Main Domestic Reservoir Research Articles

Mediterranean Spotted Fever in Children: Study of a Portuguese Endemic Region.

Mediterranean spotted fever is an infectious disease included in the human rickettsiosis group, with its main distributionin the Mediterranean and South European countries. It is an endemic disease in Portugal, with dogs acting as the main domesticreservoir. Children are a particularly vulnerable group due to their close contact with household animals and by frequently playingoutdoors. In this study, we aim to describe the local epidemiology and characterize the clinical features and treatment options in aPortuguese endemic region. We performed a prospective descriptive study of the Mediterranean spotted fever cases admitted to thepaediatrics emergency department of a of a group II hospital, between January 1st 2013 and December 31st 2015. All patients wereexamined by a physician, who was later asked to fill out a detailed questionnaire regarding clinical presentation, diagnostic attitudesand treatment of their patients. Parents were later interviewed and questioned about post-discharge disease evolution. We registered 32 cases (93.9% reported between July and October). After fever, the most frequent reported symptoms weremyalgia (37.5%), abdominal pain (25%) and headache (25%). Exanthema was present in 84.4% of cases by the third day of fever:maculonodular (53.1%), papular (37.5%) and macular (9.4%). Eschars were found in 59% of patients, with regional lymphadenopathypresent in 46.9% of cases. Azithromycin (84.4%) and doxycycline (15.5%) were the selected treatments in our population, with nocases of therapeutic failure or side-effects reported. The incidence of Mediterranean spotted fever is higher in our population in comparison with the rest of the country.Fever, myalgia, abdominal pain and headache were the most common presenting symptoms, while exanthema was the predominantcutaneous finding. Azithromycin was the preferred treatment and it proved to be successful and safe in all cases.

Risk analysis and prediction of visceral leishmaniasis dispersion in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important neglected disease caused by a protozoan parasite, and represents a serious public health problem in many parts of the world. It is zoonotic in Europe and Latin America, where infected dogs constitute the main domestic reservoir for the parasite and play a key role in VL transmission to humans. In Brazil this disease is caused by the protozoan Leishmania infantum chagasi, and is transmitted by the sand fly Lutzomyia longipalpis. Despite programs aimed at eliminating infection sources, the disease continues to spread throughout the Country. VL in São Paulo State, Brazil, first appeared in the northwestern region, spreading in a southeasterly direction over time. We integrate data on the VL vector, infected dogs and infected human dispersion from 1999 to 2013 through an innovative spatial temporal Bayesian model in conjunction with geographic information system. This model is used to infer the drivers of the invasion process and predict the future progression of VL through the State. We found that vector dispersion was influenced by vector presence in nearby municipalities at the previous time step, proximity to the Bolívia-Brazil gas pipeline, and high temperatures (i.e., annual average between 20 and 23°C). Key factors affecting infected dog dispersion included proximity to the Marechal Rondon Highway, high temperatures, and presence of the competent vector within the same municipality. Finally, vector presence, presence of infected dogs, and rainfall (approx. 270 to 540mm/year) drove the dispersion of human VL cases. Surprisingly, economic factors exhibited no noticeable influence on disease dispersion. Based on these drivers and stochastic simulations, we identified which municipalities are most likely to be invaded by vectors and infected hosts in the future. Prioritizing prevention and control strategies within the identified municipalities may help halt the spread of VL while reducing monitoring costs. Our results contribute important knowledge to public and animal health policy planning, and suggest that prevention and control strategies should focus on vector control and on blocking contact between vectors and hosts in the priority areas identified to be at risk.

Analysis of the transmission of Trypanosoma cruzi infection through hosts and vectors

Calculating epidemiological measures of infection by Trypanosoma cruzi, the causative agent of Chagas disease, is complex, because it involves several species, different stages of infection in humans and multiple transmission routes. Using the next-generation matrix method, we analysed a model which considers the three stages of human infection, triatomines and dogs (the main domestic reservoirs of T. cruzi when triatomines are present) and the main transmission routes. We derived R 0 and type-reproduction numbers T. We deduced formulas for the number of new infections generated through each transmission route by each infected individual. We applied our findings in Argentine Gran Chaco. The expressions achieved allowed quantifying the high infectivity of dogs and emphasizing the epidemiological importance of the long and asymptomatic chronic indeterminate stage in humans in the spread of the infection. According to the model, it is expected that one infected human infects 21 triatomines, that 100 infected triatomines are necessary to infect one human and 34 to infect a dog, and that each dog infects on average one triatomine per day. Our results may allow quantifying the effect of control measures on infected humans, triatomines and dogs (or other highly infected vertebrate) or on a specific route of transmission, in other scenarios.

Autochthonous Canine Visceral Leishmaniasis in Urban Area of Porto Alegre, RS, Brazil

Background: Leishmaniasis is a chronic infectious disease caused by intracellular protozoan Leishmania that affects canine and human. The visceral leishmaniasis (VL) is caused by the Leishmania donovani complex, in which canines are the main reservoir for human VL. In Southern Brazil, case reports of this disease have increased, especially when canines are infected in endemic areas in the country. Canines usually present a systemic disease, characterized by cutaneous lesions, weight loss, generalized lymphadenopathy, hepatomegaly and splenomegaly. This report aims to describe the clinicalpathological features of a case of autochthonous VL in a canine of an urban area of Porto Alegre.Case: A male, mixed breed, dog , 7 year-old, resident at the East Side of Porto Alegre, living together with two other canines, presented at clinical examination progressive weight loss, associated with hyporexia and hyperthermia, evolving to lateral recumbency, paralisys, and death. At necropsy, the dog was cachectic with diffusely pale mucosae. Gross findings included liver enlarged, with multifocal firm brownish areas, spleen enlarged, showing multifocal firm round dark-red areas, and kidneys diffusely pale with evidentiated tranversal striations on cortical zone. At microscopic examination, there was on the spleen diffuse inflammatory infiltrate of macrophages with large cytoplasm containing multiple amastigotes. The liver, exhibited atrophy of hepatocytes and moderate multifocal inflammatory infiltrate in sinusoids of macrophages containing multiple amastigotes. These features were also observed moderately on lymph nodes and lamina propria of large intestine. Immunohistochemistry examination showed marked positive staining for Leishmania spp. in amastigotes located whitin the cytoplasm of macrophages of spleen, liver, lymph nodes and large intestine.Discussion: Canine leishmaniasis is a disease that affects both internal organs and skin. However, the condition is called VL mostly because it is associated with the same species that cause the visceral form in humans. In this case the canine presented liver, spleen and lymph node lesions; however no skin lesion was observed during the clinical examination. A serological evaluation of canids in East Side of Porto Alegre performed in 2005 showed that 3.5% of these dogs were positive for Leishmania; yet neither of these animals presented clinical signs. This demonstrates that the agent was already present, however only 3-10% of the canines infected develop clinical disease. In non-endemic areas VL is related to the migration of canids from endemic areas, where VL is common. However the canine here described was born, raised, and was living in Porto Alegre. Lutzomyia longipalpis is the main vector of VL and despite its wide distribution it was identified only in 2009 in Rio Grande do Sul State. Autochthonous cases of VL were reported in canines and humans in this state, confirming the hypothesis that the vector was widely distributed and it is now present in Porto Alegre, keeping the epidemiological cycle of that disease active. Canine VL occurs prior in humans, since canines are the main domestic reservoirs and are critical for the maintenance of this disease cycle. This is the first autochthonous VL in a canine of an urban area of Porto Alegre; therefore authorities should be alert, and new control measures must be taken to avoid the canine leishmaniasis outbreak due to its potential for zoonotic transmission.Keywords: leishmaniasis, canine, protozoan, immunohistochemistry.

Leishmaniose Canina em Brasília, DF: Uma Revisão da Literatura.

A leishmaniose é uma doença infecto-parasitária que acomete seres humanos e animais, causada por protozoários do gênero Leishmania. As leishmanioses são importante problema de saúde pública em vários países e estão incluídas entre as seis endemias de maior relevância mundial. São classificadas em tegumentar (LT) cutânea e mucocutânea - e visceral (LV). A transmissão ocorre através de picada de insetos flebotomíneos pertencentes aos gêneros Lutzomyia (Novo Mundo) e Phlebotomus(Velho Mundo). A Leishmaniose Tegumentar Americana (LTA) é uma zoonose distribuída no território brasileiro. Possui evolução crônica e acomete a pele e mucosas, isoladamente ou em associação, de seres humanos, cães, gatos e eqüinos. Nos cães, se caracteriza pela presença de lesões cutâneas ulceradas, recobertas por crostas, localizadas nas orelhas, bolsa escrotal, focinho e membros. A Leishmaniose visceral é uma zoonose que ocorre nas regiões subtropicais e tropicais, causada pela Leishmania (Leishmania) chagasi, no Novo e no Velho Mundo. No Brasil, a Leishmaniose Visceral Canina (LVC) coexiste com a doença humana e os cães são seu reservatório doméstico. A eutanásia do cão é criticada por isso enfrenta limitações. Apesar das ações de vigilância e controle de LV adotadas no DF, a doença permanece em áreas urbanas e rurais.

Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

Epidemiological aspects of vector, parasite, and domestic reservoir in areas of recent transmission and no reported human cases of visceral leishmaniasis in Brazil

About 97% of the human cases of the American visceral leishmaniasis (VL) occur in Brazil. In the last few years, the disease expanded to medium- and large-sized cities, in which surveillance and control actions have been intensified, in an effort to control VL spreading. Our two-year study was conducted in Belo Horizonte, the sixth most populous city in Brazil, which is endemic for VL. We focused in two particular districts of recent transmission of the disease, with no reported human cases and submitted to minor surveillance and control actions. Our aim was to draw an epidemiological profile of the local situation concerning Lutzomyia vector, Leishmania parasites, and the main domestic reservoirs (dogs). Lutzomyia longipalpis comprised 96.5% of the total phlebotomine sand flies captured and displayed an expressive minimal infection rate by Leishmania infantum (16.7%). Positive correlations were found between the population densities of L. longipalpis, rainfall and temperature. L. infantum was also detected in the cortelezzii complex and, for the first time, in Lutzomyia lloydi. Leishmania braziliensis, an etiological agent of the American cutaneous leishmaniasis, was also identified in L. longipalpis. Among the 1408 dogs serologically tested by standard enzyme-linked and fluorescence immune assays (ELISA/IFA) 3.6% were positive for VL. L. infantum DNA and Leishmania parasites were identified in 100% and 72.5% of the seropositive dogs, respectively. The co-positivity of other diagnostic tests for VL—Leishmania-nested PCR, imprint and myeloculture—was compared to the standard serology. Both symptomatic or asymptomatic dogs displayed an equal average number of positive diagnostic tests for VL. The districts studied display favorable conditions for the rapid spreading of human infection, in terms of L. longipalpis population density, and presence of L. infantum in both vector and main reservoir.

Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

BackgroundThe early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.Methodology/Principal FindingsWe mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.Conclusions/SignificanceThe recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

Immunofluorescent Antibody Test (IFAT) for Trypanosoma cruzi in dogs from urban and rural areas of Pelotas, RS

Chagas disease (CD) is a zoonosis with the protozoan Trypanosoma cruzi as the causative agent. Dogs are considered the main domestic reservoir for T. cruzi in most Latin American countries and in some areas of the United States. In southern Brazil, despite being an endemic area of the disease, the prevalence in dogs is still unknown. This study aimed to evaluate the frequency of T. cruzi antibodies in dogs from urban and rural areas of Pelotas, RS, Brazil. A total of 227 canine sera were used for serological tests, of which 99 were from urban areas and 128 were from rural areas of Pelotas. Information regarding the environment and the possible risk factors (origin, rural contact, age, breed, confinement and gender) to which the dogs were exposed were recorded. Indirect immunofluorescence was used to assess the presence of specific immunoglobulins (IgG) anti-T. cruzi in the serum of dogs. Of the 227 sera analyzed, 81 (35.7%) exhibited anti-T. cruzi, which represented 34.3% of the dogs from the urban area and 36.7% of the dogs from the rural area. Among the variables analyzed, rural contact and male gender showed an association with seropositivity for T. cruzi. The detection of T. cruzi antibodies in the serum of these dogs emphasizes the need to study trypanosomiasis in this important domestic reservoir of Chagas disease.

Immunological profile of resistance and susceptibility in naturally infected dogs by Leishmania infantum

Visceral leishmaniasis has a great impact on public health, and dogs are considered the main domestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animals naturally infected by L. infantum from an endemic area of Brazil were evaluated through an analysis of cellular responses, using flow cytometry, and of the hematological parameters. The results confirmed that disease progression is associated with anemia and reductions in eosinophils, monocytes and lymphocytes. The investigation of the immune response, based on the immunophenotypic profile of peripheral blood, showed declines in the absolute numbers of T lymphocytes CD5+ and their subsets (CD4+ and CD8+) and a drop of B lymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs. Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesis of interferon (IFN)-γ+ in AD-II and SD groups, with decreased production of interleukin (IL)-4+ in asymptomatic seronegative dogs positive for L. infantum infection based on polymerase chain reaction testing (AD-I group). In the AD-II and SD groups, subpopulations of stimulated lymphocytes (CD4+ and CD8+) also exhibited greater synthesis of IFN-γ+ and IL-4+ in culture. These results suggest that the animals of the AD-II and SD groups exhibited a mixed immune response (Type 1 and 2) and the AD-I group presenting an immune profile very similar to normal control animals.

Epidemiological aspects of visceral leishmaniasis in Jaciara, Mato Grosso, Brazil, 2003 to 2012.

Visceral leishmaniasis (VL) is caused by Leishmania (L.) infantum chagasi in the Americas. In Brazil, it is transmitted by sandflies of the species Lutzomyia longipalpis and L. cruzi, and dogs are the main domestic reservoirs. The aim of this study was to analyze data relating to VL transmission in Jaciara, state of Mato Grosso, and discuss vector distribution, domestic reservoirs, and human cases that occurred between 2003 and 2012. The data for analysis were obtained from the Notifiable Diseases Information System (human), the State Health Department's Environmental Monitoring Agency (canine data) and the State Health Department's Entomology Laboratory (sandfly data). Over this period, Jaciara had 19 autochthonous human cases (12 males and seven females), with one death 2,273. Out of the 7,545 dogs tested by enzyme immunoassay and indirect immunofluorescence were positive. The sandflies collected comprised 5,015 individuals belonging to 24 species, with a predominance of L. whitmani followed by L. cruzi. The results showed that the parasite has frequent circulation and that the vector L. cruzi is widely distributed over all months, thus suggesting that transmission may occur at any time of the year.

Modelling American Trypanosomiasis in an Endemic Zone: Application to the Initial Spread of Household Infection in the Argentine Chaco

The complex dynamics of Trypanosoma cruzi infection (Chagas disease) involves different actors and multiple transmission routes. Based on the information currently available, here, we propose a new and more comprehensive model to better understand the dynamics of the infection. This mathematical deterministic model was formulated considering: (i) the three clinical forms in humans: acute, chronic indeterminate and chronic with determinate pathology, (ii) the three main modes of transmission in the human population: vector-borne, congenital and transfusional, (iii) populations of triatomines and dogs as the main domestic reservoirs of T. cruzi and (iv) open populations. A numerical simulation was also performed to estimate the initial spread of the infection in a typical rural household in the endemic zone of the Argentine Gran Chaco. We also analysed the incidence of infected individuals corresponding to each of the three species (humans/triatomines/dogs) over times until the appearance of the first case in the other species. The model predicts that, in the absence of control measures, a few infected individuals are sufficient for the establishment and dispersion of the infection in all the inhabitants of the household. The model proposed and the results obtained allow describing the consequences of the presence of infected individuals in any of the three species considered in the dynamics and the output of the infection.

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n = 31) compared to those from vaccinated dogs (n = 21), experimentally infected dogs with cross-reactive parasites (n = 23), and healthy controls (n = 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

Experimental Infection of Dogs with Leishmania and Saliva as a Model to Study Canine Visceral Leishmaniasis

BackgroundCanine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches.Methodology/Principal FindingsIn this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1×107 parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35×105 parasites in their lymph nodes. An increase of IFN-γ and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection.ConclusionThe infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.

Alta prevalencia de infección por Leishmania (Kinetoplastidae: Trypanosomatidae) en caninos del norte de Colombia

Although the dog is considered to be the main domestic reservoir of Leishmania infantum in several Latin American countries, the rate of infection with Leishmania parasites remains unknown in dogs from foci of visceral leishmaniasis of northern of Colombia. To determine the prevalence of Leishmania infection in canine population from department of Sucre in northern Colombia. The canine population of rural areas of the municipalities of Sincelejo, Sampués and Ovejas was studied. Parasites of the genus Leishmania were detected by amplifying a DNA segment of the small ribosomal subunit (rSSU). The relationship among sex, age group and clinical signs with infection by Leishmania was analyzed. A 561 bp fragment of the Leishmania rSSU was amplified in 34.9% (29/83), 35.7% (15/42) and 11.1% (1/9) of the dogs analyzed from Sincelejo, Sampués and Ovejas, respectively. Overall prevalence of Leishmania infection was 33.6%. A 650 bp band, characteristic of parasites of the genus Trypanosoma, was amplified in two dogs from Sincelejo and Sampués and in one dog from Ovejas. The 64.2% of the dogs that showed clinical signs compatible with canine leishmaniasis, was PCRpositive. A statistically significant relationship was found between dogs over 33 months old and positivity to Leishmania (p=0.043). No relationship was observed between sex and positivity to Leishmania (p=0.85) . A third of the canine population studied is infected with Leishmania , which could support its potential role as a source of infection for the insect vector.

Seroprevalence of canine visceral leishmaniasis in southeast of Iran.

Visceral leishmaniasis is an endemic disease in many parts of Iran and infected dogs constitute the main domestic reservoirs that play a key role in transmission to humans. The objective of this study was to assess the seroprevalence of canine visceral leishmaniasis (CVL) by enzyme linked immunosorbent assay (ELISA) in southeast of Iran. This survey was carried out from 2009 to 2011 in Kerman, Bam and Baft districts in Kerman province and Zabol in Sistan-Baluchestan province. Blood samples were taken from 201 dogs after complete clinical examination. Following hematological evaluation; collected sera were tested by indirect ELISA method for the presence of anti Leishmania infantum antibodies. Overall seroprevalence was 15.4%, including 6.4, 3.5, 3 and 2.4% in Bam, Zabol, Baft and Kerman, respectively. However, seroprevalence of disease was not significantly related to age, gender, presence of clinical signs and hematological disorders. Based to the results of the present study, CVL is endemic in southeastern Iran. Delayed diagnosis and euthanasia of potentially infectious animals may occur with an increased transmission risk to sand flies and subsequently to humans. Implementation of potent screening tests with high validity is essential for rapid detection and successful dog elimination programs in endemic parts of Iran.

Effect of the plasmid-DNA vaccination on macroscopic and microscopic damage caused by the experimental chronic Trypanosoma cruzi infection in the canine model.

The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD). Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP) and an amastigote-specific glycoprotein (TcSSP4) were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.

Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin.

Effects of fipronil on dogs over Triatoma infestans, the main vector of Trypanosoma cruzi, causative agent of Chagas disease

Chagas disease is the most important endemic disease in Latin America, mainly transmitted by Triatoma infestans in the Southern Cone countries of South America. Dogs are one of the main domestic reservoirs of Trypanosoma cruzi, the etiological agent of Chagas disease. The presence of dogs in rural households of endemic areas significantly increases the likelihood of the vectorial transmission of the parasite. We studied the mortality and blood intake of T. infestans exposed to dogs treated with different doses and formulations of fipronil. Two doses, two formulations, and different distances to the application point of fipronil were compared. Third instar nymphs of T. infestans were fed at different time intervals after the insecticide application up to 45 days post-application. No significant difference was found between the blood intake of nymphs fed on control and treated dogs with different doses and formulations (p > 0.05). The spray formulation showed lower effect and persistence than the spot-on formulation. The mortality rate caused by the spot-on formulation in the 26.8-mg active ingredient (a.i.)/kg dose was higher (48%) than with the 13.4-mg a.i./kg dose (25%), 24 h after the insecticide application. The effect was highly heterogeneous among replicates of the same treatment. The mortality rate of nymphs fed over the point of the insecticide application was higher than the mortality of nymphs fed over places 12 cm apart from the fipronil application point, suggesting that the distribution of fipronil over the dog body is lower than the needed one to obtain a persistent triatomicide effect.

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26μg/ml against promastigotes and amastigotes of 0.27μg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests.