Safety and efficacy of a salmonella gallinarum cobScbiA strain with potential to prevent chicken infections by salmonella gallinarum and salmonella enteritidis

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 cobScbiA 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 SGcobScbiA 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.

The contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica serovar Gallinarum for chickens

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.

Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates

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.

Efficacy of several Salmonella vaccination programs against experimental challenge with Salmonella gallinarum in commercial brown layer and broiler breeder hens

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.

Efficacy of several Salmonella vaccination programs against experimental challenge with Salmonella gallinarum in commercial brown layer and broiler breeder hens

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.

Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates

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.

Organic acids and/or compound with defined microorganisms to control Salmonella enterica serovar Enteritidis experimental infection in chickens

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.

Organic acids and/or compound with defined microorganisms to control Salmonella enterica serovar Enteritidis experimental infection in chickens

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.

Isolamento de salmonella: comparação das etapas de pré- enriquecimento e enriquecimento direto de amostras de fezes armazenadas por 24 e 96 horas

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

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).

Time required to protect the intestinal tract of chicks against Salmonella enterica serovar enteritidis using competitive exclusion

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.

Time required to protect the intestinal tract of chicks against Salmonella enterica serovar enteritidis using competitive exclusion

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.