In ovo administration of Bacillus subtilis serotypes effect hatchability, 21-day performance, and intestinal microflora.

Recent research has tried to maximize broiler chick health and performance by utilizing commercial in-feed probiotics to inoculate fertile hatching eggs, and thus expose birds earlier to beneficial bacteria. However, the in ovo inoculation of a specific serotype of Bacillus subtilis was detrimental for broiler hatchability. Therefore, the objective of this study was to determine if other B. subtilis serotypes negatively affect hatchability or if it is associated with a specific serotype. It was also of interest to determine if the B. subtilis serotype influence chick performance and intestinal microflora. On d18 of incubation, 1886 fertile broiler eggs were in ovo inoculated with the following treatments (T): T1 = Marek's vaccine (MV), T2 = MV + B. subtilis (ATCC 6051), T3 = MV + B. subtilis (ATCC 8473), and T4 = MV + B. subtilis (ATCC 9466). It should be noted that in a previous study, T2 was detrimental to hatchability. Inoculated eggs were transferred to 3 hatchers/T. At hatch, chicks were weighed, feather sexed, and hatch residue analysis was conducted. Male chicks were randomly assigned to 40 raised wire cage so that there were 10 birds/cage. On d 0, 7, 14, and 21 of the grow-out, chicks and feed were weighed to calculate performance data. On these days, the ileum and ceca were aseptically collected to enumerate total aerobes and coliforms. No differences were observed for percentage of mid dead embryos, cracked eggs, and cull chicks (P > 0.05). However, hatch of transfer was significantly reduced by T2 compared to T1, T3, and T4 (P < 0.001). T2 had significantly higher percentages of late dead embryos and pips when compared to the other treatments (P = 0.002 and P < 0.001, respectively). Chicks hatched from T2 were not vigorous and, thus, not used for the grow-out trial. No differences were observed for growth performance characteristics for any of the treatments (P > 0.05). For bacterial enumeration, the ileum had equal or fewer bacterial counts for T3 and T4 when compared to T1 on most sampling days, except on d21 where T4 had higher aerobic and coliform counts (P ≤ 0.0001). For the ceca, T3 and T4 had equal or fewer bacterial counts than T1 on every sampling day (P ≤ 0.0001). These data demonstrate that not all B. subtilis evaluated are detrimental to hatchability, but rather, serotype dependent. In addition, different B. subtilis serotypes can modify the intestinal microflora with potential to reduce pathogenic bacteria present in young broiler, without impacting overall performance.

Effects of in ovo probiotic administration on the incidence of avian pathogenic Escherichia coli in broilers and an evaluation on its virulence and antimicrobial resistance properties.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, which has been traditionally controlled by the prophylactic in-feed supplementation of antibiotics. However, antibiotics are being removed from poultry diets owing to the emergence of multidrug-resistant (MDR) bacteria. Therefore, alternatives to control APEC are required. This study aimed to evaluate the effects of in ovo inoculation of probiotics on the incidence of APEC in broilers and evaluate the virulence and antimicrobial resistance properties of the APEC isolates. On embryonic day 18, 4 in ovo treatments (T) were applied: T1 (Marek's vaccine [MV]), T2 (MV and Lactobacillus animalis), T3 (MV and Lactobacillus reuteri), and T4 (MV and Lactobacillus rhamnosus). A total of 180 male broilers per treatment were randomly placed in 10 pens. The heart, liver, spleen, and yolk sac were collected on day 0, 14, 28, and 42. Presumptive E. coli isolates were confirmed by real-time PCR. The positive isolates were screened for the APEC-related genes (iroN, ompT, hlyF, iss, and iutA), and E. coli isolates containing one or more of these genes were identified as APEC-like strains. A total of 144 APEC-like isolates were isolated from 548 organ samples. No differences (P > 0.05) among treatments were observed for the incidence of APEC-like strains in all organs when averaged over sampling days. However, when averaged over treatments, the incidence in the heart, liver, and yolk sac was different among sampling days; a significant increase was observed in these organs on day 14 compared with day 0. Twenty-five antimicrobial resistance genes were evaluated for all APEC-like isolates, and 92.4% of the isolates carried at least one antimicrobial resistance gene. Thirty-seven isolates were then selected for antimicrobial susceptibility testing; MDR strains accounted for 37.8% of the isolates. In conclusion, the in ovo inoculation of a single probiotic strain did not confer protection against APEC strains in broilers. The high prevalence of MDR isolates indicates that further research on antibiotic alternatives is required to prevent APEC infections in broilers.

Complete genome sequence of multidrug-resistant avian pathogenic Escherichia coli strain APEC-O2-MS1170.

OBJECTIVES: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, one of the leading causes of mortality and morbidity associated with significant economic losses in the poultry industry. This study aimed to determine antimicrobial resistance and to characterise the genome sequence of a multidrug-resistant (MDR) APEC strain isolated from a broiler chicken. METHODS: Strain APEC-O2-MS1170 was isolated from the broiler yolk sac of a 14-day-old broiler. Antimicrobial susceptibility testing was performed using a Sensititre National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative panel. Whole-genome sequencing was performed using both the long-read sequencing approach with a Nanopore GridION sequencer and short-read sequencing with an Illumina HiSeq X-Ten sequencer to obtain a complete scaffold of the genome and an accurate sequence. RESULTS: The genome size of strain APEC-O2-MS1170 is 4,993,909 bp with a GC content of 50.7% and 4,651 protein-coding sequences. Public databases were used to identify the virulence-associated gene and antimicrobial resistance gene cargo. Plasmid comparison showed that pAPEC-O2-MS1170-R is a large multidrug resistance IncB/O/K/Z plasmid, while pAPEC-O2-MS1170-ColV shares homology with the APEC ColV virulence plasmid. CONCLUSION: The genome sequence of APEC-O2-MS1170 provides valuable information on resistance mechanisms and virulence characteristics of pathogenic E. coli as well as information for tracing the potential spread of this MDR strain.

In ovo inoculation of an Enterococcus faecium-based product to enhance broiler hatchability, live performance, and intestinal morphology.

Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium-based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 µL of Marek's vaccine (MV), 2) MV and 1.4 × 105 cfu GH/50 µL, 3) MV and 1.4 × 106 cfu GH/50 µL, 4) MV and 1.4 × 107 cfu GH/50 µL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 105 and 107 GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 107 GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 107 GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 105 and 107 GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study.

Evaluating bacterial colonization of a developing broiler embryo after in ovo injection with a bioluminescent bacteria.

In ovo injection of probiotics has been of interest for achieving early health benefits. However, there is limited research demonstrating where bacteria could migrate within the embryo after injection. The objective of this study was to evaluate bacterial colonization or migration after in ovo injection of broiler embryo with bioluminescent Escherichia coli. Injection using 106 CFU/mL nonpathogenic E. coli was applied to amniotic and air cell regions on day 18 of incubation. On days 18, 19, 20, and 21 the amnion, skin, lung, gastrointestinal tract (GIT), bursa, and spleen were collected. On day 21, the GIT was separated into crop, duodenum, jejunum, ileum, and ceca sections. All tissues were visualized using anin vivo imaging system to confirm the presence of bioluminescent E. coli. Samples were homogenized, 10-fold serially diluted, and spread onto appropriate agar to determine bacterial loads in all tissues. Results indicated that eggs injected into the amnion had significantly high numbers of E. coli cells in all tissues compared to air cell injected and control treatments 2 h post-injection (P < 0.0001). E. coli was also found on the lungs, spleen, and bursa of eggs injected either in the amnion or air cell (P < 0.05). Results indicated that in ovo injection into the amnion was more efficient than air cell injection, yielding a higher bacterial concentration in the evaluated tissues, specifically the ileum and ceca. Future research using bioluminescent probiotic bacteria may establish sites of preference for different probiotics leading to site-specific application that can maximize their overall impact when in ovo injected.