The aim of the present study was to describe the identification and characterization (physiological properties) of two strains of lactic acid bacteria (LAB 18 and 48) present in a commercial probiotic culture, FloraMax®-B11. Isolates were characterized morphologically, and identified biochemically. In addition, the MIDI System ID, the Biolog ID System, and 16S rRNA sequence analyses for identification of LAB 18 and LAB 48 strains were used to compare the identification results. Tolerance and resistance to acidic pH, high osmotic concentration of NaCl, and bile salts were tested in broth medium. In vitro assessment of antimicrobial activity against enteropathogenic bacteria and susceptibility to antibiotics were also tested. The results obtained in this study showed tolerance of LAB 18 and LAB 48 to pH 3.0, 6.5% NaCl and a high bile salt concentration (0.6%). Both strains evaluated showed in vitro antibacterial activity against Salmonella enterica serovar Enteritidis, Escherichia coli (O157:H7), and Campylobacter jejuni. These are important characteristics of lactic acid bacteria that should be evaluated when selecting strains to be used as probiotics. Antimicrobial activity of these effective isolates may contribute to efficacy, possibly by direct antimicrobial activity in vivo.

Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable.

A posthatch fasting period of 24 to 72 h is a common and inevitable practice in commercial poultry production. This delay in start of feed intake has been reported to negatively affect yolk utilization, gastrointestinal development, slaughter weight, breast meat yield, performance, and to also depress immunological development, making the birds more susceptible to infection from pathogens such as Salmonella. Furthermore, public concerns regarding the considerable human rates of illness reported and the emergence of antibiotic-resistant strains of Salmonella have doubled the challenge on the poultry industry to find alternative means of Salmonella control. In the present study, we evaluated the effects of a combination of early feeding with probiotic supplementation on morphological development of mucosa, control of Salmonella, and overall performance in broiler chickens. We used a blend of a commercially available perinatal supplement, EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR), and a successful probiotic supplement, FloraMax-B11 (FM; Pacific Vet Group USA Inc.), to evaluate the effects on gut morphology, Salmonella intestinal colonization, and horizontal transmission, along with its effects on BW and related performance in broiler chickens under simulated commercial hatching management and shipping conditions. Morphometric analysis showed increased villus height, villus width, villus to crypt ratio, and villus surface area index in chickens treated with EB + FM groups. Significant reductions in Salmonella recovery, incidence, and horizontal transmission were also observed among the same groups, suggesting beneficial effects of early feeding and competitive exclusion by probiotic bacteria. Improved gut morphology and Salmonella exclusion was very well supported by BW data with significantly lower early BW loss and overall BW gains in birds treated with EB + FM mixture. The results of this study demonstrated that the combination of EB and FM improved gut morphology, reduced the amount of Salmonella that could be recovered, as well as improved BW when compared with controls and each product individually. These data address both animal welfare and food safety concerns faced by the poultry industry.

Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (106 cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.

Public concern with the incidence of antibiotic-resistant bacteria, particularly among foodborne pathogens such as Salmonella, has been challenging the poultry industry to find alternative means of control. The purposes of the present study were to evaluate in vitro and in vivo effects of chitosan on Salmonella enterica serovar Typhimurium (ST) infection in broiler chicks. For in vitro crop assay experiments, tubes containing feed, water, and ST were treated with either saline as a control or 0.2% chitosan. The entire assay was repeated in three trials. In two independent in vivo trials, 40 broiler chicks were assigned to an untreated control diet or dietary treatment with 0.2% chitosan for 7 days (20 broiler chicks/treatment). At day 4, chicks were challenged with 2 · 10 5 colonyforming units (CFU) ST/bird. In a third in vivo trial, 100 broiler chicks were assigned to untreated control diet or dietary treatment with 0.2% chitosan for 10 days (50 broiler chicks/treatment) to evaluate ST horizontal transmission. At day 3, 10 birds were challenged with 10 5 CFU ST/bird, and the remaining nonchallenged birds (n = 40) were kept in the same floor pen. In all three in vitro trials, 0.2% chitosan significantly reduced total CFU of ST at 0.5 and 6 h postinoculation compared with control ( p < 0.05). In two in vivo trials, at 7 days, dietary 0.2% chitosan significantly reduced total CFU of recovered ST in the ceca in both experiments. Dietary 0.2% chitosan significantly reduced total ST CFU recovered in the ceca of horizontally challenged birds in the third in vivo trial. Chitosan at 0.2% significantly reduced the CFU of recovered ST in vitro and in vivo, proving to be an alternative tool to reduce crop, ceca, and consequently carcass ST contamination as well as decreasing the amount of ST shed to the environment.