Experimental infection of chickens by a flagellated motile strain of Salmonella enterica serovar Gallinarum biovar Gallinarum.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT), a septicaemic disease which can result in high mortality in poultry flocks. The absence of flagella in SG is thought to favour systemic invasion, since bacterial recognition via Toll-like receptor (TLR)-5 does not take place during the early stages of FT. In the present study, chicks susceptible to FT were inoculated with a wild type SG (SG) or its flagellated motile derivative (SG Fla(+)). In experiment 1, mortality and clinical signs were assessed, whereas in experiment 2, gross pathology, histopathology, systemic invasion and immune responses were evaluated. SG Fla(+) infection resulted in later development of clinical signs, lower mortality, lower bacterial numbers in the liver and spleen, and less severe pathological changes compared to SG. The CD8(+) T lymphocyte population was higher in the livers of chicks infected with SG at 4 days post-inoculation (dpi). Chicks infected with SG had increased expression of interleukin (IL)-6 mRNA in the caecal tonsil at 1 dpi and increased expression of IL-18 mRNA in the spleen at 4 dpi. In contrast, the CD4(+) T lymphocyte population was higher at 6 dpi in the livers of birds infected with SG Fla(+). Therefore, flagella appeared to modulate the chicken immune response towards a CD4(+) T profile, resulting in more efficient bacterial clearance from systemic sites and milder infection.

Detection of chromosomal bla(CTX-M-2) in diverse Escherichia coli isolates from healthy broiler chickens.

The rise of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in food-producing animals is a growing concern for public health. We investigated ESBL producers isolated from broiler chickens in Brazil and characterized 19 CTX-M-2-producing E. coli. The ISCR1 was detected upstream of the chromosome-located gene bla(CTX-M-2), associated with sul-1 type integron structure. CTX-M-2-producing E. coli exhibited different PFGE-types and phylogenetic groups, showing a non-clonal dissemination. The sequence types found (ST93, ST155 and ST2309) have been associated with humans and animals worldwide. Herein, we report the chromosomal location of bla(CTX-M-2) on E. coli, highlighting the risks of multidrug-resistant bacteria in food-producing animals.

Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes.

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to d-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source.

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.

Passive immunity of progeny from broiler breeders vaccinated with oil-emulsion bacterin against Salmonella enteritidis.

Young poultry are very susceptible to Salmonella Enteritidis (SE) infections because of the absence of complete intestinal flora colonization and an immature immune system. This study evaluated the role of passive immunity on the resistance of young birds against early infections caused by SE. The progeny of broiler breeders vaccinated with an oil-emulsion bacterin was compared to the progeny of unvaccinated birds. Efficacy was determined by challenging birds at 1 and 14 days of age with SE Nal Spc strain, phage type 4. After challenge at 1 day of age, the progeny of vaccinated birds presented a significantly lower number (log10) of SE Nal Spc reisolation (P < 0.05) in liver (2.21), spleen (2.31), and cecal contents (2.85) compared with control groups (2.76, 3.02, and 6.03, respectively). The examination of the internal organs, 3 days after infection, revealed that 28% of the birds (7/25) from vaccinated breeders were positive, whereas 100% (25/25) of the chicks derived from unvaccinated birds were positive. Birds challenged at 14 days of age presented a lower number of positive samples compared with those challenged at 1 day of age, and the progeny of vaccinated birds presented statistically lower numbers (log10) of colony-forming units/ml of SE Nal Spc only in the cecal contents compared with nonvaccinated breeder progeny (2.11 vs. 2.94). Age seems to influence the susceptibility of birds to SE infections: in control groups, the number of positive birds at 14 days of age (9/25) was lower when compared with the group infected at 1 day of age (25/25). The number of positive fecal samples of the progeny of vaccinated birds was significantly lower (36) than those of the control group (108) after challenge at 1 day of age. Unchallenged progeny of vaccinated birds presented passive antibodies detectable by enzyme-linked immunosorbent assay (ELISA) up to 21 days of age. On the other hand, antibodies of the control group were detected by ELISA 14 days after challenge. These results show a significant contribution of breeder vaccination by increasing the resistance of the progeny against early SE infections. However, the bacteria were not completely eliminated, suggesting that additional procedures are needed to effectively control SE infections.

Evaluation of isopathic treatment of Salmonella enteritidis in poultry.

BACKGROUND: Salmonellosis is a common problem worldwide in commercially reared poultry. It is associated with human Salmonellosis. No fully satisfactory method of control is available. METHOD: Nosodes to an antibiotic-resistant strain of Salmonella enterica serovar Enteritidis in D30 (30X) potency were prepared. One day old chicks (N = 180) were divided into four groups: two control and two different preparations of the nosode. Treatments were administered in drinking water for 10 days. The birds were challenged by a broth culture of the same Salmonella, by mouth, on day 17. Cloacal swabs were taken twice weekly for Salmonella enterica serovar Enteritidis. RESULTS: Birds receiving active treatment were less likely to grow the strain of Salmonella from cloacal swabs compared to control. CONCLUSION: Isopathy is low cost and non-toxic. It may have a role to play in the widespread problem of Salmonella in poultry. Further research should be conducted.

Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens.

Salmonella enterica serovar Pullorum is worldwide a poultry pathogen of considerable economic importance, particularly in those countries with a developing poultry industry. In addition to the characteristic high mortality rates among young chicks, one of the features of Salmonella serovar Pullorum infection is that it persists for long periods in convalescent chicks in the absence of clinical disease. This can lead to colonization of the reproductive tract of chickens and at sexual maturity can result in infected progeny through transovarian transmission to eggs. The sites of Salmonella serovar Pullorum persistence in convalescent birds are not known, and the mechanisms of persistence are not understood. Here we show that Salmonella serovar Pullorum can persist in both the spleen and the reproductive tract for over 40 weeks following experimental infection in chickens. During the period of sexual maturity, Salmonella serovar Pullorum colonized both the ovary and the oviduct of hens and led to 6% of laid eggs being infected by Salmonella serovar Pullorum. The colonization of several different sites of the reproductive tract suggests that Salmonella serovar Pullorum may employ more than one mechanism of egg infection. Persistence occurred despite a strong humoral response, suggesting an intracellular site of infection. By use of a Salmonella serovar Pullorum strain containing a plasmid stably expressing green fluorescent protein, we demonstrated that the main site of carriage in the spleen is within macrophages. This raises interesting questions about the biology of Salmonella serovar Pullorum, including why there is an increase in bacterial numbers when birds become sexually mature and in particular how Salmonella serovar Pullorum avoids clearance by macrophages and whether it modulates the immune system in other ways.

Experimental Salmonella enterica serovar Pullorum infection in two commercial varieties of laying hens.

An experiment was carried out to investigate the biology of Salmonella Pullorum in two varieties of laying hens, from 5 days of age up to 9 months. One variety was resistant to systemic salmonellosis (light layers producing white eggs) and the other was considered susceptible (brown layers producing brown eggs). The brown birds were more affected by the infection, showing signs of clinical disease in the first month of life. Later, these signs disappeared, but postmortem examination revealed persistent gross pathological changes in the liver, spleen, heart and ovary. The rapid agglutination test detected reactors throughout the experiment, with the strongest agglutination from 1 to 7 months post-infection. S . Pullorum was isolated from some of the organs and the eggs laid throughout the experiment. The relationship between white birds and S . Pullorum was less intense, and there were no noticeable signs of disease. There were few gross pathological changes, and the bacteria were isolated infrequently and only for a brief period after infection, although contaminated eggs were laid by these birds. The strongest serological response in the white chickens occurred between the second and the fifth month post-infection.

Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background.

Commercial laying hens inoculated with a strain of Salmonella enterica ser. Pullorum when they were 4 days old showed no morbidity, but harboured infection until they came into lay, and then produced S. Pullorum-contaminated eggs and infected progeny. There was limited evidence of transmission of maternal immunity to the progeny. Attempts were made to set up similar infections in hens with Salmonella Gallinarum, but without success. Infection either resulted in clinical disease or elimination of the pathogen. Infection of birds when in lay produced a similar result. The possibility of eggs becoming contaminated with S. Gallinarum after they were laid in the nest box was evaluated but there was no evidence for this. In-bred chicken lines with a SalI-susceptible phenotype showed greater localization of S. Pullorum in the reproductive tract than did a SalI-resistant line. In addition, in-bred birds, which were SalI resistant but showed greater susceptibility to intestinal colonization by Salmonella, infected with S. Gallinarum when they were 1 week old, showed longer term persistence in the liver and spleen than did a resistant line.

Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens.

One-week-old commercial layers were infected orally with 10(8) colony forming units of Salmonella enterica serovar Enteritidis phage type 4. No mortality was observed. The inoculated organism was isolated in decreasing viable numbers from a number of tissues, particularly the spleen, liver and caeca. Organisms present in the spleen were primarily localized within macrophages. No Salmonella Enteritidis organisms were isolated between 10 and 24 weeks of age, when the experiment was terminated after several weeks of lay. When two groups of adult hens, housed with males, were infected, contaminated eggs were found within 2 weeks of infection in one of the experiments only. Progeny hatched from these eggs showed no mortality unless they were infected artificially with the S. Enteritidis strain. In this case, the percentage mortality fell as the hatches progressed, indicating increasing immunity to infection. The faecal excretion of the inoculated phage type 4 strain by infected but healthy progeny was followed. Although most birds ceased to excrete by 11 to 12 weeks of age, a small number of the birds continued to excrete until they themselves came into lay. The small numbers of birds in which this occurred indicates that tolerance to infection does not occur readily following infection of hens laying fertile eggs or in progeny birds infected before or within hours of hatching. Birds infected when they were less than 24 h old remained persistently infected until they were well into lay. However, control birds infected when 1 week old, on this occasion, showed a high level of excretion until the birds began to lay at 18 weeks. Inbred lines of chickens showing differences in their susceptibility to systemic salmonellosis did not show significant differences in the extent to which S. Enteritidis localized in the organs of the reproductive tract or in the number of infected eggs produced.

Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves.

A lytic bacteriophage, which was previously isolated from sewage and which attaches to the K1 capsular antigen, has been used to prevent septicemia and a meningitis-like infection in chickens caused by a K1+ bacteremic strain of Escherichia coli. Protection was obtained even when administration of the phage was delayed until signs of disease appeared. The phage was able to multiply in the blood. In newly borne colostrum-deprived calves given the E. coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span. With some provisos there is considerable potential for this approach to bacterial-disease therapy.

The antibacterial effects for Salmonella Enteritidis phage type 4 of different chemical disinfectants and cleaning agents tested under different conditions.

A selection of commercially available disinfectants, sanitizers and water sanitizers based on iodophor, quaternary ammonium compounds (QACs) and phenolic compounds were tested for their activity against a phage type 4 strain of Salmonella serotype Enteritidis in the presence of a variety of organic materials. In general the phenolic preparations were the most effective followed by the QACs and the iodophors. They were all inactivated to different degrees by chick fluff, chicken faeces, feed and wood shavings. The inactivation was greatest when Salmonella organisms were pre-dried in feed. Under these conditions formaldehyde and glu-taraldehyde were still active. There was some evidence that induced resistance to stress conditions including culture at 42 degrees C and anaerobic culture increased resistance to one of the water sanitizers.

[Inhibition phenomena between Salmonella strains--a new aspect of salmonella infection control in poultry]. / Hemmphänomene zwischen Salmonella-Stämmen--ein neuer Aspekt der Bekämpfung der Salmonellose beim Geflügel.

Freshly hatched chickens show a very high susceptibility to Salmonella infections and control measures are therefore frequently focused on the period shortly after hatching. Experimental investigations using one strain against itself, differentiated by different antibiotic resistance markers, have shown that colonisation with Salmonella prevents the establishment of subsequently inoculated challenge organisms in the chicken gut. The inhibition effect lasts for several days and is detectable even when a challenge dose of 10(8) organisms is used. It is dependent of the breed of bird. Chickens colonised with Salmonella shed a subsequently inoculated challenge strain with significant lower numbers for several weeks than do non colonised control birds. The phenomenon is strain specific but not serovarspecific as has been shown in investigations using different strains of the same and other serovars for colonisation and challenge. The phenomenon shows a large variability between strains. Using other Enterobacteriaceae strains comparable inhibition against Salmonella was not observed. One important topic for further investigation is the capability of Salmonella live vaccines given orally to establish a protection effect, based on the inhibition phenomenon in the first few days of live, developing into a long-lasting immunity when birds reach immunological maturity.

Reduction in incidence of experimental fowl typhoid by incorporation of a commercial formic acid preparation (Bio-Add) into poultry feed.

The experiment described evaluated the effect of a commercial in-feed preparation (Bio-Add) involving a mixture of formic acid and propionic acid on the incidence of experimental fowl typhoid in groups of 41 and 42 1-wk-old Rhode Island Red chickens. The chickens were infected through contact with 12 identical chickens that had been inoculated orally with 10(8) cfu of Salmonella gallinarum strain 9. The incidence of mortality and morbidity due to fowl typhoid was 31/41 (76%) in birds given untreated feed and 14/42 (33%) in birds given feed treated with Bio-Add.

Examination by ELISA of sera obtained from chicken breeder and layer flocks showing evidence of fowl typhoid or pullorum disease.

An indirect ELISA using soluble whole cell antigen was used to screen serum samples obtained from breeder and layer flocks some of which had shown clinical or bacteriological evidence of infection with Salmonella Gallinarum or S. Pullorum. There was good correlation between Salmonella infection and the presence of serum samples showing high optical density (OD) values. Sera from seven flocks showing high values were retested using group D (0-1, 9, 12) lipopolysaccharide (LPS) and g,m-H flagella as detecting antigens. Sera from six flocks produced high OD values with LPS and low values with flagella confirming infection with a non-flagellate, group D Salmonella while one produced high values with both antigens indicating mixed infection with another group D serotype.

Studies on the use of a formic acid-propionic acid mixture (Bio-add) to control experimental Salmonella infection in broiler chickens.

Experiments were carried out on the antibacterial effects of a commercial formic acid-propionic acid mixture (Bio-add) against different Salmonella serotypes. The preparation exerted a strong antibacterial effect on S. typhimurium strain F98 in artificially contaminated feed. After 28 days storage, the bactericidal effect was still considerable. When chickens were reared on feed that had been treated with Bio-add and artificially contaminated with different serotypes, S. enteritidis, S. typhimurium and S. agona were not isolated from the caecal contents, but S. infantis was. No organisms of this strain were isolated when a lower feed-contamination rate of bacteria was used.

Salmonella em raçäo para aves no Brasil / Salmonella in poultry feeds in Brazil

This experiment was undertaken to determine the possible presence of Salmonella in poultry diets.A total of two hundred samples of ration fr4om 4 commercial poultry feed industries were examined.The results revealed the presence of salmonellae in 10 cents of the samples studied and 14 serotypes were identified.The produce for Salmonella isolation included the pre-enrichment step and the strains were submited to antimicrobial tests.The 29 strains were resistant to the followings antimicrobial agents(porcents

Serological response of chickens to infection with Salmonella gallinarum-S. pullorum detected by enzyme-linked immunosorbent assay.

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.

The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella enteritidis phage type 4.

Laying hens aged 24 weeks were vaccinated twice, separated by a 2-week interval, with spectinomycin-resistant mutants of either the Salmonella gallinarum 9R vaccine or a rough, aroA insertion mutant of a S. enteritidis phage type 4 strain. The 9R strain was given intramuscularly, the rough, aroA mutant both intramuscularly and orally. Two weeks after the second vaccination the immunized and a control group of unimmunized birds were challenged with a nalidixic acid-resistant mutant of a fully virulent phage type 4 strain. Full post-mortem examinations were carried out and eggs were examined bacteriologically for 3 weeks after challenge. Immunization with the 9R strain produced a marked reduction in the number of isolations of the challenge strain from a number of organs, including the ovaries. In contrast, immunization with the rough, aroA mutant produced little change in the isolation rate from the ovaries with small reductions for the liver and spleen. Both candidate vaccines produced a reduction in the number of isolations from laid eggs. The 9R strain was itself isolated from the ovaries throughout the period of examination whereas the aroA strain was not. Sera taken from the birds immediately prior to challenge were examined by slide agglutination using live S. enteritidis cells. Sera from 9R vaccinated birds contained agglutinins whereas sera from most of the chickens immunized with the rough, aroA mutant did not.

In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium.

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20 degrees C and 40 degrees C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A TnphoA insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.