Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens.

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.     

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.     

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 anaerobic cecal microflora and dietary lactose on colonization resistance of layer chicks to invasive Salmonella enteritidis.

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.     

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.     

Loss of virulence by heterophil-adapted Salmonella pullorum.

We report that reduction of virulence for day-old chicks was achieved after eight-times-repeated heterophil passage of Salmonella pullorum (SP). The virulent source strain SP-V caused 64% mortality and 89% internal organ as well as 89% cecal colonization 10 days after administration of 10(7) colony-forming units (CFU) to day-old chicks. Eight-times-repeated passage of SP in heterophils resulted in attenuated strain SP-A that was nonlethal and reduced colonization of internal organs from 89% for SP-V strain to 4.3% for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks. Cecal colonization was reduced from 89% for SP-V strain to 0 for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks.     

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.     

Colonization control of lactose-fermenting Salmonella typhimurium in young broiler chickens by use of dietary lactose

Inclusion of lactose in the diets of chickens has been determined to reduce cecal colonization with Salmonella typhimurium. We hypothesized, therefore, that dietary lactose may be a practical means for reducing the prevalence of Salmonella contamination of chicken products. Because some strains of Salmonella are atypical and ferment lactose, we investigated the effects of dietary lactose on cecal colonization with lactose-fermenting S typhimurium. Broiler chicks were inoculated intracloacally with Lac+ S typhimurium selected for resistance to novobiocin and rifampicin. The chicks also were inoculated orally with certain anaerobes that do not effectively inhibit colonization by S typhimurium, but do appear essential for lactose mediated inhibition of cecal colonization. Control chicks were not given dietary lactose, and chicks in the experimental group were fed a diet containing 7% lactose. Enumeration of Lac+ S typhimurium in cecal contents revealed dietary lactose to be effective at controlling this organism. Control was correlated with changes in cecal pH and increases in undissociated volatile fatty acids, especially propionic acid.     

Effect of feeding selected short-chain fatty acids on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two groups of 20 chicks each were fed 1% fatty acid continuously starting at 1 day of age, while a control group of 20 chicks received unsupplemented feed. At 2 days of age, chicks were inoculated orally with 1 ml of Salmonella typhimurium (1 x 10(6) colony-forming units/ml). Ceca were obtained from six chicks of each group at 7, 14, and 21 days of age. At 14 days of age, formic and propionic acids had statistically reduced Salmonella recovery by 2.56 logs and 3.09 logs, respectively, compared with controls. At 21 days of age, both test groups showed significant reductions of approximately 3.6 logs compared with controls. There were no statistical differences in body weights among the groups at 21 days of age.     

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.     

Differences among six Salmonella serovars in abilities to colonize reproductive organs and to contaminate eggs in laying hens

The abilities of Salmonella serovars to colonize the reproductive organs of chickens and to contaminate eggs were compared. Mature laying hens were inoculated intravenously with 10(5) colony-forming units of Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to cause the systemic infection. Salmonella enteritidis was recovered from three yolks of the laid eggs (7.0%), suggesting egg contamination from the transovarian transmission of S. enteritidis. The liver, spleen, and cecum were colonized by each serovar similarly at 4 or 7 days postinoculation (PI), whereas the ovary and preovulatory follicles were colonized by S. enteritidis with significantly (P < 0.05) higher levels than by the other serovars at 4 and 7 days PI. Salmonella enteritidis was recovered from the cloaca and vagina at 2, 4, and 7 days PI and from the other portions of the oviduct at 4 and 7 days PI. In addition, S. enteritidis had been persistent in the peripheral blood for 7 days PI. These results suggest that S. enteritidis is the predominant serovar to colonize the reproductive organs of mature laying hens among six serovars used in this study, reflecting the field situatibn in which the predominant outbreaks of human salmonellosis were caused by S. enteritidis-contaminated eggs recently. The ability of S. enteritidis to colonize the reproductive organs may be one of the reasons that egg contamination with S. enteritidis has increased.     

Influence of temperature, social, and dietary stress on development and stability of protective microflora in chickens against S. typhimurium

In the day-old chick, physiological stress in the form of high or low temperatures or feed and water deprivation either interfered with colonization or altered the protection provided by normal intestinal microflora against subsequent challenge with Salmonella typhimurium. The response was more obvious after individual exposure of chicks to salmonella than after exposure to infected seeders. Physiological and dietary stress at 2 weeks of age did not appreciably alter the S. typhimurium excretion pattern in treated chicks. The protective microflora appears to be quite stable under these stress conditions.     

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.     

Dietary conjugated linoleic acids alleviate early inflammatory response caused by lipopolysaccharide injection in male broiler chicks

An experiment was undertaken to determine if dietary conjugated linoleic acid (CLA) influences the early stage of the inflammatory response caused by a single injection of Salmonella enteritidisi lipopolysaccharide (LPS) in male broiler chicks. Chicks (7 days old) were fed either 0 or 10 g CLA/kg in their diet for 21 days. At 28 days old, birds were injected intraperitoneally with 1.5 mg LPS/kg body weight after 12 h fasting. Changes in feed intake, rectal temperature and plasma α‐1 acid glycoprotein and ceruloplasmin concentrations were measured during 24 h after LPS injection. Chicks fed the CLA diet were fed more feed for 24 h, had a lower rectal temperature at 9 and 24 h and a lower plasma α‐1 acid glycoprotein concentration at 24 h after LPS injection than chicks fed the basal diet. Plasma ceruloplasmin concentration tended to be lower in chicks fed the CLA diet than in chicks fed the basal diet. The results suggest that dietary CLA alleviate undesirable early inflammatory response due to LPS injection in male broiler chicks.     

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).     

Comparison of Salmonella enterica serovar enteritidis levels in crops of fed or fasted infected hens

Long-term feed withdrawal has been shown to increase ileocecal intestinal colonization and fecal shedding of Salmonella enterica serovar Enteritidis in challenged hens. Less information is available regarding effects of fasting on crop colonization. Two trials were conducted to compare effects of 14-day feed withdrawal vs. full feed on crop colonization in hens challenged with Salmonella Enteritidis. The levels of Salmonella Enteritidis in the crops of fasted hens were significantly higher than in nonfasted hens on days 3 and 10 and days 3, 9, and 16 postinfection (PI) in trials 1 and 2, respectively. Fecal shedding of Salmonella Enteritidis was significantly increased in the fasted hens on day 10 PI in trial 1. Analysis of crop IgA anti-Salmonella Enteritidis lipopolysaccharide levels in crop lavage samples of hens in trial 1 revealed a humoral response PI in both treatment groups with no significant differences, although peak response for fasted hens occurred 1 wk later. Histologic evaluation of hematoxylin and eosin-stained crop sections from trial 1 birds revealed mild to moderate heterophilic infiltration within the crop lamina propria (LP) or LP and epithelium of nonfasted infected hens at 24 and 96 hr PI. In comparison, heterophils in crops of fasted hens infected at this time point were sparse, indicating a possible diminished heterophil response in the fasted birds. Multifocal areas of tissue inflammation, as indicated by marked heterophil infiltration, with necrosis and sloughing of epithelium, were observed in crops from fasted hens at day 11 PI (14th day of feed withdrawal) but not in the fed groups. This severe heterophilic inflammation was observed in both challenged and nonchallenged fasted hens, suggesting that some factor other than Salmonella Enteritidis was responsible. These results indicate that feed withdrawal can have a dramatic effect on the integrity of the crop and its ultimate response to infection.     

Colonisation studies using Campylobacter jejuni in chicks

White Leghorn chicks used in this study were hatched from specific pathogen-free eggs. The colonizing capability of Campylobacter (C.) jejuni strains was investigated in 6 experiments. The formation of specific antibodies associated to colonization was also detected. In each experiment, day of hatch chicks were randomly separated into three groups of 24 birds each: two groups colonized experimentally and one control group. Chicks were reared on the floor in three separated, adjacent rooms with sterilized wood shavings as litter. At 2 or 8 days of age, respectively, the chicks in the experimentally colonized groups received between 3.3 x 10(7) and 2.0 x 10(8) colony-forming units (CFU) of C. jejuni via oesophageal gavage. Furthermore, 7, 14, 21, 28, 42 and 56 days after inoculation, 4 chicks of each group were sacrificed by cervical dislocation, at which time blood, liver and faeces were collected for processing. Serum was centrifuged and Campylobacter-specific IgG, IgA and IgM antibodies were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Altogether, the colonizing capability of 11 C. jejuni strains was examined. Surprisingly, there were large differences between the C. jejuni isolates. After these experiments, we could divide the isolates into three groups. 4 out of 11 isolates could not be reisolated, 2 isolates caused weak or delayed colonization and 5 C. jejuni produced strong, long-lasting colonization. In the first days of life (9 days), the C. jejuni-free SPF chicks (control animals) had high IgG titres in sera, which decreased markedly up to the age of 15 days. During the experiments the IgM and IgA titres remained nearly at the same level, i.e., the amounts of maternal antibodies were low and there was no evidence for antibody formation in the chicks themselves. Two- and 8-day-old chicks were inoculated with C. jejuni strain Penner 1. Two-day-old chicks were colonized 3 weeks after inoculation. In comparison with these animals, 8-day-old chicks were colonized already 2 weeks after inoculation. There is the assumption, that the higher maternal antibodies in 2-day-old chicks could be responsible for this delay. In chicks the C. jejuni colonization resulted in a marked IgG (but not IgM and IgA) increase. Apparently, there is a positive relationship between the counts of this pathogen in caeca and the IgG increase.     

The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.     

Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars

In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S. enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs. Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%). The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S. enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI). In the vagina, the positive rates were 90%-100% in hens inoculated with S. enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI. The ceca were colonized similarly by each serovar at 7 days PI. The spleen and ovary were infected with S. enteritidis in three and one hen, respectively. No Salmonella was recovered from liver and peripheral blood in any hen. Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct. In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants. The mean number of S. enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars. These results suggest that S. enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S. enteritidis to the vagina may play a significant role in the production of many S. enteritidis-contaminated eggs.     

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.