Estabelecimento de um protocolo de Nested-PCR para detecção do vírus da anemia das galinhas e análise filogenética de cepas brasileiras.

O vírus da anemia das galinhas (CAV) pode causar imunodepressão em galinhas de todas as idades e doença nos frangos jovens, a qual é caracterizada por severa anemia, atrofia da medula óssea e hemorragias. A doença clínica é rara hoje, porém a forma subclínica é freqüentemente encontrada em criações comerciais e resulta num considerável decréscimo do desempenho. O CAV apresenta variabilidade genética, entretanto, em relação às amostras brasileiras do CAV, pouco ou quase nada desta variabilidade é conhecida. O presente trabalho descreve um protocolo de Nested-PCR para a detecção do CAV diretamente de amostras clínicas e analisa filogeneticamente as seqüências nucleotídicas das amostras positivas buscando verificar se a patologia apresentada por estas está relacionada com a variabilidade genética. Para a extração de DNA, o método baseado em tiocianato de guanidina mostrou-se mais eficiente e prático de executar que os demais testados. Foi selecionado um par de primers que amplifica uma região de 664 pb do gene vp1 e outro par que amplifica uma região interna de 539 pb para a realização da Nested-PCR. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV e 30 diferentes isolados de vírus e bactérias causadoras de doenças em galinhas, as quais não geraram produto de amplificação. A sensibilidade foi determinada a partir de diluições seriadas da vacina comercial para o CAV. A Nested-PCR mostrou ser mais sensível do que a PCR e foi capaz de detectar 0,16 DICC50% da cepa vacinal. Além disso, a Nested-PCR detectou DNA viral em tecidos, soro e cama aviária de lotes com e sem sintomas clínicos.O produto de amplificação de 539 pb do gene vp1 de 44 amostras, provenientes de diferentes Estados produtores de frangos do Brasil, foi seqüenciado e foram encontradas 10 novas seqüências nucleotídicas do CAV. Estas 10 seqüências nucleotídicas foram analisadas filogeneticamente pelo método de distância neighbour joining com 1000 replicações o qual, mostrou que não houve correlação entre a patogenia apresentada nos animais e os grupos genéticos. Estas seqüências nucleotídicas também foram comparadas com 30 cepas de CAV isoladas em outros países e não foi observada correlação entre a distribuição geográfica e a variabilidade genética. Substituições de amino ácidos foram observadas em 9 posições sendo que, 65R substituindo o resíduo Q e 98F substituindo o resíduo Y ainda não haviam sido observadas. Conclui-se que, como técnica de detecção do CAV, o protocolo de Nested-PCR aqui descrito é mais sensível e menos trabalhoso do que o isolamento viral. As amostras Brasileiras de CAV possuem características filogenéticas similares às isoladas em outros países.

Glomerulonefrite na fasciolose bovina e bubalina

A fasciolose é uma zoonose parasitária de importância econômica principalmente para ruminantes. O dano causado pela morte dos animais é só uma fração das perdas econômicas, que é produzido pelos estados subclínico e crônico, que se manifesta com redução na produção de carne, leite e lã, condenação de fígados parasitados, infecções secundárias por bactérias, interferências na fertilidade e gastos com tratamento. O envolvimento de complexo antígeno-anticorpo causando reação de hipersensibilidade tipo III, decorrente de parasitoses é bem documentado. Porém não existem relatos do dano renal associado à Fasciola hepatica . Esta tese apresenta a comprovação da hipótese de glomerulonefrite associada à complexos imunes, em bovinos e búfalos naturalmente infectados..Foi necessária a obtenção dos parasitos para a produção de antígenos e de soro hiperimune, e de biópsias renais para a verificação in situ da reação antígeno – anticorpo. Esta tese consta de quatro artigos que descrevem estudos sobre a prevalência de fasciolose em bovinos e búfalos e os diagnósticos histopatológico e imunológico para evidenciação da reação de hipersensibilidade tipo III nos rins dos animais parasitados. O Artigo um descreve a prevalência de fasciolose em 10, 34 % (39) dos 377 fígados bovinos, obtidos de animais de 11 municípios do estado do Rio Grande do Sul. Dos municípios incluídos no estudo, em 63,9 % (7) deles houve o registro do parasito. O Artigo dois relata a prevalência de 20 % (21) de fasciolose hepática em 105 búfalos procedentes do estado do Rio Grande do Sul. Dos 5 municípios incluídos no estudo, em 80 % (4) deles houve o registro do parasito. Por faixa etária, para os búfalos de até 2 anos de idade a prevalência foi de 81 %. Para os animais acima de 2 anos de idade, a prevalência foi de 19 %.

A rastreabilidade de carnes segundo os atributos valorizados pelos consumidores : o caso da cadeia avícola do Rio Grande do Sul

Problemas de saúde pública causados pelo consumo de carnes envolvem diversos países e obrigam a cadeia agroalimentar a repensar o modo de produção e os sistemas de garantia de qualidade. Devido a este fato, esta pesquisa teve como objetivo estudar a rastreabilidade na cadeia avícola, tendo como foco o consumidor. Para isso foi realizado um survey, no qual foram realizadas entrevistas com 393 consumidores da cidade de Porto Alegre. Os questionários foram aplicados no período de abril a julho de 2004 em supermercados de pequeno, médio e grande porte, além do mercado público da cidade. Os atributos sanitários que o consumidor considera importantes para se rastrear na carne de frango são: Influenza Aviária (Gripe do Frango), Salmonela, Hormônios, Controle do Índice de Absorção de Água e Resíduos de Antimicrobianos. Além disso, os entrevistados consideram que as seguintes informações devam constar na embalagem da carne de frango: Data de Validade, o número do SIF, Certificado de Qualidade, Advertência quanto a Riscos de Toxinfecção Alimentar, Preço, Data de Abate e Origem/Procedência. São exploradas na pesquisa as correlações de diversas variáveis com o nível de escolaridade, renda e faixa etária. Com base nestes dados e utilizando a análise de filière, buscou-se propor maneiras de se atingir na rastreabilidade os pontos cruciais indicados pelos consumidores. Apesar da cadeia avícola ser bem organizada, foi evidenciada por esta pesquisa a necessidade de melhorar a circulação da informação na cadeia; para isso, sugere-se um sistema integrado de informação. Finalmente, foi proposto um modelo de rastreabilidade que contempla toda a cadeia avícola, incluindo todos os elos, desde o consumidor até o matrizeiro, indicando-se possíveis fatores que podem dificultar a construção do mesmo.

Estudo sobre a participação de rodedores na cadeia de transmissão de Leishmania infantum (Protozoa: Trypanosomatidae) no Rio Grande do Norte

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by the bite of the sand flies Lutzomia longipalpis.The main domestic reservoir is the dog, while foxes and opposums are the known wild reservoirs. However, identification of natural infections with L. infantum in rodents appears for need of investigating the participation of these rodents how source of infection of the parasite. In the present work the Leishmania infantum infection was investigated in rodents captured in Rio Grande do Norte, aiming at to offer subsidies to the understanding of the epidemic chains of LVA in the State. Thirteen Galea spixii were distributed in four groups, being G1 the group control with four animals and the others, G2, G3 and G4, with three animals each. Those animals were intraperitoneally inoculated with 107 promastigotas of L. infantum and accompanied for, respectively, 30, 90 and 180 days. Weekly the animals were monitored as for the corporal weight and rectal temperature. At the end of each stipulated period the animals were killed. Blood were used for determination of the parameters biochemical and haematological, PCR, ELISA, microscopic examination and cultivation in NNN medium. Liver, spleen and lymph node were used in Giemsa-stained impression and cultivation in NNN medium. Liver and spleen fragments were still used in PCR and histopathological, respectively. At the same time 79 rodents of the species Rattus rattus, Bolomys lasiurus, Oligoryzomys nigripis, Oryzomys subflavus and Trichomys apereoides were captured in the Municipal districts of Brejinho, Campo Grande, Coronel Ezequiel, Passa e Fica and Vázea for identification of natural infection with L. infantum. Evidence of infection was checked by direct examination of Giemsa-stained impression of liver, spleen and blood and culture of these tissues in NNN medium. Antibodies were researched by ELISA. They were not found differences among the weigh corporal final, rectal temperature and biochemical and haematological parameters of the Galea spixii controls and infected. The rectal temperature of the animals varied from 36OC to 40OC. For the first time values of the haematocrit (33,6% to 42,8%), hemoglobin (10,2 to 14,5g/dl), erythrocyts number (4,67x106 to 6,90x106/mm3), total leukocytes (0,9x103 to 9,2x103/mm3), platelets (49x103 to 509x103/mm3) total proteins (1,56 to 6,06 g/dl), albumin (1,34 to 3,05 g/dl) and globulins (0,20 to 3,01 g/dl) of the Galea spixii were determined. The lymphocytes were the most abundant leucocytes. Infection for L. infantum was diagnosed in two animals euthanasied 180 days after the infection. In one of the animals was also identified antibodies anti-Leishmania. The parasite was not found in none of the five other species of rodents captured. Galea spixii are resistant to the infection for L. infantum and they are not good models for the study for visceral leishmaniose, although they can act as infection sources. More studies are necessary to determine the paper of the rodents in the epidemic chain of transmission of the visceral leishmaniose in the State of Rio Grande do Norte

Estudo clínico-laboratorial e histopatológico de cães naturalmente infectados por Leishmania chagasi com diferentes graus de manifestação física

Canine Visceral Leishmania (CVL) is an important zoonotic disease that has a world wide distribution and has a large impact on public health on the American Continent, especially in Brazil, where the nature of endemic diseases in humans affects a large part of the nation. The influence of the prevalence of CVL in the increased rate of human cases in endemic areas and in the unleashing of epidemic outbreaks shows the need for a more profound understanding, that would generate significant advances in the current measures used to control the reservoirs of sickness that are practiced by the Programa Nacional de Vigilância e Controle da Leishmaniose Visceral. The present work describes and compares the clinical-laboratorial and histopathological findings of twenty-three dogs that were naturally infected by Leishmania chagasi, from endemic areas in metropolitan Natal, Rio Grande do Norte, Brazil. These animals, that were selected and given physical and serological exams (IFI and ELISA rK-39), were classified according to the degree of clinical severity and had blood samples drawn (whole blood and serum) for a complete hemogram and a coagulogram to be done as well as biochemical tests for kidney and liver function. The confirmation of infection by L. chagasi was done after the euthanasia of the animals, through the direct demonstration of the parasite in the impression of the spleen and liver crowned with GIEMSA and through a cultivation by means of NNN/Schneider. According to the clinical evaluation, the animals were classified as asymptomatic (7), oligosymptomatic (7) and polysymptomatic (9). Among the animals that were chosen to be autopsied, there were 2 asymptomatic, 3 oligosymptomatic and 3 polysymptomatic, for the purpose of studying their histopathology, having collected fragments of the spleen, liver, kidneys and skin and were fixed in 10% tamponed formol. The comparison between the average parameters of the clinical-laboratory tested animals in the groups was done through the Student t test (a<0.05). The main clinical signals observed were lymphadenomegaly, alopecy, dermatitis, exfoliation, cutaneous ulcers, onicogriphosis and emaciation. The main clinical-laboratorial alterations established, mainly in the polysymptomatic group, were anemia, hyperproteinemia, hyperglobulinemia, alterations in the albumin/globulin ratio and increased ALT activity. Renal alterations were not verified (urea and creatinine levels were normal). Thrombocytopenia was observed in three clinical groups. However, the other indicators of coagulation function (TAP and TTPA) did not have abnormal variations. There were inflammatory infiltrations and leishmania amastigotes in the skin of polysymptomatic dogs, however, they were not found in the skin of asymptomatic animals. Hypertrophy and hyperplasia of the phagocyte mononuclear system, leishmania amastigote parasites were found in the macrophages, extramedullary hematopoiesis and degenerative alterations were detected in the spleen and liver of 8 of the animals submitted to histopathological exams. In accord with these results, it was demonstrated that the expected alterations in the hematological and biochemical parameters in function of their viscerotropic nature of CVL are mainly observed in the more advanced stages of the disease. The absence of inflammatory infiltration and parasite load in the skin suggest that infected animals without symptoms may have an importance irrelevant to the infectiousness of the vector

Efetividade da doxiciclina na profilaxia contra letptospirose

Leptospirosis is a worldwide zoonosis of considerable medical and economical importance that affects humans in both urban and rural contexts, as well as domestic animals and wild fauna. Leptospira interrogans is the causative agent and is transmitted to humans by indirect contact with contaminated soil or water. The clinical syndromes include sub clinical infection, self-limited anicteric febrile illness, and severe and potentially fatal illness, known as Weil´s syndrome. In developed countries, leptospirosis is related to occupational or recreational activities while in developing countries, outbreaks occur during floods. In those regions, traditional strategies to prevent the transmission are difficulties to be implemented because of costs and lack of community acceptance. In addition, no efficient vaccine is available for human use. Several studies have suggested that chemoprophylaxis with doxycycline pre and post-exposure may be effective to prevent leptospirosis. Leptospirosis has been reported in rural areas of the State of Rio Grande do Norte, Brazil since 1985 in rice farmers who present the anicteric illness. The disease cause great social and economics impact. The study was conducted in São Miguel where an epidemic of leptospirosis in rice farmers was reported. The main objective was to determine the efficacy of doxycycline in preventing Leptospira exposure. A taxa de soroprevalência de leptospirose na população estudada antes e após a colheita foi de 14,2% (n=22) e de 16.6% (n=27) respectivamente. Anti-Leptospira serology was determined for 61 subjects in two instances, pre and post-exposure to potential contaminated water. There was an increased risk of 29.0 per cent in acquiring infection for individuals that did not use doxycycline. In addition, an increased risk of 30.0 % observed in farmers who did not use protection when exposed to Leptospira. The adhesion to preventive chemoprophylaxis was 55.7%. Therefore doxycycline, under specific circunstances appears to be an effective alternative to protect against leptosprirosis infection. A large sample composed of individuals to adhere to preventive therapy is needed to define time, dosage and length of use of doxycycline in this area

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Frequência da infecção por Toxoplasma gondii em galinhas caipira e frangos de corte em regiões dos Estados do Rio Grande do Norte e Paraíba

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a protozoan that has a cosmopolitan geographic distribution and low host specificity. Usually a benign and selflimiting, infection can manifest itself in a severe systemic becoming overwhelming in fetuses and patients with immunosuppression. Domestic fowl are considered one of the most important hosts in the epidemiology of toxoplasmosis, since they are potential sources of infection for humans, in addition to playing the role of important indicators of environmental contamination by oocysts of T. gondii. We studied the prevalence of infection by the protozoan in chickens of different breeding systems mesoregions from the states of Rio Grande do Norte and Paraiba: broilers from commercial farms (200/PB) and free-range chickens of small farms (322/RN and PB). Were standardized IFAT and ELISA techniques for detecting specific antibodies in blood samples of birds, and commercial kit was used to determine the prevalence by IHAT. There was no seropositive reaction by T. gondii in the samples of broilers tested, indicating that the particularities of intensive management limit the chances of infection for these animals. Among the hens, the frequency of IgG anti-T. gondii diagnosed by the techniques of IHAT, IFAT and ELISA, respectively, were 3.73% (12/322), 37.88% (122/322) and 40.37% (130/322), for both young and adult animals. Amongst the seropositive samples by IFAT, 33 (27.05%) were positive at a dilution of 1:16, in 1:32, 31 (25.41%), in 1:64, 24 (19.67%), 15 (12.29%) in 1:128, and 19 presented titer greater than or equal to 1:256 (15.57%). The evaluation of the presence of anti-T. gondii should be careful, and reagents IHAT provided erratic results in this measure for the specie studied. This suggests the need for own standardization of the kit before the use in epidemiological studies in animal species. On the other hand, substantial agreement observed between IFAT and ELISA techniques (Kappa = 0.62) enables these methods as effective methodologies for the diagnosis of toxoplasmosis in chickens. The high prevalence of specific antibodies among poultry in the region studied attempts to the potential risk of transmission of toxoplasmosis to humans

Proteínas SAG1, SAG2, SAG3 de toxoplasma gondii como perspectivas para o desenvolvimento de protótipo vacinal contra a toxoplasmose

Toxoplasmosis is a zoonosis of worldwide distribution caused by the protozoan Toxoplasma gondii, triggering dangerous complications in immunocompromised patients and pregnant women, as well as having great economic impact for the livestock. So far the control of toxoplasmosis is made primarily by chemotherapy. However, most drugs used routinely have some limitations. In order to control this disease, several research groups, including ours, has been working to develop a medical-veterinary vaccine based on parasite antigens, vectors and protocols of immunization. In this study were implemented and standardized methodologies for amplification and cloning of recombinant immunogens in the system for the development of a prototype vaccine, based on the surface antigens of T. gondii and recombinant adenovirus encoding these antigens. Genes encoding BAG1, GRA2 and SAG1 proteins were amplified. We established a strategy for cloning SAG1, SAG2, SAG3 and TgAMA1- genes in recombinant system. The genes encoding SAG1 and SAG2 were cloned and their sequences showed high similarity with sequences from GenBank. The virtual translation of these proteins showed polymorphisms in the amino acid sequence, which can be correlated with levels of antigenicity. Simultaneously, the adenovirus encoding the SAGs (HAdSAGs) were expanded, purificated and characterizated. Immunization of C57bl/6 mice, using viral supernatant was not enought to elicit immune responses at high levels, being required HAdSAGs titration for future immunizations. Therefore, this study allowed the cloning of the two genes important for the development of a prototype vaccine. Besides, implementations methodologies that permit advancements in the development of a vaccine against toxoplasmosis using adenovirus to express proteins of the parasite

Apoptose na infecção experimental de cães domésticos com Ehrlichia canis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Frações Nitrogenadas, Glicídicas e Amônia Liberada pela Cama de Frangos de Corte em Diferentes Densidades e Tempos de Confinamento

O presente trabalho foi conduzido com o objetivo de estudar a liberação de amônia pela cama de frangos de corte, bem como o teor de umidade e de compostos glicídicos (carboidratos redutores) e nitrogenados (nitrogênio total, nitrogênio solúvel total e nitrato) presentes na mesma, em função da densidade populacional, tempo de confinamento e sexo das aves. Realizou-se o experimento em delineamento inteiramente casualizado. A cama de frango foi submetida às densidades de 10; 14; 18 e 22 aves/m² de piso, para ambos os sexos, e analisada em três épocas diferentes (24, 38 e 42 dias de idade das aves), utilizando-se de esquema fatorial 4x2x3 (densidades x sexos x épocas). de modo geral, observou-se que o sexo não influenciou o comportamento das variáveis estudadas. O aumento da densidade e o avanço da época de amostragem induziram a aumentos da quantidade de amônia liberada, do teor de umidade e dos teores de nitrogênio total e de nitrogênio solúvel, bem como diminuição do teor de carboidratos redutores. Estes resultados sugerem a necessidade de um controle rigoroso da amônia no ar dos galpões, principalmente em densidades elevadas e no período final de criação.

Estudo epidemiológico da Toxoplasmose em gestantes atendidas na Maternidade Escola Januário Cicco, Natal, Rio Grande do Norte

Congenital Toxoplasmosis results in severe systemic disease. If mother is infected for the first time during gestation, she can infect the fetus causing substantial damage. However, relatively little is known about the seroprevalence and epidemiological and economic factors of Toxoplasmosis infection in pregnancy in the most state in northeastern Brazil and knowledge about this can be essential in determining effective and acceptable prevention strategies. Our aim was to determine the prevalence of Toxoplasmosis in pregnant woman consulted by reference Maternity Escola Januário Cicco in Natal, a city in Northeastern Brazil, which belongs to the public health system, correlating to the risk factors involved in the infection and to accomplish active Search in the Hospital of Pediatrics Profº Heriberto Bezerra of the damages caused by the Toxoplasmic infection in children up to 12 years of age. The study was conducted from March to December 2007 and sera obtained from 190 pregnant women were tested for IgM and IgG antibodies avidity to Toxoplasma by Microparticle enzyme immunoassay (Abbott AxSYM system - Abbott Laboratories, Chicago, IL, USA). Data were examined with univariate analysis. Chi-squared (x2) and Odds ratio was calculated (IC 95% p 0,05). Of these women, 126 (66,3%) had only IgG antibodies high-avidity against T. gondii; 01 (0,52%) had a IgM and IgG high-avidity antibodies against T. gondii and 63 (33,1%) have neither IgM nor IgG against T. gondii. Our studies shown that the direct contact with cats or dogs was highly associated with the Toxoplasma gondii infection (OR, 2.72, p<0.001, 95% CI 1.46 5.02). The years school (p<0,001), socioeconomic status and knowledge about the disease (both p value 0.05) also were associated with Toxoplasmosis. The pattern of risk factors for infection presents regional variations, however our data corroborate others studies in Brazil. In children up to 12 years, one case of Congenital Toxoplasmosis was just registered in seven years (2000 - 2006). There were several suggestive cases, with signs and characteristic symptoms, but that the infection was not confirmed due to lack in the researches through laboratorial and images exams that addressed that it zoonosis

Esporotricose do gato doméstico (Felis catus): transmissão humana

No presente trabalho relata-se caso de paciente, funcionário de hospital veterinário, infectado através de arranhadura de gato doméstico portador de esporotricose. Inquérito domiciliar junto aos proprietários do animal fonte de infecção, revelou dois outros casos presuntivos de esporotricose humana transmitida por gatos, e confirmou o diagnóstico, por cultivo do Sporotrix schenckii, em 3 gatos domésticos adicionais. A esporotricose felina caracteriza-se por lesões cutâneas ulceradas e tendência à disseminação sistêmica e evolução fatal. A transmissão intra e inter-espécie é facilitada pela exuberância de fungos nas lesões cutâneas de felinos infectados.