ABSTRACT: We describe an outbreak of leishmaniasis in seven guinea pigs (Cavia porcellus) in which nodular ulcerated skin lesions of varying sizes were observed in the nasal cavity, upper lip, pinnae, vulva, and periarticular region of the limbs. Cytologic exam of collected samples of the lesions in the auricle of one of the animals revealed macrophages containing parasitophorous vacuoles of approximately 4.0μm in diameter in their cytoplasm with morphology suggestive of Leishmania sp. Although skin lesions spontaneously regressed in two of the Guinea pigs, only one survived. All six animals that died were necropsied. Grossly, all animals showed bloody nodular cutaneous lesions with crusts. One of the guinea pigs had distended dark red and firm lungs. Histopathology of the skin lesions revealed histiocytic interstitial acanthotic dermatitis associated with a myriad of Leishmania organisms within macrophages cytoplasm. In the lung, the lesions were characteristic of broncho-interstitial pneumonia with focal infiltrates of neutrophils, epithelioid macrophages, and multinucleated giant cells containing 2µm basophilic amastigotes with morphology compatible with Leishmania spp. A focal granulomatous lesion ,associated with the causal agent in the lung is a novel description of leishmaniasis in guinea pigs caused by L. enriettii. The polymerase chain reaction (PCR) technique with mini-exon primer performed in samples of lesions from two affected guinea pigs was positive and equal to the reference strain, identifying Leishmania enriettii. The cytological, macroscopic, and histological lesions associated with the PCR technique allowed the diagnosis of leishmaniasis and the identification of the specie L. enriettii.

RESUMO: Descrevemos um surto de leishmaniose em sete cobaias (Cavia porcellus), com lesões cutâneas nodulares ulceradas de tamanhos variados observadas na cavidade nasal, lábio superior, pavilhões auriculares, vulva e região periarticular dos membros. No exame citológico foram encontrados macrófagos contendo vacúolos parasitóforos no citoplasma de aproximadamente 4.0μm em diâmetro com morfologia sugestiva de Leishmania sp. Apesar de regressão espontânea das lesões cutâneas terem ocorrido em duas das sete cobaias, apenas um sobreviveu. Seis dos sete animais afetados morreram e foram necropsiados. Macroscopicamente, todos os animais apresentaram lesões cutâneas nodulares, crostosas e sanguinolentas. Uma das cobaias tinha pulmões vermelho-escuros, distendidos e firmes. A histopatologia das lesões cutâneas revelou dermatite acantótica intersticial histiocítica associada a miríades de organismos de Leishmania no citoplasma de macrófagos. Nos pulmões as lesões eram características de pneumonia bronco-intersticial com infiltrado focal de neutrófilos, eosinófilos, macrófagos epitelioides e células gigantes multinucleadas contendo amastigotas basofílicos de 2µm com morfologia compatível com Leishmania spp. Lesões granulomatosas focais associadas ao agente no pulmão são um achado inédito na leishmaniose causada por L. enriettii em cobaias. A técnica de reação em cadeia da polimerase (PCR) com primer mini-exon realizada em amostras de lesões de duas cobaias afetadas foi positiva, identificando Leishmania enriettii. Os aspectos macroscópicos, citológicos, e histológicos associados à técnica da (PCR), permitiram o diagnóstico da leishmaniose e a identificação da espécie L. enriettii.

RESUMEN Lutzomyia intermedia (Diptera: Psychodidae) es uno de los principales vectores que participan en la transmisión de leishmaniasis cutánea americana (LCA) en la región Neotropical. A pesar de que aún los estudios genéticos que involucran a este taxón son incipientes, tienen una gran importancia para comprender el nivel de variabilidad de las diferentes poblaciones y sus implicaciones en su papel vectorial. El objetivo de este estudio fue determinar el nivel de diversidad genética de L. intermedia presente en el Valle del Río Ribeira, área de transmisión de LCA en el estado de Paraná, Brasil, mediante RAPD (ADN polimórfico amplificado aleatoriamente). Los flebótomos fueron recolectados en los municipios Cerro Azul (nueva área de transmisión de LCA) y Adrianópolis (área endémica de LCA), donde fueron capturados en ambientes residenciales, en el peridomicilio y en el bosque. Dos de los marcadores utilizados permitieron estimar el polimorfismo en las poblaciones estudiadas con la obtención de 40 genotipos, la mayoría de ellos en el peridomicilio. El dendrograma generado por el análisis con el cebador A10 mostró diferentes grados de similitud, lo que sugiere que puede haber flujo gènico en las poblaciones. El Análisis de Coordenadas Principales (PCO) con el cebador A2 fue útil para agrupar L. intermedia según su origen ecológico y geográfico. No hubo distinción entre los linajes que componen el complejo L. intermedia. Los resultados de este estudio, con el registro de gran diversidad genotipica en L. intermedia, pueden contribuir a explicar el mantenimiento del ciclo biológico de Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) en la región.

ABSTRACT Lutzomyia intermedia (Diptera: Psychodidae) features as one of the main vectors that are involved in the transmission of American cutaneous leishmaniasis (ACL) in the Neotropical region. However, genetic studies involving this taxon are still incipient and important for understanding the level of variability of different populations, their role, and implications as vectors. The aim of this study was to determine the level of genetic diversity of L. intermedia present in the Ribeira River Valley, an area of ACL transmission in the state of Paraná, Brazil, through the Random amplified polymorphic DNA (RAPD). Two municipalities were chosen to collect sand flies: Cerro Azul (new transmission area of the ACL) and Adrianópolis (endemic area of the ACL). The insects were captured in the house, in the peridomicile and in the wild (forest). Two of the used markers made it possible to estimate the polymorphism of the studied populations, resulting in 40 genotypes, most of them from peridomicile. The dendrogram generated by the analysis with the primer A10 showed different degrees of similarity, suggesting that there may be gene flow in the studied populations. The Principal Coordinate Analysis (PCO) with the A2 primer, was useful in grouping L. intermedia according to its ecological and geographical origin. There was no distinction between the lineages composing the L. intermedia complex. The results of this study, with the record of great genotypic diversity in L. intermedia, may contribute to explain the maintenance of the life cycle of Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) in the region.

Abstract The rapid and accurate diagnosis of tuberculosis (TB), especially considering limited resources, is still a challenge. Development of new methodologies and tests are needed to overcome several disadvantages of the available standard tests. We evaluated the diagnostic potential of two antigens specific for Mycobacterium tuberculosis, the CFP10 and ESAT6 recombinant proteins, and developed stable formulations thereof. Sensitivity and specificity of the delayed-type hypersensitivity (DTH) skin testing and the induction of gamma interferon production (IFN-γ) by lymphocytes, as a non-invasive test, were evaluated using the CFP10 and ESAT6 protein formulations. The recombinant proteins produced by our group presented a high DTH response and the ability to differentiate between tuberculosis infection, BCG vaccination, and the contact with non-tuberculous mycobacteria (NTM). The production of IFN-γ by stimulation with individual and combined proteins was detected in a panel of 40 individuals and showed a specificity of 100% and a sensitivity of 90% when the two proteins were used together. Lyophilized formulations were stable under all conditions, while soluble formulations were stable under freezing at -20 ºC and -80 ºC. The proposed formulations containing the ESAT6 and CFP10 recombinant antigens constitute satisfactory tools for TB testing, suitable to be developed and implemented in a large-scale trial.

Resumo O objetivo deste estudo foi confirmar a emergência da leishmaniose visceral canina em Foz do Iguaçu próximo à fronteira com a Argentina e ao Paraguai, por meio do isolamento e identificação molecular de Leishmania infantum. Em um primeiro estágio de coleta de animais (2012), três amostras de aspirados de linfonodos e 46 camadas leucocitárias foram obtidas de cães soropositivos para leishmaniose. Em um segundo estágio de coleta (2013), foram coletadas amostras de camada leucocitária de 376 cães de 20 localidades próximas à fronteira com o Paraguai, rio Paraná, centro e periferia da cidade. Seis isolados foram obtidos, quatro da primeira etapa (4/49) e dois da segunda etapa (2/376); estes isolados foram submetidos à amplificação com iniciadores K26F/R, e a análise de sua sequência confirmou a espécie como L. infantum. A autoctonia dos casos foi confirmada, pois 100% dos cães nunca haviam saído da cidade. O estudo confirma a emergência de leishmaniose visceral canina no Paraná com identificação de L. infantum em cães da cidade de Foz do Iguaçu. Assim, medidas de controle devem ser elaboradas e implementadas por órgãos públicos e instituições de pesquisa do Brasil, Argentina e Paraguai em parceria com o objetivo de controlar a disseminação de zoonoses e os casos humanos de LV.

Abstract The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.

ABSTRACT The objective of this study was to determine the abundance and diversity of the species and their presumed vectorial role in the transmission of Cutaneous Leishmaniasis (CL). Research on sand flies (Diptera: Psychodidae) was carried out in two municipalities in the Ribeira River Valley in the State of Paraná, southern Brazil. One, Adrianópolis, is an endemic area of CL and the other is a recent outbreak area (Cerro Azul). A total of 432 specimens were collected from residential, peri-domestic and wild environments. According to the data obtained, for each ecotope studied, the statistical analysis has shown that two lines of the Lutzomyia intermedia population coexist and that the quantity of specimens from L. intermedia s.l. is significantly different from L. intermedia s.s. in all environments (χ2=9.943; DF=2; p=0.07). The prevalence of L. intermedia (93.28%) in the ecotopes studied suggests that it is the main vector of leishmaniasis. The succession of vector species and their involvement in the epidemiological cycle of L. braziliensis is discussed in this article.

RESUMEN El objetivo de este trabajo fue determinar la abundancia y diversidad de las especies y su supuesto papel vectorial en la transmisión de Leishmaniasis Cutánea (LC). La investigación sobre flebótomos (Diptera: Psychodidae) se llevó a cabo en dos municipios en el Valle del Río Ribeira en el Estado de Paraná, sur de Brasil. Las regiones estudiadas fueron Adrianópolis, área endémica de LC, y Cerro Azul, que se reporta como zona de brote reciente. Se recolectaron un total de 432 especímenes de ambientes residenciales, peri-domésticos y silvestres. El análisis estadístico realizado, a partir de los datos obtenidos para cada ecotopo estudiado, mostró que coexistían dos linajes de la población de Lutzomyia intermedia y que la cantidad de especímenes de L. intermedia s.l. fue significativamente diferente de L. intermedia s.s. en todos los entornos (χ2=9,943; GL=2; p=0,07). La prevalencia de L. intermedia (93,28%) en los ecotopos estudiados sugiere que es el principal vector de la leishmaniasis. La sucesión de especies de vectores y su participación en el ciclo epidemiológico de L. braziliensis se discute en este artículo.

Resumo Este estudo avaliou a soroprevalência de Toxoplasma gondii, Neospora caninum e Leptospira spp. em cães de Foz do Iguaçu, Paraná, Brasil. O teste de imunofluorescência indireta foi utilizado para detectar anticorpos anti-T. gondii e anti-N. caninum. Ensaio imunoenzimático e soroaglutinação microscópica foram utilizados para pesquisa de anticorpos anti-T. gondii e anti-Leptospira spp., respectivamente. Os resultados obtidos foram: 67,02% (435/649) das amostras reativas para T. gondii e, 1,38% (9/649) para N. caninum, ambas com ausência de variáveis significativas estatisticamente. Para Leptospira spp. os resultados indicaram soroprevalência de 23,11% (153/649). A análise das variáveis sem distinção de sorovar mostraram associação para caraterísticas intrísecas como raça, idade, estado nutricional e categoria de cães. Para as variáveis extrínsecas, a região da cidade e ter acesso à rua mostraram associação estatística (p<0,05). Os sorovares mais prevalentes foram: Canicola com 59,47% (91/153); Bratislava 13,07% (20/153) e Butembo 15,68% (24/153). As variáveis que compõem o modelo de análise multivariada ajustada usando como desfecho Leptospira spp. foram: idade, raça, estado nutricional e área. Para o sorovar Canicola, as variáveis significantes (p<0,05) foram sexo, estado nutricional e área; para o sorovar Bratislava, as variáveis significantes (p<0,05) foram linfadenomegalia e presença de pulgas. Dos resultados obtidos, cães podem ser usados como sentinelas para infecção por T. gondii e Leptospira spp. na cidade de Foz do Iguaçu e em outras cidades com desfechos similares. Além disso, medidas preventivas devem ser tomadas pelas autoridades de saúde, pois são zoonoses e os seres humanos também estão em risco.

Abstract This study evaluated the seroprevalence of Toxoplasma gondii, Neospora caninum and Leptospira spp. in dogs from Foz do Iguaçu, Paraná, Brazil. Indirect immunofluorescent antibody test was used to detect antibodies anti-T.gondii and anti-N. caninum. Immunoenzymatic assay and microscopic serum agglutination were used for screening antibodies anti-T.gondii and anti-Leptospira spp., respectively. The results were: 67.02% of the samples reactive for T.gondii and 1.38% for N. caninum, both without statistically significant variables. For Leptospira spp. the results indicated seroprevalence of 23.11%. The analysis of the variables without distinction of serovar showed association for intrinsic characteristics as breed, age, nutritional status and dog category. The extrinsic variables as city region and access to the street presented association (p<0.05). The most prevalent serovars were: Canicola 59.47%; Bratislava 13.07% and Butembo 15.68%. Variables that make up the adjusted multiple analysis model using Leptospira spp. were: age, breed and nutritional status; serovar Canicola, sex, nutritional status and area (p<0.05); serovar Bratislava, lymphadenomegaly and presence of fleas (p<0.05). Given the results obtained, dogs can be used as sentinels for toxoplasmosis and leptospirosis in Foz do Iguaçu and other cities with similar outcomes. In addition, preventive measures should be taken by health authorities because they are zoonoses and humans are also at risk.

Resumo O objetivo deste estudo foi investigar a ocorrência de anticorpos anti- Leishmania spp. em cães da cidade de Foz do Iguaçu, estado do Paraná, Brasil, fronteira com a Argentina e o Paraguai. Amostras de sangue de cães foram coletadas para realização dos seguintes testes sorológicos: teste rápido imunocromatográfico DPP®, ensaio imunoenzimático indireto (ELISA) e ensaio de imunofluorescência indireta (IFI). Em 2012, 285 cães foram analisados na fronteira com Argentina e, em 2013, amostras de soro de 396 cães na fronteira com o Paraguai. Utilizando ELISA para triagem e IFA para o teste confirmatório, os resultados mostraram uma prevalência de anticorpos de 1,8% (5/285) na fronteira da Argentina e 3,0% (12/396) na fronteira com o Paraguai. Ao usar o DPP® para triagem e ELISA como uma análise confirmatória, observou-se uma prevalência de cães sororreagentes de 2,5% (7/285) na fronteira com a Argentina e 5,1% (20/396) na fronteira com o Paraguai. A não coleta pública de lixo doméstico (p = 0,0004) mostrou-se associada à leishmaniose. Este estudo demonstra a presença de leishmaniose e sugere a emergência da leishmaniose visceral canina no estado do Paraná devido à ocorrência de cães sororreagentes na fronteira Argentina e Paraguai, que possuem características ambientais e geográficas que favorecem a disseminação do parasito.

Abstract The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.

Abstract INTRODUCTION: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production. METHODS: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis , and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis , and, after 45 days, they were skin tested with standard Montenegro antigen. RESULTS: Cavia porcellus immunized with L. amazonensis or L. braziliensis , and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test. CONCLUSIONS: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen.

ABSTRACT The genetic study of sandfly populations needs to be further explored given the importance of these insects for public health. Were sequenced the NDH4 mitochondrial gene from populations of Nyssomyia neivai from Doutor Camargo, Lobato, Japira, and Porto Rico, municipalities in the State of Paraná, Brazil, to understand the genetic structure and gene flow. Eighty specimens of Ny. Neivai were sequenced, 20 from each municipality, and 269 base pairs were obtained. A total of 27 haplotypes and 28 polymorphic sites were found, along with a haplotypic diversity of 0.80696 and a nucleotide diversity of 0.00567. Haplotype H5, with 33 specimens, was the most common among the four populations. Only haplotypes H5 and H7 were present in all four populations. The population from Doutor Camargo showed the highest genetic diversity, and only this population shared haplotypes with those from the other municipalities. The highest number of haplotypes was sheared with Lobato which also had the highest number of unique haplotypes. This probably occurred because of constant anthropic changes that happened in the environment during the first half of the twentieth century, mainly after 1998. There was no significant correlation between genetic and geographical distances regarding these populations. However, the highest genetic and geographical distances, and the lowest gene flow were observed between Japira and Porto Rico. Geographical distance is a possible barrier between these municipalities through the blocking of haplotype sharing.

ABSTRACTThe aim of this work was to develop and evaluate an indirect enzyme-linked immunosorbent assay (ELISA) and made a serological screening for specific antibodies to BoHV-4. Bovine serum samples were collected from different Brazilian states and evaluated for the presence of antibodies for BoHV-4 and BoHV-1. The serological results obtained showed that the indirect ELISA assay could be applied for the detection of specific antibodies for BoHV-4. The ELISA test allowed concluding that BoHV-4 is present in bovines in every Brazilian state from which serum samples were collected. The ELISA assay here standardized proved to be useful for the epidemiological studies and showed a positivity range from 1.8 to 66% in Brazil.

The aim of this study was to evaluate the viability of bull spermatozoa collected from the cauda epididymis stored at 18-20°C, which were compared with semen collected by electro-ejaculation method and preserved at 5°C. Ten pairs of testes from Tabapuã bulls were removed by orchiectomy and stored for 6 (G6), 12 (G12), 18 (G18), 24 (G24) and 30 (G30) h at room temperature (18-20°C). Seven days before orchiectomy, semen was collected by electro-ejaculation method. The sperm parameters evaluated were: sperm motility, vigor, concentration, morphology and acrosome defects. Sperm motility declined (p<0.05) when spermatozoa were stored for 30 h in the epididymis. The spermatozoa from the epididymis showed lower sperm motility than that of spermatozoa collected via electro-ejaculation. There was a little expressive decrease in sperm vigor and increased in morphological defects with storage time, but the acrosome integrity was not affected. Cold storage (5°C) maintained sperm viable for 15 to 40.8 h. Thus, it was possible to recover viable sperm with 41.25% of motility from the cauda epididymis stored at room temperature of 18-20°C for 30 h. There were differences between the ejaculated and epididymal sperm for the bulls and the conservation at 5°C allowed short-term preservation of the gametes.

Introduction Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity.

The study aimed to investigate the antihyperglycemic and antilipidperoxidative effects of ethanol extract of the dry matter from culture broth of Coriolus versicolor (ECBC) in streptozotocin-induced diabetes mice. Blood glucose level, insulin level, total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol(LDL-C), high density lipoprotein -cholesterol (HDL-C) in serum and reduced glutathione level (GSH), lipid peroxidation, glycogen, antioxidant enzymes in liver were evaluated. Moreover, histopathological observation was conducted. Streptozotocin treatment (150 mg/kg body weight) induced the decrease of GSH level, antioxidant enzymes activities, glycogen content in liver, HDL-C content and insulin level in serum, accompanied by the elevation of the lipid peroxidation in liver, serum blood glucose level, contents of TC, TG and LDL-C. Treatment with ECBC restored the changes in the above parameters up to the basal level. The protective effects were further supported by the attenuation of the degree of pancreas damage in ECBC treated mice.

The aim of the present work was to study the clinical aspects and relevance of molecular diagnosis in late mucocutaneous leishmaniasis patients in Parana, Brazil. Twenty one suspected cases of mucocutaneous leishmaniasis (MCL) in patients from the endemic areas of leishmaniasis were assessed. Different methods used in diagnosing the disease and the polymerase chain reaction (PCR) technique were compared in order to establish the sensitivity of each method. Out of the 21 patients analyzed, 14.3% presented other etiologies such as vasculitis, syphilis, and paracoccidioidomycosis, with all tests negative for leishmaniasis. Out of the remaining 15 patients, 6.7% cases were confirmed for leishmaniasis by direct examination; 46.67% were positive for culture, which allowed isolating and identifying the parasite and - with the PCR technique - it was possible to diagnose 100% MCL patients for all the three repetitions of exams. The PCR optimized for the present work proved to be an auxiliary method for diagnosing leishmaniasis applicable in the patients carrying MCL due to Leishmania (Viannia) braziliensis and did not need culture to be performed, resulting in a faster diagnosis.

A extração de fitases produzidas por fermentação em estado sólido de polpa cítrica foi estudada utilizando um processo de extração sólido-líquido em varias etapas. A adição de EDTA permite manter durante 24 horas a temperatura ambiente 90% da atividade inicial do caldo com a enzima extraída. Um planejamento fatorial 2², com 4 replicas no ponto central, foi desenvolvido para testar os valores de ph e agitação convenientes para a extração das enzimas. A interação entre ambos os fatores foi estadisticamente significativa. A atividade da enzima foi otimizada nos valores onde o pH (5.0) e a agitação (350 rpm) resultaram ser as melhores condições para extrair a enzima da matriz sólida. O ajuste do modelo matemático obtido mostra que é possível considerar a difusividade como o mecanismo que controla o processo de transferência de massa. A constante cinética que descreve este processo e a concentração de saturação foram 0.039 min-1 e 4.01 IU/mL respectivamente. A extração em varias etapas mostrou que nas duas primeiras etapas é possível recuperar 85% da fitase produzida.

The extraction of phytase produced by solid-state fermentation of citrus peel was studied employing a multistage leaching process. It was observed that the extracts containing EDTA retained over 90% of phytase activity at room temperature after 24 h after the leaching. A fractional design 2² (with 4 replicates at the central point) was carried out for testing the pH and agitation as process independent factors. Only the interaction between the pH and agitation showed a significant influence. These factors were optimized with a central composite design. Agitation at 300 rpm and pH at 5.0 were the best conditions to extract the enzyme from solid matrix. The modeling of the process indicated that diffusivity of the enzyme in the solvent was the controlling mechanism. The corresponding kinetic constant and saturation concentration in this process were 0.89 min-1 and 4.0 IU/mL, respectively. The multistage process indicated that after two steps, it was possible to recover 85% of total enzyme produced.