Concerns about the use of antimicrobials in swine herds and alternative trends.

Pig productivity in Brazil has advanced a lot in recent decades. Specialized breeds are more vulnerable to pathogens, which has boosted the use of antimicrobials by farmers. The selective pressure generated favors the emergence of resistant bacteria, which compromises the effectiveness of this treatment and limits therapeutic options. In addition to increasing costs and mortality rates in the production system, public awareness of this issue has increased. The authorities have imposed restrictive measures to control the use of antimicrobials and have banned their use as growth promoters. This literature review highlights biosecurity and animal welfare to prevent pig diseases. Hence, we describe alternatives to the use of antimicrobials in pig production for the selection of effective non-antibiotic feed additives that help maintain good health and help the pig resist disease when infection occurs.

Impact of Aspergillus fumigatus inoculation on the composting of wood shaving bedding for horses.

Equine farming generates a significant amount of waste, prompting the need for effective management. Composting enhanced by filamentous fungi holds promise for this purpose. This study focused on inoculating Aspergillus fumigatus isolates in composting horse bedding made with wood shavings (Pinus elliottii). The experiment lasted 90 days, with two treatment groups, control and inoculated, analyzing temperature, pH, electrical conductivity, total organic carbon and nitrogen content, and cellulose, hemicellulose, and lignin contents. Both treatments entered the thermophilic phase by the fourth day, reaching temperatures above 55°C and mesophilic maturation at 35 days (41 ± 0.2°C). The inoculated treatment exhibited higher electrical conductivity after 30 days and a more pronounced reduction in the total carbon content (42.85% vs. 38.29%) compared to the control. While there was no significant nitrogen difference, the inoculated treatment had a sharper reduction in carbon/nitrogen ratio, and cellulose and hemicellulose contents. Both treatments showed low coliform counts, no Salmonella sp., and reduced Strongyloides sp. larvae. Inoculating A. fumigatus in saturated horse bedding made from wood shavings improved compost quality, providing a possibility for sustainable equine farming waste treatment.

Molecular characterization and biofilm-formation analysis of Listeria monocytogenes, Salmonella spp., and Escherichia coli isolated from Brazilian swine slaughterhouses.

This study aimed to verify the presence of Listeria monocytogenes, Salmonella spp., and Escherichia coli in two Brazilian swine slaughterhouses, as well as to perform antibiograms, detect virulence and antimicrobial resistance genes, and evaluate the in vitro biofilm-forming capability of bacterial isolates from these environments. One Salmonella Typhi isolate and 21 E. coli isolates were detected, while L. monocytogenes was not detected. S. Typhi was isolated from the carcass cooling chamber's floor, resistant to several antimicrobials, including nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline, and sulfonamide, and contained resistance genes, such as tet(B), tet(C), tet(M), and ampC. It also showed moderate biofilm-forming capacity at 37°C after incubating for 72 h. The prevalence of the 21 E. coli isolates was also the highest on the carcass cooling chamber floor (three of the four samplings [75%]). The E. coli isolates were resistant to 12 of the 13 tested antimicrobials, and none showed sensitivity to chloramphenicol, an antimicrobial prohibited in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), and blaSHV, as well as the virulence genes stx-1, hlyA, eae, tir α, tir ß, tir γ, and saa were detected in the E. coli isolates. Moreover, 5 (23.8%) and 15 (71.4%) E. coli isolates presented strong and moderate biofilm-forming capacity, respectively. In general, the biofilm-forming capacity increased after incubating for 72 h at 10°C. The biofilm-forming capacity was the lowest after incubating for 24 h at 37°C. Due to the presence of resistance and virulence genes, multi-antimicrobial resistance, and biofilm-forming capacity, the results of this study suggest a risk to the public health as these pathogens are associated with foodborne diseases, which emphasizes the hazard of resistance gene propagation in the environment.

Uterine Inflammatory Response After Prostaglandin E1 (Misoprostol) Infusion Prebreeding or Immediately After Embryo Flushing in Commercial Donor Mares.

Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +200 µg) or sham (3 mL of lactate Ringer's solution) infusions, bilaterally deposited via deep-horn, at least 72 hours prebreeding (experiment 1) or immediately after embryo flushing (experiment 2). Each mare had one cycle for misoprostol and sham in both experiments and a breeding cycle (no sham or misoprostol) between experiments. Uterine edema, fluid accumulation, and the number of uterine PMN were assessed before each infusion and then daily for 72 hours. Uterine lavage was performed the day after each infusion across groups and experiments. Ovulation was hastened with a GnRH agonist and confirmed at 24 hour-intervals. Mares were bred with semen from one of six stallions per owner's choice. Embryo flushing was performed 8 to 9 days postovulation. In either experiment, misoprostol did not affect uterine edema or fluid accumulation (P > .05). However, both the sham and misoprostol infusions increased the number of PMN up to 48 hours postinfusion in both experiments. Embryo recoveries were similar between sham (45%, 5/11) and misoprostol cycles in experiments 1 (45%, 5/11; P > .05) and 2 (sham, 68%, 7/11; misoprostol, 45%, 5/11; P > .05). In conclusion, misoprostol did not induce exacerbated uterine inflammation in mares or systemic adverse reactions when infused prebreeding or immediately after embryo flushing.

Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil.

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Assessment of Internalin A Gene Sequences and Cell Adhesion and Invasion Capacity of Listeria monocytogenes Strains Isolated from Foods of Animal and Related Origins.

Listeria monocytogenes is a foodborne pathogen of global relevance that causes outbreaks and sporadic cases of listeriosis, acquired through the consumption of contaminated products, including milk or meat products and ready-to-eat meat products subjected to intensive handling. The objective of the present study was to classify L. monocytogenes isolated from various food-related sources in the Federal District of Brazil and surrounding areas to sequence internalin A (inlA) genes from these isolates and assess their adhesion and invasion capacity using Caco-2 cells. In addition, 15 were classified as group I, 3 as group II, and 7 classified as group IV. Premature stop codons (PMSCs) at the nucleotide position 976 (GAA→TAA) of the inlA gene were identified in 5 of the 25 isolates. Adhesion and invasion tests in Caco-2 cells showed that all the isolates were capable of adhesion and cellular invasion, with isolates containing PMSCs exhibiting on average higher invasion capacity than those without PMSCs (p = 0.041) and a median of adhesion very distinctive from those without stop codons. These results are the first report of PMSCs in the inlA gene of L. monocytogenes from the Federal District of Brazil and Brazil.

Evaluation of the anti-apoptotic activity of bovine alphaherpesvirus type 5 US3 protein kinase in insect cells using a recombinant baculovirus.

Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPV anti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

Efeito da luz ultravioleta na eclodibilidade e viabilidade de pintos provenientes de ovos férteis sujos / Effect of ultraviolet light in hatchability and viability of chicks from dirty fertile eggs

Considerando a necessidade de se desenvolver estratégias para a incubação de ovos sujos e a demanda de se utilizar métodos renováveis de sanitização, a utilização da luz ultravioleta para desinfecção de ovos incubáveis sujos foi o objetivo desta pesquisa. Foram avaliados ovos sem desinfecção (C-), desinfetados com paraformaldeído (C+), e expostos a tempos diferentes de exposição ultravioleta: UV 3’30”, UV 4’30” e UV 5’30”. Os ovos C(+) e os expostos à UV por 3’30” e 5’30” tiveram uma redução significativa na quantidade de colônias na casca dos ovos em relação ao C(-), variando de 0.64 a 1 logUFC/g. Os tratamentos C(+), UV 3’30” e UV 5’30” foram aplicados para avaliação da incubabilidade dos ovos. A eclodibilidade do tratamento UV 3’30” (70,51%) foi superior ao UV 5’30” (51,16%) e similar ao C(+) (55,81%). Conclui-se que o tratamento UV 3’30” é eficaz na redução da contaminação microbiológica de ovos sujos e não afeta negativamente a eclodibilidade e a viabilidade dos pintinhos nascidos.

Considering the need to develop strategies for dirty eggs incubation and the demand to use renewable methods of sanitation, the use of ultraviolet light for disinfecting hatching dirty eggs was the objective of this research. Eggs not disinfected (C-), disinfected with paraformaldehyde (C+) and exposed to different times of UV radiation (UV 3’30”, UV 4’30” and UV 5’30”) were tested. The eggs C(+) and those exposed to UV for 3’30” and 5’30” presented a significant reduction on the number of colonies of eggshells comparing to C(-), with a 0.64 to 1 logUFC/g variation. The treatments C(+), UV 3’30” and UV 5’30” were applied in an incubation phase. The hatchability of treatment UV 3’30” (70,51%) was superior to UV 5’30” (51,16%) and alike C(+) (55,81%). In conclusion, the treatment UV 3’30” is effective in reducing microbiologic contamination of dirty eggs and do not affect negatively hatchability and quality of chicks.

Bacterial culture and antibiotic sensitivity from the ocular conjunctiva of horses / Cultura e sensibilidade bacteriana da conjuntiva ocular de equinos

ABSTRACT: The aim of this study was to identify the conjunctival bacterial flora of healthy horses in Brasilia (Distrito Federal, Brazil), and to evaluate the antimicrobial susceptibility of the isolated strains. We examined 200 eyes of 100 healthy adult horses without any eye problems, belonging to the 1st Regiment of the Cavalry Guard (RCG) of the Brazilian Army in Brasilia. Samples were collected from the inferior conjunctival fornix of both eyes. Drug sensitivity test was performed with the antibiotics gentamicin (10µg), tobramycin (10µg), chloramphenicol (30µg) and ciprofloxacin (5µg). Of the 200 samples collected, 131 (65.5%) were considered positive for bacterial growth. A total of 208 bacterial strains belonging to 19 genera were isolated, where there was prevalence of gram-positive bacteria (65%), with Staphylococcus sp. being the species of greatest incidence. It was observed that 94, 85, 81 and 68% of the isolates were sensitive to ciprofloxacin, gentamicin, chloramphenicol and tobramycin, respectively. These results can guide the empirical selection antimicrobial therapy for infections of the ocular surface of horses, pending the identification of the etiologic agent.

RESUMO: Objetivou-se identificar a flora bacteriana conjuntival de cavalos saudáveis, residentes de Brasília (Distrito Federal - Brasil), e avaliar a susceptibilidade antimicrobiana das cepas isoladas. Foram avaliados 200 olhos de 100 equinos hígidos, adultos, saudáveis e sem alterações oculares, provenientes do 1o Regimento de Cavalaria de Guarda (RCG) do Exército de Brasília. As amostras foram coletadas do fórnix conjuntival inferior de ambos os olhos. O teste de sensibilidade foi realizado para os antimicrobianos: gentamicina (10mcg), tobramicina (10mcg), cloranfenicol (30mcg) e ciprofoxacino (5mcg). Das 200 amostras coletadas, 131 (65,5%) foram consideradas positivas para o crescimento bacteriano. Um total de 208 bactérias pertencentes a 19 gêneros foram isoladas, verificando-se prevalência de bactérias gram-positivas (65%), sendo Staphylococcus sp, a espécie de maior incidência. Foi observado que 94%, 85%, 81% e 68% das bactérias isoladas foram sensíveis ao ciprofloxacino, à gentamicina, ao cloranfenicol e à tobramicina, respectivamente. Tais resultados podem direcionar a escolha empírica da terapêutica antimicrobiana nas afecções da superfície ocular de equinos, enquanto se aguarda a identificação do agente etiológico.

Deficiência de cobre em suínos: caracterização clínico-patológica / Copper deficiency in swine: clinical-pathologic characterization

São relatados dois casos de deficiência de cobre em suínos (A e B), fêmeas, sem raça definida, de três meses de idade, provenientes de propriedade rural em Planaltina, GO. Os animais apresentavam incoordenação, paresia de membros pélvicos, com progressão para os membros torácicos e andar com flexão da articulação metacarpo-falangeana. Na propriedade de origem dos animais, outros dois suínos apresentavam atrofia da musculatura da coxa e posição sentada quando parados. O rebanho recebia dieta não-balanceada constituída por milho, soro de leite e sobras de lavoura. Na necropsia, não foram observadas alterações significativas, mas histologicamente havia vacuolização periaxonal multifocal no tronco encefálico e na medula espinhal, quantidade discreta de esferoides axonais e macrófagos no interior dos vacúolos. A coloração com azul rápido de luxol (ARL) para mielina demonstrou que as áreas de vacuolização caracterizavam focos de desmielinização. Amostras de fígado dos dois suínos apresentaram níveis de cobre muito abaixo do normal, de 13,1 partes por milhão (ppm) (suíno A) e 5,7ppm (suíno B). Este trabalho descreve dois casos de desmielinização medular e de tronco encefálico devido à deficiência de cobre em suínos jovens.

Two cases of copper deficiency are described in three-month-old female crossbred piglets from a swine herd in Planaltina, GO. The animals presented incoordination, hindlimbs paresis progressing to the forelimbs, and gait with flexion of the metacarpofalangeal articulation. On the farm of origin of the animals, other two piglets presented atrophy of thigh muscle and sitting position when in station. The herd was fed with unbalanced diet consisting of corn, whey and leftover crop. On necropsy no significant changes were observed, but histologically there was multifocal periaxonal vacuolization in the brainstem and in spinal cord, discrete amount of axonal spheroids and macrophages in the vacuoles. The luxol fast blue (LFB) stain for myelin showed that the vacuolated sites represent demyelinated areas. Liver sample of the two piglets had very low copper levels, 13.1 parts per million (ppm) in swine A and 5.7 (ppm) in swine B. This paper describes two cases of spinal cord and brainstem demyelination due to copper deficiency in piglets.

Análise microbiológica da placa bacteriana da doença periodontal em cães e o efeito da antibioticoterapia sobre ela / Microbiological analysis of bacterial plaque of periodontal disease on dogs and effects of antibioticotherapy on it

Objetivou-se determinar a microbiota da placa bacteriana subgengival de cães com doença periodontal (DP) e estabelecer o efeito da antibioticoterapia. Avaliaram-se 20 cães com graus variados de DP e coletaram-se amostras da placa bacteriana subgengival antes e após antibioticoterapia. Preconizou-se antibioticoterapia distinta em dois grupos, com 10 animais cada: clindamicina (G1) e metronidazol + espiramicina (G2). Observou-se crescimento bacteriano subgengival na maioria dos cães com DP e correlação entre a severidade da DP e a idade dos animais. Houve redução significativa no crescimento bacteriano após a antibioticoterapia e o antibiograma demonstrou maior sensibilidade à clindamicina, seguido da espiramicina; todos os microrganismos foram resistentes ao metronidazol.

The objective was to determine microbiote of the subgingival bacterial plaque of dogs with periodontal disease (PD) and establish the effect of antibioticotherapy on its reduction. Twenty dogs with varied stages of PD were evaluated and samples of their subgingival bacterial plaque were collected. Distinct antibiotic protocols were used in two groups with ten animals each: clindamycin (G1) and metronidazole + espiramycin (G2). New subgingival samples were collected 15 days after antibiotic therapy started. There were observed subgingival bacterial culture on most dogs with PD and correlation between severity of PD and age. There was reduction of bacterial growth in 20 percent of the samples after treatment and antibiogram showed higher sensibility to clindamycin, followed by espiramycin - all microorganisms were resistant to metronidazole.

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses.

BACKGROUND: Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricultural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed, and their insecticidal properties analysed against Spodoptera frugiperda larvae. RESULTS: Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. CONCLUSIONS: Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat.

The occurrence of foodborne diseases is increasing throughout the world. Bacteria of the genus Salmonella are responsible for food poisoning and, in some cases, may be fatal. The aim of this study was to adapt the multiplex PCR technique (mPCR) on the rapid and direct identification of the presence of Salmonella sp. as well as serotypes Enteritidis, Typhi and Typhimurium in poultry carcasses (n=127) and viscera (n=73). The implementation of the standard technique using positive controls was successfully adapted. The results of Salmonella sp. detection in refrigerated viscera showed that the mPCR was able to detect Salmonella genus in 2.74% of these samples. Traditional microbiological analysis also identified the same positive samples for Salmonella sp. but was not able to differentiate the serotype. The serotype Enteritidis was detected by mPCR in 1.37% of the samples. Our conclusion was that the mPCR was able to detect the presence of these bacteria in a short period of time and enabled the identification of serotype Enteritidis in one of the samples found positive for Salmonella sp.

Síndrome do abscesso pituitário em bezerros na Região Centro-Oeste / Pituitary abscess syndrome in calves from Mid-Western Brazil

A síndrome do abscesso pituitário é uma doença neurológica responsável por casos esporádicos e surtos, principalmente em bezerros, ocasionando alto índice de mortalidade. Descreve-se a ocorrência e os achados clínicos, laboratoriais e anátomo-patológicos em três bezerros com síndrome do abscesso pituitário no Centro-Oeste do Brasil. Os animais tinham 8-11 meses de idade e os sinais clínicos mais marcantes relacionaram-se aos sinais nervosos de origem cerebral e do tronco encefálico com evolução clínica de 7-20 dias. A hematologia revelou leucocitose por neutrofilia e hiperfibrinogenemia. A análise do líquido céfalo-raquidiano apresentou pleocitose neutrofílica. Arcanobacterium pyogenes foi isolado do líquido céfalo-raquidiano. Um dos bezerros apresentou recuperação após antibioticoterapia. A mortalidade foi de 66,6 por cento (2/3). Os achados de necropsia consistiram em um único abscesso de localização parapituitária ou situado no parênquima da glândula; um dos bezerros apresentou rinite necrosante e outro, broncopneumonia abscedativa. O exame histológico do sistema nervoso central revelou ausência quase completa do tecido hipofisário normal, devido à necrose extensa e infiltrado inflamatório neutrofílico difuso concomitante. Reitera-se a importância da realização de práticas de manejo adequadas a fim de reduzir a incidência de inúmeras enfermidades, principalmente em bezerros, dentre elas a síndrome do abscesso pituitário.

Pituitary abscess syndrome is a neurologic disease responsible for sporadic cases and outbreaks especially in calves leading to high mortality rates. This paper aimed to report the occurrence and the clinical, laboratorial and pathologic findings in three 8 to 11-month-old calves with pituitary abscess syndrome from Mid-Western Brazil. The most important clinical findings were nervous signs of cerebral and brainstem origin with clinical evolution of 7-20 days. Hematology revealed leucocytosis by neutrophilia and hyperfibrinogenemia. Cerebrospinal fluid analysis showed neutrophilic pleocytosis. Arcanobacterium pyogenes was isolated from cerebrospinal fluid. One calf recovered after antibiotic treatment. Mortality rate was 66.6 percent (2/3). Necropsy findings included single para-hypophyseal abscesses or located in the glandular parenchyma; one calf showed necrotizing rhinitis and another abscedative pneumonia. Histological exams of the central nervous system reveal complete absence of normal pituitary tissue due to the wide necrosis and neutrophilic inflammatory infiltrate. The authors reiterate the importance of adequate management practices to reduce incidence of several diseases especially in calves, including the pituitary abscess syndrome.

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.

A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA), um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE) é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da amplificação foi clonado no vetor pGem®-T Easy e subclonado no plasmídeo de expressão pFastBac™1. O DNA recombinante pFastBac-gE-VDA foi usado para a transposição sítio-específica no baculovírus recombinante (bacmid). Após seleção por antibióticos e cor, as colônias com o recombinante bacmid-pFastBac-gE-VDA foram selecionadas e a presença do gene da gE foi confirmada por PCR. O DNA recombinante viral, bacmid-pFastBac-gE-VDA, foi usado para cotransfecção de células de inseto Trichoplusia ni e a presença do recombinante e a proteína gE foi determinada por PCR, por SDS-PAGE e Western blotting, respectivamente.

Utilização de técnicas direta (cultivo) e indireta (coloração de DNA pelo DAPI) para detecção de micoplasmas contaminantes de cultivos celulares

Trinta cultivos celulares de cinco Instituições de Ensino e Pesquisa do Estado do Rio de Janeiro foram testadas por duastécnicas diferentes, cultivo em caldo e agar SP4 e coloração de DNA, para a detecção de micoplasmas contaminantes decultivos celulares, que são, na atualidade, os causadores de maior prejuízo para laboratórios, mas não foram encontradosresultados positivos.

Utilizaçäo de técnicas direta (cultivo) e indireta (coloraçäo de DNA pelo DAPI) para detecçäo de micoplasmas contaminantes de cultivos celulares / Utilization of direct (culture) and indirect (DNA colorization by DAPI) tecniques for Mycoplasma contaminants detection in cell culture

Trinta cultivos celulares de cinco Instituiçöes de Ensino e Pesquisa do Estado do Rio de Janeiro foram testadas por duas técnicas diferentes, cultivo em caldo e agar SP4 e coloraçäo de DNA, para a detecçäo de micoplasmas contaminantes de cultivos celulares, que säo, na atualidade, os causadores de maior prejuízo para laboratórios, mas näo foram encontrados resultados positivos.