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...

Chemical characteristics of beddings for swine: effects of bedding depths and of addition of inoculums in a pilot-scale.

The effect of depths and of addition of inoculums on the chemical content of swine beddings was evaluated. For beddings 0.25m (25D) and 0.50m (50D) deep, three treatments were tested in two repeats with the same beddings: control (no inoculums); T1 (250g of Bacillus cereus var. toyoii at 8.4×10(7)CFU/g); and T2 (250g of a pool of Bacillus sp. at 8.4×10(7)CFU/g) (250g for 25D and 500g for 50D). For 25D, the C:N ratio was lower, but N, K and C contents were greater than for 50D (P<0.05). The inoculums did not benefit any chemical parameter (P>0.05). In the second repeat, beddings presented lower C:N ratio and greater N, P and K contents than in the first repeat (P<0.05). Thus, the compost produced after using 25D twice had greater fertilizer value than that of 50D.

Addition of Bacillus sp. inoculums in bedding for swine on a pilot scale: effect on microbial population and bedding temperature.

Thermal and microbiological characteristics of beddings for swine were compared according to their depth and of addition of inoculums. Bedding was added to boxes at 0.25 (25D) and 0.50 m (50D), with three treatments: control (no inoculums); T1, with 250 g of Bacillus cereus var. toyoii at 8.4 × 10(7) CFU; and T2, with 250 g of a pool of B. subtilis, Bacillus licheniformis and Bacillus polymyxa at 8.4 × 10(7) CFU (250 g for 25D and 500 g for 50D). Mean temperatures were 28.5 ± 3.9 at the surface and 35.2 ± 8.9 inside the beddings. The most probable number (MPN) of thermophilic bacteria was higher for T1 and T2 than for the control (P<0.05). The MPN of thermophilic bacteria and fungi was greater for D50 than for D25 (P<0.05). The use of 25D without inoculums is recommended due to the reduction of thermophilic microbiota.

Chemical and microbiological characteristics of rice husk bedding having distinct depths and used for growing-finishing swine.

This study compared the effects of different bedding depths on the chemical and microbiological characteristics of the bedding material used to raise pigs during growing and finishing. The experiment was conducted in two pens housing 5 pigs from 60 to 145 days of age, with rice husk beddings 0.50 or 0.25 m deep. Four lots of pigs (replicates) were raised over time in each bedding depth: each bedding was used by two consecutive lots. Bedding samples were collected quarterly to determine the most probable number (MPN) of thermophilic and mesophilic bacteria, fungi and actinomycetes. Contents of N, P, K, C, organic, mineral and dry matter, C:N ratio and pH were also determined. The MPN of thermophilic bacteria was higher for the 0.50 m than for the 0.25 m bedding (p<0.05). The compost of 0.25 m deep bedding had a higher N, P and K content than that from the 0.50 m bedding (p<0.05). Thus, the use of the 0.25 m deep bedding would be recommended due to its greater agronomical value in comparison with the deeper bedding.

Bacillus cereus var. toyoii and Saccharomyces boulardii increased feed efficiency in broilers infected with Salmonella enteritidis.

1. The effect on feed efficiency of two probiotics, one prepared with Saccharomyces boulardii and the other with Bacillus cereus var. toyoii, was tested in broilers infected with Salmonella enteritidis. 2. One-day-old chicks were divided at random into three groups and fed commercial feed devoid of antibiotics: group 1 was fed with non-supplemented feed, group 2 was supplemented with S. boulardii and group 3 with B. cereus. At 14 d of age the animals were challenged by the oral route with 1 x 10(7) viable S. enteritidis. 3. At d 47, average live weights were: group 1, 1.77 kg, group 2, 1.89 kg and group 3, 2.06 kg, and were significantly different. Feed conversion rates were 2.61 for group 1, 2.35 for group 2 and 2.30 for group 3. 4. We conclude that both probiotics improved feed efficiency in broilers.

Avaliaçäo de testes de ELISA para diagnóstico sorológico de infecçöes pelos sorotipos 3, 5 e 7 de Actinobacillus pleuropneumoniae em suínos / Evaluation of ELISAs for the diagnosis of Actinobacillus pleuropneumoniae infections serotypes 3, 5 e 7 in pigs

Compararam-se dois antígenos no teste de ELISA para o diagnóstico sorológico dos sorotipos 3, 5 e 7 de A. pleuropneumoniae prevalentes no Brasil. Um compunha-se de da fase aquosa da extraçäo fenólica de suspensäo bacteriana (FAF) e o outro de lipopolissacarídeos de cadeia longa (LPS-CL). Com esses antígenos foram padronizados ELISAs monovalente e polivalente para os sorotipos prevalentes no Brasil. Com os resultados dos testes de um conjunto de amostras de soro de suínos livres de infecçäo para A. pleuropneumoniae e um conjunto de amostras de soro obtidas de leitöes inoculados com os sorotipos citados e que apresentaram soroconversäo, determinaram-se as equaçöes discriminantes para os conjuntos de soros e compararam-se os testes quanto à distância generalizada de Mahalanobis, ao coeficiente de determinaçäo, ao teste F, ao coeficiente global, à sensibilidade e à especifidade. Na análise desses parâmetros observou-se que o antígeno FAF foi superior. Com o ELISA PFAF reaçöes positivas näo esperadas foram observadas com animais inoculados com os sorotipos heterólogos 2 e 9, näo observadas com o PLPS-CL. A sensibilidade dos testes polivalentes ficou entre 91,5 e 95,7 por cento com especifidade similar, indicando que ambos os testes säo adequados para triagem sorológica uma vez que detectam os sorotipos 3, 4, 5, 6, 7 e 8

Effect of probiotic CenBiot on the control of diarrhoea and feed efficiency in pigs.

The production and testing of a probiotic formulated with a strain of Bacillus cereus (Probiotic CenBiot) is reported. The strain was grown in fed batch fermenters, desiccated, and mixed with cornflour. To test the effect of the probiotic in controlling piglet diarrhoea, two groups of sows and their respective litters received feed supplemented with probiotic CenBiot or Furazolidone; the control group received the same basic feed but not supplemented. Probiotic CenBiot was as effective as Furazolidone, reducing the prevalence of diarrhoea to half that in the control group. It also significantly improved feed conversion, daily weight gain and total weight gain in pigs in the nursery phase. Feed conversion ratios of weaned pigs were 1.904, 2.146 and 2.099 for the groups that received Probiotic CenBiot, a commercial probiotic, and basic feed, respectively. In terms of food consumption, the Probiotic CenBiot group consumed 12.6% less than the commercial probiotic group, and 10% less than the control group, to achieve the same weight.

Generation of monoclonal antibodies against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.

Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies.

Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.

Studies on the antigenic relationships of six adherent isolates of Bordetella bronchiseptica.

Six isolates of Bordetella bronchiseptica recovered from swine with atrophic rhinitis were studied. All hemagglutinated swine red blood cells, autoagglutinated in saline and showed fimbriae by electron microscopy. Hyperimmune sera against each were produced in rabbits and the antigenic relationships between the isolates were studied by cross-absorption and by the determination of the cross-reactivity indices of pairs of sera. Three isolates seemed to be identical by both methods, while 2 others showed close antigenic relationships. Hemagglutination titers with heterologous antigens and cross-reactivity indices greater than 0 suggest some degree of cross-immunity among the isolates studied, even when antigenic heterogeneity was demonstrated.

Moraxella bovis hemagglutinins: effect of carbohydrates, heating and erythrocytes.

Several properties of the adhesins of eight isolates of Moraxella bovis recovered from cattle suffering from infectious keratoconjunctivitis, were studied. Adhesions were detected through autoagglutination in saline and hemagglutination. Autoagglutinating strains agglutinated red blood cells of the chicken, rabbit, sheep and swine, but not those of the guinea pig. The adhesins were not inhibited by D-mannose or D-galactose and resisted heating at 100 degrees C for 15 minutes. Magnesium chloride at a final concentration of 10% inhibited autoagglutination and hemagglutination. The value of the hemagglutination test for monitoring synthesis of fimbriae by M. bovis, is discussed.

Serotypes and antibiotic sensitivity of Moraxella bovis isolated from an outbreak of infectious bovine keratoconjunctivitis.

Samples of conjunctival fluid of four calves, from a herd with infectious bovine keratoconjunctivitis, were collected five times at monthly intervals and from another 16 calves in the initial stages of the disease, were collected only once. Moraxella bovis was recovered from 82.3% of the samples. Twenty-six isolates were typed with polyvalent sera and 25 with monovalent sera. The antibiotic sensitivity of 13 isolates was also studied. Twenty of the 26 isolates reacted with polyvalent sera and 16 of 25 tested isolates, reacted with monovalent sera. The isolates recovered from the same animal in a different collection or from a different animal in the same collection did not always belong to the same serogroup. Different serotypes were prevalent in each collection. Differences in the antibiotic sensitivity of the isolates were also detected.

Hemagglutination, autoagglutination and pathogenicity of Moraxella bovis strains.

Three isolates of Moraxella bovis, recovered from cattle with signs of infectious bovine keratoconjunctivitis, were tested for autoagglutinating activity, hemagglutinating activity and pathogenicity in young calves. Only the autoagglutinating and hemagglutinating isolates were pathogenic in calves. Treatment of the pathogenic isolates with magnesium chloride eliminated their pathogenic effects.

Serological characterization of strains of Moraxella bovis using double immunodiffusion.

Sera were produced in rabbits against nine Moraxella bovis strains isolated in Brazil and three in the United States. Antigens were prepared for double immunodiffusion tests by thawing concentrated suspensions of the strains. Sera were tested against homologous and heterologous antigen preparations by the double immunodiffusion method. Sera showing precipitin bands with heterologous antigens were absorbed. Antigenic differences were detected between the strains and a provisional grouping of strains of M. bovis was suggested on the basis of antigenic composition. Differences between isolates from different geographical locations were found and some strains appeared antigenically more complex than others. The relevance of this work to vaccine production was suggested.