Visceral leishmaniasis in a captive crab-eating fox Cerdocyon thous.

Visceral leishmaniasis (VL) is a zoonotic disease with worldwide distribution. The crab-eating fox (Cerdocyon thous) is considered a wild reservoir of many zoonotical diseases, particularly VL. This study reported the presence of Leishmania infantum amastigotes in different organs of one captive C. thous found dead in a zoo. This animal was positive by the indirect fluorescence antibody test and had many clinical signs of VL. Intracellular amastigote forms of L. infantum were seen in neutrophils and macrophages in sample tissues from skin, lymph nodes (popliteal, submandibular, prescapular, and mesenteric), spleen, and liver. The numbers of positive cells and intracellular parasites were higher in macrophages than in neutrophils. In addition, polymerase chain reaction demonstrated extensive distribution of Leishmania DNA in C. thous tissues from multiple organs. The presence of intracellular amastigotes in neutrophils and macrophages as well as DNA of the parasite in tissues, specifically skin demonstrate that this crab-eating fox is an adequate host for L. infantum and reinforce the importance of VL for symptomatic wild canids kept in captivity in endemic areas.

Diversity of Blastocystis subtypes in wild mammals from a zoo and two conservation units in southeastern Brazil.

The enteric protist Blastocystis is one of the most commonly parasite reported in humans and a variety of animal hosts worldwide. Regarding genetic diversity, at least 17 subtypes (STs) have been identified in mammals and birds, with eight of them (ST1-8) infecting both humans and animals. Recently, isolates from wild mammalian species have been genetically characterized, however data is still scarce, mainly in Latin America. Here, we aimed to verify the occurrence and genetic diversity of Blastocystis in captive wild mammals kept in one zoo and in two units of protection and conservation in southeastern Brazil. A total of 78 fecal samples (14 pooled and 64 individual samples) were recovered from 102 wild mammals of 35 species included in the following orders: Primates, Carnivora, Artiodactyla, Pilosa, Rodentia and Marsupialia. Zoo and units staff were invited to participated but only 16 fecal samples could be screened. Based on the sequence analyses of SSUrDNA gene, out of 29 PCR products from animal samples, 51.7% (15/29) were successfully sequenced and five Blastocystis subtypes were identified as follows: ST1 (2/15; 13.3%), ST2 (2/15; 13.3%), ST3 (4/15; 26.6%), ST5 (2/15; 13.3%) and ST8 (5/14; 33.3%). Only four isolates from humans were sequenced and identified as ST1 (2 isolates), ST2 and ST3. It was observed that Blastocystis infecting non-human primates belong to ST1 and ST2 and mainly to ST3 and ST8, artiodactyls ST5, carnivores ST1 and ST5 and rodents ST1. In addition, this present study reports some interesting findings: (1) 63% (12/19) of Blastocystis isolates from animals and employees belonged to the potentially zoonotic subtypes ST1-ST3; (2) most of these isolates displayed high identity with publicly available DNA sequences from non-human primates and humans, including primate handlers; (3) Blastocystis ST5 was found infecting the northern tiger cat, a native South American felid and one of the species facing a high risk of extinction in Brazil.

Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis.

The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.

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.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil.

Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania) infantum and Leishmania (Leishmania) chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.

Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis / Avaliação comparativa entre vários métodos para o diagnóstico da Leishmaniose Visceral Canina

The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.

O objetivo do presente estudo foi avaliar os métodos sorológicos usando ELISA (Ensaio Imunoenzimático Indireto) com o antígeno recombinante rK39 (ELISA-rK39) e o antígeno extrato solúvel bruto (ELISA-ES) e a RIFI (Reação de Imunofluorescência Indireta) em comparação com o método imunocromatográfico rápido (RDT-rK39) e o parasitológico direto (PA), para o diagnóstico da Leishmaniose Visceral Canina (LVC) em cães de Ilha Solteira, São Paulo, Brasil. Os resultados mostraram que 89% (60/67) dos cães foram positivos por pelo menos um teste diagnóstico sorológico (RIFI, ELISA-ES, ELISA-rk39 ou RDT-rK39) e somente 40,3% (27/67) foram positivos pelo PA. O ELISA-ES foi o teste que detectou anticorpos anti-Leishmania em maior número de cães (71,6%) seguido por ELISA-rK39, RIFI (65,7%) e por RDT-rK39 (55,2%). No total de cães analisados (assintomáticos e sintomáticos), o índice Kappa de concordância (p ≤ 0,05) foi considerado de boa concordância entre ELISA-ES e IFAT (88,1%; k= 0,7237) e entre RDT-rK39 com ELISA-ES (83,6%, k= 0,6561), RIFI (83,5%, k= 0,6605) e PA (85,0%, k= 0,7074). O índice de concordância ruim foi observado em qualquer associação de ELISA-rk39 com todos os outros testes nos animais sintomáticos e nos assintomáticos. Tanto o ELISA como o RDT com proteínas recombinantes (rK39) detectaram a menor porcentagem de cães assintomáticos (33,3%) em relação aos outros testes sorológicos. Em conclusão, somente um método diagnóstico não foi suficiente para identificar todos os cães positivos com LVC, principalmente os assintomáticos e por isso foi necessário a associação de dois ou mais métodos. Em função da boa concordância do teste RDT-rK39 com ELISA-ES, RIFI e PA, o mesmo foi sugerido como um teste complementar ao ELISA-ES ou RIFI para o diagnóstico da LVC, principalmente dos cães sintomáticos. No entanto, novos estudos são recomendados para melhorar a sensibilidade dos testes principalmente para cães assintomáticos.

[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.

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.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil / Detecção sorológica e molecular de Leishmania spp. em animais selvagens do zoológico de Ilha Solteira, SP, Brasil

Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania) infantum and Leishmania (Leishmania) chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.

Leishmaniose é uma doença zoonótica que afeta cerca de 12 milhões de pessoas no mundo todo. Várias espécies mamíferas podem servir de reservatório para a doença. Os cães são considerados os principais reservatórios para a leishmaniose visceral em áreas urbanas, o que tem se tornado um sério problema de saúde pública no Brasil. O objetivo deste trabalho foi avaliar a presença de Leishmania spp. em animais selvagens mantidos no zoológico de Ilha Solteira, São Paulo, Brasil. Foram coletados amostras de sangue e tecidos de cinco espécies diferentes: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, e Procyon cancrivorus. Anticorpos contra Leishmania spp. foram detectados em três canídeos pelo teste de imunofluorescência indireta (RIFI) e pelo ensaio imunoenzimático indireto (ELISA-teste). A análise de PCR das amostras de sangue e medula óssea foi negativa para todas as amostras, mas DNA de Leishmania foi encontrado em tecidos e pele de animais soropositivos. As amostras de PCR positivas também foram positivas para o complexo Leishmania donovani. Análise de sequenciamento dos produtos de PCR mostrou similaridade com diferentes regiões do cinetoplasto de Leishmania (Leishmania) infantum e Leishmania (Leishmania) chagasi. Medidas de controle de leishmaniose visceral em animais selvagens mantidos em zoológicos brasileiros devem ser estabelecidas, uma vez que não há nenhum programa de controle disponível.

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.