ABSTRACT Poultry products may be a source of foodborne human salmonellosis. The use of alternatives to antimicrobials that are not harmful to humans may reduce the presence of Salmonella spp. in poultry production. Among the products used, organic acids stand out. In the present study, three different organic acid (OA) blends were evaluated for the control of Salmonella Heidelberg (SH) in commercial broilers. Day-old chicks (n = 114) were randomly assigned to four treatments, with three replicates of 12 birds each. Birds in treatments A and B received SCFA (0.2mL/L) and SCFA + MCFA (0.2mL/L), respectively, in the drinking water, while birds in treatment C received SCFA + MCFA in the feed (2g/Kg of feed). Birds from treatment D did not receive OAs (control group). At 8 days of age, each bird was orally inoculated with SH at 108 CFU/mL, and cloacal swabs and SH enumeration of the cecal content were performed 24-, 48-, and 72-hours post-inoculation (hpi). The results show a reduction of both SH shedding and counts in the birds fed OAs at all pi times relative to the control birds. Fecal shedding was significantly lower in the OA-treated groups compared with the control group. As for SH presence in the cecum, significant differences were detected between groups C and D at 24 and 72 hpi, and between groups B and D at 72 hpi. The results of this study indicate that the use of feeding OAs to broilers may contribute to reduce the incidence of SH in the poultry production chain, allowing better flock health management, provided an efficient biosecurity program is employed.

ABSTRACT Fowl paratyphoid infections are caused by different Salmonella serovars that can affect a wide range of hosts. Due to its complex epidemiology, Salmonella serovar identification is crucial for the development and implementation of monitoring and control programs in poultry farms. Moreover, the characterization of the antimicrobial resistance profiles of Salmonella strains isolated from livestock is relevant to public health because they are a common causative agent of foodborne diseases. The objective of this study was to investigate the presence of Salmonella spp. and to identify the antimicrobial resistance profiles of strains isolated in the midwestern region of São Paulo state, which accounts for the highest production of table eggs in Brazil. For this purpose, 2008 fecal samples were collected on 151 commercial layer farms and submitted to microbiological analyses. Twenty-two serovars were isolated from 80 (52.9%) farms, among which S. Mbandaka and S. Braenderup were the most prevalent. All isolates expressed resistance to at least one of the 23 antimicrobials tested, and the highest resistance rates were determined against streptomycin (93.5%) and sulfonamide (84.6%). Moreover, multidrug resistance was observed in 41% of the isolates and the maximum drug resistance profile was against ten different antimicrobials. Therefore, the identification of Salmonella serovars in poultry production provides epidemiological knowledge to develop prevention and control measures in order to ensure poultry health and to prevent human infection by multiresistant strains.

ABSTRACT The incidence of foodborne diseases caused by the genus Salmonella spp. in industrialized countries is often high in epidemiological surveys. Obtaining a rapid diagnostic test for identification of bacteria is crucial in order to rapidly implement control measures to contain bacterial spread, to reduce losses in animal production and to avoid risks from food-borne infections to human health. The aim of this study was to standardize duplex real-time PCR using SYBr Green I for differential and quantitative diagnosis of S. Typhimurium and S. Enteritidis. According to the experiment, the melting temperature of 85°C was observed for a 206bp amplified product when S. Enteritidis DNA was added to the reaction. S. Typhimurium DNA showed that the melting temperature of 79°C when observed for a 62bp amplified product. The standard curve showed the high sensitivity of the proposed test, since it was possible to obtain eight quantification points, starting at 108 CFU/mL and ending at 101 CFU/mL. As a result of the present study, a real-time PCR duplex reaction with high sensitivity, specificity and based on the fluorescence of SYBr Green I was standardized. In addition, this methodology aligns low cost to the faster diagnostic result, in relation to other molecular tests, making it attractive for application in routine laboratory analyzes.

The aim of the present study was to comparatively evaluate hemogram, blood serum components and anatomopathologic alterations in commercial layers experimentally challenged with an attenuated vaccine candidate strain (SG∆cobS∆cbiA) and other two pathogenic strains (SGDcobS and SGNalr) of Gallinarum (SG). In total, 280 commercial layers were randomly divided into 4 groups (G1, G2, G3 and G4). At five days of age, birds from groups G1 received approximately 107 colony forming units (CFU) of SGDcobS; meanwhile birds from group G2 and G3 received the same dose of SGNalr and SG∆cobS∆cbiA, respectively. Birds from G4 were not infected. At 24 hours before (DBI) and 24 hours after (1 DAI), and three (3 DAI), five (5 DAI), seven (7 DAI) ten (10 DAI), and fifteen (15 DAI) days after the infection, 10 birds of each group were humanely killed and blood samples collected to hematological and serum tests. Samples of liver, spleen, thymus, bursa of Fabricius, kidney and heart were also collected for the histological examination. Birds inoculated with SGDcobS and SGNalr showed similar alterations in hemogram, blood serum components and anatomopathologic exams. On the other hand, the exams of birds inoculated with SG∆cobS∆cbiA strain were similar to those of the uninfected birds. However, changes could be noticed in levels of uric acid and cholesterol during the course of the infection of birds from G3. Decrease in levels of light IgG 3 DAI was also observed in birds from this group. Pyknosis in kidney cells was a microscopic alteration found in birds from G3. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SG∆cobS∆cbiA strain.

Salmonella enterica serovar Gallinarum (SG) is an intracellular pathogen of chickens. To survive, to invade and to multiply in the intestinal tract and intracellularly it depends on its ability to produce energy in anaerobic conditions. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. In this study mortality rates of chickens challenged with mutants of Salmonella Gallinarum, which were defective in utilising anaerobic electron acceptors, were assessed in comparison to group of bird challenged with wild strain. The greatest degree of attenuation was observed with mutations affecting nitrate reductase (napA, narG) with additional attenuations induced by a mutation affecting fumarate reductase (frdA) and a double mutant (dmsA torC) affecting DMSO and TMAO reductase.

The association of human foodborne salmonellosis with poultry products enhanced the efforts to control Salmonella Enteritidis in poultry farms. Dietary organic acid supplementation is one of the measures currently used to reduce the presence of undesirable microorganisms. Another method to control enteric Salmonella in poultry is competitive exclusion using defined or undefined microorganisms products. Organic acids and microbiological methods to accelerate the development of the intestinal microbiota can be used individually or in combination. The present study evaluated the effect of dietary supplementation of an acidifier and of a defined multi-strain microbial mixture (Biomin® PoultryStar) via drinking water in the control of the intestinal colonization of broilers by Salmonella Enteritidis. Four experiments were performed. The first experiment showed that the organic acids mixture was able to prevent Salmonella Enteritidis colonization of ceca in both inclusion rates applied (p<0.05). In the second and third experiments the probiotic either individually or in combination the acidifier, both in high and low doses reduced the incidence of Salmonella Enteritidis in the cecal contents (p<0.05). In these three experiments, birds were orally challenged. Similar results were obtained in a fourth trial, in which challenge was made by contact.

This study evaluated two enzyme-linked immunosorbent assays (ELISA) in the detection of chicken serologic response against Salmonella enterica sorotype Typhimurium. The assays have used as detecting antigen the soluble bacterial proteins of a non-flagellated strain of Salmonella Typhimurium (AgTM), and antibody conjugated to peroxidase or alkaline phosphatase. According to the results, optimal dilutions of antigen (concentration 5.49 mg/mL) and serum samples in both assays were 1:20,000 and 1:1,000, respectively. In such conditions, the ELISA/AgTM was able to detect serological response to Salmonella Typhimurium. Cross-reactions to Salmonella serotypes Gallinarum and Pullorum were seen, but not with other serotypes such as Enteritidis.

The strain used in this work was a Salmonella enterica subsp enterica serovar Kottbus (6,8:e,h:1,5) isolated from imported day-old ducklings in Laboratório Nacional Agropecuário (LANAGRO/SP) of the Ministry of Agriculture of Brazil (MAPA). In view of the lack of information available about this Salmonella isolate and also because it was detected in day-old imported birds, this study was carried out to investigate the dissemination of S. Kottbus among newly hatched chicks. The birds were placed in three groups: one group of 20 birds received 0.1 mL of S. Kottbus culture containing 1.2 x 10(8) CFU/mL, the second group of 20 birds was inoculated with 1.2 x 10(5) CFU/mL and the third group of 10 birds was untreated (control group). Results were similar for both infected groups. The bacterium was recovered from cloacal swabs collected from the first day following the experimental infection until the end of the trial (42 days post-inoculation). At 15 and 42 days post-inoculation (dpi), half of the birds of each group were killed for bacteriological examination of cecal contents, liver and spleen. At 15 dpi, viable cell counts of S. Kottbus were obtained in all kinds of samples. At 42 dpi, Salmonella was present in the liver and spleen of few birds, but in large amounts in the cecal contents of almost all birds.

This study was carried out to investigate the presence of Salmonella sp in flocks of white laying hens. In different farms, the transport boxes of twelve flocks were inspected at arrival for the presence of Salmonella. Four positive (A, B, L and M) and one negative (I) flocks were monitored at each four weeks using bacteriological examination of cecal fresh feces up to 52 weeks. Birds were also evaluated at 52 weeks, when 500 eggs were taken randomly, and at 76 weeks, after forced molt. Salmonella enterica serovar Enteritidis and S. enterica rough strain were isolated from the transport boxes of the four positive flocks (flocks A, B, L and M). Salmonella sp was not isolated from the transport boxes or from the feces after 76 weeks-old in flock I. Salmonella sp was isolated in the 1st, 11th, 34th, 42nd and 76th weeks from flock A; in the 1st, 4th, 11th and 76th weeks from flock B; in the first week and in the 17th to 52nd weeks from flock L; and in the 1st and 76th weeks from flock M. S. Enteritidis, S. enterica rough strain and Salmonella enterica serovar Infantis were isolated from the four positive flocks. Besides, Salmonella enterica serovar Javiana was isolated from flocks B and L, and Salmonella enterica serovar Mbandaka was isolated from flock L. Eggs produced by flock A and by flock L were contaminated with S. Enteritidis and S. enterica rough strain. According to these results, Salmonella-infected flocks may produce contaminated eggs.

Este trabalho foi desenvolvido para avaliar, comparativamente, os caldos de enriquecimento Rapapport-novobiocina (RVN), selenito-cistinanovobiocina (SCN) e tetrationato-novobiocina (TN) e os meios para plaqueamento ágar Hektoen (HE), ágar MacConkey (MC), ágar Salmonella-Shigella (SS), ágar verde brilhante (VB) e ágar xilose lisina desoxicolato (XLD), utilizados no isolamento de Salmonella em carcaças de frango e fezes de aves. O procedimento bacteriológico consistiu das etapas de pré-enriquecimento, enriquecimento em caldos seletivos, plaqueamento, testes bioquímicos presuntivos e confirmação sorológica com soros polivalentes anti antígenos somáticos e flagelares de Salmonella.. As fezes foram experimentalmente contaminadas com 8 sorotipos de Salmonella (Agona, Anatum, Enteritidis, Havana, Infantis, Owakam, Schwazengrund e Typhimurium) e a concentração final foi aproximadamente de 1,2 x 10² ufc/g. As fezes foram inoculadas nos caldos enriquecedores e a partir daí, seguiu-se o mesmo procedimento utilizado para as carcaças. Os resultados referentes às carcaças de frango não foram estatisticamente diferentes (p>0,01) para os caldos e as placas. Todavia, verificou-se superioridade numérica em relação aos caldos SCN e TN sobre o caldo RVN, e em relação ao ágar XLD sobre os demais. Verificou-se também que com o emprego de dois caldos de enriquecimento e de dois meios para plaqueamento pode-se obter maior positividade. Quanto ao exame das fezes, o caldo TN mostrou-se superior aos demais (p>0,01), não havendo diferença (p>0,01) de resultados para os meios de plaqueamento. Os resultados sugerem que a utilização de mais de um meio de enriquecimento e de plaqueamento poderia aumentar as chances de isolamento de Salmonella.

This study was undertaken to compare selenite-cystine-novobiocin (SCN) broth, tetrationate-novobiocin (TN) broth and Rappaport-Vassiliadisnovobiocin (RVN) broth as enrichment and MacConkey (MC) agar, brilliant green (BG) agar, Hektoen (HE) agar, Salmonella-Shigella (SS) agar and xylose Iysine desoxycholate (XLD) agar as plating media for isolating Salmonella from broiler carcasses and chicken feces. The carcasses were washed with 300mL of Buffer Peptone Water 0.1% (BPW). The BPW solation was incubated 6h at ambient temperature and up to 24 h/43ºC. From this, 2 mL were inoculate in 20 mL of enrichment broth, incubated at 43ºC/24h. The enrichment broth was plated on the five tested selective agars, also incubated at 43ºC/24h. Up to five colonies from each plate were inoculated in TSI agar and LIA agar, incubated at 37ºC/24h. The genus of Salmonella was confirmed by slide agglutination test using poly serum anti-somatic Salmonella antigens (O) and poly serum anti-fiagellar (H) Salmonella antigens. The feces were experimentally contaminated with eight Salmonella serotypos (Agona, Anatum, Enteritidis, Havana, Intantis, Owakam, Schwazengrundand Typhimurium). Each serotype was tested five times. Salmonella serotypes were added in the sample of feces individually so that the final concentration of the bacterium was 1,2 x iO³ ufclg. Two grams of each sample were inoculated in 20 mL of the enrichment broth. The proceduring from this point wes the same adopted to carcasses examination. To broiler carcasses there was no statistic difference (p>0, 001) among the enrichment broth and among plating media. However the use of two enrichment broth (SCN and TN) and two agars between XLD agar, SS agar and HE agar gave the bost results. The assay done with feces showed that the TN broth was much better than the others and, in this case it can be plated either HE agar, or VB agar or SS agar The results from the broiler carcasses examination and the feces examination showed that it is not easy to determine the better bacteriologic routine to search for Salmonella and suggest that the association of two eorichments broth and two plating media may improve the test.

Em quatro experimentos foram avaliados como agentes antibacterianos os produtos própolis em solução alcoólica e álcool etílico, adicionados às rações artificialmente contaminadas com os respectivos sorotipos: Salmonella typhimurium Nalr - Specr, (resistentes ao ácido Nalidíxico e a Spectinomicina) nos três primeiros experimentos e Salmonella agona Nalr - Specr, Salmonella infantis Nalr - Specr e Salmonella enteritidis Nalr - Specr no quarto experimento. As rações foram fornecidas a grupos de 10-16 pintos de corte de um dia. Em todos os experimentos os produtos testados foram adicionados na base de 2% da ração. Quando se utilizou solução hidroalcóolica de própolis (exp. 1), seguidas 120 horas após o desafio, detectou-se a presença da bactéria nos cecos. No segundo experimento, testou-se a solução de própolis e seu diluente, o álcool etílico; seguidas 96 horas após o desafio, não foi observada a presença da bactéria nos cecos (< 2,0 log10). Avaliou-se, no terceiro experimento, a ação da solução de própolis e do álcool etílico no tempo, adicionados na ração 14 dias e 28 dias antes do fornecimento às aves. Após 72 horas do desafio, a leitura nas placas acusou a presença da bactéria nos cecos. Dentro deste último período, também se avaliou a ação da própolis em pó (extrato seco) e esse mesmo extrato em uma solução aquosa, adicionados à ração 48 horas antes do fornecimento às aves sendo que os resultados confirmaram a presença da bactéria nos cecos. No quarto experimento avaliou-se somente o álcool etílico nas rações artificialmente contaminadas com os sorotipos S. agona, S. enteritidis e S. infantis, registrando-se contagem zero (<2,0 log10) apenas com o último sorotipo. Os resultados obtidos permitem concluir que o tratamento com a solução de própolis apresentou ação sobre a S. typhimurium somente quando em solução alcóolica, dentro de um período de 48 horas, indicando que o efeito bactericida se deveu ao álcool etílico presente na solução. A ação do tratamento com o álcool etílico sobre os demais sorotipos demonstrou resultado parcial sendo observado efeito bactericida nos sorotipos S. typhimurium e S. enteritidis artificialmente inoculados na ração.

Four similar trials were conducted to evaluate the effects of dietary inclusion of an alcoholic solution of propolis and ethyl alcohol on the control of salmonella artificially added to the feed offered to groups of 10-16 day-old broiler chicks. Salmonella typhimurium Nalr - Specr (resistant to Nalidixic acid and Spectinomicin) were used in the first three experiments and Salmonella enteritidis Nalr - Specr in the fourth. In every experiment the antibacterial agent was added in the proportion of 2%of the feed. When using hydroalcoholic solution of propolis (experiment 1), 120 hours after the challenge on the chicks, the presence of bacteria was in detected cecal contents. In the next experiment (experiment 2) an alcoholic solution of propolis and ethyl alcohol was tested: 96 hours after the challenge on the chicks the presence of bacteria in cecal content of the birds was not observed (< 2.0 log10 FCU/g). In the third experiment, a propolis solution and ethyl alcohol was evaluated when added to the feed 14 days and 28 days before the chicks consumed the experimental ration. Seventy-two hours after the chicks consumed the Salmonella contaminated ration, the plaque counts showed the presence of bacteria in cecal contents of the chicks. Within the last period (72 hours), a powdered propolis sample was evaluated (dehydrated extract) and, this extract in an aqueous solution, added to the feed 48 hours before the birds started ration consumption; the results confirmed the presence of the bacteria in cecal contents. In the fourth experiment, only ethyl alcohol in the feed artificially contaminated with the following serotypes: S. agona, S. enteritidis and S. infantis was evaluated . The results indicated zero count (< 2.0 log10 , FCU/g) only with the last serotype. Under this experimental conditions, propolis showed action over S. typhimurium only when in alcoholic solution and 48 hours before the birds consumed the contaminated ration, showing that bactericidal effect was due to ethyl alcohol present in the solution rather than to the propolis action per se. Ethyl alcohol showed bactericidal effect over two of the serotypes S. typhimurium and S. enteritidis artificially added to the feed, pointing that a standardized response did not occur.