Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp, and Listeria monocytogenes DNA in milk / [Padronização de um método rápido de PCR quadriplex para a detecção simultânea de DNA de bovino, bubalino, Salmonella spp. e Listeria monocytogenes em leite]

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.

Padronização de uma PCR para a autenticação do Salmo salar em pratos da culinária japonesa / Standardization of a PCR for the authentication of Salmo salar in dishes of Japanese cuisine

O objetivo deste trabalho foi padronizar uma PCR para a detecção do Salmo salar, a qual possa ser usada na autenticação do salmão utilizado em pratos da culinária japonesa e do pescado comercializado in natura. Para isso, dois lotes de sushi foram produzidos experimentalmente. Além disso, foram visitados 38 estabelecimentos que comercializam comida japonesa e 10 peixarias na região metropolitana de Belém, visando à coleta do sushi, do temaki e do pescado pertencente à espécie Salmo salar. Os dados demonstraram que a técnica foi eficiente para a autenticação de Salmo salar, visto que a espécie foi detectada tanto nas amostras de sushis preparados experimentalmente quanto nas alíquotas de pescados isolados, utilizados para a preparação do sushi. Em contrapartida, a espécie Salmo trutta não foi detectada nas amostras de sushis preparados com esta espécie nem nas alíquotas de pescado isolado. Além disso, foi possível a confirmação da utilização da espécie Salmo salar no preparo das amostras de sushi, temaki e de pescado. Portanto, concluiu-se que a técnica foi capaz de amplificar o DNA da referida espécie e não gerou identificação inespecífica quando a espécie Salmo trutta foi analisada, podendo ser uma ferramenta adequada para a autenticação do Salmo salar.(AU)

The objective of this work was to standardize a PCR for the detection of Salmo salar, which can be used in the authentication of salmon used in Japanese dishes and fish commercialized in natura. For this, two batches of sushi were produced experimentally. In addition, 38 establishments that sell Japanese food and 10 fishmongers in the metropolitan area of Belém were visited, aiming to collect sushi, temaki and fish belonging to the species Salmo salar. The data demonstrated that the technique was efficient for the authentication of Salmo salar, since the species was detected in both the experimentally prepared sushi samples and the isolated fish aliquots used for the preparation of sushi. In contrast, the species Salmo trutta was not detected in the sushi samples prepared with this species nor in the isolated fish aliquots. In addition, it was possible to confirm the use of the Salmo salar species in the preparation of sushi, temaki and fish samples. Therefore, it was concluded that the technique was able to amplify the DNA of this species and did not generate nonspecific identification when the species Salmo trutta was analyzed, being able to be a suitable tool for the authentication of Salmo salar.(AU)

Imunogenicidade da proteína M recombinante de Streptococcus equi subsp. equi coadministrada com um adjuvante molecular / Immunogenicity of recombinant M protein of Streptococcus equi subsp. equi co administered with a molecular adjuvant

The strangles is an infectious disease that affects horses from all ages and causes important economic losses in the equine-related business. The aim of this work was to evaluate the immunogenicity of the recombinant M protein from Streptococcus equi (rSeM) co-administered with the recombinant heat-labile enterotoxin B subunit from Escherichia coli (rLTB) in mice and horses. A total of 72 female Balb-c mice were divided into eight groups and 18 horses were divided into six groups. The animals were inoculated by intramuscular (IM) or intranasal (IN) routes with different treatments of rSeM, rLTB and/or Al(OH)3. The results obtained in both species, independent of administration routes, demonstrated that rSeM + rLTB had higher levels of specific serum immunoglobulins, however, in mucosal immunity the increase was not identified. Thus, the use of rSeM as vaccine antigen and rLTB as adjuvant can be a potential tool in the control of equine strangles.(AU)

Cloning, expression and characterization of SeM protein of Streptococcus equi subsp. equi and evaluation of its use as antigen in an indirect ELISA / Clonagem, expressão e caracterização de proteína SeM de Streptococcus equi subsp. equi e avaliação de sua utilização como antígeno em um ELISA indireto

Strangles is an economically important horse disease caused by Streptococcus equi subsp. equi. The diagnosis can be confirmed either directly by bacterial isolation and PCR or by ELISA, which is an indirect method based on the detection of serum antibodies. The aim of this study was to clone, express and characterize the SeM protein of Streptococcus equi subsp. equi, evaluate its use as antigen in indirect ELISA and determine its performance to distinguish sera of negative, vaccinated and positive animals. This was initially performed by cloning the gene encoding the SeM protein and its expression in Escherichia coli. Subsequently, the protein produced was characterized and used as antigen in ELISA. Serum samples for evaluation were taken from 40 negative foals, 46 horses vaccinated with a commercial vaccine against strangles and 46 horses diagnosed with the disease. The test showed high specificity and sensitivity, allowing discrimination between negative and positive, positive and vaccinated animals, and vaccinated animals and negative sera. Thus, it was concluded that the protein produced rSeM, which can be used as antigen for disease diagnosis, and the described ELISA might be helpful to evaluate the immune status of the herd...

A adenite equina é uma enfermidade economicamente importante de equinos, causada por Streptococcus equi subsp. equi. Seu diagnóstico pode ser confirmado de forma direta, por meio de isolamento bacteriano e de PCR, ou de forma indireta, por meio de ELISA, método baseado na detecção de anticorpos séricos. O objetivo deste estudo foi clonar, expressar e caracterizar a proteína SeM de Streptococcus equi subsp. equi, avaliar sua utilização como antígeno em um ELISA indireto e determinar a capacidade do teste de distinguir soros de animais negativos, vacinados e positivos. Para tal, foi inicialmente realizada a clonagem do gene que codifica para a proteína SeM e sua expressão em Escherichia coli. Posteriormente, a proteína produzida foi caracterizada e utilizada como antígeno em um teste de ELISA indireto. Para avaliação do teste, foram utilizadas amostras de soro de 40 potros negativos, de 46 equinos vacinados com uma vacina comercial contra adenite equina e de 46 equinos com diagnóstico da doença. O teste demonstrou alta sensibilidade e especificidade, permitindo discriminar entre soros negativos e positivos, positivos e de animais vacinados, e negativos e de animais vacinados. Assim, conclui-se que a proteína rSeM produzida pode ser usada como antígeno para o diagnóstico da enfermidade e que o ELISA descrito pode ser útil para avaliar o estado imunológico do rebanho...

Development and evaluation of a trapping system for Anoplophora glabripennis (Coleoptera: Cerambycidae) in the United States.

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), commonly known as the Asian longhorned beetle, is an invasive wood-boring pest that infests a number of hardwood species and causes considerable economic losses in North America, several countries in Europe, and in its native range in Asia. The success of eradication efforts may depend on early detection of introduced populations; however, detection has been limited to identification of tree damage (oviposition pits and exit holes), and the serendipitous collection of adults, often by members of the public. Here we describe the development, deployment, and evaluation of semiochemical-baited traps in the greater Worcester area in Massachusetts. Over 4 yr of trap evaluation (2009-2012), 1013 intercept panel traps were deployed, 876 of which were baited with three different families of lures. The families included lures exhibiting different rates of release of the male-produced A. glabripennis pheromone, lures with various combinations of plant volatiles, and lures with both the pheromone and plant volatiles combined. Overall, 45 individual beetles were captured in 40 different traps. Beetles were found only in traps with lures. In several cases, trap catches led to the more rapid discovery and management of previously unknown areas of infestation in the Worcester county regulated area. Analysis of the spatial distribution of traps and the known infested trees within the regulated area provides an estimate of the relationship between trap catch and beetle pressure exerted on the traps. Studies continue to optimize lure composition and trap placement.

Evidence of local adaptation in plant virus effects on host-vector interactions.

Recent research suggests that plant viruses, and other pathogens, frequently alter host-plant phenotypes in ways that facilitate transmission by arthropod vectors. However, many viruses infect multiple hosts, raising questions about whether these pathogens are capable of inducing transmission-facilitating phenotypes in phylogenetically divergent host plants and the extent to which evolutionary history with a given host or plant community influences such effects. To explore these issues, we worked with two newly acquired field isolates of cucumber mosaic virus (CMV)-a widespread multi-host plant pathogen transmitted in a non-persistent manner by aphids-and explored effects on the phenotypes of different host plants and on their subsequent interactions with aphid vectors. An isolate collected from cultivated squash fields (KVPG2-CMV) induced in the native squash host (Cucurbita pepo) a suite of effects on host-vector interactions suggested by previous work to be conducive to transmission (including reduced host-plant quality for aphids, rapid aphid dispersal from infected to healthy plants, and enhanced aphid attraction to the elevated emission of a volatile blend similar to that of healthy plants). A second isolate (P1-CMV) collected from cultivated pepper (Capsicum annuum) induced more neutral effects in its native host (largely exhibiting non-significant trends in the direction of effects seen for KVPG2-CMV in squash). When we attempted cross-host inoculations of these two CMV isolates (KVPG2-CMV in pepper and P1-CMV in squash), P1-CMV was only sporadically able to infect the novel host; KVPG2-CMV infected the novel pepper host with somewhat reduced success compared with its native host and reached virus titers significantly lower than those observed for either strain in its native host. Furthermore, KVPG2-CMV induced changes in the phenotype of the novel host, and consequently in host-vector interactions, dramatically different than those observed in the native host and apparently maladaptive with respect to virus transmission (e.g., host plant quality for aphids was significantly improved in this instance, and aphid dispersal was reduced). Taken together, these findings provide evidence of adaption by CMV to local hosts (including reduced infectivity and replication in novel versus native hosts) and further suggest that such adaptation may extend to effects on host-plant traits mediating interactions with aphid vectors. Thus, these results are consistent with the hypothesis that virus effects on host-vector interactions can be adaptive, and they suggest that multi-host pathogens may exhibit adaptation with respect to these and other effects on host phenotypes, perhaps especially in homogeneous monocultures.

Dynamics of short- and long-term association between a bacterial plant pathogen and its arthropod vector.

The dynamics of association between pathogens and vectors can strongly influence epidemiology. It has been proposed that wilt disease epidemics in cucurbit populations are sustained by persistent colonization of beetle vectors (Acalymma vittatum) by the bacterial phytopathogen Erwinia tracheiphila. We developed a qPCR method to quantify E. tracheiphila in whole beetles and frass and used it to assess pathogen acquisition and retention following variable exposure to infected plants. We found that (i) E. tracheiphila is present in frass in as little as three hours after feeding on infected plants and can be transmitted with no incubation period by vectors given brief exposure to infected plants, but also by persistently colonized vectors several weeks following exposure; (ii) duration of exposure influences rates of long-term colonization; (iii) frass infectivity (assessed via inoculation experiments) reflects bacterial levels in frass samples across time; and (iv) vectors rarely clear E. tracheiphila infections, but suffer no apparent loss of fitness. These results describe a pattern conducive to the effective maintenance of E. tracheiphila within cucurbit populations.

Emission of alarm pheromone in aphids: a non-contagious phenomenon.

In response to attack by natural enemies, most aphid species release an alarm pheromone that causes nearby conspecifics to cease feeding and disperse. The primary component of the alarm pheromone of most species studied is (E)-beta-farnesene. We recently demonstrated that the production and accumulation of (E)-beta-farnesene during development by juvenile aphids is stimulated by exposure to odor cues, most likely by (E)-beta-farnesene emitted by other colony members. Here, we tested whether the release of (E)-beta-farnesene can be triggered by exposure to the alarm pheromone of other individuals, thereby amplifying the signal. Such contagious emission might be adaptive under some conditions because the amount of (E)-beta-farnesene released by a single aphid may not be sufficient to alert an appropriate number of individuals of the colony to the presence of a potential threat. By using a push-pull headspace collection system, we quantified (E)-beta-farnesene released from Acyrthosiphon pisum aphids exposed to conspecific alarm signals. Typical avoidance behavior was observed following exposure to (E)-beta-farnesene (i.e., aphids ceased feeding and dropped from host-plant); however, no increase in alarm pheromone amount was detected, suggesting that contagious release of (E)-beta-farnesene does not occur.

Preparação e caracterização inicial de complexo de inclusão entre nitrofurazona e 2-hidroxipropil-Beta-ciclodextrina / Preparation and initial characterization of inclusion complex between nitrofurazone and 2-hydroxypropyl-Beta-cyclodextrin

Nitrofurazona(NF), 5-nitro-2-furaldeído semicarbazona, é um antibiótico de amplo espectro, que apresenta diversos efeitos tóxicos e baixa solubilidade aquosa. A complexação da NF com ciclodextrinas é de grande interesse para o desenvolvimento de uma formulação para este antibiótico que seja mais segura e eficiente. Neste trabalho foi realizada a preparação e caracterização inicial do complexo de inclusão entre NF e hidroxipropil-Beta-ciclodextrina (HP-Beta-CD) através de experimentos para determinação da cinética de complexação, medidas de fotoestabilidade, medidas de constante de afinidade fármaco: ciclodextrina, ensaios de liberação in vitro, estequiometria de formação do complexo e morfologia do complexo por microscopia eletrônica de varredura. Os ensaios de cinética de complexação mostram que para o complexo atingir o equilíbrio são necessárias 17,3h. As isotermas de solubilidade determinadas para a NF em função da temperatura mostraram perfis do tipo A e B indicando que a temperatura é um fator importante na complexação da NF com ciclodextrina. Os experimentos de fotoestabilidade indicam que a inserção da molécula de NF na cavidade interna da ciclodextrina protege o fármaco da fotodecomposição. A cinética de liberação mostra que o perfil de liberação do fármaco é modificado pela presença da ciclodextrina no meio. A estequiometria de complexação entre NF e HP-Beta-CD determinada foi de 1:1 NF:HP-Beta-CD. Os resultados demicroscopia eletrônica de varredura indicam alterações na estrutura cristalina da NF em presença deciclodextrina. Este estudo está baseado na caracterização físico-química da complexação entre NF e HP-Beta-CD podendo ser uma nova potencial opção para utilização terapêutica do NF.

Nitrofurazone (NF), 5-nitro-2-furaldehyde semicarbazone, a broad-spectrum antibiotic, has reported toxic effects and low solubility in water. It would be of great interest to form inclusion complexes between NF and a cyclodextrin, to develop more effective and safer antibiotic formulations. This paper focuses on the preparation of inclusion complexes of NF with 2- hydroxypropyl- -cyclodextrin (HP- -CD) and their initial characterization by evaluating rates of complex formation, photostability, solubility isotherms, release rate profiles, stoichiometry of the complexes and their morphology, as revealed by scanning electron microscopy. The kinetic tests of complex formation revealed that 17,3 h is enough for stabilization of the NFcyclodextrin complex. The solubility isotherm studies showed that the isotherm changes from type A to type B, as a function of temperature. The photostability experiments showed that the insertion of the NF in the HP- -CD cavity protects the drug from photodecomposition. The release kinetic tests showed that the profile of NF release from the complex is altered by the presence of HP- -CD in the medium. A Job's plot indicated that the stoichiometry of the complex was 1:1 NF:HP- -CD. The scanning electron micrographs showed changes in the crystal structure of NF in the complex. This study focused on the physicochemical properties of drug-delivery formulations that could potentially be developed into a novel type of therapy with NF.

Inclusion complex of S(-) bupivacaine and 2-hydroxypropyl-beta-cyclodextrin: study of morphology and cytotoxicity

Local anesthetics (LA) belong to a class of pharmacological compounds that attenuate or eliminate pain by binding to the sodium channel of excitable membranes, blocking the influx of sodium ions and the propagation of the nerve impulse. S (-) bupivacaine (S(-)bvc) is a local anesthetic of amino-amide type, widely used in surgery and obstetrics for sustained peripheraland central nerve blockade. This article focuses on the characterization of an inclusion complex of S(-) bvc in2-hydroxypropyl- beta-cyclodextrin (HP-beta -CD). Differential scanning calorimetry, scanning electron microscopy andX-Ray diffraction analysis showed structural changes inthe complex. In preliminary toxicity studies, the cellviability tests revealed that the inclusion complex decreased the toxic effect (p smaller that 0.001) produced by S(-) bvc.These results suggest that the S(-) bvc:HP- beta-CD inclusion complex represents a promising agent for the treatment of regional pain.

Caterpillar-induced nocturnal plant volatiles repel conspecific females.

Plants respond to insect herbivory by synthesizing and releasing complex blends of volatile compounds, which provide important host-location cues for insects that are natural enemies of herbivores. The effects of these volatile blends on herbivore behaviour have been investigated to only a limited extent, in part because of the assumption that herbivore-induced volatile emissions occur mainly during the light phase of the photoperiod. Because many moths-whose larvae are some of the most important insect herbivores-are nocturnal, herbivore-induced plant volatiles have not hitherto been considered to be temporally available as host-location cues for ovipositing females. Here we present chemical and behavioural assays showing that tobacco plants (Nicotiana tabacum) release herbivore-induced volatiles during both night and day. Moreover, several volatile compounds are released exclusively at night and are highly repellent to female moths (Heliothis virescens). The demonstration that tobacco plants release temporally different volatile blends and that lepidopteran herbivores use induced plant signals released during the dark phase to choose sites for oviposition adds a new dimension to our understanding of the role of chemical cues in mediating tritrophic interactions.

Solvolysis and aminolysis on peptidyl-Kaiser oxime resin assisted by Ca2+ and Eu3+: a mild procedure to prepare alpha-methyl and -ethyl esters of protected peptides.

Ca2+ and Eu3+ were able to assist solvolysis on peptidyl-Kaiser oxime resins generating alpha-methyl and -ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca2+ or Eu3+ was needed to enhance this reaction efficiency. Ca2+ also assisted aminolysis on peptidyl-Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of the C-terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the beta-benzyl or cyclohexyl esters, was detected in the reaction media. The alpha-methyl and -ethyl esters of Ac-Ala-Gly-X [where, X = Gly, Ala, Phe or Lys (2-Cl-Z)] and of Ac-Ile-Ser (Bzl)-Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg(HCl)-OMe and Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg (HCl)-OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin-cleavage yields were > 50%. In their absence, they were < 15%.

Composiçäo de leite de éguas de P.S.I e desenvolvimento ponderal de suas crias / Milk composition of the hroroughbred mares and weight gain of the foals

Com o objetivo de analisar a composiçäo do leite, foram coletadas amostras de nove éguas da raça Puro Sangue Inglês. A composiçäo foi determinada em amostras antes da primeira mamada de cada um dos dias considerados. A matéria seca foi determinada por gramivetria, a proteína bruta pelo micro-Kjedahl, a gordura, pelo milko-Tester, a lactose e o fósforo por colorimetria e o cálcio por permanganometria. Os constituintes, com exceçäo da lactose, decresceram durante o período. Os potros atingiram o desenvolvimento ponderal esperado, com um ganho de peso de 1,14 kg/dia durante o periodo observado