In order to maintain the high production and export rates achieved by the Brazilian poultry industry, it is necessary to prevent and control certain disease agents, such as Salmonella spp. Using bacterial cultures, the aim of the present study was to investigate the prevalence of Salmonella spp. in specimens collected from broiler facilities. Local wild birds were also sampled, as well as the feces of swine housed on the poultry farm. After sample collection, the isolated serotypes were subsequently inoculated into broiler chicks to determine their effects. Positive samples were collected from the following locations in the poultry facilities: poultry litter (S. serotype 4,5,12:R:-; S. Heidelberg; S. Infantis), broiler feces (S. Heidelberg; S. serotype 6,7:R:-; S. serotype 4,5,12:R:-; S. Tennessee), water (S. Glostrup; S. serotype 6,8:d:-;), and lesser mealworms (Alphitobius diaperinus) found in the litter (S. Tennessee). Among the 36 wild birds captured, S. Heidelberg was isolated from one bird's organs and intestinal contents (Colaptes campestris), and S. Enteritidis was isolated from another bird's intestinal contents (Zenaida auriculata). Salmonella Panama and Salmonella Typhimurium were isolated from swine feces. One-day-old chicks (150) were divided into 10 groups of 15 animals each. Each group was orally inoculated with a previously isolated serotype of Salmonella. Soft stools were observed on the cage floor and around the birds' cloaca between 3 and 12 days post-infection (dpi). The different serotypes of Salmonella used to inoculate the chicks were re-isolated from the spleen, liver, and cecal content samples of the infected birds on 15 and 21 dpi.

In the present study Salmonella spp. was surveyed in four flocks of meat-type quails reared in a farm that also had processing plant on site, located in the region of Bastos, state of São Paulo, Brazil. Meconium samples of one-day-old quail chicks were collected from transport cardboard boxes. Cecal content was collected on days 7, 14, 21, 28 and 35 of rearing. At 36 days of age, birds were slaughtered in the farm's processing plant, where two samples of water from the scalding and the chilling tanks and four carcasses per flock were collected. All samples were examined for Salmonella spp. using traditional bacteriological methods. Salmonella spp. was present in meconium samples of three flocks and in cecal feces of the four flocks. This bacterium was also isolated in the chiller water and in the carcasses of three of the evaluated flocks and in the scalding water of one flock. In this study, S. enterica subspecies enterica 4, 5, 12; S. Corvalis; S. Give; S. Lexington; S. Minnesota; S. Schwarzengrund; S. Rissen and S. Typhimurium were the eight serovars identified.

New vaccine design techniques have allowed the development of effective vaccine strains against Salmonella infections inwhich the risks of reversion to the wild type and virulence is null. The mutant strain Salmonella Gallinarum ΔcobSΔcbiA was previously shown to be avirulent in chickens. In this study, this strain was tested as a vaccine against Salmonella Gallinarum (SG) and S. Enteritidis (SE) infections, and its protection levels, safety and possible risks of reversion to virulence after vaccination of layers were evaluated. Birds were vaccinated at five days of age or at five and 25 days of age. At 45 days of age, brown and white layers were challenged with SG and SE wild strains, respectively. Two assays to test the possibility of reversion to virulence were performed. Five successive bacterial passages in brown layers were carried out in the first assay. In the second assay, brown layers received a ten-fold concentrated inoculum of the SGΔcobSΔcbiA strain and were evaluated for clinical signs and mortality. In both experiments, no birds that received the inoculation of the attenuated strain died. Additionally, the use of the mutant strain as a vaccine provided good protection levels against both challenge strains.

Salmonellosis is a worldwide disease caused by bacteria of the genus Salmonella. Currently, there are over 2,500 identified serovars of Salmonella. A reduced number of these serovars, about eighty, are implicated in most animals and human diseases. Most cases of salmonellosis in humans are associated with the consumption of contaminated food products such as beef, pork, poultry meat, eggs, vegetables, juices and other kind of foods. It may also be associated with the contact between humans and infected pet animals. Therefore, the chain of human salmonellosis is very complex and in most cases the origin of the infection is difficult to establish. The use of antimicrobial agents to treat and to prevent bacterial infections in humans and animals, as well as as growth promoters in animal production, has favoured the selection and transference of resistance genes between different bacteria, including Salmonella serovars. Many studies have confirmed the role of foods of animal origin as a source of multi drugresistant Salmonella serovars. For this reason, continuous surveillance of these pathogens along the food chain together with the responsible use of antimicrobial agents is necessary.

Although Salmonella Pullorum and Salmonella Gallinarum cause different diseases in poultry, they are very similar. Both are non-motile and present the same somatic antigenic structure. They are differentiated by biochemical tests. Certain atypical strains are very difficult to distinguish. They do not produce the expected results when dulcitol and ornithine descarxboxylase tests are performed. Therefore, additional tests could be helpful. Many studies have chose the part I of the gene that encodes flagellin (fliC) to differentiate serotypes. Most Salmonella strains have two structural genes (fliC and fliB) that encode flagellins. Non-motile strains generally present these structural genes, but are not able to build a functional flagellum. It was demonstrated that enzymatic restriction of the amplified fliC gene using Hinp1I enzyme can differentiate SG from SP. In the present study, this method was adopted to analyze 14 SP and 22 SG strains, including some strains with atypical results in biochemical tests assessing the utilization of dulcitol and ornithine. The results showed that all SG strains were broken by the enzyme, whereas the 14 SP strains were not.

The protective effect of various Salmonella vaccines regimens against an experimental Salmonella Gallinarum challenge (SGNalr strain at 12 wk of age) was evaluated in two experiments. In Experiment 1 commercial brown layers were vaccinated according to one of the following programs: (i) two doses of a SE bacterin (Layermune SE; group 1); (ii) a first dose of a live SG9R vaccine (Cevac SG9R) followed by a SE bacterin (Layermune SE; group 2); (iii) one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4 & Corymune 7; group 3) or (iv) not vaccinated (group 4). In Experiment 2, broiler breeders were given the same vaccination treatments except for the group vaccinated with the multivalent vaccines. Overall, in both experiments, all vaccination schemes were effective in reducing mortality after challenge with a SG field strain. Primary vaccination with an initial dose of a live SG9R vaccine followed some weeks later by a dose of an inactivated SE bacterin was the most effective (p<0.05) vaccination program against mortality induced by field SG experimental challenge in both experiments. In conclusion, Salmonella vaccination programs containing SE bacterins alone or in combination with a live SG9R vaccine are effective in preventing mortality induced by infection of field SG. Nevertheless, it is important to emphasize that any vaccination program against any Salmonella serotype will only be effective if it is part of a sound and comprehensive biosecurity program designed for Salmonella control in poultry farms.

RESUMO Este trabalho foi desenvolvido para avaliar comparativamente o isolamento de Salmonella sorotipos Enteritidis (SE) e Typhimurium (STM) a partir do enriquecimento direto (ED) ou processamento com pré-enriquecimento (PE) de amostras de fezes de aves adultas, armazenadas em água peptonada tamponada a 1% (APT) por 24 ou 96h a 4º C. Utilizou-se os caldos de enriquecimento Rapapport-Vassiliadis novobiocina (RVN), tetrationato-novobiocina (TN) e selenitonovobiocina (SN) e os meios para plaqueamento ágar verde brilhante (VB), ágar de MacConkey (MC), ágar de Hektoen (HE), ágar Salmonella-Shigella (SS), ágar xilose lisina desoxicolato (XLD) e ágar xilose lisina tergitol 4 (XLT4). O procedimento bacteriológico incluiu as etapas de pré-enriquecimento, enriquecimento em caldo seletivo, plaqueamento, testes bioquímicos presuntivos e confirmação sorológica com utilização de soros polivalentes anti-antígenos somáticos e anti-antígenos flagelares de Salmonella. Não houve diferença estatisticamente significativa (p > 0,05) para as amostras armazenadas por 24h submetidas tanto ao PE quanto ao ED. Entretanto, em armazenagem por 96h o número de isolamentos nas amostras submetidas ao PE foi estatisticamente superior às submetidas ao ED (p < 0,05). Quanto aos caldos enriquecedores, não houve diferença estatística de número de isolamentos (p > 0,05) entre os caldos SN e TN, mas o caldo RVN mostrou-se estatisticamente superior aos demais (p < 0,05). Para os meios de plaqueamento, o XLD destacou-se por promover maior número de recuperações, embora sem significado estatístico (p > 0,05) para as amostras estocadas por 24h. Entre os dois sorotipos de Salmonella (SE e STM) não houve diferença estatística no número de recuperações (p > 0,05).

ABSTRACT This work was carried out to assess the preenrichment (PE) and enrichment (DE) steps for isolating Salmonella serotypes Enteritidis (SE) and Typhimurium (STM) from chicken feces kept at 4° C for 24 and 96h. The samples were artificially contaminated and kept in 1% peptone water at 4° C for 24 or 96h. After that, part of them was incubated at 37° C/24h and part was inoculated into enrichment broth, selenite broth plus novobiocin (SN) and tetrathionate broth plus novobiocin (TN) incubated at 37°C/24h. The PE culture was inoculated in SN, TN and Rapapport-Vassiliadis novobiocin (RVN), also incubated at 37° C/24h. The enrichment broth was plated on brilliant green agar (BGA), MacConkey agar (MCA), Hektoen agar (HEA), Salmonella-Shigella agar (SSA), xylose-lysine desoxicholate agar (XLDA) and xylose-lysine tergitol 4 (XLT4), which were incubated at 37° C/24h. Salmonella-like colonies were submitted to TSI agar and LIA agar, and incubated at 37° C/24h, as well as to slide agglutination tested with poly O and poly H Salmonella antiserum. When the samples were stored for 24h there was no difference between PE and DE (p > 0.05). However after 96h the PE was superior to DE (p < 0.05). For enrichment, better results were seen with RVN broth (p < 0.05). The XLD yielded

Competitive exclusion (CE) has been designed to accelerate the colonization of the alimentary tract of young commercial birds and it has been also used to repopulate the digestive tract after antibiotic therapy. The method has been successfully adopted as a means to prevent enteric salmonellosis. The present study was carried out to evaluate if CE is able to prevent this kind of infection. Newly hatched chicks were given a CE culture and at different intervals of time birds infected with Salmonella Enteritidis were placed together with the group of treated birds. CE culture was prepared from feces of adult laying hens incubated overnight at 37C under aerobic conditions. Birds were killed 4 and 8 days after challenge and viable counts of Salmonella Enteritidis were assessed in the cecal contents. The results showed that Salmonella infection was reduced even if CE culture administration was concomitant with the inclusion of the infected bird in the group.

Cultura de fezes (Método de Exclusão Competitiva - EC) utilizada para prevenir a colonização cecal de aves por Salmonella enterica serovar Enteritidis (SE) foi submetida a cultivos seriados para evitar a presença de patógenos e, após o tratamento mais adequado, foi armazenada em temperatura de refrigeração antes do seu uso por até 63 dias. Os resultados mostraram que o cultivo repetido por 14 vezes não prejudica a ação protetora da cultura (CE), a qual continua inibindo a colonização cecal por SE. O produto submetido a 12 cultivos e armazenado durante 28 dias em temperatura de refrigeração também continua eficaz.

The competitive exclusion method (EC) was used to protect day-old birds against colonisation of the intestinal tract by Salmonella enterica serovar Enteritidis (SE). The culture prepared in nutrient broth incubated at 37ºC/24 h inhibited SE growth in the caecal contents. The beneficial effect was also observed after serial passage of the feces culture for up to 14 times. The material obtained after 12 serial sub-cultures after storage for 28 days at 4-6ºC also kept its action on SE.