Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil.

INTRODUCTION: We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil. METHODS: Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners' knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher's exact tests, followed by logistic regression, was used. RESULTS: It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog's size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023). CONCLUSIONS: These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil

Abstract INTRODUCTION: We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil. METHODS: Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners' knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher's exact tests, followed by logistic regression, was used. RESULTS: It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog's size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023). CONCLUSIONS: These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

Biological control of Anopheles darlingi, Aedes aegypti and Culex quinquefasciatus larvae using shrimps.

Mosquitoes can act as vectors of important diseases such as malaria, dengue, Zika virus, yellow fever, Chikungunya and Mayaro fever, in addition to filariasis. The use of insecticides, larvicides, bed nets and repellents, besides the use of drugs as chemoprevention and the treatment of the sick are currently the pillars of the control of these vectors. We studied the biological control against of Anopheles darlingi, Aedes aegypti and Culex quinquefasciatus larvae using shrimps of the species M. pantanalense, M. amazonicum, M. brasiliense and M. jelskii. Larvae of mosquitoes were collected from the breeding environment and placed in a 500 and 1000 l tank containing 60 shrimps/m2. The predatory activity was evaluated for 30 days and, in all groups it was observed that 100% of the larvae were consumed in few minutes. In the environment, these same species of crustaceans were released in water bodies with the presence of larvae of these insects. In just 72 h there was a marked reduction of the larvae in the release sites of shrimps. Similarly, there was a reduction in the number of adult mosquitoes caught near the breeding sites, allowing to infer that, in places where the crustaceans were released, the predatory activity on the larvae of mosquitoes was sufficient to reduce the number of adult mosquitoes p ≤ 0,05. This is the first description of the predatory activity of M. pantanalense, M. amazonicum, M. brasiliense and M. jelskii on An. darlingi, A. aegypti and C. quinquefasciatus larvae, constituting an important tool of biological control of these parasites-vectors.

Are Aedes aegypti mosquitoes potential vectors for leishmaniasis? ­ Case report / Mosquitos Aedes aegypti são vetores potenciais de leishmaniose? ­ Relato de caso

In Brazil dipters of the Lutzomyia genus are the main vectors of leishmaniasis for humans and animals. However, other hematophagous insects such as ticks, fleas, and horse flies may also be considered potential vectors of this protozoon. This paper, regarding an endemic area for visceral leishmaniasis, is the the first description of the Leishmania spp. presence in Aedes aegypti mosquitoes. Two A. aegypti mosquitoes were captured: one of them was feeding on a polysymptomatic dog with leishmaniasis, confirmed by parasitic demonstration and positive PCR for Leishmania spp., and the other was collected in the environment where the dog was isolated. The mosquito engorged with dog's blood was crushed between two microscopic slides and the other one was processed by the polymerase chain reaction assay (PCR) searching for the presence of Leishmania spp. DNA. Amastigote forms of Leishmania sp, were observed in the smear prepared from one mosquito by microscopic examination, as well as other protozoa's flagellated forms. In the other insect it was observed Leishmania DNA amplification. This observation reinforces the role of dogs as sources of infection of Leishmania spp. even to other potential vector species.(AU)

No Brasil, os dípteros do gênero Lutzomyia são os principais vetores da leishmaniose para humanos e animais. No entanto, tem sido constatado que outras espécies de invertebrados hematófagos, como carrapatos, pulgas e mutucas, também podem ser vetores desse protozoário. Este trabalho, realizado em uma área endêmica de leishmaniose visceral, é a primeira descrição da presença de Leishmania spp. em mosquitos da espécie A. aegypti. Dois mosquitos A. aegypti foram capturados no local onde estava isolado um cão polissintomático acometido por leishmaniose visceral, confirmada pela demonstração do parasita em biópsias de órgãos e por resultado positivo na prova de PCR para Leishmania spp. Um dos mosquitos estava sugando o sangue do cão e o outro estava livre no ambiente. O mosquito ingurgitado com o sangue do animal foi esmagado entre duas lâminas de microscopia e o outro foi processado por meio da reação em cadeia pela polimerase (PCR) aplicada à pesquisa do ADN de Leishmania spp. Ao exame microscópico do esfregaço preparado com o mosquito que estava parasitando o cão foram observadas formas amastigotas de Leishmania spp., bem como formas flageladas de outra espécie de protozoário. No outro inseto foi detectada amplificação de ADN do gênero Leishmania. Esta constatação reforça o papel dos cães como fontes de infecção de Leishmania spp. até mesmo para outras espécies de vetores potenciais.(AU)

Histochemical and molecular evaluation of the prevalence of Leishmania spp. in hematophagous insects.

The prevalence study of Leishmania spp. in hematophagous insects captured from the environment in bat roosts and pigeon nests, or feeding their hosts (cattle, pigs, horses, dogs and humans) in urban, peri-urban and rural areas, between 2012 and 2014. For this study, the amastigotes present in these insects were detected by histochemical and PCR techniques. Positive gene amplification for Leishmania was found in two horseflies of the species Tabanus importunus collected in the environment, and amastigote forms of Leishmania spp., as well as erythrocytes and leukocytes, were histochemically detected in one of that insect. The other analyzed insects were not positive by PCR our by direct parasitological examination. Only horseflies captured in urban and peri-urban areas were positive. During the collection, no phlebotomine sand flies were captured in rural areas far from the city limits. It can be concluded that the discovery of horseflies positive for Leishmania spp. in urban and peri-urban areas indicates the likelihood that urban areas and their surroundings provide vector parasites with an environment suitable for the spread and consequent perpetuation of the biological cycle of this protozoan.

Seroprevalence rates of antibodies against Leishmania infantum and other protozoan and rickettsial parasites in dogs.

Canine visceral leishmaniasis (CVL) is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT) to analyze L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii and Neospora caninum infection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.

Seroprevalence rates of antibodies against Leishmania infantum and other protozoan and rickettsial parasites in dogs / Soroprevalência de anticorpos contra Leishmania infantum e outras espécies de protozoários e rickettsia em cães

Canine visceral leishmaniasis (CVL) is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT) to analyze L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii and Neospora caninum infection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.

Leishmaniose Visceral Canina (LVC) é causada pelo protozoário Leishmania infantum, podendo infectar cães e humanos em várias regiões do Brasil. O presente estudo teve por objetivo realizar a reação de imunofluorescência indireta (RIFI) para analisar os índices de infecção parasitária para L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii e Neospora caninum, em 93 amostras de soro de cães de um assentamento rural no município de Ilha Solteira, SP, Brasil. A taxa de soroprevalência de cães com anticorpos anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii e anti-N. caninum foi de 37,6%, 75,3%, 72%, 47,3% e 6,4%, respectivamente. Pelo exame microscópico direto dos parasitas nos esfregaços de aspirados de linfonodos poplíteos dos cães, a positividade para LVC foi de 26,9%. Pelos exames sorológicos, 17,2% dos cães estavam positivos com um único parasita, 29% com dois, 33% com três, 16,1% com quatro e 1,1% com cinco parasitas. Além disso, 3,2% eram soronegativos para todos os cinco agentes parasitários. A presença de anticorpos aos parasitos em amostras sorológicas confirmam a exposição dos cães às doenças parasitárias nesse assentamento rural. Devido ao potencial risco zoonótico destas doenças, principalmente leishmaniose, erliquiose e toxoplasmose, atenção especial deve ser dada aos programas que objetivam o aprimoramento de testes diagnósticos e de medidas de controle dessas parasitoses.

[Comparative study of diagnostic methods for visceral leishmaniasis in dogs from Ilha Solteira, SP]. / Estudo comparativo dos métodos diagnósticos para Leishmaniose Visceral em cães oriundos de Ilha Solteira, SP.

The purpose of the present work was a comparative study of diagnostic methods for Canine Visceral Leishmaniasis (CVL) using serological methods, enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT), histochemical (HE) and immunohistochemical (IMHC) tests using spleen, lymph node and liver canine tissues. In addition, Polymerase Chain Reaction (PCR) was done in blood and in tissues in order to compare and confirm no conclusive and negative diagnosis by the methods above. For this study, 34 dogs were divided according to clinical signs in asymptomatic, oligosymptomatic and polisymptomatic Leishmania-infected dogs euthanized by Zoonotic Disease Control Center (CCZ) from Ilha Solteira, SP, Brazil. The positivism indexes of ELISA, IMHC, IFAT and HE were 65.0, 62.0, 56.0 and 56.0%, respectively with the highest numbers of positive dogs in polisymptomatic (92.0%) followed by oligosymptomatic (57.0%) and asymptomatic dogs (12.5%). Furthermore, PCR confirmed the positive results and detected DNA in tissues from 100% of negative dogs and 89.0% suspects raising the animal positivism index up to 97.0%. In conclusion, PCR was the most sensitive and a valuable method for a definitive CVL diagnosis.

Estudo comparativo dos métodos diagnósticos para Leishmaniose Visceral em cães oriundos de Ilha Solteira, SP / Comparative study of diagnostic methods for visceral leishmaniasis in dogs from Ilha Solteira, SP

O objetivo da presente pesquisa foi avaliar comparativamente os métodos diagnósticos da Leishmaniose Visceral Canina (LVC), utilizando-se o ensaio imunoenzimático indireto (ELISA), a reação de imunofluorescência indireta (RIFI), a histoquímica (HE) e a imunoistoquímica (IMIQ) em tecidos de órgãos, como o baço, linfonodo e fígado. Além disso, a Reação em Cadeia pela Polimerase (PCR) das amostras de sangue e dos tecidos foi utilizada para comparar e confirmar os diagnósticos negativos e não conclusivos pelos métodos acima. Para esse estudo, foram utilizados 34 cães com diferentes sintomas da LVC, classificados em polissintomáticos, oligossintomáticos e assintomáticos e eutanasiados no Centro de Controle de Zoonoses (CCZ) de Ilha Solteira, SP. Os índices de positividade para os testes ELISA, IMIQ, RIFI e HE foram de 65,0, 62,0, 56,0 e 56,0 por cento, respectivamente, sendo a maior positividade detectada nos cães polissintomáticos (92,0 por cento), seguida pelos oligossintomáticos (57,0 por cento) e assintomáticos (12,5 por cento). A PCR confirmou os resultados positivos pelas outras técnicas e ainda detectou DNA do parasita nos tecidos de 100 por cento dos cães negativos e em 89,0 por cento dos suspeitos, elevando para 97,0 por cento a positividade. Em conclusão, a PCR demonstrou ser o método mais sensível e preciso para o diagnóstico definitivo da LVC.

The purpose of the present work was a comparative study of diagnostic methods for Canine Visceral Leishmaniasis (CVL) using serological methods, enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT), histochemical (HE) and immunohistochemical (IMHC) tests using spleen, lymph node and liver canine tissues. In addition, Polymerase Chain Reaction (PCR) was done in blood and in tissues in order to compare and confirm no conclusive and negative diagnosis by the methods above. For this study, 34 dogs were divided according to clinical signs in asymptomatic, oligosymptomatic and polisymptomatic Leishmania-infected dogs euthanized by Zoonotic Disease Control Center (CCZ) from Ilha Solteira, SP, Brazil. The positivism indexes of ELISA, IMHC, IFAT and HE were 65.0, 62.0, 56.0 and 56.0 percent, respectively with the highest numbers of positive dogs in polisymptomatic (92.0 percent) followed by oligosymptomatic (57.0 percent) and asymptomatic dogs (12.5 percent). Furthermore, PCR confirmed the positive results and detected DNA in tissues from 100 percent of negative dogs and 89.0 percent suspects raising the animal positivism index up to 97.0 percent. In conclusion, PCR was the most sensitive and a valuable method for a definitive CVL diagnosis.

[Parasitological, immunohistochemical and histopathological study for Leishmania chagasi detection in splenic tissues of dogs with visceral leishmaniasis]. / Exames parasitológicos, imunoistoquímicos e histopatológicos para detecção de Leishania chagasi em tecidos esplênicos de cães com leishmaniose visceral.

The purpose of this work was a Canine Visceral Leishmaniasis--CVL study by parasitological direct examination of Leishmania (L.) chagasi (imprinting and histological), immunohistochemical test and histopathological analysis using spleen tissues from 34 dogs euthanized by the Zoonotic Disease Control Centre from Ilha Solteira, SP, Brazil. According to the clinical signs, the dogs were divided in three groups: asymptomatics (8 dogs), oligosymptomatics (17 dogs) and symptomatics (9 dogs). After the accomplishment of all diagnostic tests, 22 dogs were considered positives (64.7%) and 12 (35.3%) were negatives to CVL. From these positive dogs, 1/22 (4.5%) was asymptomatic, 12/22(54.5%) were oligosymptomatics and 8/22 (40.1%) were symptomatics. The histopathological study in spleen tissues from positive, especially symptomatic dogs, showed a diffuse chronic inflammation with thickness of capsular and trabecular regions and there was extensive morphologic alteration of the red and white pulp by the presence of abundant macrophages full with amastigotes, the granulomatous inflammatory reaction and haemorrhagic areas. The data of this work from histopathologic examination and direct microscopic visualization of L. (L.) chagasi showed that the spleen was an useful organ to collect sample tissues for CVL diagnosis. The immunostaining detected the highest number of positive dogs and were considered an important and conclusive method to be used in addition to parasitological methods for CVL, particularly in asymptomatic or oligosymptomatic dogs.

Exames parasitológicos, imunoistoquímicos e histopatológicos para detecção de Leishmania chagasi em tecidos esplênicos de cães com leishmaniose visceral / Parasitological, immunohistochemical and histopathological study for Leishmania chagasi detection in spleenic tissues of dogs with visceral leishmaniasis

O propósito do presente trabalho foi o estudo da Leishmaniose Visceral Canina - LVC por meio de métodos parasitológicos e imunoistoquímicos para a detecção de formas amastigotas de Leishmania (L.) chagasi em baço, além de descrever a histopatologia das lesões esplênicas em 34 cães, com diferentes manifestações clínicas da LVC, eutanasiados pelo Centro de Controle de Zoonoses de Ilha Solteira, SP. Esses animais foram examinados clinicamente antes da eutánásia e de acordo com os sinais clínicos da LVC, foram classificados em três grupos: assintomáticos (8 cães), oligossintomáticos (17 cães) e sintomáticos (9 cães). Após a realização desses exames, dos 34 cães, 22 (64,7 por cento) estavam positivos e 12 (35,3 por cento) negativos. Desses cães positivos, 1/22 (4,5 por cento) era assintomático, 12/22 (54,5 por cento) eram oligossintomáticos e 9/22 (40,1 por cento) sintomáticos. Pela histopatologia, os cães, especialmente os sintomáticos apresentavam o baço com inflamação crônica e espessamento na região capsular e trabecular, além de extensa alteração morfológica na polpa vermelha e branca pela presença de grande quantidade de macrófagos repletos de amastigotas, pela reação granulomatosa inflamatória e pelas áreas hemorrágicas. Os exames histopatológicos e a detecção microscópica direta da L. (L.) chagasi revelaram que o baço é um órgão útil para auxiliar no diagnóstico da LVC. A coloração imunoistoquímica foi a que detectou o maior número de tecidos esplênicos positivos com amastigotas, além de elucidar os casos suspeitos pelos exames parasitológicos, principalmente, nos animais assintomáticos ou oligossintomáticos.

The purpose of this work was a Canine Visceral Leishmaniasis - CVL study by parasitological direct examination of Leishmania (L.) chagasi (imprinting and histological), immunohistochemical test and histopathological analysis using spleen tissues from 34 dogs euthanized by the Zoonotic Disease Control Centre from Ilha Solteira, SP, Brazil. According to the clinical signs, the dogs were divided in three groups: asymptomatics (8 dogs), oligosymptomatics (17 dogs) and symptomatics (9 dogs). After the accomplishment of all diagnostic tests, 22 dogs were considered positives (64.7 percent) and 12 (35.3 percent) were negatives to CVL. From these positive dogs, 1/22 (4.5 percent) was asymptomatic, 12/22 (54.5 percent) were oligosymptomatics and 8/22 (40.1 percent) were symptomatics. The histopathological study in spleen tissues from positive, especially symptomatic dogs, showed a diffuse chronic inflammation with thickness of capsular and trabecular regions and there was extense morphologic alteration of the red and white pulp by the presence of abundant macrophages full with amastigotes, the granulomatous inflammatory reaction and haemorragic areas. The data of this work from histopathologic examination and direct microcopic visualization of L. (L.) chagasi showed that the spleen was an useful organ to collect sample tissues for CVL diagnosis. The immunostaining detected the highest number of positive dogs and were considered an important and conclusive method to be used in addition to parasitological methods for CVL, particularly in asymptomatic or oligosymptomatic dogs.

Prevalência de cisticercose bovina em animais abatidos em frigoríficos sob inspeçäo federal, da 9ª regiäo administrativa de Araçatuba, SP / Prevalence of Cysticercus bovis in dead animals slaughtery and beef industries under Federal Inspection regulations which were operating on 9th Administrative region of Araçatuba, State of Säo Paulo

Foi analisada a prevalência da cisticercose bovina em 1.976.824 bovinos no período de janeiro de 1990 a junho de 2000, nos frigoríficos sob regime de Inspeçäo Federal, instalados na 9ª regiäo administrativa de Araçatuba, Estado de Säo Paulo, procedentes dos Estados de Säo Paulo, Mato Grosso do Sul, Paraná, Minas Gerais, Goiás, Rondônia, Tocantins e Santa Catarina, constituindo-se numa amostragem de grande importância para o desempenho de serviços de vigilância epidemiológica. Os dados foram obtidos através dos mapas nosográficos mensais: de destinaçäo de carcaças de bovinos, de localizaçäo das lesöes, de condenaçöes de órgäos, nosográfico de cisticercose e de origem dos bovinos abatidos. Observou-se índice de 4,18 por cento de prevalência de cisticercose bovina, sendo que a forma calcificada (2,99 por cento) foi superior a forma viva (1,19 por cento), com predileçäo pelos músculos do coraçäo e os mastigadores (masseteres) em 97,46 por cento dos casos. Todos os municípios que compöem a 9ª regiäo administrativa de Araçatuba, forneceram animais para o abate no período e 100 por cento deles apresentaram casos positivos de cisticercose bovina; o município de Lourdes apresentou o maior índice (19,90 por cento) e Gastäo Vidigal o menor (2,95 por cento). De acordo com estes dados conclui-se que os índices de prevalência da cisticercose bovina para a regiäo estudada foram altos e isto constitui um sério problema para a saúde pública e acarreta prejuízos econômicos para a pecuária regional.