Canine Leishmaniasis: An Overview of the Current Status and Strategies for Control.

Canine leishmaniasis (CanL) is a vector-borne disease caused by Leishmania infantum and 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.

Host modulation by a parasite: how Leishmania infantum modifies the intestinal environment of Lutzomyia longipalpis to favor its development.

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.

Relationship between clinical and pathological signs and severity of canine leishmaniasis.

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.

Relationship between clinical and pathological signs and severity of canine leishmaniasis / Relação entre sinais clínico-patológicos e gravidade da leishmaniose visceral canina

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 analisadas 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 frequência 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.

Leishmania metacyclogenesis is promoted in the absence of purines.

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.

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in 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.

First report of infection of Lutzomyia longipalpis by Leishmania (Leishmania) infantum from a naturally infected cat of Brazil.

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.

First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil.

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.

Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein.

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.

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

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

Comparison of paraffin-embedded skin biopsies from different anatomical regions as sampling methods for detection of Leishmania infection in dogs using histological, immunohistochemical and PCR methods.

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.

Transmission of Leishmania infantum via blood transfusion in dogs: potential for infection and importance of clinical factors.

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.

An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues.

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.

Canine visceral leishmaniasis: a histopathological study of lymph nodes.

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.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin ).

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin ), produced by Biobr s (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 30 g of each protein at 15-day intervals combined with 250 microg 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-gamma 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.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

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 30æg of each protein at 15-day intervals combined with 250æg 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 percent were demonstrated for each purified protein

Tratamento da leishmaniose tegumentar americana utilizando vacina / Treatment of american cutaneous leishmaniasis by means of vaccine

Foi realizado o tratamento de 62 pacientes portadores de leishmaniose tegumentar utilizando como agente imunoterápico uma vacina constituída de promastigotas mortas. O esquema terapêutico utilizado mostrou ser uma alternativa adequada ao tratamento de leishmaniose tegumentar já que houve sucesso terapêutico em 75,8% dos casos, näo se observando efeitos colaterais. A eficiência terapêutica foi semelhante nos casos de lesöes cutâneas ou mucosas

Histological observations on Montenegro's reaction in man

Foi realizado o estudo histológico do material obtido nas biópsias do local de inoculaçäo do antígeno para teste de Montenegro (T.M.) nos seguintes grupos de indivíduos: I) Seis pacientes com leismaniose cutânea comprovados parasitologicamente, com Montenegro positivo; II) Cinco indivíduos normias, näo residentes em zona endêmica, com Montenegro negativo; III) Nove soldados participantes de ensaios clínicos com vacina anti-LTA - MAYRINK e cols. 1979 e que tiveram o TM positivo 35 dias após vacinaçäo. IV) Um último grupo constituído de quatro soldados, também participantes de ensaio clínico com a mesma vacina acima, dois vacinados que näo mostraram TM positivo 35 días após vacinaçäo dois que receberam placebo. As biópsias foram realizadas 48 horas após a inoculaçäo do antígeno. O material foi fixado em formol à 10% (pH 7.2). Histologicamente, excetivando o grupo II (controle negativo), os grupos I-III-IV mostraram diferenças quantitativas no infiltrado mononuclear. Os quadros histológicos de cada grupo säo descritos e discutidos

Leishmaniose tegumentar canina: infecçäo experimental / Canine cutaneous leishmaniases: experimental infection

Os autores promoveram inoculaçöes de promastigotas de Leishmania braziliensis em cäes, verificando que o inóculo de 10 elevado a 5 formas foi capaz de infectar 100 por cento dos animais. As lesöes, que eram inicialmente nodulares, ulceravam após aproximadamente 70 dias da infecçäo. Parasitos foram demonstrados nas lesöes, 6 meses após a inoculaçäo.