, this review proposes a short overview of the main points of CanL.

American visceral leishmaniasis is a zoonosis of the New World. Dogs are the main reservoir of the disease and there is much interest in the understanding of mechanisms implicated in protection against canine infection. Nevertheless, most studies in dogs have not been carried out in organs that are targets of infection. This work is first to report the profile of cytokines and parasite burdens, as determined by real-time PCR, in the lymph nodes of dogs naturally infected with Leishmania chagasi. With this purpose, 18 mongrel dogs were divided in three groups: control non-infected dogs (n=6) and naturally infected animals with L. chagasi, asymptomatic (n=6) and symptomatic (n=6). Parasite burden in lymph nodes was 73-fold greater in symptomatic than asymptomatic animals. Prescapular lymph nodes of asymptomatic dogs had the highest expression of IFN-gamma and TNF-alpha and low parasite burden, indicating that these cytokines play a role in protection against infection. Highest expression of IL-10 and TGF-beta and high parasite burden were observed in symptomatic dogs, suggesting a role for these cytokines in the progression of disease. Hence, the balance of expression of IFN-gamma and TNF-alpha (protective) and IL-10 and TGF-beta (disease progression) in lymph nodes determine parasite burden and clinical expression in naturally infected dogs.

The aim of this study was to evaluate the diffuse intralobular fibrosis in dogs naturally infected with Leishmania (Leishmania) chagasi. One hundred five infected animals with positive serologic tests for Leishmania were divided into two clinical groups: 69 symptomatic animals and 36 asymptomatic. Special staining with Gomori, Heidenhain, Silver, and Picrosirius Red was applied to characterize fibrilopoesis. The tissue parasite load was measured by immunohistochemistry and associated histomorphometric analyses. Intralobular fibrosis was observed in all dogs, and more collagen deposition was confirmed in the infected animals than in the controls by these histomorphometric studies. There were significant differences among the distinct clinical groups. In fact, symptomatic dogs showed an increased collagen deposition in the liver compared with asymptomatic ones. A peculiar diffuse intralobular fibrosis, where the collagen fibers encircled small groups of hepatocyte(s), was observed in two cases (1.9%).

The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG(2000)-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non-long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non-pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum.

A novel liposomal formulation of meglumine antimoniate (MA), consisting of vesicles of reduced size, has been evaluated in dogs with visceral leishmaniasis to determine its pharmacokinetics as well as the impact of vesicle size on the targeting of antimony to the bone marrow. Encapsulation of MA in liposomes was achieved through freeze-drying of empty liposomes in the presence of sucrose and rehydration with a solution of MA. The resulting formulation, with a mean vesicle diameter of about 400 nm, was given to mongrel dogs with visceral leishmaniasis as an i.v. bolus injection at 4.2 mg Sb/kg of body weight. The pharmacokinetics of antimony were assessed in the blood and in organs of the mononuclear phagocyte system and compared to those achieved with the free drug and the drug encapsulated in large sized liposomes (mean diameter of 1200 nm). The targeting of antimony to the bone marrow was improved (approximately three-fold) with the novel liposomal formulation, when compared to the formulation of MA in large sized liposomes. This study provides the first direct experimental evidence that passive targeting of liposomes to the bone marrow of dogs is improved by the reduction of vesicle size from the micron to the nanometer scale.

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan by the species Leishmania infantum in the Old World and Leishmania chagasi in the New World. Several methods are currently employed for the diagnosis of CVL including microscopic detection of the parasite in bone marrow and lymph node aspirates, demonstration of specific antibodies anti-Leishmania in sera from infected animals, and isolation of the parasite by in vitro culture or by inoculation of laboratory animals. However, a definitive diagnosis is based on the actual detection of the parasite, which is conventionally achieved by examining Giemsa-stained smears or histopathological sections stained with hematoxylin and eosin. These methods have a low sensitivity, and therefore, they are often inconclusive. This is particularly true in canine organs that have a low level of parasitism such as kidneys, lungs, central nervous system, and testis, or, in some cases, the skin. The technique for immunohistochemical detection of leishmanial amastigotes in canine tissues has been reported previously and has proved to be undoubtedly efficient for the diagnosis. In this paper, we describe a straightforward and inexpensive immunohistochemical approach for Leishmania detection in formalin-fixed paraffin-embedded canine tissues. Amastigote forms of Leishmania were easily observed within macrophages in several organs from naturally infected dogs using the streptavidin–biotin immunohistochemical method with canine hyperimmune serum as the primary antibody. In addition, the secondary antibody used was not specific to canine immunoglobulin, characterizing a cross-immune reaction. Our results indicate that this technique could be a useful tool for epidemiological, clinical, and histopathological studies.

Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed. O crescente uso das nanotecnologias nas terapias avançadas tem permitido a observação de reações adversas específicas relacionadas às nanoestruturas. A toxicidade de uma nova formulação lipossomal de antimoniato de meglumina após dose única foi avaliada em cães com leishmaniose visceral. Grupos de 12 animais receberam por via intravenosa uma dose única de antimoniato de meglumina lipossomal (grupo I [GI], 6,5 mg Sb/kg), lipossomas vazios (GII) ou solução salina isotônica (GIII). A avaliação de parâmetros hematológicos e bioquímicos não revelou alterações significativas quatro dias após a administração. Nenhum efeito indesejável foi registrado no GIII. No entanto, reações adversas foram observadas em 67,7% dos cães de ambos os grupos que receberam formulações lipossomais. Os efeitos colaterais iniciaram momentos após a administração em "bolus" e desapareceram no decurso dos primeiros 15 minutos após o tratamento. Prostração, sialorréia e defecação foram os sinais clínicos mais frequentes, registrados em 33,3% e 41,6% dos animais dos grupos GI e GII, respectivamente. Taquipnéia, midríase, miose, vômitos e cianose também foram registrados em ambos os grupos. As reações adversas observadas neste trabalho foram atribuídas à ativação do sistema complemento pelas vesículas lipídicas em fenômeno conhecido como Pseudoalergia Relacionada à Ativação do Complemento (PARAC). A influência das características físico-químicas da formulação lipossomal no desencadeamento de PARAC é abordada.

We propose that canine visceral leishmaniasis (CVL) is a systemic fibrotic disease, as evidenced by the wide distribution of fibrosis that we have found in the dogs suffering from chronic condition. The inflammatory cells apparently direct fibrosis formation. Twenty-four cases (symptomatic dogs) were identified from a total of one hundred and five cases that had been naturally infected with Leishmania chagasi and had been documented during an epidemiological survey of CVL carried out by the metropolitan area of the municipality of Belo Horizonte, MG, Brazil. The histological criterion was intralobular liver fibrosis, as has been described previously in dogs with visceral leishmaniasis. In addition to the findings in the liver, here we describe and quantify conspicuous and systemic deposition of collagen in other organs, including spleen, cervical lymph nodes, lung and kidney of all the infected symptomatic dogs. Thus we report that there is a systematic fibrotic picture in these animals, where inflammatory cells appear to direct fibrosis in all organs that have been studied. Therefore we propose that CVL is a systemic fibrotic disease.

An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os ) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines., Author Summary Leishmania parasites are the causative agent of a spectrum of diseases characterized by severe lesions in skin or life threatening visceral infections. In the parasite life cycle, a range of morphological transitions can be found such as amastigotes (hosted in humans and others mammals) and promastigotes (located in the sand fly vector). The disease begins when the infective non-dividing promastigote metacyclic form is transmitted to mammalian hosts by the bite of an infected sand fly. The conditions for the development of these metacyclic promastigotes are still not fully understood. Here we tested the hypothesis that the presence or absence of purine will determine whether the parasite will proliferate or differentiate into the metacyclic form. Our experiments indicate that the presence of purines in the culture medium of Leishmania parasites interfere with the development of metacyclic promastigotes. Our results also show that although decreased proliferation and metacyclic differentiation occur simultaneously these two phenomena are independently regulated. Finally we show, for the first time in a natural vector, that metacyclogenesis is inhibited in vivo by adenosine, suggesting a new level of cell differentiation control in these protozoan parasites which is driven by purine sensing.

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT>50) and 36 (32.7%) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P50) e 36 (32,7%) para T. gondii (RIFI>16). Os títulos de anticorpos anti-L. chagasi em cães assintomáticos (n=10) foram significativamente (P

The achievement of parasitological cure of dogs with visceral Leishmaniasis (VL) remains a great challenge, since dogs act as main reservoir for transmission of Leishmania infantum to humans and respond poorly to conventional drugs including pentavalent antimonials. Liposome-encapsulated antimonials are hundreds of times more effective than the free drugs against VL based on parasite suppression in the liver. However, complete parasite elimination in dogs seems to depend on the ability of liposomes to reach less accessible infection sites such as the bone marrow and the skin. Recently, the reduction of liposome size from 1200- to 400-nm diameter was found to improve the targeting of Sb to the bone marrow of dogs with VL. In the present work, the influence of further reduction of vesicle diameter from 400- to 175-nm on the pharmacokinetics of Sb in dogs with VL and on the distribution of Sb in the liver, spleen and bone marrow were investigated. For this purpose, two liposome formulations of meglumine antimoniate with the same lipid composition but different mean hydrodynamic diameters were prepared. The formulations were given to mongrel dogs with VL as a single intravenous bolus injection and Sb concentrations were determined by graphite furnace atomic absorption spectroscopy. Surprisingly, much more prolonged blood levels of Sb were achieved from small size (175 nm) than medium size (400 nm) liposomes. Small size vesicles were also less effective than medium size ones in targeting Sb to the liver. On the other hand, similar Sb concentrations were achieved in both spleen and bone marrow. In conclusion, the prolonged blood circulation time of liposomes with 175-nm diameter makes this nanosystem suitable for passive drug targeting to the less accessible infection sites in dogs with VL.

In recent years, cases of feline visceral leishmaniasis (FVL) have been described in different countries. In urban areas, domestic cats are suggested as possible alternative reservoirs of Leishmania (L.) infantum, the causal agent of visceral leishmaniasis (VL). This paper reports the first case of infection of Lutzomyia longipalpis by L. infantum of a naturally infected cat from Brazil through xenodiagnosis. The presence of a cat with FVL and its infectivity to the natural vector in Belo Horizonte city, an endemic area of VL in Brazil, suggests the need for further studies to determine the rate of occurrence of FVL among domestic cats and the infectivity ratio of L. longipalpis in endemic areas, and what is the role of these animals in the epidemiology of the disease.

Dogs are the most important reservoir of Leishmania (L.) infantum, the causal agent of visceral leishmaniasis (VL) in Brazil. Vectorial infection is the main route of transmission of the parasites. This paper reports the first case of vertical transmission of L. infantum in Brazil, confirmed by PCR and immunohistochemistry techniques in samples from spleen and liver of two stillborn pups from a bitch naturally infected with L. infantum in Belo Horizonte city, endemic area of VL. This result confirms the existence of transplacental transmission of Leishmania between dogs, and suggests the need for further studies to determine the rate of occurrence of this fact in endemic areas and what is their role in the epidemiology of the disease.

In this study, we investigated in dogs the immunogenicity and protective immunity against Leishmania (Leishmania) chagasi infection induced by vaccination with a formulation containing the recombinant A2 protein, an amastigote specific antigen, and saponin. Vaccinated animals produced significantly increased levels of total IgG and IgG2, but not IgG1 anti-A2 antibodies, and remained negative in conventional leishmaniasis serodiagnostic methods. Significantly increased IFN-gamma and low IL-10 levels were detected in vaccinated animals before and after challenge, as compared to control animals. Importantly, while the symptoms onset appeared as early as three months after infection in most control dogs, 14 months after challenge, 5 out of 7 vaccinated dogs remained asymptomatic. Therefore, immunization with rA2 antigen was immunogenic and induced partial protection in dogs, and allowed the serological differentiation between vaccinated and infected animals, an important requirement for a canine visceral leishmaniasis (CVL) vaccine.

Background There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi. Results In the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages. Conclusion These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phle-botomine 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, oligosymp-tomatic 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 oligo symptomatic 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. (c) 2007 Elsevier Ltd. All rights reserved.

Blood transfusion is an important routine practice in veterinary medicine that generally involves the use of whole blood. Permanent blood donors must be vaccinated against viral infections that affect dogs and submitted periodically to clinical and serological examinations to detect blood-transmitted diseases. There is a very high risk of transmission of infectious agents, particularly protozoans due to their long incubation periods, subclinical persistence in infected animals and likelihood of remaining viable in bloodstocks. The aim of the present study was to identify the potential of asymptomatic and oligosymptomatic dogs for Leishmania infantum transmission as a result of transfusional practice. Nineteen Leishmania-seropositive adult dogs of both sexes and indeterminate breeds were selected as donors. The animals were classified as symptomatic, oligosymptomatic or asymptomatic after clinical examination and evaluated by ELISA, IFAT and bone marrow puncture biopsies. Whole blood and monocyte cells were collected and used for dog's serological evaluation and inoculation in culture medium as well as in hamsters. All but three dogs were positive for IFAT, ELISA and parasite demonstration in bone marrow aspirates, irrespective of their clinical conditions. Parasites were detected in 77% of the whole blood and 90% of the monocyte cultures. Six months after inoculation with whole blood or monocytes, hamsters developed infection and clinical symptoms of visceral leishmaniasis, as well as positive titres measured by ELISA. These results suggest that blood donors should be monitored periodically and rigorously for Leishmania infection, to prevent dissemination of the disease through blood transfusion.

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30µg of each protein at 15-day intervals combined with 250µg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein. Proteínas imunogênicas da vacina polivalente de promastigotas mortas de leishmanias (Leishvacin®) produzida pela Biobrás Bioquímica do Brasil, Montes Claros, Minas Gerais, Brasil foram identificadas e purificadas por eletroforese em gel de poliacrilamida e eletroeluição. Camundongos C57BL/10 foram vacinados com proteínas de pesos moleculares estimados em 42, 46, 63, 66, 73, 87, 97 e 160kDa em três doses de 30µg de cada proteína combinada com 250µg de Corynebacteriumparvum em intervalos de 15 dias e desafiados com uma infecção desafio de 10(5) promastigotas infectantes de Leishmania (Leishmania) amazonensis na base da cauda. Foram avaliadas a habilidade dessas proteínas em induzir resposta imune e proteção dos animais vacinados após a infecção desafio. Nenhuma diferença estatística foi observada nos níveis de IFN-g nos grupos vacinados em comparação ao grupo controle. Proteção de 28,57; 42,86; 57,14; 42,86; 42,86, 57,14; 42,86; 57,14% foi demonstrado para cada proteína.

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan of the genus Leishmania. In the New World, the disease is caused by the species Leishmania (Leishmania) chagasi. There are only a few studies on the histopathology of lymph nodes in canine leishmaniasis. In the present paper, we report a histopathological description of lymph nodes considering animals with a defined clinical status and the parasite burden of lymph node tissues. Forty-eight mongrel dogs naturally infected with L. chagasi, were obtained from two endemic areas of Brazil. Cervical, axillary and popliteal lymph nodes were analyzed. The parasite burden, expressed as “Leishman–Donovan units”, was variable among the defined types of clinical condition. Asymptomatic dogs can show higher parasitism than oligosymptomatic or symptomatic animals. Grossly, a generalized lymphadenopathy was found, but it was mainly observed in cervical and popliteal nodes. Histologically, the increased number and size of lymphoid follicles, and the marked hypertrophy and hyperplasia of medullary macrophages (cords and sinus) explained the lymphadenopathy. In addition, the clinical status or the tissue parasitism load might not be directly related to the intensity of the lesions.

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ−/XENO−). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ+/XENO+) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b+TLR2+ and CD11b+MHCII+, higher values were obtained from dogs with IHQ−/XENO− than dogs with IHQ+/XENO+ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b+TLR2+ and NO with higher values for dogs with IHQ−/XENO− than dogs with IHQ+/XENO+), led to the conclusion that IHQ−/XENO− dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.

Foi realizado um estudo comparativo da reação de imunofluorescência indireta em eluatos de sangue de cães infectados experimentalmente com diferentes tripanosomatídeos. Utilizaram-se como antigenopromastigotas de L. mexicana, L. braziliensis e L. chagasi. Os resultados mostraram que a sensibilidade do método foi de 87,5% para o diagnóstico do calazar canino, independentemente do antigeno empregado; e que ocorre reação cruzada com Leishmaniose tegumentar em 75% dos casos e com doença de Chagas em 83,3%. Levantamento epidemiológico em área de leishmaniose confirma que a reação de imunofluorescência em eluatos de sangue canino fornece reações cruzadas em cães infectados com Leishmania braziliensis e L. chagasi. Não se verificou reação cruzada pela RFC. Sugere-se a utilização da reação de imunofluorescência nas campanhas de saúde pública, mas é de se chamar a atenção para o fato de que as taxas de positividade não devem ser utilizadas como indicadores da prevalência do calazar canino.A comparative study was made of eluates of the blood of dogs experimentally infected with different trypanosomatids. Using antigens prepared from promastigotes of Leishmania mexicana, L. braziliensis and L. chagasi, assessments were made by the indirect immunofluorescence test. The results showed a sensitivity of 87,5% in the diagnosis of canine visceral leishmaniasis, independent of antigen used. Cross-reactions occurred in 75% of cases of cutaneous leishmaniasis and 83,3% of dogs with chagas' disease. An epidemiological survey in an area of leishmaniasis confirmed that immunofluorescence tests on eluates of dogs' blood give cross-reactions between L. braziliensis and L. chagasi. The results suggest that such testing could be useful in public health campaigns but attention is drawn to thefact that the level ofpositive reactions cannot be used as an indicator of the prevalence of canine kala-azar.

Background We compared skin biopsy samples from different anatomical regions for detecting Leishmania in dogs, using histological (HE), immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques. Results The sensitivity was 82.8 percent for PCR, 62.1 percent for IHC and 44.8 percent for HE. These methods do not appear to depend on the clinical status of the animal or the anatomical source of the skin sample; there is no "best region" for any method. However, PCR was more effective than IHC and HE for ear and nose skin samples whereas IHC was better than HE for nose samples. There was weak agreement between PCR and HE for all tissue samples; good agreement between PCR and IHC for ear and abdomen samples, and weak agreement for nose; and optimal agreement between IHC and HE for ear and abdomen and good agreement for nose samples. Conclusion The PCR on ear skin could be the best procedure for diagnosing canine visceral leishmaniasis. The good agreement between PCR and IHC indicates that IHC can be used as an alternative method. Finally, tissue samples from ears, nose and abdomen, particularly ears and nose, are potentially useful for diagnosing canine visceral leishmaniasis independently of the animal's clinical status.

Two controlled, double blind field trials of a non-living promastigote vaccine against New World Cutaneous Leishmaniasis (NWCL) were conducted in 1981 and 1983 in Brazil. Brazilian Army conscripts were randomly assigned to the vaccine or placebo groups and tested during their training in the Amazon jungle, a high risk area for NWCL. The results obtained showed: no significant differences between the vaccine and the placebo groups with respect to a number of characteristics (age, race, previous contact with the jungle, etc.); no significant differences between the participants who got and who did not get NWCL during the trial, with respect to length of exposure, contact with the jungle, etc. and a reduction of 67.3 and 85.7% in the annual incidence rate of NWCL, in 1981 and 1983 respectively (although the difference between incidence rates of the disease in vaccinated and control groups in the 1983 trial was not statistically significant), among those vaccinated who had converted to a positive leishmanin skin test as compared with the placebo groups.

Canine visceral leishmaniasis (CVL) is a zoonotic disease that presents variable clinical and laboratory aspects. The aims of this study were to identify the main biochemical/hematological status of dogs naturally infected with Leishmania (Leishmania) infantum and to associate theses parameters with clinical forms of CVL. Blood samples were analyzed from 51 dogs, 15 uninfected (control group) and 36 infected, which were classified clinically in three groups: asymptomatic (n=12), oligosymptomatic (n=12) and symptomatic (n=12). All the infected dogs showed lower albumin/globulin ratios (A-G ratio) than the limit of reference. The mean values of total protein, urea, α-globulin 2, globulin and A-G ratio of infected dogs were outside the reference interval and differed significantly from those of the controls. Anemia was detected only in groups that showed clinical signs of the disease, and a statistical analysis indicated a significantly higher frequency of lower eritrogram in these groups than in the asymptomatic group. In addition, a significant association was observed between anemia and the presence of the symptoms, with dogs displaying higher erythrogram values showing better clinical conditions. These results provide additional evidence that the clinical forms of CVL may reflect on the erythrogram status. A leishmaniose visceral canina (LVC) é uma zoonose com aspectos clínicos e laboratoriais variáveis. O objetivo deste trabalho foi identificar os principais achados hematológicos e bioquímicos em cães naturalmente infectados com Leishmania (Leishmania)infantum e associar esses parâmetros com as formas clínicas da LVC. Foram analizadas amostras sanguíneas provenientes de 51 cães, sendo 15 cães não infectados (grupo controle) e 36 infectados, os quais foram classificados clinicamente em três grupos: assintomáticos (n=12), oligossintomáticos (n=12) e sintomáticos (n=12). Todos os cães infectados apresentaram valores na relação albumina/globulina (A/G) abaixo do limite inferior de referência. Os valores médios de proteína total, uréia, α-2 globulina, globulina e A/G dos grupos de cães infectados permaneceram fora dos intervalos de referências e significativamente diferente quando comparados aos do grupo controle. Anemia foi registrada somente nos grupos de animais que manifestavam sinais clínicos da enfermidade, sendo que nas análises estatísticas constatou-se frequencia significativamente maior de alterações no eritrograma quando comparados ao grupo assintomático. Associação significativa foi observada entre anemia e a presença de sinais clínicos, onde os cães com os maiores valores de eritrograma apresentavam a melhor condição clínica. Os resultados fornecem evidência adicional que as formas clínicas da LVC podem refletir no eritrograma.

Background The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT), including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania. Methods Twenty mongrel dogs naturally infected with Leishmania (Leishmania) infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG) state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry) evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania). The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs. Results The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal) irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT. Conclusion The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance).

Os estudos das alterações da matriz extracelular (MEC) na leishmaniose visceral são, sobretudo, aqueles relacionados a deposição de colágeno (fibropoese). Na leishmaniose visceral canina (LVC) há estudos mostrando intensa fibropoese associada à inflamação crônica que se instala em vários órgãos como, pulmões (pneumonite intersticial), baço (esplenite esclerosante granulomatosa), fígado (hepatite granulomatosa), linfonodos (linfadenite granulomatosa) e rins (glomerulonefrites e nefrite intersticial). Todavia, de acordo com a literatura, não há uma caracterização mais detalhada da fibropoese renal, tanto glomerular ou túbulo-intersticial em cães natural ou experimentalmente infectados com parasitos do gênero Leishmania da espécie L. (L.) infantum. E mais ainda, não há descrição detalhada da anatomiapatológica de rins de cães experimentalmente infectados com L. (L.) infantum. No Brasil, o principal agente etiológico da LVC é a espécie L. (L.) infantum, mas trabalhos recentes têm demonstrado cada vez mais a participação de outra espécie viscerotrópica: a L. (L.) amazonensis. Assim, os objetivos desse trabalho foram os de estudar alguns aspectos anatomopatológicos de rins obtidos de cães naturalmente infectados com L. infantum quanto de cães experimentalmente infectados com duas diferentes cepas de L. infantum e uma única cepa de L. amazonensis, com ênfase especial ao processo da fibropoese. Oitenta e sete amostras de fragmentos de rim emblocados em parafina (acervo) foram assim distribuídas: (1) sessenta e duas amostras de rins de cães, sem raça definida, naturalmente infectados com L. infantum, com diagnóstico sorológico e parasitológico positivos, obtidos de Centros de Zoonoses, da região metropolitana de Belo Horizonte, MG, denominado grupo CNI; (2) cinco amostras de rins cães experimentalmente infectados com L. infantum cepa MCAN/BR/2002/BH401, na dose de 5x107 promastigotas/ml de L. infantum, via endovenosa, denominado grupo BH401; (3) onze amostras de rins de cães experimentalmente infectados com L. infantum cepa MCAN/BR/2000/BH400, na mesma dose e via do grupo anterior, denominado grupo BH400. (4) cinco amostras de rins de cães experimentalmente co-inoculados com L. infantum cepa MCAN/BR/2002/BH401 (BH401) e L. amazonensis cepa PH8 (FLA/BR/1967/PH8), denominados grupo Coinoculados Os cães desse grupo foram infectados com formas promastigotas de L. infantum na dose de 2,5x107parasitos /ml e de formas promastigotas de L. amazonensis na dose de 2,5x107parasitos/ml, ambas por via endovenosa, em uma única supensão. A infecção experimental ocorreu aos 11 meses de vida de cada animal para todos os grupos, e o intervalo de tempo da infecção experimental foi de dois anos para os grupos BH401 e co-infectados, e de dezoito meses para o grupo BH400. Todos os animais com exames parasitológicos positivos para Leishmania aos 6-8 meses após a infecção, e que se mantiveram positivos até o final do ensaio experimental; (5) quatro amostras de rim de cães da raça Beagle, dois anos e meio de idade, todos com exames sorológicos e parasitológicos negativos para Leishmania. A eutanásia desses animais ocorreu juntamente com as dos grupos BH401 e os Co-inoculados. Após análises histológicas pela microscopia óptica convencional, todos os animais, revelaram a fibropoese glomerular e intersticial, em menor ou maior intensidade, associadas respectivamente aos diferentes tipos de glomerulonefrites e a nefrite intersticial crônica. Entretanto, a frequência das lesões foi distinta entres os grupos. A fibrose foi nitidamente mais intensa no grupo BH401, seguido dos animais do grupo CNI. Estudo imuno-histoquímico para investigar alguns aspectos dos mecanismos dessa fibrose foi realizado tendo como alvo marcadores celulares para miofibroblastos (marcadores mesenquimais) como a alfa-actina (-SMA) e a vimentina. A citocina fator transformador do crescimento do tipo â(TGF-â) e as metaloproteinases (MMPs), enzimas celulares secretadas de forma inativa relacionadas ao remodelamento da MEC, do tipo 2 (MMP-2), 7 (MMP7) e 9 (MMP-9), foram também investigadas. Novamente, o grupo BH401, seguido dos Co-inoculados, foi o mais marcante para a visualização da expressão de todos esses marcadores quando comparados aos demais grupos. As glomerulonefrites do tipo membranoproliferativas foram mais frequentes nos grupos BH401 (75%) e CNI (75%). As glomerulonefrites do tipo membranosa (mediante diagnostico diferencial pela prata metenamina de Jones) ocorreram em todos os animais do grupo Co-inoculados e em alguns animais do grupo BH400 (33,3%). As glomerulonefrites do tipo esclerosante apareceram apenas nos animais do grupo BH401 (100%). Estudo morfométrico da celularidade glomerular e deposição de matriz mesangial visualizadas pelas colorações especiais do periodic acid shiff (PAS) e o tricromico de Masson confirmaram lesões mais marcantes no grupo BH401. Macrófagos mesangias L1 e CD163 positivos foram visualizados por microscopia confocal preferencialmente no grupo BH401 reforçando os achados descritos. A presença de formas amastigotas intracelulares de Leishmania, visualizadas por imuno-histoquímica, foi observada em especial no grupo BH401, seguido do grupo CNI, mas não visualizados no grupo BH400 e nos cães CC. Os resultados confirmam a fibrose renal com marcações intensas para -SMA, vimentina e TGF-beta, e MMPs, em especial do grupo BH401, indicando ser a cepa MCAN/BR/2002/BH401 uma boa escolha para o estudo das alterações renais no modelo experimental da LVC. Extracellular matrix (ECM) changes studies, in visceral leishmaniasis, are mainly those related to collagen deposition (fibropoese). There are studies in canine visceral leishmaniasis (LVC) showing intense fibropoiesis associated to a chronic inflammation that occurs in various organs such as lungs (interstitial pneumonitis), spleen (granulomatous sclerosing splenitis), liver (granulomatous hepatitis), lymph nodes (granulomatous lymphadenitis) and kidneys (glomerulonephritis and interstitial nephritis). However, according to the literature, there are no renal fibropoiesis detailed studies, either glomerular or tubulointerstitial renal lesions in dogs naturally or experimentally infected with parasites of the genus Leishmania, L. (L.) infantum specie. Furthermore, there is no detailed description of the pathological aspects of the kidneys of dogs experimentally infected with L. (L.) infantum. In Brazil, the main etiological agent of the LVC is the species L. (L.) infantum, but recent works have increasingly demonstrated the participation of another viscerotropic species: L. (L.) amazonensis. The objectives of this work were to study some pathological aspects of kidneys obtained from dogs naturally infected with L. infantum and from dogs experimentally infected with two different strains of L. infantum and a single strain of L. amazonensis, with special emphasis on fibrotic process. Eighty-seven specimens of paraffin-embedded kidney fragments were collected as follows: (1) sixty-two kidney samples of adult mongrel dogs, naturally infected with L. infantum, with positive serological and parasitological diagnosis, obtained from Centers of Zoonoses of the metropolitan region of the municipality Belo Horizonte, MG, denominated group CNI; (2) five kidney samples of adult Beagles experimentally infected with L. infantum strain MCAN / BR / 2002 / BH401 at the dose of 5x107 promastigotes/ml of L. infantum, endovenous route, denominated group BH401; (3) eleven kidney samples of adult Beagles dogs experimentally infected with L. infantum strain MCAN / BR / 2000 / BH400, at the same dose and same route of the previous group, denominated group BH400. (4) five kidney samples of adult Beagles experimentally co-inoculated with L. infantum strain MCAN / BR / 2002 / BH401 (BH401) and L. amazonensis strain PH8 (FLA / BR / 1967 / PH8), denominated Co-inoculated group. The dogs of this group were infected with promastigote forms of L. infantum at the dose of 2.5 x 107 parasites / ml and promastigotes forms of L. amazonensis at the dose of 2.5 x 107 parasites / ml, both intravenously, in a single strain. The experimental infection occurred at 11 months of life of each animal for all groups, and the time interval of the experimental infection was two years for the BH401 and co-infected groups, and eighteen months for the BH400 group. All animals with Leishmania-positive parasitological examinations at 6-8 months post-infection and remained positive until the end of the experimental trial; (5) four kidney samples from adult Beagle dogs, two and a half years old, all with negative serological and parasitological tests for Leishmania, denominated control group (CC). . The euthanasia of these animals occurred along with those of the BH401 and Co-inoculated groups. After histological analysis by conventional optical microscopy, all animals revealed glomerular and interstitial fibropoiesis, discrete to intense, associated with different types of glomerulonephritis and chronic interstitial nephritis. However, the frequency of lesions was different between groups. Fibrosis, for example, was markedly more intense in the BH401 group, followed by animals in the CNI group. Immunohistochemical study to investigate some aspects of the mechanisms of this fibrosis was carried out targeting cell markers for myofibroblasts (mesenchymal markers) such as alpha-actin (áSMA) and vimentin. The cytokine transforming growth factor beta (TGF-â) and metalloproteinases (MMPs), cellular enzymes secreted inactivated related to ECM remodeling, type 2 (MMP-2), 7 (MMP-7) and 9 (MMP9), were also investigated. Again, the BH401 group, followed by the Co-inoculated group, was the most striking for the visualization of the expression of all these markers when compared to the other groups. Membranoproliferative glomerulonephritis was more frequent in the BH401 (75%) and CNI (75%) groups. Membranous glomerulonephritis (by means of a differential diagnosis of Jones silver metenamine) occurred in all animals of the Co-inoculated group and in some animals of the BH400 group (33.3%). Sclerosing glomerulonephritis appeared only in animals of group BH401 (100%). Morphometrical study of glomerular cellularity and mesangial matrix deposition visualized by the special staining as periodic acid shiff (PAS) and Masson triochrome confirmed more striking lesions in the BH401 group. Mesangial macrophages, L1 and CD163 positive cells, were visualized by confocal microscopy preferably in the BH401 group reinforcing the described findings. The presence of intracellular amastigotes forms of Leishmania, visualized by immunohistochemistry, was observed especially in the BH401 group, followed by the CNI group, but not visualized in the BH400 group and in the CC dogs. The results confirm renal fibrosis with intense labeling for á-SMA, vimentin and TGF-beta, and MMPs, especially the BH401 group, indicating that the MCAN / BR / 2002 / BH401 strain is a good choice for the study of renal LVC experimental model.

As leishmanioses constituem um complexo de enfermidades que atingem o homem, causadas por protozoários flagelados intracelulares obrigatórios que residem predominantemente em células do sistema mononuclear fagocitário dos hospedeiros vertebrados. Nas Américas, os cães (Canis Lupus familiares) são considerados os principais reservatórios domésticos para a leishmaniose visceral (LV) humana. No Brasil, as medidas preconizadas, referentes ao controle da LV, consideram o diagnóstico precoce e tratamento dos casos humanos; aspersão de inseticidas residuais, para combate do inseto vetor, e inquérito sorológico dos cães, com posterior eutanásia daqueles soropositivos. Entretanto, a eliminção dos cães, por muitos, não é aceita por questões econômicas, sociais e éticas. O tratamento do cão infectado, com a terapêutica convencional é discutível, assim, a utilização de formulações lipossomais carreando fármacos com ação leishmanicida constitui uma estratégia promissora. Somado a isso, o co-tratamento com fármacos com ação leishmaniostática constituem uma boa estratégia no tratamento da leishmaniose visceral canina (LVC). Dessa forma, nesse estudo, foram utilizados quarenta e seis cães naturalmente infectados com Leishmania (Leishmania) chagasi, classificados como sintomáticos. Esses foram alocados, aleatoriamente, em seis grupos experimentais: grupo antimoniato de meglumina encapsulado em lipossomas nanométricos (Ant); grupo lipossomo vazio (LipV); grupo antimoniato de meglumina encapsulado em lipossoma associado ao alopurinol (Ant+Alo); grupo lipossoma vazio associado ao alopurinol (LipV+Alo); grupo alopurinol (Alo) e controle (C), sendo esse último mantido como controle da infecção. O protocolo terapêutico preconizado consitiu em seis administrações, a cada 96h, por via endovenosa, de 6,5mg/Kg de antimoniato de meglumina encapsulado em lipossomas nanométricos; seis administrações, a cada 96h, por via endovenosa, em volume correspondente de lipossomas nominados vazios; sendo que, metade dos cães, de cada um dos grupos supracitados, receberam conjuntamente 20mg/Kg de alopurinol, uma vez ao dia, por um período de 140 dias. Outro grupo recebeu apenas o alopurinol, por um período similar ao anterior. Avaliações clínicas foram realizadas antes do início do tratamento, nominado tempo (T0) e nos momentos correspondentes a 50, 80, 110, 140, 170 e 201 dias após o início do tratamento (T50, T80, T110, T140, T170 e T201). De forma semelhante, amostras de sangue periférico foram coletadas no momento T0, T60, T140 e T200. Procedeu-se à imunomarcação das células mononucleares do sangue periférico com anticorpos anti-CD11b, anti-MHCII, anti-TL-2, anti-CD4 e anti-CD8 avaliados pela técnica de citometria de fluxo. Biópsias de pele, da face interna da pina direita, foram coletadas nos momentos T0, T60, T140. No T201 amostras de pele, fígado, baço e linfonodo cervical superficial foram coletadas durante a avalição necroscópica e processadas para avaliações histológicas rotineiras. Estudos semi-quantitativos das alterações histológicas e qualitativas do parasitismo tecidual (imuno-histoquímica) foram realizados utilizando-se microscópia optica convencional. O tratamento envolvendo o alopurinol (Alo) principalmente associado ao antimonial pentavalente encapsulado com lipossomos manométricos (Ant+Alo) promoveu significativa redução do parasitismo tecidual (pele, fígado e baço) e melhora clínica de todos os animais. Entretanto, apesar dessa melhora não houve modulação da resposta imune do tipo celular (frequência da população linfocitária CD4/CD8) nos cães estudados. Alem disso, a expressão de CR3 e TLR-2 nos monócitos CD14+, apesar das variações observadas ao longo da cinética do diferentes protocolos de tratamento, não apresentaram correlação com o quadro clinico. Esses mesmos grupos (Alo e Ant+Alo) mostraram remissão dos sinais dermatológicos clássicos da LVC; além de diminuição das alterações histológicas na pele caracterizada principalmente pela menor frequência de dermatite crônica. Quando comparado ao grupo LipV, esses grupos apresentaram peso relativo do fígado significamente inferior. Cães tratados com alopurinol, com as diversas associações, e cães tratados com antimoniato de meglumina encapsulado com lipossomos manométricos, quando comparados ao grupo de cães tratados com lipossomas vazios, apresentaram diminuição no número de granulomas intralobulares hepáticos caracterizando uma resolusão da hepatite crônica. Cães do grupo Ant+Alo apresentaram peso relativo do baço significamente menor em relação ao grupo Alo e C. O tratamento nas diferentes condições experimentais não interferiu significativamente nas lesões esplênicas clássicas como a depleção da área T-dependente da polpa branca, evidenciada microscopicamente na maioria dos animais estudados. Cães do grupo Ant+Alo e Alo apresentaram uma redução significativa da celularidade dos seios medulares em comparação ao grupo LipV, com tendência a um menor parasitismo tecidual. Podemos dizer que os animais sob os diferentes protocolos terapêuticos, apesar da melhora clínica e parasitológica, devam ser acompanhados por um maior período de tempo, visto que alterações histológicas como fibrose e degeneração foram notadamente encontradas no fígado dos animais tratados com antimoniato associado ao alopurinol e alopurinol sozinho. Leishmania are intracellular obligate parasites that reside predominantly in mononuclear phagocyte cells of vertebrate hosts. In the Americas, dogs (Canis lupus familiaris) are considered the main domestic reservoirs for human visceral leishmaniasis (HVL). In Brazil, the measures proposed for the LV control, consider early diagnosis and treatment of human cases, residual spraying insecticides to combat the insect vector and dogs serological survey, with following euthanasia of positive animals. However, dogs euthanasia is not sustained by economic, social and ethical concerns. The treatment of infected dog with conventional therapy is questionable thus use of liposomal formulations with leishmanicidal activity is a promising strategy. Added to this, the co-treatment with leishmaniostatic drugs represents a good strategy in canine visceral leishmaniasis (CVL) treatment. In our study, treated dogs were divided in six groups as follow: Group I composed by eight dogs treated with meglumine antimoniate encapsulated in liposomes (LMA) associated with allopurinol (Allop) denominated LMA+Allop; Group II composed by eight dogs treated with LMA; Group III composed by eight dogs treated with allopurinol (Allop); Group IV composed by eight dogs treated with empty liposome (LEMP) associated with Allop denominated group LEMP+Allop; Group V composed by eight dogs treated with LEMP. Group VI composed by twelve dogs treated with saline solution (SG). The recommended therapeutic protocol consisted in six administrations every 96h, of 6.5 mg/kg/i.v. LMA; six administrations every 96h, intravenously, into the corresponding volume of empty liposome; and 20mg/kg of allopurinol, once daily for a period of 140 days. Clinical assessments were done before treatment begins, named time (T0) and at moments corresponding to 50, 80, 110, 140, 170 and 201 days after establishment of treatment (T50, T80, T110, T140, T170 and T201). Similarly, peripheral blood samples were collected at time T0, T60, T140 and T200. The procedure was peripheral blood mononuclear immunostaining cells with anti-CD11b, anti-MHCII, anti-TL-2, anti-CD4 and anti-CD8 evaluated by the flow cytometry technique. Skin biopsies, of the inside region of the right pina, were collected at T0, T60, T140. In T200 samples of skin, liver, spleen, superficial cervical lymph nodes were collected during necropsy, which was processed for routine histological evaluations. Semi-quantitative and qualitative histologic studies and parasitism evaluation (immunohistochemistry) were performed using conventional optical microscopy. The Allop group concerning mainly associated with LMA promoted significantly reduced parasitis load (skin, liver and spleen) and clinical improvement of all animals. However, despite this improvement, in dogs studied, was not observed immune response modulation (CD4/CD8 lymphocyte population frequency). Furthermore, the TLR-2 and CR3 expression, in CD14+ monocytes, despite variations observed, along the kinetics of different treatment protocols, did not correlate with the clinical picture. Allopurinol treated dogs mainly associated with LMA showed remission of dermatological classic signs of LVC. Those Allop group, mainly LMA associated showed histological alterations decreased mainly characterized by a lower chronic dermatitis frequency. Allopurinol treated dogs and meglumine antimoniate encapsulated in liposomes treated groups showed significantly lower relative liver weight than in the empty liposomes group. Allopurinol treated dogs with the various associations and treated dogs with encapsulated meglumine antimoniate liposomes when compared to empty liposomes group, showed a decrease of intralobular hepatic granulomas number and hepatic chronic signs. Meglumine antimoniate encapsulated liposomes group associated with allopurinol showed significantly lower in relative weight of the spleen than in the allopurinol-treated group and control. The treatment in the different experimental conditions did not showed significantly alteration in conventional splenic lesions as white pulp T-dependent deplation zone that was observed in most animals studied. The LMA+Allop and Allop groups showed a significant sinnus medullary cellularity reduction as compared to the empty liposome group with a lower trend tissue parasitism. We could say that the animals under different therapeutic protocols, despite the clinical and parasitological improvement, must be accompanied by a longer period of time, since histological changes such as fibrosis and degeneration were mainly found in the liver of LMA + Allop and Allop groups.

A leishmaniose tegumentar americana (LTA), como conhecida nas Américas, trata-se de uma infecção dermatológica resultante da multiplicação intracelular de formas amastigotas de Leishmania em células do sistema fagocítico mononuclear. O diagnóstico precoce e correto da LTA é fundamental para o estabelecimento de um tratamento efetivo, porém, os métodos diagnósticos atualmente disponíveis apresentam baixa sensibilidade, custo elevado ou demandam ampla estrutura técnica e laboratorial. Assim, o objetivo principal desse trabalho foi padronizar um método parasitológico imuno-histoquímico para o diagnóstico de certeza da LTA. A padronização baseou-se em um protocolo previamente descrito por TAFURI et al., (2004), na literatura, utilizando-se o método imuno-histoquímico da estreptoavidina peroxidase, que empregou como anticorpo primário, soro hiperimune de cão naturalmente infectado por Leishmania infantum chagasi. As amostras foram obtidas de 73 pacientes com LTA, não submetidos a qualquer tratamento, procedentes do Ambulatório de Leishmanioses Dr. Paulo de Araújo Magalhães, Caratinga/MG, Todos os pacientes apresentavam lesões típicas da forma localizada da LTA, que variou de uma a quatro lesões, localizadas, sobretudo, em áreas expostas do corpo e com tempo de evolução de 15 a 180 dias. Em sua maioria eram lavradores, residentes na zona rural, pertenciam a ambos os sexos e à faixa etária de 16 a 45 anos. Fragmentos de pele obtidos de biópsias cutâneas foram fixados em solução de formol tamponado e incluídos em parafina. Para a visualização da ligação do anticorpo primário com antígenos de espécies de Leishmania que causam LTA, utilizou-se o complexo da estreptoavidina peroxidase e um polímero livre de biotina. Como exames complementares ao método imuno-histoquímico proposto, foram realizados estudos histológicos por Hematoxilina e Eosina (HE), imuno-histoquímicos empregando-se anticorpo monoclonal anti-lipofosfoglicano (LPG) de Leishmania e a reação em cadeia da polimerase (PCR). As principais alterações histopatológicas apresentadas foram acantose e papilomatose, além de hiperceratose e paraceratose. Na derme observou-se moderado a acentuado infiltrado inflamatório crônico difuso de células mononucleares, principalmente plasmócitos, macrófagos e linfócitos, além de algumas células gigantes do tipo Langhans. Não foi observada reação granulomatosa típica (granuloma completo) e, em algumas amostras foram identificadas áreas de necrose fibrinoíde. A técnica proposta foi padronizada e as formas amastigotas de Leishmania foram facilmente identificadas, com coloração acastanhada, conformação arredondada, em 91,78% dos fragmentos de pele da amostra, sem a ocorrência de coloração de fundo inespecífica (background). A técnica padronizada apresentou elevada sensibilidade para a identificação das amastigotas, que ocorreu de forma mais fácil e rápida, quando comparada com a pesquisa do parasito através da coloração pela Hematoxilina e Eosina, com 17,81% de positividade e pela técnica de imuno-histoquímica, que usou anticorpo monoclonal anti-LPG, que apresentou 71,23% de positividade. O resultado da PCR, embora tenha revelado 100% de positividade, não mostrou diferença estatisticamente significativa quando comparado ao método de imuno-histoquímica (p=0,7671). O uso do polímero livre de biotina como sistema de visualização na técnica de imuno-histoquímica, com soro hiperimune de cão infectado, permitiu reduzir de forma notável o tempo de realização da técnica (de 24 horas para 06 horas), sem elevação do custo final da reação. Conclui-se que o método de imuno-histoquímica padronizado, permitiu o diagnóstico de certeza da LTA com, baixo custo, rapidez e fácil execução quando comparado com outras técnicas de imuno-histoquímica e a PCR. Human Cutaneous Leishmaniasis, in America is known as American Tegumentar Leishmaniasis (LTA), is a cutaneous disease due an infectious agent, a protozoan of genus Leishmania where the amastigotes forms are able to multiply inside cells of mononuclear system of vertebrate host. The earlier and definitive diagnostic for LTA is fundamental to define the the patient treatment. At present, the diagnostic methods have shown low sensibility, laborious and time consuming. Thus, the aim of this study was to patronize an immunohistochemical parasitological method to LTA diagnostic. This study was based on another previously published immunohistochemistry method where it uses serum of a dog naturally infected with Leishmania infantum as primary antibody anti-Leishmania. 73 dermal biopsies samples of patients with LTA, from Caratinga/MG, were fixed in formol buffer solution (10%, pH=7,0) and included in paraffin. Previous parasitological exams were confirmed by positive dermal imprints stained by Giemsa and positive Montenegro Intradermal Reaction (IRM) test, All adult patients of both sex, living in rural zone nearest the forest, showed one to four classical cutaneous lesions of LTA, with 15 to 180 days ears old, mainly localized in external regions of the body as members and face. The immunohistochemistry method using serum hiperimune of dogs naturally infected with Leishmania infantum chagasi as a primary antibody anti-Leishmania was employed in all 73 skin samples. The immune reaction was revealed by the avindin-biotin peroxidase system. In parallel, histological and another immunohistochemistry method using monoclonal antibody anti-lipophosphoglican (LPG) of Leishmania, as a primary antibody, and the polymerase chain reaction (PCR) were carried out. The main histological epithelial alterations were acanthosis, papilomatois, hiperceratosis and parakeratosis. A mononuclear chronic and diffuse exudate composed by plasma cells, macrophages and lymphocytes were observed. In some cases giant cells (Langhans giant cells) could be found, but we did not find a classical granulomatous reaction in any case. The immunohistochemistry method proposal using was patronized because amastigotes forms of Leishmania where easily found in 91.78% of the skin biopsies samples wihout any kind of background. The parasitological diagnosis of LTA carried out the immunohistochemistry method patronized in this work showed higher sensibility than histology in comparison to Hematoxilin-Eosin analysis with 17.81% positive of cases and immunohistochemistry, using monoclonal anti-LPG with 71.23% positive of cases. PCR method revealed 100% of positive of cases, but these results were not statistical significant than the immunohistochemistry method patronized (p=0.7671) here. In addition, we use the polymer system free of biotin in the immunohistochemistry method patronized. It reduced the time consuming of the reaction in at least 06 hours in comparison to the classical protocol. We concluded the immunohistochemistry method patronized to define the parasitological diagnosis for LTA revealed high sensibility, lower time consuming and also less laborious than other diagnostic methods

No Brasil, dentre as medidas de controle preconizadas pelo Ministério da Saúde para controle da Leishmaniose visceral, a identificação de cães positivos para leishmaniose visceral canina (LVC), é recomendada, integradas com outras, visto ser este animal um dos principais elos da cadeia epidemiológica. Os métodos sorológicos utilizados para identificação dos animais, não são 100% eficazes que pode comprometer esta medida de controle. Visando à padronização de um antígeno que permita separar de maneira adequada animais acometidos ou não pela LVC, cento e três animais oriundos do Centro de Controle de Zoonoses da cidade de Betim foram categorizados como positivos ou negativos para LVC através de testes parasitológicos e sorológicos, resultando em cinqüenta e quatro positivos e quarenta e nove negativos para esta patologia. Estes animais foram doadores de soro para os ensaios de padronização de um antígeno para o teste de ELISA. Duas cepas de Leishmania (L.) chagasi, BH46 e BH400, isoladas de paciente humano e canino respectivamente, e uma cepa de Leishmania (L.) amazonensis, BH6, isolada de paciente humano foram cultivadas em meio LIT e Alfa-MEM e as massas antigênicas utilizadas para preparar antígenos para sensibilização de placas de ELISA e lâminas de RIFI, assim como para obtenção de um extrato antigênico a ser utilizado na eletroforese em gel bidimensional seguida de Immunobloting. As placas de ELISA foram testadas com 24 horas, 3 meses e 6 meses de armazenamento em freezer a -20ºC. As lâminas de RIFI foram armazenadas da mesma forma e utilizadas após 30 dias. Em todas as fases foram determinados para ELISA e para RIFI, os valores de sensibilidade, especificidade, valor preditivo positivo, valor preditivo negativo e índice de concordância de Youden. Os resultados foram comparados com aqueles obtidos com os kits de ELISA e RIFI de Biomanguinhos. A cepa BH400, independente do meio de crescimento, foi a que apresentou melhor desempenho em caracterizar corretamente animais positivos e negativos. Esta cepa mostrou, durante o experimento, valores de sensibilidade e especificidade entre 98-100%, valor preditivo positivo de 100%, valor preditivo negativo entre 98-100% e índice de concordância de Youden de 98-100%. Houve reação cruzada com soros de cães com Trypanosoma cruzi e com Leishmaniose tegumentar, mas o antígeno não apresentou reação cruzada com Babesia canis e Ehrlichia canis. O meio Alfa-MEM foi escolhido para cultivo desta cepa e caracterização do perfil proteômico-imunogênico, devido à facilidade de obtenção comercial e preparo do mesmo. O Immunobloting foi realizado com pool de soros de alto e baixo títulos de anticorpos e pool de soros negativos, e revelou mais de cinqüenta spots reativos sem reação com soros negativos, mostrando alta imunogenicidade do antígeno. No gel bidimensional foram localizados 37 spots que poderão futuramente ser identificados por espectrometria de massa, visando obtenção de proteínas que possam ser utilizadas como antígenos para ELISA e conferir aumento da sensibilidade e especificidade deste teste.

A Leishmaniose é uma doença antiga, negligenciada e reicidivante que merece uma solução nova. Distribui-se entre os trópicos e os subtrópicos, apresenta-se nas formas visceral, muco-cutâna e cutânea; ocorre em pessoas e especialmente em cães. A interação hospedeiro-parasito é afetada pelo sexo devido à influência hormonal na imunidade. O agente etiológico utilizado neste estudo é o protozoário Leishmania (Leishmania) chagasi, conhecido também por Leishmania infantum, cepa MHON/BR/1972/BH400 originária da forma visceral canina na concentração de 1 x 106 promastigotas. O hamster é um dos modelos clássicos e foi utilizado neste estudo porque é susceptível à doença, apresenta sinais clínicos, histológicos e parasitológicos com alta letalidade. Os tratamentos estabelecidos para humanos apresentam resistência, efeitos colaterais, são tóxicos aos rins e fígado, mas se não for tratada é fatal. A Terapêutica FAO (Fatores de Auto-organização) é uma modalidade da Homeopatia concebida como pesquisa-ação e trata diversas patologias clínicas para humanos e animais. As medicações ultra diluídas que compõe a série são: Antimonium crudum (sulfeto de antimônio); Kali carbonicum (carbonato de potássio); Mercurius solubilis (nitrato de mercúrio); Sulphur (enxofre); Natrum muriaticum (cloreto de sódio); Aurum metallicum (ouro); Ammonium muriaticum (cloreto de amônia). As potências 5 (cinco) e 3 (três); e 12 (doze) e 3 (três) da escala cinquenta milesimal, popularmente conhecida como LM, foram utilizadas nesta ordem e administradas em 2 (duas) séries penta, realizadas aos 17 (dezessete) e 20 (vinte) dias após a inoculação do agente. Os sinais clínicos avaliados foram sobrevida, evolução da massa corporal, consumo de ração, alopecia, diminuição das atividades, desidratação, caquexia e ascite. Os sinais da carga parasitária para o baço e fígado foram imunoistoquímica para amastigotas de Leishmania, índice de LDU de baço e de fígado, índice esplênico e hepático. Os sinais das alterações macroscópicas e microscópicas avaliados por análise semi-quantitativa ou qualitativa para a histopatologia do baço foram: parasitismo tecidual, presença de granulomas esplênicos, hiperplasia dos macrófagos da polpa vermelha, e presença de depleção da polpa branca; e para o fígado foram: parasitismo tecidual, presença de granulomas intralobulares, inflamação portal, hiperplasia das células de Kupffer, amiloidose, hiperemia (congestão). As comparações foram realizadas entre os grupos experimental e controle positivo do mesmo sexo e foram considerados significantes valores de p < 0,05. Foram utilizados os testes de Lilliefors para normalidade, teste t, Mann-Whitney, análise da variância de duas vias, Log-Rank test e teste exato de Fisher. Este estudo teve aprovação prévia do comitê de ética em experimentação animal da UFMG (CONCEA) de registro 210/2007. O sucesso da infecção no modelo foi confirmado por demonstração direta do parasito no esfregaço. Os animais foram acompanhados a partir dos 3 (três) meses de vida até o 106º dia de infecção, e o fator sexo influenciou o desenvolvimento da doença e o efeito do tratamento. Significativamente, a sobrevida e a redução da alopecia foram menor e a imunoistoquímica maior, todos para as fêmeas medicadas; houve aumento da evolução da massa corporal para machos e fêmeas medicados; e houve redução na formação de granulomas para os machos medicados; todos comparados em relação aos seus respectivos controles positivos. Os parâmetros: consumo de ração, imunoistoquímica de baço para machos, depleção, parasitismo, células de Kupffer, índice hepático, apresentaram ausência de significância com valor de p entre 0,05 e 0,10. Ao considerar que a relação hospedeiro-parasito será influenciada pelo sexo através da ação de hormônios; a determinação para evolução da doença no baço ou a resolução com formação de granulomas no fígado, em última instancia, será determinada por um ajuste fino na resposta do sistema imunológico; e que as medicações ultra diluídas são capazes de causar imunomodulação e cicle arrest, observamos que a medicação alterou o curso da doença de forma significativa, em especial favorável a fêmeas. Futuros trabalhos podem identificar os mecanismos deste fenômeno; avaliar o risco de transmissão do parasito na pele; os efeitos em relação à ampla gama de escalas e potências; associada ou não a outros tratamentos, a fim de determinar a melhor relação entre efeito-escala-potência para esta ferramenta terapêutica.

Este trabalho teve como objetivo estudar as alterações da matriz extracelular em fígado, baço e linfonodos cervicais em cães naturalmente infectados com Leishmania (Leishmania) chagasi correlacionando estes achados com os aspectos clínicos, histopatológicos, parasitológicos e imunológicos. Para este estudo foram utilizados 30 cães, divididos em três grupos: dez animais não infectados (grupo controle) e vinte animais infectados. Todos sem raça e idade definidas, provenientes da região do Município de Sabará/MG e de Belo Horizonte/MG. Os animais infectados foram divididos em dois grupos: grupo denominado assintomático composto por dez animais que não apresentavam sinais clínicos da doença; grupo denominado sintomático: composto por 10 animais que apresentavam sinais clínicos clássicos da doença como lesões de pele (alopecia, eczemas, seborréia, ulcerações), perda de peso e linfadenopatias. Foram coletados em necropsia, fragmentos de baço, fígado, linfonodos cervicais, fixados em solução de formol a 10% tamponado e em seguida processados pelas técnicas rotineiras de histopatologia. Cortes parafinados dos diversos tecidos foram corados pela Hematoxilina e Eosina (H&E); Prata amoniacal de Gomori, para marcação das fibras reticulares e pela técnica imuno-histoquímica da estreptoavidina-peroxidase para detecção de formas amastigotas de Leishmania. Os cortes dos mesmos tecidos foram criopreservados e corados pela técnica imuno-histoquímica estrepto-avidina-peroxidase para marcação de laminina (LN) e imuno-histoquímica de imunofluorescência a qual foi utilizada para marcação da fibronectina (FN) tecidual. As análises morfométricas foram feitas utilizando o programa KS300 e o pelo sistema de análise de imagens Kontron Elektronic/Carl Zeiss, Germany. Os resultados mostram que há um aumento significativo da deposição de fibras colágenas no fígado, baço e linfonodos cervicais dos animais infectados quando comparadas aos animais controles, revelando diferenças significativas entre os animais sintomáticos e assintomáticos. Foram encontradas correlações positivas entre a presença do parasitismo tecidual e a deposição de colágeno no fígado, baço e na região medular dos linfonodos cervicais dos animais infectados. De fato, animais sintomáticos apresentaram uma maior deposição de colágeno nestes órgãos que pode estar associado ao maior parasitismo tecidual encontrado. A expressão das fibras adesivas laminina (LN) e fibronectina (FN) no fígado, baço e linfonodos cervicais foi também mais elevada nos animais infectados, principalmente, no grupo de animais sintomáticos. Não houve diferença estatística significativa quando comparamos a expressão de LN no baço e linfonodos cervicais entre os grupos de animais sintomáticos e assintomáticos, mas a expressão de FN foi significante estes grupos. Nossos resultados demonstram que na leishmaniose visceral canina há uma fibrogênese no fígado, baço e linfonodos, associada ao parasitismo tecidual e a processos degenerativos. The aim of this work was study the extracellular matrix alterations in liver, spleen and cervical lymph nodes in dogs naturally infected with Leishmania (Leishmania) chagasi correlating with clinical aspects, histological, parasitological and immunological. This study was carried out with 30 dogs, divided at three groups: ten not infected animals (group control) and twenty infected animals. All them was mongrel dogs with undefined age, obtained from the municipality of Belo Horizonte, MG, metropolitan area. Infected animals were divided in two groups: asymptomatic group composed by ten animals without clinical signs of the disease; group denominated symptomatic: composed by ten animals with classical clinical signals of the disease as skin lesions (alopecia, eczemas and ulcers), loss weight and lymphopathy. During necropsy, spleen, liver and cervical lymph nodes fragments were collected and fixed in buffer formaldehyde solution to 10% for histological analyses. Paraffined sections of the tissues were stained by Hematoxylin-Eosin (HE); Gomori's Ammoniacal Silver staining for reticular fibers and strepto-avidin peroxidase Immunohistochemical method for tissue Leishmania amastigotes detection. Frozen tissue sections of organs were stained by strepto-avidin peroxidase Immunohistochemical method for laminin (LN) tissue characterization and immunofluorescence technique for fibronectina (FN). The tissue images were transferred to a computer video screen by means of the software KS300 and relayed to a computer-assisted image analysis system (Kontron Elektronic/Carl Zeiss, Germany) for morphometrical analysis. Significant increase collagens deposition in liver, spleen and cervical lymph nodes of infected dogs when compared to controls animals. There was significant difference between symptomatic and asymptomatic dogs collagen deposition in organs. Positive correlation between the parasite load and collagen deposition in liver, spleen and cervical lymph nodes of infected animals. In fact, symptomatic animals showed increase collagen deposition in these organs, its can be associate to parasite burden. Adhesive fibers LN and FN expression in liver, spleen and cervical lymph nodes was higher in symptomatic animals than in asymptomatic. No significant statistical difference when we compare LN expression in the spleen and cervical lymph nodes between symptomatic and asymptomatic groups. While in all organs studied of infected animals FN expression demonstrated significant differences in these groups. Our results demonstrate that in canine visceral leishmaniasis induces fibrogenesis in liver, spleen and lymph nodes attached the parasite load and degenerative processes

Vários aspectos da história natural da doença de Chagas foram avaliados em cães Beagle, experimentalmente infectados com três cepas do Trypanosoma cruzi, com características biológicas e genéticas distintas. Para isso, 34 cães foram inoculados com 2000 ou 4000 tripomastigotas sangüíneos (TS) das cepas, Be-78, Y ou ABC do T. cruzi/kg de peso corporal. Os animais foram avaliados clinicamente durante as fases aguda e crônica da infecção e a permanência do parasito no sangue e nos tecidos desses animais foi monitorada ao longo de 24 meses, por meio de exames parasitológicos, moleculares e imuno-histoquímico. Além de ter sido avaliada a sensibilidade das técnicas utilizadas, a positividade dos testes parasitológicos foi relacionada com a positividade da PCR em tecidos cardíacos. As alterações histopatológicas foram avaliadas pela análise semi-quantitativa da presença de infiltrado de células adiposas, inflamação e fibrose em fragmentos do coração. Além disso, os parasitos isolados na fase aguda e crônica foram avaliados geneticamente por quatro marcadores moleculares distintos.A avaliação clínica dos animais, durante a fase aguda da infecção, mostrou alterações clínicas discretas. Nos animais inoculados com as cepas Be-78, Y ou ABC, o exame de sangue a fresco (ESF) apresentou 86% (19/22) de positividade. Entretanto, a freqüência de testes positivos foi variável entre os animais inoculados com essas cepas. Durante a fase crônica da infecção foram realizadas 88 hemoculturas (Hc) em 22 animais, sendo observado uma taxa de positividade de 46,6% (41/88). A freqüência de testes positivos, na fase crônica da infecção foi similar entre os animais inoculados com as cepas Be-78 (75%) e ABC (62.5%), e significativamente menor (6,2%) naqueles inoculados com a cepa Y. Durante a fase crônica da infecção a taxa de positividade da PCR foi de 72,7% (64/88), superior a da Hc. Nos testes de PCR realizados no sangue dos animais inoculados com as cepas Be-78, Y ou ABC a positividade foi de 94,4%, 41,6% e 93,7%, respectivamente. O T. cruzi foi demonstrado, pela análise imuno-histoquímica (IMH), nos fragmentos cardíacos de todos os animais avaliados durante a fase aguda da infecção, porém, na fase crônica foi negativa em todos os fragmentos analisados. Entretanto, a presença do parasito foi demonstrada, na fase crônica da infecção, em todos os animais infectados com as três cepas, por meio da PCR. O parasitismo tecidual médio avaliado na fase crônica, pela PCR, em seis fragmentos cardíacos, foi semelhante entre os animais inoculados com as diferentes cepas. Porém, o padrão histopatológico das lesões cardíacas, observado na fase crônica, foi relacionado com a cepa do T. cruzi. Os resultados das avaliações realizadas nos animais inoculados com 2000 TS foram similares aos inoculados com 4000 TS. A caracterização molecular dos isolados do T. cruzi obtidos de todos os cães infectados foi realizada na fase aguda (30 dias após a inoculação) e na fase crônica (até 24 meses após a inoculação). O perfil eletroforético de isoenzima foi idêntico entre as cepas parentais Be-78 ou ABC e seus respectivos isolados. Entretanto, o perfil de isoenzima da cepa Y pode ser alterado durante a fase crônica da infecção de cães, sendo esta mudança acompanhada pela alteração nos perfis de RAPD e DNA mitocondrial (CO II). O fenograma construído pela UPGMA permitiu separar as cepas Be-78, Y e ABC e os respectivos isolados em dois grupos distintos, correspondendo a T. cruzi II e T. cruzi III ou híbrido. Os dados obtidos pelo rDNA mostraram perfil do grupo 1 (T. cruzi II) para todas as cepas parentais e seus isolados. Entretanto, o DNA mitocondrial (CO II) mostrou perfil de T. cruzi II para as cepas Be-78 e Y e de T. cruzi III ou híbrido para dois isolados da cepa Y e para a cepa ABC e seus isolados. Esses dados demonstraram que a cepa do T. cruzi exerce um papel importante no curso da história natural da doença de Chagas em cães e reforçam a importância da presença do parasito na formação/gênese das lesões cardíacas, cujo padrão histopatológico estaria relacionado especialmente com a população do T. cruzi presente nos tecidos.

As leishmanioses representam um grupo de doenças transmitidas por flebotomíneos que afetam animais domésticos, silvestres e humanos. No contexto urbano, o cão é considerado o principal reservatório do agente etiológico da leishmaniose visceral (LV), ocupando posição estratégica nos programas de controle da doença. Entretanto, o tratamento convencional dos animais é discutível. Os lipossomas representam grande avanço na terapia das doenças parasitárias, especialmente daquelas que envolvem os componentes do sistema fagocítico mononuclear, como é o caso da LV. O presente estudo teve como objetivo avaliar a biodistribuição de Sb, toxicidade e eficácia terapêutica de uma formulação composta de antimoniato de meglumina (AM) encapsulado em lipossomas nanométricos, em cães naturalmente infectados com Leishmania (Leishmania) chagasi. Foram utilizados 50 cães SRD com diagnóstico parasitológico positivo. Para a avaliação da biodistribuição e toxicidade, 14 animais receberam uma única dose intravenosa da formulação e foram sacrificados 24 (n = 3), 48 (n = 3), 72 (n = 3) e 96 (n = 5) horas após. Os cães foram monitorados clinicamente durante todo o experimento. Além disso, parâmetros hematológicos e bioquímicos foram avaliados antes do tratamento e na eutanásia. Para o estudo da eficácia, 36 cães foram estratificados segundo peso, sexo e classificação clínica e alocados aleatoriamente em três grupos (GI, GII e GIII), os quais receberam protocolo de quatro injeções intravenosas a cada 96 horas: GI - 6,5mg de Sb/kg de AM encapsulado em lipossomas; GII - lipossomas na ausência de Sb (vazios); GIII - solução salina. Foram realizadas, em diferentes tempos após o tratamento, avaliações clínicas, hematológicas, bioquímicas e sorológicas. A carga parasitária medular e esplênica foi investigada respectivamente aos quatro e 150 dias, quando então foram realizados o xenodiagnóstico e a eutanásia dos animais. Os resultados mostraram maior direcionamento de Sb para o baço e fígado, quando comparado à medula óssea. Não houve, entretanto, variações significativas nas concentrações de Sb em relação ao tempo para os três órgãos. Embora não tenham sido registradas alterações laboratoriais após administração da formulação em única ou múltipla dose, 57% dos cães tratados com dose única manifestaram reações adversas de caráter clínico-transitório, as quais incluíram prostração, sialorréia, defecação e taquipnéia. O regime de múltiplas doses não modificou o perfil nem a intensidade das reações, as quais puderam ser relacionadas à estrutura vesicular, mas não ao metal. A formulação foi capaz de proporcionar melhora clínica parcial entre os cães. Aos 60 dias após o tratamento, os cães do GI apresentaram normalização dos valores médios de hematócrito, proteínas totais, globulinas, ã-globulina, albumina e da relação A/G, bem como elevação significativa dos níveis de á-1, á-2 e â-globulinas. A formulação proporcionou redução significativa da carga parasitária medular e esplênica, quando comparado aos demais grupos. Cinco meses após o tratamento, foi verificada redução significativa da capacidade infectante destes cães para fêmeas de Lutzomyia (Lutzomyia) longipalpis, o que representa o primeiro relato na literatura da interferência de uma formulação lipossomal nanométrica de Sb sobre a infectividade de cães naturalmente infectados com Leishmania (L.) chagasi.

Este trabalho teve como objetivo estudar alguns aspectos clínicos, histopatológicos, parasitológicos e imunológicos, de cães naturalmente infectados com Leishmania (Leishmania) chagasi. O enfoque principal foi o de comparar a expressão do receptor do complemento do tipo 3 (CR3 - CD11b/CD18) no baço, fígado, linfonodos (axilares, cervicais e poplíteos) com alguns aspectos clínicos, histológicos e parasitológicos.Para isso, foram utilizados dez animais não infectados (grupo controle) e trinta animais infectados. Todos eram provenientes da região do Município de Sabará/MG sem raça e idade definidos. Os animais infectados foram subdivididos em dois grupos: grupo denominado assintomático composto por dez animais que não apresentavam sinaisclínicos da doença; (2) grupo denominado sintomático: composto por vinte animais que apresentavam sinais clínicos clássicos da doença como lesões de pele (alopecia, eczemas, seborréia, ulcerações), perda de peso e linfadenopatias. Em necropsia, fragmentos de baço, fígado, linfonodos (axilares, cervicais e poplíteos) e pele (orelha, espelho nasal e abdômen) foram coletados, fixados em solução de formol a 10%tamponado e em seguida processados pelas técnicas rotineiras de histopatologia. Cortes parafinados dos diversos tecidos foram montados em lâminas histológicas e corados pela Hematoxilina e Eosina (H&E), e pela técnica Imuno-histoquímica da estrepto -avidina- peroxidade para análises microscópica e parasitológica, respectivamente. Cortes criopreservados dos mesmos tecidos, com a exceção dapele, foram corados pela técnica imuno-histoquímica da estrepto-avidina-peroxidade para marcação tecidual das proteínas CD11b e CD18 (CR3). A expressão tecidual e celular de CR3 (CD11b/CD18) foi acessada por técnicas de morfometria digital (avaliação do número e técnicas de densitométrica óptica). Nossos resultados revelaram uma expressão densitométrica e celular do CR3 aumentada em animais infectados quando comparada a animais controle, mas não diferente entre os animais assintomáticos e sintomáticos. Correlações positivas foram encontradas entre a presença do parasitismo tecidual e a expressão das proteínas CD11b e CD18 no baço e linfonodo cervical e negativa para e o parasitismo hepático. De fato, animais sintomáticos apresentaram uma maior expressão de CR3 no baço e nos linfonodos cervicais, associada também a um maior parasitismo tecidual. Por outro lado, no fígado ocorreu uma maior expressão tecidual de CR3 em animais assintomáticos do que os sintomáticos. Entretanto, isto foi associado a um menor parasitismo nesses animais. Podemos dizer, então que a resposta imune parece ser órgão-específica (compartimentalização da resposta imune), em que as células inflamatórias exercem um papel imune distinto em diferentes órgãos. Às vezes, podem atuar como fonte de perpetuação da infecção, ou às vezes, como células efetoras capazes de controlar a infecção por Leishmania. Seguindo nossos resultados, em que os animais sintomáticos apresentam maior expressão de CR3 e maior taxa de parasitismo no baço e linfonodos, podemos permite atribuir a essas células (monócitos-macrófagos) papel na manutenção da infecção. The aim of this work was to evaluate some clinical, histopathological, parasitological and immunological aspects of dogs naturally infected with Leishmania chagasi. The principal aim was to compare the third receptor of complement CR3 (CD11b/CD18) in spleen, liver, lymph nodes (axilar, cervical and polpliteal) with clinical, histological and parasitological findings. This study was carried out with ten control dogs (uninfectedanimals) and thirty infected dogs. All them was mongrel dogs with undefined age and was obtained from the municipality of Belo Horizonte, metropolitan area. The infected animals were divided in two groups: (1) group denominated asymptomatic composed by ten animals without clinical signs of the disease; (2) group denominated symptomatic composed by twenty animals with classical clinical signs of the disease as skin lesions (alopecia, eczema and ulcers), loss weight and lymphopathy. During necropsy, fragments of spleen, liver, lymph nodes (axillary, cervical, and popliteal) and skin (ears, nose and abdomen) were collected and fixed with formaldehyde solution 10% for histological analysis. Paraffined sections of all tissues were mounted on histological slides and stained by Hematoxylin-Eosin (H&E) and the strepto-avidinperoxidase immunohistochemical method for microscopical and parasitological analysis, respectively. Frozen tissue sections of all organs, except skin samples, were stained by the strepto-avidin peroxidase immunohistochemical method for CR3 (CD11b/Cd18) tissue characterization. The tissue and cellular CR3 expression was evaluated by morphometrical digital analysis. Densitometrical CR3 expression washigher in infected animals than controls, but it was not different when compared between animals with different defined clinical status. CR3 cell expression in spleens was higher in symptomatic animals than in asymptomatic ones where the parasite load was higher. In another side, CR3 cell expression in livers was higher in asymptomatic animals than in symptomatic ones, where the tissue parasite load was lower. However,there was a positive correlation between the parasite load and cellular CR3 expression in the spleens and cervical lymph nodes of infected dogs and there is a negative correlation in liver. We can say, that the immune response appears to be organ specific (immune response compartmentalization) whereas inflammatory cells play a distinctrole. Sometimes, they could act to perpetuate the infection or they could act as effector cells in order to control the infection. Following our results where the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their lymphoid organs as spleen and lymph nodes, we can conclude that these cells (monocyte-macrophages) might serve to perpetuate intracellular infection.