Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks

One-day-old broiler chicks were inoculated with volatile fatty acid producing cecal flora from adult chickens. The chicks were divided into four groups and provided 1) no lactose, 2) 2.5% lactose in water, 3) 5% lactose in feed, or 4) 10% lactose in feed, until 10 days of age. All groups were challenged at 3 days of age with 10(6) or 10(8) S. typhimurium. At 10 days, the number of Salmonella in the ceca of the chicks challenged with 10(6) Salmonella was significantly decreased (P less than 0.01) in the groups provided lactose as compared with the controls. A significant decrease (P less than 0.01) in Salmonella numbers occurred in the chicks challenged with 10(8) Salmonella and provided 10% lactose. Providing 2.5% lactose or 5% lactose failed to inhibit Salmonella growth in chicks challenged with 10(8) Salmonella. The pH of the ceca of the groups provided lactose decreased significantly (P less than 0.05) and was accompanied by significant increases (P less than 0.01) in the concentrations of bacteriostatic acetic and propionic acids. Results showed that providing dietary lactose to broiler chicks and inoculation with normal cecal flora decreased cecal pH, increased the concentrations of bacteriostatic volatile fatty acids, and inhibited Salmonella colonization.     

Effect of dietary lactose on Salmonella colonization of market-age broiler chickens

The effect of providing lactose in feed and inoculation with volatile fatty acid-producing anaerobic cultures (AC) of cecal flora on Salmonella typhimurium colonization was evaluated in broilers. One-day-old chicks were divided into four groups and provided 1) no lactose, no AC; 2) AC, no lactose; 3) AC and lactose on days 1-10; or 4) AC and lactose on days 1-40. All groups were challenged per os with 10(6) Salmonella on day 3 and with 10(8) Salmonella on day 33. Salmonella growth in the cecal contents was significantly decreased (P less than 0.01) on day 10 in the chicks provided lactose from day 1-10. However, after the removal of lactose from the diet, the chicks were susceptible to Salmonella colonization. The number of Salmonella in the ceca was significantly reduced (P less than 0.05) in the chicks provided lactose throughout the 40-day growing period. Dietary lactose decreased the pH of the cecal contents and was accompanied by marked increases in the concentrations of undissociated bacteriostatic volatile fatty acids in the cecal contents.     

Effect of nitrofurazone or novobiocin on Salmonella enteritidis cecal colonization and organ invasion in Leghorn hens.

Nitrofurazone (433 mg/liter drinking water) administration to leghorn laying hens for 72 hours with a 48-hour withdrawal period before nitrofurazone-sensitive Salmonella enteritidis challenge resulted in a S. enteritidis culture-positive rate significantly (P less than 0.05) higher than that of unmedicated controls when hens were cultured 6 days following challenge. In a similar experiment, simultaneous nitrofurazone administration and S. enteritidis challenge resulted in no significant differences in S. enteritidis isolation frequency. However, unchallenged nitrofurazone-medicated contact control hens showed a significantly higher S. enteritidis culture-positive rate than the unchallenged unmedicated controls. This increase in apparent S. enteritidis susceptibility was associated with significantly lower cecal propionic acid and fewer anaerobic colony-forming units (CFU). In a third experiment, nitrofurazone treatment had no effect when the antibiotic was withdrawn 72 hours before S. enteritidis challenge. In a subsequent experiment, novobiocin (385 mg/kg) or nitrofurazone (300 mg/kg) was administered to adult hens for 7 days before challenge with nitrofurazone and novobiocin-resistant S. enteritidis. Both nitrofurazone and novobiocin administration resulted in a significantly higher S. enteritidis culture-positive rate than unmedicated controls at end of the experiment 7 days post-challenge. Results indicate that administration of some antibiotics may enhance susceptibility to S. enteritidis in leghorn hens.     

Intracloacal Salmonella typhimurium infection of broiler chickens: reduction of colonization with anaerobic organisms and dietary lactose

The combined effect of treatments with dietary lactose plus anaerobic organisms on cecal colonization of broiler chicks by Salmonella typhimurium was evaluated. Chickens treated with a combination of anaerobic organisms and 7% dietary lactose were resistant to cecal colonization by S. typhimurium. The number of recoverable S. typhimurium cells per gram of cecal contents taken on days 10 and 15 after infection was significantly reduced. Treatment with anaerobes without the addition of lactose did not effectively control cecal colonization. Intracloacal inoculations with bacterial concentrations that varied by 10,000-fold resulted in roughly similar levels of colonization. The treatments resulted in reduced cecal pH and elevated levels of undissociated volatile fatty acids. Statistically significant correlations (P less than 0.01) were observed between the S. typhimurium concentrations in cecal material and the concentrations of undissociated fatty acids (r = -0.79, and between the bacterial counts and pH (r = 0.72).     

Effects of rearing conditions on the colonization of Salmonella enteritidis in the cecum of chicks.

Salmonella enteritidis is the cause of human salmonellosis associated with contaminated eggs. In this study, we artificially challenged S. enteritidis to chicks just after hatching, and the effects of breeding conditions on the intestinal carriage of S. enteritidis were examined. S. enteritidis was not directly detected from spleen, liver and blood, but were constantly isolated from the cecal contents throughout the experiment. When chicks were reared in the unsanitary conditions and in the high housing density, the numbers of S. enteritidis increased. The subsequent experiment was undertaken to examine whether the antibacterial additive in a feed would have any impact on S. enteritidis colonization in chicks. Some antibiotic effective on the growth promotion had an influence on S. enteritidis colonization.     

Use of cecal microflora cultured under aerobic or anaerobic conditions in the control of experimental infection of chicks with Salmonella Enteritidis

One-day-old broiler chicks received cecal microflora (CM) cultured under aerobic, anaerobic conditions, or both (mixed) and were then infected with Salmonella Enteritidis, in order to compare the efficacy of these different types of culture in terms of the number of chicks infected, cecal colonization and faecal excretion of the challenging bacteria. Regardless of culture type, CM always led to a smaller number of S. Enteritidis for any of the parameters studied compared to untreated chicks. Aerobic CM demonstrated better efficacy in reducing the number of infected chicks and cecal colonization by S. Enteritidis, followed by mixed CM. No difference was observed in faecal excretion of S. Enteritidis between the chicks that received different types of CM culture.     

Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces

Chicks were treated orally on the day of hatch with either fresh or frozen competitive exclusion (CE) cultures (native gut microflora). Chicks were fed either unmedicated feed or one of five commercial broiler starter rations or nine experimental feed mixtures containing varying amounts and combinations of anticoccidial and antimicrobial medicaments. After 2 days, they were challenged with approximately 10(6) colony-forming units of a nalidixic-acid-resistant strain of Salmonella typhimurium. Six days later, chicks were sacrificed and ceca were analyzed for S. typhimurium. Colonization of 2-day-old chicks was prevented or at least greatly reduced in most instances by treatment of chicks with a CE culture, but the efficacy of CE broth cultures stored at -70 C diminished over time. Not all CE cultures tested gave equal protection against Salmonella colonization, and CE cultures were more susceptible to some feed additives than others. Of the commercial or experimental feed tested, only the feed containing the combination of nicarbazin and bacitracin interfered with the protective effect of the CE culture.     

Influence of dietary lactose on the gut flora of chicks

1. The gut microflora of chicks fed on a purified diet containing 300 g lactose plus 300 g starch/kg was compared with that of control birds receiving a diet containing 600 g starch/kg.

2. In 14‐d‐old conventional chicks, lactose in the diet decreased the incidence of lactobacilli and clostridia in the caecal contents, although when present in lactose‐fed chicks the counts of lactobacilli exceeded those of control chicks.

3. High counts of Proteus sp. were present in the caeca of control birds but they were completely suppressed in conventional birds fed on the lactose diet. In vitro tests showed that this inhibition was partially due to Escher‐ichia coli and Streptococcus faecalis.

4. The growth of Lactobacillus acidophilus was inhibited by lactose when gnotobiotic chicks were monoassociated but not when polyassociated. The protective effect was shown in vitro to be due to L. salivarius.

5. The pH was markedly lowered in the caecum of conventional and polyassociated chicks receiving dietary lactose. Of the strains used in gnotobiotic experiments E. coli, S. faecalis and L. salivarius produced the lowest pH values in the caeca.     

Effect of sample selection from experimentally contaminated hatching eggs and freshly hatched chicks on Salmonella enteritidis detection rate.

The conventional culture methods of hatching eggs using shell and/or egg contents for detection of salmonella organisms give mostly unsatisfactory results. The aim of the present study is to evaluate selection of other samples and techniques of culturing hatching eggs and freshly hatched chicks. This current study provides the best evidence of Salmonella enteritidis in artificially contaminated eggs (Layer type) by using enrichment broth in empty egg shell samples in comparison to culturing samples from yolk, albumen or from shell above the air cell (with the outer shell membrane). The isolation rates could be enhanced if empty egg shell was initially filled with Buffered Pepton Water as pre-enrichment broth. Examination of organs from freshly hatched chicks revealed that crop samples give mostly higher reisolation rates.     

Candidate genes for resistance to Salmonella enteritidis colonization in chickens as detected in a novel genetic cross

Salmonellosis is a zoonotic disease that is problematic for both animal production and food safety. A novel genetic cross, named the Iowa Salmonella response resource population (ISRRP), was established to elucidate the genetic control of resistance to Salmonella enteritidis (SE) colonization in young chicks, to characterize unique resistance alleles, and to estimate gene interaction effects. Outbred broiler sires were mated with dams of diverse, highly inbred, light-bodied lines to produce an F(1) generation that was informative for all heterozygous alleles of the sires. Mating F(1) sires back to dams of the corresponding inbred line produced a backcross generation. To mimic the natural route of exposure and thus afford the opportunity to investigate mucosal immunity, pathogenic SE were inoculated into the esophagus of day-old chicks. After 1 week, the SE colonizing the cecal lumen and the spleen were enumerated. Candidate genes were selected for analysis based upon one of the two criteria. Functional candidates were genes with reported activity related to the tested traits. Positional candidates were genes mapped near microsatellites that were linked, in other phases of this project, with antibody levels to SE vaccine. Broiler sire alleles of the MHC class I, NRAMP1, PSAP, and IAP1 genes showed association with SE colonization in the F(1) generation of this novel disease resistance resource population.     

柔嫩艾美尔球虫与肠炎沙门菌混合感染对雏鸡盲肠菌群的影响

为探讨柔嫩艾美尔球虫与肠炎沙门菌单独感染及混合感染对盲肠肠道菌群的影响,12日龄莱航鸡口服单独或混合感染柔嫩艾美尔球虫或肠炎沙门菌,18日龄通过16SrRNA Miseq高通量测序检测鸡盲肠菌群组成。结果表明,18日龄莱航鸡盲肠内主要菌群为厚壁菌门和拟杆菌门。与柔嫩艾美尔球虫或肠炎沙门菌单独感染相比,混合感染导致肠杆菌科明显增加,毛螺菌科明显减少;因此,柔嫩艾美尔球虫与肠炎沙门菌混合感染对盲肠肠道菌群的影响与两者单独感染明显不同。     

Efficacy of maternal Salmonella antibodies and experimental oral infection of chicks with Salmonella enteritidis

Distribution of maternally transmitted Salmonella antibodies and their protective effects were studied in the progeny of broiler breeder birds which had been vaccinated with live S. Typhimurium and inactivated S. Enteritidis vaccines. Vaccination resulted in a significant increase of the antibody concentration in yolk of hatching eggs and in serum and jejunum of the progeny of immunized breeder birds. Higher antibody titres for isotypes IgG and IgA were still seen on day 21 of age. Antibody production of isotypes IgA and IgM by the chickens themselves was found between 14 and 21 days of age. Two challenge models (10(2) cfu/bird on day 1 of age and a seeder bird model, respectively) were used to evaluate the efficacy of maternal antibodies against challenge with S. Enteritidis. Using both models numbers of challenge organisms were lower in the caeca of the progeny of immunized parent birds between day 7 and day 21 of age (maximum about 1.5 log10 units) compared with control chicks. The results indicate the efficacy of maternally transferred antibodies but it remains the question of their practical relevance. The effects of acquired maternal antibodies on an active immunization of the progeny of immunized breeder birds with live Salmonella vaccines are discussed.     

Effect of used litter from floor pens of adult broilers on Salmonella colonization of broiler chicks.

The effect of used pine-shaving litter from broiler floor pens on Salmonella colonization resistance was evaluated in broiler chicks. One-day-old chicks were placed in floor pens on fresh unused litter or on used litter. All chicks were challenged orally with 10(4) S. typhimurium at 3 days of age. The study was replicated in three trials with used litter that was collected and stored for 1 day (Trial 1), 4 days (Trial 2), or 50 days (Trial 3) before the start of each trial. Cecal concentrations of volatile fatty acids (VFAs) were significantly higher (P < 0.05) in chicks placed on used litter than in chicks on new litter. In all three trials, the number of Salmonella in the cecal contents and the number of Salmonella cecal-culture-positive chicks was significantly lower (P < 0.01) at 10 days and 20 days of age in the chicks on used litter than in the chicks on new litter. The results indicated that newly hatched chicks reared on used litter had higher cecal VFA concentrations and higher resistance to Salmonella colonization than chicks reared on new litter.     

Effects of probiotics and maternal vaccination on Salmonella enteritidis infection in broiler chicks

The effects of probiotics and maternal vaccination with an inactivated Salmonella Enteritidis (SE) vaccine on day-old chicks challenged with SE were evaluated. A 2 X 3 factorial arrangement was used (with or without probiotics; breeders nonvaccinated, vaccinated intramuscularly, or vaccinated intraperitoneally). Three trials were conducted in isolation cabinets and SE challenge was different between trials. The number of SE organisms per chick and the time interval between housing and introduction of seeder birds (hereafter called challenge) were 1.6 X 10(8) and 1 hr (Trial I), 1.8 X 10(6) and 12 hr (Trial II), and 1.2 X 10(4) and 24 hr (Trial III). SE recovery was assessed in ceca and liver at 3, 5, and 7 days postchallenge, and the number of colony-forming units (CFU) in ceca was evaluated at 5 and 7 days postchallenge. The number of SE (log CFU) in the ceca reduced 0.56 log (from 7.59 to 7.03) and 1.45 log (7.62 to 6.17) because of the treatment with probiotics in Trials II and III, respectively. The greater reduction in Trial III indicates the importance of the early use of probiotics on the prevention of SE infection. Treatment with probiotics resulted in a smaller number of SE-positive livers after 5 days postchallenge on Trial III. Although there was no significant effect of maternal vaccination on the number of SE CFU in the ceca, a significant effect of maternal vaccination on the SE CFU was observed in the liver, but not in the ceca at 5 days after challenge.     

Effect of prolonged heat stress in single-comb white leghorn hens on progeny resistance to Salmonella enteritidis organ invasion.

In our laboratory, we have often had difficulty infecting neonatal chickens with invasive salmonellae when ambient temperatures exceed 30 C. We hypothesized that this increased resistance in chicks during warmer months may be associated with heat stress-associated maternal factors. Presently, single-comb white leghorn hens were separated into a non-heat-stressed group, reared under temperatures from approximately 10 to 24 C, and a heat-stressed group, in which environmental temperature was incrementally elevated to near 37 C and maintained for the duration of the 13-wk study. For Expt. 1, eggs from heat-stressed or control hens, collected on days 8-14 of the study, were pooled respective to treatment and incubated. At the time of egg collection, mean hen-day egg production was 51.83% or 65% for heat-stressed or control hens, respectively. On day of hatch, progeny from hens in each group were orally challenged with 0.9 x 10(4) colony-forming units (CFU) Salmonella enteritidis (SE). Rates of SE organ invasion of 97.3% or 94.4% were obtained in progeny from heat-stressed or control hens, respectively. In Expt. 2, eggs from heat-stressed or control hens from days 30-42 of the study were collected and pooled by treatment for incubation. Mean hen-day egg production was 46.5% or 72.85% for heat-stressed or control hens, respectively. On day of hatch, progeny were orally challenged with either 2.2 x 10(3) or 2.2 x 10(4) CFU SE. A 100% incidence in SE organ invasion was observed in all groups. In Expt. 3, eggs were collected from days 43 through 56 of the study. Mean hen-day egg production was 19.8% or 76.8% for heat-stressed or control hens, respectively. On day of hatch, progeny were orally challenged with 2 x 10(3) CFU SE. Rates of SE organ invasion of 95.8% or 95.6% were obtained in progeny from heat-stressed or control hens, respectively. These data suggest that factors other than elevated temperature may be responsible for seasonal resistance to invasive salmonellae infection in neonatal chickens observed in our laboratory during warmer months in Texas.     

Effect of immunosuppression with cyclophosphamide, cyclosporin, or dexamethasone on Salmonella colonization of broiler chicks

One-day-old broiler chicks were divided into four groups: untreated controls and three groups treated with the immunosuppressive drugs cyclophosphamide, cyclosporin, or dexamethasone on days 1-4 of age. On day 5, all groups were challenged orally with 10(4) Salmonella typhimurium. Suppression of immunocompetency was demonstrated in the treatment groups by significant decrease (P less than 0.05) in weights of bursas of Fabricius, and diminished mitogen-induced T and B lymphocyte blastogenesis and cutaneous basophil hypersensitivity responses. There were no significant differences among the four groups in the number of Salmonella in the cecal contents or in the number of cecal-culture-positive chicks on day 10 post-challenge at 15 days of age. Results indicated that impairment of B or T lymphocyte responses had little influence on the resistance of young chicks to Salmonella cecal colonization.     

Effect of administration of Lactobacillus crispatus,Clostridium lactatifermentans and dietary lactose on the development of the normal microflora and volatile fatty acids in the caeca of broiler chicks

1. Lactobacillus crispatus and Clostridium lactatifermentans, both isolated from the caeca of chickens, grown together in an in vitro model system are able to ferment lactose to acetate and propionate. In this study, the capabilities of these organisms were studied in vivo. 2. The effect on concentrations of volatile fatty acids and lactate, together with the development of some bacterial groups in the caeca of chicks, was studied after oral inoculation with L. crispatus and C. lactatifermentans together with dietary lactose. For this purpose, chicks were divided into 4 groups: (i) control group, (ii) dietary lactose, (iii) L. crispatus and C. lactatifermentans, and (iv) dietary lactose together with L. crispitaus and C. lactatifermentans. 3. In general, concentrations of (undissociated) volatile fatty acids in the caeca were not significantly different in broilers receiving both bacteria and dietary lactose compared with control broilers. Concentrations of lactate in the caeca of 14-d-old broilers treated with any of the three treatments were significantly higher than in the caeca of control broilers. 4. This indicates that L. crispatus or other lactate-producing organisms were responsible for the elevated concentrations of lactic acid. Clostridium lactatifermentans has probably not colonised the caeca sufficiently to ferment this lactate further to acetate and propionate. 5. Numbers of Enterobacteriaceae and enterococci in the caeca of broilers receiving both bacteria and dietary lactose were not different from control broilers. 6. We conclude from these results that under the conditions applied in this study a mixture of L. crispatus and C. lactatifermentans with dietary lactose was able to increase lactate concentrations but was unable to increase concentrates of acetate and propionate in the caeca of broiler chicks.     

Pathogenicity of Salmonella enteritidis phage types 4, 8, and 23 in broiler chicks.

Four hundred fifty day-old Hubbard broiler chicks were subdivided into 15 groups of 30 chicks each. Six groups of chicks received 0.5 ml of broth culture containing 5 x 10(6) colony-forming units (CFU) of Salmonella enteritidis (SE) phage types (PTs) 4, 8, and 23 by crop gavage. Similarly, six other groups received 0.5 ml containing 5 x 10(8) CFU of SE. One group was inoculated with 0.5 ml containing 5 x 10(6) CFU of Salmonella pullorum, and another group received 0.5 ml containing 5 x 10(8) CFU of S. pullorum. A group of 30 chicks were kept as uninoculated controls. Chicks were observed daily for clinical signs and mortality. All birds were weighed at 7, 14, and 21 days postinoculation 21 (DPI). Four chicks were randomly selected from each treatment group, euthanatized, and necropsied at 7 and 14 DPI. Gross lesions were recorded and selected tissues were collected for histopathology. The higher rates of illness and mortality were observed in chicks inoculated with 5 x 10(6) and 5 x 10(8) CFU of S. pullorum, followed by SE PT4 of human origin and SE PT4 of chicken origin. Moderate to high mortality was observed in chicks inoculated with the higher dose of SE isolates that belonged to PT8 and one SE of PT23. Variable mortality was evident in groups inoculated with the lower dose of salmonella. The most consistent gross and histopathologic changes, including fibrinous pericarditis and perihepatitis, were seen in the dead birds from various treatment groups. The lower mean body weights were present in all treatment groups compared with uninoculated controls. No illness or mortality was observed in uninoculated control groups.     

Use of competitive exclusion to protect newly-hatched chicks against intestinal colonisation and invasion by Salmonella enteritidis PT4.

1. The recommended dose of a commercial competitive exclusion (CE) product (BROILACT) was given orally to newly-hatched broiler chicks to protect them against oral challenge by Salmonella enteritidis PT4. 2. In 5 replicate trials, half of the birds thus treated and half from untreated control groups were examined for salmonellas at 5 d and the other half at 12 d after challenge. 3. Caecal contents were examined quantitatively while heart, liver and spleen samples were examined qualitatively by enrichment. 4. The treatment effectively prevented both colonisation of the caeca and invasion of the other organs by S. enteritidis PT4; the average number of salmonellas was less than 10 colony forming units (cfu)/g of caecal contents in the treated birds and more than 10 million cfu/g in the untreated birds. 5. Infection of organs other than the caeca was completely prevented by protective treatment, whereas 38% of the untreated birds were still infected at the end of the trial.