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Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and swab samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.Highlights*Using 28S primers it was possible to identify L. similis’ DNA with high sensitivity;*LAMP was 62-fold more sensitive than PCR and detected as low as 0.32 pg of DNA;*LAMP detected L. intermedia DNA from hair, serum, and exudate from experimentally-envenomed rabbits;*LAMP presents an excellent potential for identifying Loxosceles spp. envenomation.

Made available in DSpace on 2022-05-01T09:47:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Universidade Federal do Paraná Fundação Araucária Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism. Department of Cell Biology Federal University of Paraná (UFPR) Production and Research Center of Immunobiological Products (CPPI) State Department of Health Veterinary Hospital Federal University of Paraná (UFPR) Department of Structural Molecular Biology and Genetics State University of Ponta Grossa (UEPG) Multiuser Center for Biomolecular Innovation Departament of Physics Universidade Estadual Paulista (UNESP) Multiuser Center for Biomolecular Innovation Departament of Physics Universidade Estadual Paulista (UNESP) Universidade Federal do Paraná: 02/2020 Universidade Federal do Paraná: 04/2019 Fundação Araucária: 057/2017 CNPq: 303868/2016-3 CNPq: 408633/2018-2

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.

Brown spider (genus Loxosceles) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms.

Made available in DSpace on 2021-06-25T11:14:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the der-monecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and character-ized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced. Departamento de Biologia Celular Universidade Federal do Paraná Centro de Produção e Pesquisa de Imunobiológicos (CPPI) Departamento de Física Centro Multiusuário de Inovação Biomolecular Universidade Estadual Paulista (UNESP) Departamento de Biologia Estrutural Molecular e Genética Universidade Estadual de Ponta Grossa Departamento de Física Centro Multiusuário de Inovação Biomolecular Universidade Estadual Paulista (UNESP) FAPESP: 2015/13765-0 FAPESP: 2019/10475-2 FAPESP: 2915/18868-2 CNPq: 303868/2016-3 CNPq: 307338/2014-2 CNPq: 309940/2019-2 CNPq: 408633/2018-2

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Accidents with venomous animals pose a health issue in Brazil, and those involving brown spiders (Loxosceles sp.) figure between the most frequent ones. The accidental envenomation by brown spiders causes a strong local dermonecrotic effect, which can be followed by systemic manifestations that in some cases lead to death. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses. In the present work, there is an attempt to reduce and optimize animal experiments with the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes previously mapped from the main proteins of the Loxosceles venom, the Sphingomyelinases D. The Lil protein contains epitopes from Sphinomyelinases D of the three-main species found in Brazil and this chimeric protein was found capable of inducing antibodies with the potential to partially neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to improve the lifespan of the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols.

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32–35 kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10(7) and 10(9) CFU g(-1). Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.

Neurocysticercosis (NCC), the cerebral presence of Taenia solium metacestode (Cysticercus cellulosae), is responsible for neurological disorders worldwide. In order to validate an immunodiagnosis for public-health patients in the State of Parana-Brazil, crude antigen of Taenia crassicepsmetacestode (Cysticercus longicollis) was used as an alternative heterologous antigen to be used in ELISA and in electroimmunotransfer blotting (EITB) for active and inactive NCC diagnosis. Indirect ELISA was able to discriminate between active and inactive samples and presented high specificity and sensitivity. Any immunodominant band was able to distinguish the NCC stages, although the EITB showed 100% specificity. The immunological results proved to be an important auxiliary toll for NCC diagnosis, mainly for public-health systems in developing countries, where either the neuroimage techniques are not accessible or the resources are scarce.Neurocisticercose (NCC), causada pela presença do metacestódeo do parasito Taenia solium (Cysticercus cellulosae) no sistema nervoso central, é uma doença mundialmente conhecida como responsável por distúrbios neurológicos. Com o objetivo de validar um imunodiagnóstico para pacientes da rede pública do estado do Paraná-Brasil, o extrato bruto do metacestódeo de T. crassiceps (C. longicollis) foi produzido e utilizado como antígeno heterólogo para o diagnóstico de NCC ativa e inativa utilizando-se ELISA e eletroimunotransferência (EITB). O ensaio de ELISA indireto foi capaz de discriminar a forma ativa e inativa da NCC, apresentando alta especificidade e sensibilidade. Ao se utilizar EITB, nenhuma proteína foi imunodominante de forma a distinguir os diferentes estágios da NCC, embora o ensaio tenha tido 100% de especificidade. Os resultados mostram que os ensaios imunológicos podem ser uma ferramenta auxiliar importante para o diagnóstico da NCC, principalmente para o sistema público de saúde, cujo diagnóstico por imagem não é acessível ou cujos recursos financeiros são escassos.

The beta-hemolytic group C streptococci (Lancefield's group) has been considered an emergent human pathogen, showing an important role as an opportunist agent, being responsible for nosocomial infections and outbreaks. This study is reporting the first outbreak of nosocomial infection caused by Streptococcus dysgalactiae subsp. equisimilis in Brazil. From January, 2002, to December, 2004, S. equisimilis was isolated in 67/207 (32.37%) samples from secretions of patients' infected wounds, interned at the Hospital of Sanitary Dermatology in the State of Paraná (HDSPR). The prevalence of this microorganism increased from 11/42 (26.19%) in 2002, 14/65 (21.54%) in 2003 to 42/100 (42.00%) in 2004. This increase was statistically significant (p=0.024), and this microorganism became the most frequently isolated in these patients, overtaking the rates of isolation of Pseudomonas aeruginosa. The S. equisimilis grew in pure culture, as a unique microorganism, in six samples (2.9%) out of 207. Fresh feces of 15 animals (horses and sheep) living in the proximities of the hospital were also examined and three of them positive for S. equisimilis. The biochemical profile of the strains isolated from the patients and from the animals was the same. These animals might have been the source of the dissemination of the outbreak in the hospital. New studies will be necessary to confirm the genetic relationship between the strains isolated from patients and animals. O estreptococo beta-hemolítico do grupo C de Lancefield tem sido considerado patógeno humano emergente, mostrando importante papel como agente oportunista, implicado algumas vezes em infecções hospitalares e surtos. Este estudo está relatando o primeiro surto de infecção hospitalar causado pelos Streptococcus dysgalactiae subsp. equisimilis no Brasil. De janeiro de 2002 a dezembro de 2004, isolou-se S. equisimilis em 67/207 (32,37%) das amostras de secreções de lesões de feridas coletadas de pacientes internados no Hospital de Dermatologia Sanitária do Paraná (HDSPR). A prevalência deste microrganismo aumentou de 11/42 (26,19%) em 2002, 14/65 (21,54%) em 2003 para 42/100 (42,00%) em 2004. Este aumento foi estatisticamente significante (p=0.024), tornando este microrganismo o mais freqüentemente isolado nos pacientes, ultrapassando as taxas de isolamento de Pseudomonas aeruginosa. Em seis amostras (2,9%) entre as 207 examinadas, S. equisimilis cresceu em cultivo puro, como único microrganismo. Também foram examinadas fezes frescas de 15 animais (cavalos e ovelhas) que vivem nas proximidades do hospital, e três amostras foram positivas para S. equisimilis. O perfil bioquímico encontrado entre os isolados dos pacientes foi o mesmo encontrado entre os isolados das fezes dos animais. Acredita-se que estes animais possam ter sido a fonte de disseminação do surto no hospital. Novos estudos serão necessários para confirmar o relacionamento genético entre os isolados dos pacientes e animais.

The beta-hemolytic group C streptococci (Lancefield's group) has been considered an emergent human pathogen, showing an important role as an opportunist agent, being responsible for nosocomial infections and outbreaks. This study is reporting the first outbreak of nosocomial infection caused by Streptococcus dysgalactiae subsp. equisimilis in Brazil. From January, 2002, to December, 2004, S. equisimilis was isolated in 67/207 (32.37%) samples from secretions of patients' infected wounds, interned at the Hospital of Sanitary Dermatology in the State of Paraná (HDSPR). The prevalence of this microorganism increased from 11/42 (26.19%) in 2002, 14/65 (21.54%) in 2003 to 42/100 (42.00%) in 2004. This increase was statistically significant (p=0.024), and this microorganism became the most frequently isolated in these patients, overtaking the rates of isolation of Pseudomonas aeruginosa. The S. equisimilis grew in pure culture, as a unique microorganism, in six samples (2.9%) out of 207. Fresh feces of 15 animals (horses and sheep) living in the proximities of the hospital were also examined and three of them positive for S. equisimilis. The biochemical profile of the strains isolated from the patients and from the animals was the same. These animals might have been the source of the dissemination of the outbreak in the hospital. New studies will be necessary to confirm the genetic relationship between the strains isolated from patients and animals.O estreptococo beta-hemolítico do grupo C de Lancefield tem sido considerado patógeno humano emergente, mostrando importante papel como agente oportunista, implicado algumas vezes em infecções hospitalares e surtos. Este estudo está relatando o primeiro surto de infecção hospitalar causado pelos Streptococcus dysgalactiae subsp. equisimilis no Brasil. De janeiro de 2002 a dezembro de 2004, isolou-se S. equisimilis em 67/207 (32,37%) das amostras de secreções de lesões de feridas coletadas de pacientes internados no Hospital de Dermatologia Sanitária do Paraná (HDSPR). A prevalência deste microrganismo aumentou de 11/42 (26,19%) em 2002, 14/65 (21,54%) em 2003 para 42/100 (42,00%) em 2004. Este aumento foi estatisticamente significante (p=0.024), tornando este microrganismo o mais freqüentemente isolado nos pacientes, ultrapassando as taxas de isolamento de Pseudomonas aeruginosa. Em seis amostras (2,9%) entre as 207 examinadas, S. equisimilis cresceu em cultivo puro, como único microrganismo. Também foram examinadas fezes frescas de 15 animais (cavalos e ovelhas) que vivem nas proximidades do hospital, e três amostras foram positivas para S. equisimilis. O perfil bioquímico encontrado entre os isolados dos pacientes foi o mesmo encontrado entre os isolados das fezes dos animais. Acredita-se que estes animais possam ter sido a fonte de disseminação do surto no hospital. Novos estudos serão necessários para confirmar o relacionamento genético entre os isolados dos pacientes e animais.

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH.

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D(121 °C) value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D(121 °C)=2.9±0.5 min and Usk=12.7±2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D(121 °C) value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.

Com o objetivo de padronizar e avaliar a campo um teste sorológico para diagnóstico de cisticercose em bovinos foram examinados 812 animais procedentes de 139 municípios do Estado do Paraná, a maioria bovinos azebuados, machos e fêmeas, de grupo etário variando de 18 meses a 60 meses. Os animais foram devidamente identificados e abatidos em frigorífico comercial na região metropolitana de Curitiba. Dos animais que apresentaram cistos durante a Inspeção post-mortem foi colhida amostra de sangue, as quais foram submetidas ao teste ELISA para pesquisa de anticorpos contra-Cysticercus bovis. O coeficiente de sensibilidade para o teste ELISA foi de 83,6% e o Coeficiente de Especificidade foi de 92,8%. Portanto, o teste sorológico (ELISA) foi mais sensível do que o exame póst-mortem, pois este deixou de detectar 06 carcaças com infecção leve por cisticercose (VPP 99,7% - ELISA).

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.

O presente trabalho teve como objetivo pesquisar a ocorrência de leishmaniose visceral em cães com sinais clínicos compatíveis, procedentes de clínicas veterinárias das diferentes regiões do Estado do Paraná e em caso positivo verificar a autoctonia dos mesmos. Dos animais com suspeita clínica e epidemiológica de LV, foi coletada amostra de sangue para realização de hemograma, provas bioquímicas, sorologia, cultura do parasito, PCR e RAPD. Os cães portadores de quadro clínico, como febre, hiperqueratose, onicogrifose e emagrecimento exagerado, tiveram gânglio poplíteo e/ou medula óssea puncionados e o conteúdo inoculado em meio de cultivo NNN. A extração de DNA do parasito em sangue e cultura foi realizada pelo método fenol/clorofórmio. A amplificação de DNA do protozoário foi feita por PCR e RAPD. Dos 24 animais analisados, o parasito foi isolado em 19 cães. As técnicas moleculares permitiram identificar 14 isolados como L. (Leihmania) infantum e cinco como L. (Viannia) braziliensis. A análise epidemiológica dos casos permitiu determinar o local de transmissão e definir que todos os cães com LV diagnosticados eram casos alóctones, ou seja eram importados de regiões endêmicas. Sugere-se que a migração dos cães de regiões endêmicas para regiões indenes poderá permitir a instalação de novos focos, desde que o agente encontre ecótopo adequado e vetor específico (Lutzomyia longipalpis). The south region is the only area in Brazil that does not present autochthonous cases of visceral leishmaniasis (VL), however, in the state of Parana, dogs and humans have been found showing a VL compatible clinical profile. In view of this problem, the present work aimed at isolating and identifying the parasite and determining the cases autochthony. All animals clinically suspect of VL were clinically evaluated, and had samples of their blood collected for hemoculture (NNN culture medium), serology, PCR and RAPD-PCR, hemogram, and biochemical assays. The dogs presenting VL clinical profile had their lymph nodes and/or bone marrow punctured and their content inoculated in NNN culture medium. The protozoan isolated was identified by PCR and PCR-RAPD. Strains of Leishmania were isolated in 19 out of the 24 studied animals. Fourteen isolates were identified as L. (Leishmania) infantum, and five were L. (Viannia) braziliensis. In the epidemiological analysis, it was possible to determine that all dogs with L. (L.) infantum being allochthonous cases. Leishmaniasis is a zoonose that has the domestic dog as reservoir, the migration ofsuch animals can disseminate the parasite to other regions, provided the agent finds an adequate ecotope and a specific vector (Lutzomyia longipalpis).

Bites by the brown spider (Loxosceles spp.) are an important health problem in South America, where three species predominate (Loxosceles laeta, Loxosceles gaucho, Loxosceles intermedia). Brown spider bites (loxoscelism) induce a block of cutaneous necrosis and, less commonly, may cause fatal systemic poisoning. A variety of controversial protocols are used to treat loxoscelism, while treatment with antivenin is the only venom specific treatment. Here we studied the action of the venom as well as the response to the antivenin for Loxosceles through an experimental study that simulates bites of L. intermedia (bites of this species are the most common in Brazil). Beneficial effects are known for antivenin applied quickly (within 4 h) after envenomation. Here we wished to examine the temporal development of the brown spider bite as well as the temporal patterns of the action of the antivenin to determine the time limits for beneficial use of the antivenin after envenomation. This information is important since most patients only appear for treatment several hours after being bitten. New Zealand rabbits were experimentally exposed to the venom from brown spiders by the injection of venom from L. intermedia (2x minimum necrotic dose), followed at regular time intervals by antivenin. The use of the loxoscelic antivenin--CPPI (4 mL per animal)--minimized the effects of envenomation when applied for up to 12 h after the injection of the venom, as evaluated by cutaneous (erythrema, edema, ecchymosis and necrosis) and systemic (blood cell and platelet counts, hematimetrics and fibrinogen dosage) criteria. Also, antivenin reduced the size of the necrotic area when applied up to 48 h after envenomation. Thus, therapy with loxoscelic antivenin, CPPI, may provide beneficial results by interfering with envenomation well after the bite occurred and therefore may become an important tool for medical treatment of brown spider bites.

The south region is the only area in Brazil that does not present autochthonous cases of visceral leishmaniasis (VL), however, in the state of Paraná, dogs and humans have been found showing a VL compatible clinical profile. In view of this problem, the present work aimed at isolating and identifying the parasite and determining the cases autochthony. All animals clinically suspect of VL were clinically evaluated, and had samples of their blood collected for hemoculture (NNN culture medium), serology, PCR and RAPD-PCR, hemogram, and biochemical assays. The dogs presenting VL clinical profile had their lymph nodes and/or bone marrow punctured and their content inoculated in NNN culture medium. The protozoan isolated was identified by PCR and PCR-RAPD. Strains of Leishmania were isolated in 19 out of the 24 studied animals. Fourteen isolates were identified as L. (Leishmania) infantum, and five were L. (Viannia) braziliensis. In the epidemiological analysis, it was possible to determine that all dogs with L. (L.) infantum being allochthonous cases. Leishmaniasis is a zoonose that has the domestic dog as reservoir, the migration of such animals can disseminate the parasite to other regions, provided the agent finds an adequate ecotope and a specific vector (Lutzomyia longipalpis).

As larvas da Dermatobia hominis provocam lesões ulcerativas, danificando o tecido subcutâneo e conseqüentemente a pele do hospedeiro. O couro é o subproduto que sofre maior depreciação, o que, muitas vezes, impossibilita seu aproveitamento na industrialização. Atualmente o controle químico é utilizado como forma de combate à dermatobiose, entretanto, leva ao acúmulo de substâncias tóxicas nos animais e no ambiente. No presente trabalho, foram avaliadas as respostas imune-humoral e celular de bovinos imunizados com extrato antigênico preparado com larvas de D. hominis. Três grupos de oito bezerras da raça Nelore com 10 meses de idade foram usados, tendo o primeiro grupo (A) recebido aplicação de extrato imunogênico de larvas de D. hominis, com intervalos de quinze dias; o grupo (B), utilizado como controle, não recebendo nenhum tipo de tratamento; e o grupo (C) recebendo o tratamento ectoparasiticida à base de Dichlorvos associado a Cypermetrina. Neste mesmo período, foram avaliados o leucograma e os níveis de IgG contra D. hominis pela técnica de enzimoimunoensaio-ELISA. Quanto à avaliação da imunidade humoral, verificou-se que os animais do grupo A apresentaram maior produção de IgG contra D. hominis, com níveis máximos de anticorpos circulantes aos 45 dias após a primeira imunização. Estes animais também apresentaram maior produção de neutrófilos, eosinófilos e monócitos que os dos grupos B e C. O número de nódulos de larvas encontrado nos animais do grupo C foi 148,3% maior que nos animais dos grupos A e B. A comprovação da resposta imune celular e humoral, parcialmente caracterizadas, bem como a redução do número de nódulos, são indicadores que a imunização contra D. hominis foi parcialmente protetora para os bovinos imunizados. Dermatobia hominis larvae cause ulcerative lesions and damage to subcutaneous tissue and skin of the host. Leather is the subproduct which undergoes major depreciation, making it difficult to industrial use. Nowadays, the chemical control is utilized against dermatobiosis, therefore it leads to rising toxic chemicals in the animals and environment. The immunological challenge with D. hominis larval extract may represent an important altervative for this parasitosis control. Humoral and cellular immune responses were tested in bovine using an antigenic extract prepared with D. hominis larvae. Three groups of 10 months old Nelore females were used. The first group (A) received immunogenic larval extract of D. hominis with fifteen-days interval between injections; the group (B) was the control and has not received any sort of treatment; and the group (C) received an ectoparasitecide treatment based on Dichlorvos associated to Cypermetrina. Aditionally, leucogram and levels of IgG against D. hominis by immunoassay technique were evaluated. As for the humoral immunity, animals from group A presented higher IgG production against D. hominis with maximum levels of circulating antibodies at the 45th day after the first injection. These animals also showed higher production of neutrophils, eosinophils and monocytes than those from groups B and C. The number of D. hominis larvae nodules observed in animals from the group C was 148.3% larger than those from group A and B. The evidence concerning both cellular and humoral immune responses as well as the reduction on nodules number are an indication that the immunization against D. hominis was partially protective for the immunized bovines.

Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the working range was0.1–11U/L and cutoff value was0.2U/L EA for nonsmokers. The intra- and interassay coefficients of variation were3.7%–6.5% (ICA-PLAP assay) and9.0%–9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes.

O presente experimento teve como objetivo avaliar a resposta imune humoral de coelhos frente a extrato de larvas (L2 e L3) de D. hominis. Vinte e quatro coelhos foram imunizados com tres diferentes extratos de larvas de D. hominis. Foram estabelecidos tres grupos imunizados da seguinte forma: grupo A, com extrato soluvel; grupo B, com extrato bruto; e grupo C, com extrato soluvel e extrato bruto. Uma dose de reforco foi aplicada no 135o dia apos a primeira inoculacao, nos animais dos grupos A, B e C. Amostras de soro foram coletadas quinzenalmente do dia 0 aos 150 dias apos a primeira imunizacao (DPI) para verificar a cinetica de producao de anticorpos por meio do ensaio de imunoadsorcao enzimatica (ELISA). Anticorpos circulantes foram encontrados em niveis maximos a partir de 45 DPI. A dose de reforco induziu o aparecimento imediato de uma resposta secundaria. O extrato soluvel foi mais eficaz na estimulacao da resposta imune humoral de machos e femeas. Constatou-se diferenca significativa (P Humoral immune response of rabbits against extracts of Dermatobia hominis larvae Abstract The purpose of this research was to evaluate the humoral immune response in rabbits immunized with extracts of D. hominis larvae (L2 and L3). Twenty four rabbits, immunized with three different larvae extracts, were studied in three groups. The immunization was carried out as following: group A, with soluble extract; group B, with raw extract; and group C, with soluble and raw extracts. In groups A, B and C a booster dose was applied on the 135th day after the first immunization. Serum samples were collected biweekly from 0 to the 150th day after the first immunization and processed by enzyme-linked immunosorbent assay (ELISA) to access the antibody production kinetics. Circulating antibodies were found at maximum levels on the 45th day after the first immunization. The booster dose induced an immediate secondary response. The soluble extract was the most efficient inducer of the humoral immune response in both females and males. A significant difference (P

Studies were carried out on the recovery rate and cysticerci location in bovines experimentally infected with Taenia saginata eggs. Three calves of 6.5 months and one with 19 months of age were infected orally with 2 x 10(4) eggs of Taenia saginata. A fifth calf served as control. After 90 days of infection, the animals were slaughtered and organs and skeletal muscles were inspected using a slicing technique every 5 mm. From the four infected calves, 702 cysticerci were recovered, of which 570 (81.2%) were alive and 132 (18.8%) were degenerated. The recovery rate ranged from 0.01 to 1.43% with an average of 0.88%. The cysticerci presented the following anatomical distribution: hioideos muscles 02 (0.28%), kidneys 03 (0.43%), tongue 07 (1.00%), liver 12 (1.71%), lungs 15 (2.14%), diaphragm 18 (2.56%), mastication muscles 25 (3.56%), heart 49 (6.98%), anterior muscle 323 (46.00%) and posterior muscle 248 (35.33%).Foi avaliada a taxa de recuperação e localização de cisticercos em bovinos experimentalmente infectados com ovos de Taenia saginata. Três bezerros de 6,5 meses e um adulto com 19 meses de idade foram infectados, por via oral, com 2 x 10(4) ovos de Taenia saginata. Um quinto bezerro serviu como testemunha. Após 90 dias da infecção, os animais foram abatidos. Fez-se inspeção, de todos os animais, por fatiamento de órgãos e musculatura esquelética, com intervalo entre os cortes de, no máximo, cinco milímetros. Dos quatros bezerros desafiados foram recuperados 702 cisticercos sendo 570 (81,20%) vivos e 132 (18,80%) degenerados. A taxa de recuperação foi de 0,01% a 1,43% com média de 0,88%. Os 702 cistos encontrados apresentaram a seguinte distribuição anatômica: músculos hióideos 02 (0,28%), rins 03 (0,43%), língua 07 (1,00%), fígado 12 (1,71%), pulmões 15 (2,14%), diafragma 18 (2,56%), músculos da mastigação 25 (3,56%), coração 49 (6,98%), musculatura dianteira 323 (46,00%) e musculatura traseira 248 (35,33%). Na infecção experimental os cistos encontraram-se distribuídos por toda a musculatura dos animais, não mostrando predileção pelos tecidos normalmente pesquisados pelo serviço de inspeção (língua, coração, diafragma, músculos mastigatórios). Os dados da inspeção de rotina pode não estimar a real incidência da cisticercose bovina. Os bovinos adultos são mais resistentes a infecção por ovos de Taenia saginata, apresentando menor número de cisticercos e com maior número de cistos calcificados.

O estudo foi realizado em Piraquara, Paraná, Brasil entre 1994 a 1996. Foi avaliada a possibilidade de bovinos terem participação no ciclo de Taenia solium. Uma cadeia de seis proglótes grávidas de T. solium foi obtida de paciente não medicado. Bovinos da raça Holstein foram infectados com 40.000 ovos de T. solium. A taxa de infecção foi de 100%. Um total de 44 cisticercos foi recuperado depois de 160 a 570 dias, 18 (40,9%) vivos e 26 (59,1%) calcificados. Dos 44 cisticercos, 19 (43,2%) foram encontrados na musculatura esquelética: 10,5% na cabeça, 36,8% nos membros torácicos, 10,5% no diafragma, 42,1% nos membros pélvicos; e 25 (56,8%) nos órgãos: 80% no fígado, 8% no coração, 8% na língua e 4% nos rins. Cisticercos vivos foram encontrados com acúleos rudimentares, com número variável de 01 a 42 e com 25,4-35,7 m de comprimento. Os resultados sugerem que bovinos poderiam servir como hospedeiros intermediários da T. solium. É necessário continuar os estudos para verificar a infectividade de cistos que se desenvolvem em hospedeiros não habituais. Abstract A study was performed in Piraquara, Parana, Brazil during 1994-1996 to evaluate the possibility of bovines participation in the epidemic chain of the Taenia solium. Six gravid segments of T. solium were obtained from a non medicated patient. Holstein bovines were infected with 40,000 eggs of T. solium. The infection rate was 100%. A total of 44 cysticerci were recovered: 18 (40.9%) alive and 26 (59.1%) calcified. Of the 44 cysticerci, 19 (43.2%) were found in the skeletal musculature: 10,5% in the head, 36.8% in the anterior members, 10.5% in the diaphragm, 42.1% in posterior members and 25 (56.8%) in the organs: 80% in the liver, 8% in the heart, 8% in the tongue and 4% in the kidneys. Living cysticerci had 1 to 42 short hooks of 25,4 to 35,7 µm. The results suggested that bovines could serve as intermediary host for T. solium.

A primeira etapa do trabalho consta de uma revisão de literatura da ciclozoonose teníase e cisticercose. Foi avaliado o comportamento imunológico de bovinos experimentalmente infectados com ovos de Taenia saginata através da cinética de produção de anticorpos anti- Cysticercus bovis. Três bezerros de 6,5 meses e um com 19 meses de idade foram infectados, por via oral, com 2 x 104 ovos de Taenia saginata. Um quinto bezerro serviu como testemunha. Após 90 a 111 dias da infecção, os animais foram abatidos. Fez-se inspeção por fatiamento de órgãos e musculatura esquelética com intervalo entre os cortes de, no máximo, cinco milímetros. Dos quatros bezerros desafiados foram recuperados 702 cisticercos sendo 570 (81,20%) vivos e 132 (18,80%) degenerados. A taxa de recuperação foi de 0,01% a 1,43% com média de 0,88%. Os 702 cisticercos encontrados apresentaram a seguinte distribuição anatômica: músculos hióideos 02 (0,28%), rins 03 (0,43%), língua 07 (1,00%), fígado 12 (1,71%), pulmões 15 (2,14%), diafragma 18 (2,56%), músculos da mastigação 25 (3,56%), coração 49 (6,98%), musculatura dianteira 323 (46,00%) e musculatura traseira 248 (35,33%). Foi padronizado o teste de ELISA indireto para pesquisa de anticorpos anti- Cysticercus bovis em bovinos, utilizando como antígeno o extrato salino parcial de Cysticercus cellulosae. O “cut-off” foi obtido com soro de 99 bovinos jovens considerados não portadores de cisticercos, ficou estabelecido como 0,310. Nos bovinos, experimentalmente infectados, foram pesquisados o desenvolvimento e a quantificação de anticorpos anti-Cysticercus bovis através da prova de ELISA indireta em 16 amostras de soros de cada animal. A primeira amostra foi colhida 97 dias antes da infecção dos animais, a segunda no dia da infecção experimental e as restantes, semanalmente, até o abate. Os anticorpos foram detectados a partir de 12 dias após a infecção, com pico inicial aos 27 dias. O ápice de reação, em todos os animais, ocorreu entre 41 e 55 dias depois da infecção. Em seguida, observou-se um lento declínio com um segundo pico de anticorpos, de menor intensidade, a partir de 75 dias. Através da prova ELISA foi comparada a reatividade dos antígenos extrato salino parcial de Cysticercus cellulosae, extrato salino total de Cysticercus bovis e antígeno total de Cysticercus longicollis com soros de bovinos portadores de cisticercos da Taenia saginata. Pesquisou-se também anticorpos anti-Cysticercus bovis, em soros de bovinos considerados como não portadores de cisticercos, pelo serviço de inspeção. De 20 amostras de soros analisadas, 2 (10%) apresentaram valores de absorbância acima do “cut-off”. Estes animais seriam possíveis portadores de cisticercos.

A cisticercose e a patologia mais encontrada na Inspecao post mortem em bovinos abatidos em estabelecimentos com Servico de Inspecao Federal (SIF). No Brasil a media da prevalencia desta parasitose e de 5%, porem, estes indices podem variar segundo a regiao anatomica avaliada, regiao geografica ou periodo de realizacao de trabalho. Para conhecer a prevalencia desta patologia em bovinos, no Estado do Parana, foi realizada uma pesquisa durante seis meses, entre julho a dezembro de 2000, em animais procedentes de 137 municipios, que foram abatidos no Frigorifico Argus, Regiao Metropolitana de Curitiba. A prevalencia de animais positivos encontrados com Cysticercus bovis variou de 0 a 27,27% com media de 3,83% entre os 26.465 bovinos inspecionados. Os municipios com maior numero de animais abatidos tiveram prevalencia aparente no lote entre 2,8 a 5%. De acordo com o sexo dos animais nao foi verificada predilecao ou especificidade parasitaria sendo que nos machos foi encontrado o indice de 3,81% e nas femeas 3,93 %. Todos os cisticercos encontrados vivos passaram pelo processo de desinvaginacao, coloracao por carmim acetico e identificacao microscopica constatando que 100% eram C. bovis . As implicacoes epidemiologicas destes achados sao discutidas no presente trabalho.

International audience; 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.

An NC-1 mimotope from Taenia solium cysticerci can help identify patients with neurocysticercosis through immunoassay. After chemical synthesis, an NC-1 peptide was coupled to bovine serum albumin (NC-1/BSA) for used as an immunogen in murine Taenia crassiceps cysticercosis, which is an experimental model of cysticercosis caused by T. solium. NC-1/BSA immunisation decreased parasitaemia by inducing 74% protection compared to the 77% protection obtained with T. crassiceps crude antigen. The influence of immunisation was also observed on the size and stage of development of the parasite. Antibodies from NC-1/BSA-immunised mice recognised proteins from the tegument and from the buddings, and intense immunostaining was observed in the final stage of the metacestode. The capacity of NC-1/BSA to induce protective antibodies which are reactive to proteins from the tegument of the metacestode suggests that this mimotope is a potential candidate for a vaccine against human and animal cysticercosis.

Background An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients’ sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Methods Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Results Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. Conclusions The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.