Final report for LNE21-425R
Project Information
Background: Poultry meat is often implicated in foodborne Salmonellosis in the US, prompting the need for effective control strategies. Hatchlings are particularly vulnerable to Salmonella colonization, with their contamination potentially leading to pathogen spread on farms. Thus, interventions targeting Salmonella in hatchlings can reduce the risk of dissemination in the flock. Hence, we evaluated the application of probiotics to i) disinfect hatching eggs, ii) incorporation in water replacer during chick transport and ii) in-feed to control Salmonella in developing embryos, hatchlings, and grow-out birds.
Hypothesis: Probiotic supplementation along the hatchery to farm continuum will significantly reduce i) delivery of Salmonella-positive chicks to the grow-out farms and ii) subsequent Salmonella dissemination in the flock, thereby improving poultry meat safety.
Materials & methods: Hatching Ross 308 eggs (n=820) inoculated with Salmonella Enteritidis (SE; ~7 log CFU/egg) were sprayed with PBS (Control), Lactobacillus rhamnosus NRRL-B-442 (LR; ~8 log CFU/egg), Lactobacillus paracasei DUP 13076 (LP; ~8 log CFU/egg) or peracetic acid (PAA; 0.4%). Eggs including internal contents were sampled throughout incubation to determine SE populations. Once hatched, chicks were held in transport boxes containing water replacers with/without probiotics for 24 h to simulate transport. Then, chicks were transferred to floor pens and reared for 10 days on starter ration with/without probiotics. At different times, chicks were sacrificed to determine SE populations in the liver, spleen, and caecum. The experiments were set out as a completely randomized design and data analysed using R with significance set at P≤0.05.
Results & Conclusion: LR, LP and PAA application significantly reduced SE populations on egg surface when compared to the control (P<0.05). Although PAA was effective in surface decontamination, trans-shell migration of SE was significantly higher compared to LR and LP. On day 18 and 20 of incubation, SE load in the embryonic gut was significantly lower in the probiotic groups (~1.75 log CFU/g) when compared to control and PAA (~3.5 log CFU/g). Similar reductions in SE populations were observed in chicks at hatch and during transport simulation. Further, in-feed supplementation with LR and LP resulted in significant reduction in SE populations in the grow out birds (> 2 log CFU/g) when compared to the control and PAA groups. These findings demonstrate that application of a multi-hurdle probiotic based approach along the hatchery to farm pipeline can help control SE in broilers and thereby improve meat safety.
Outreach: Results of the study were shared with producers and stakeholders in the region through two in-person workshops (April 26, 2023, and May 24, 2024). Topics related to sustainable poultry production, poultry diseases, beneficial bacteria in poultry production, the future of sustainable poultry production, and fostering sustainable poultry innovation in Connecticut through Academic-Community Collaboration were discussed.
In the pre-harvest environment, the population most vulnerable to Salmonella colonization are the hatchlings. Reducing Salmonella colonization at this stage will reduce subsequent transmission and pathogen prevalence in the flock. Therefore, our goal is to control Salmonella in broilers along the hatchery to farm continuum. We aim to do this by supplementing probiotics i) in-ovo to hatching eggs, ii) in water replacement during transport to hatchlings and iii) as an on-farm supplement to chicks. Ultimately, inclusion of these pre-harvest hurdles will help improve food safety while promoting the viability and sustainability of the enterprise.
Need and Justification: Poultry meat is one of the leading causes of foodborne Salmonellosis in the US. Thus, to promote poultry meat safety, the USDA-FSIS announced new performance standards for controlling Salmonella on raw poultry. However, despite advancements in food safety, Salmonella-contaminated poultry meat continues to pose a significant threat to public health. This national trend is also reflected in the Salmonella outbreaks in the Northeast. Therefore, there is an ongoing need to develop effective and feasible antimicrobial interventions to control Salmonella in chickens. Towards this, most pre-harvest Salmonella-control strategies primarily target breeder flocks and grow-out birds. However, the population most vulnerable to Salmonella colonization are the hatchlings. Contamination at this stage can result in horizontal spread of the pathogen on the farm. Therefore, early interventions to reduce Salmonella in hatchlings will help reduce the subsequent risk for pathogen dissemination in the flock.
Approach: We hypothesized that probiotic (Lactobacillus rhamnosus - LR and Lactobacillus paracasei - LP) administration along the hatchery to farm continuum will significantly reduce the delivery of Salmonella-positive chicks and control subsequent Salmonella dissemination at the grow-out farm. Specifically, we investigated the efficacy of in-ovo probiotic spray application and incorporation in water replacer to reduce Salmonella population in developing embryos and hatchlings. Further, to control Salmonella in the grow-out birds, in-feed probiotic supplementation was provided to the birds following hatch. Through these approaches our goal was to promote probiotic establishment in the growing chick gut and prevent Salmonella colonization. Also, working through our stakeholder advisory committee and our extension partners we shared our results and obtained feedback from poultry producers in the region.
Results: During incubation (day 0 – day 18): The results of our study demonstrated that approximately 3 log CFU/ml of SE was recovered from egg surface following inoculation and spray treatment in all treatment samples on Day 0. However, on all following sampling days, SE population in the treatment samples was significantly reduced compared to control (P<0.05). Specifically, with the surface counts, spray application of LR, LP, and PAA reduced SE populations to below detection limit (< 1 log CFU/ml) by day 3 of incubation. It is significant to mention that although 0.3% PAA was able reduce SE populations on the shell surface, it was less effective in reducing SE population inside the egg when compared to LR and LP. This is significant since internalization of SE is an important source for contamination of the growing embryo and eventual colonization in the hatchling. Further, on day 18, ~80% of the eggs (internal contents) were positive for SE in the control group whereas SE was detected in only 20% of the eggs in the probiotic treated groups. Besides SE counts, our study also revealed that the LR and LP could survive in significant numbers on the eggs throughout the study. Overall, these data demonstrate that in-ovo spray application of LR and LP could potentially be used to reduce SE in embryonated eggs.
Late incubation period and hatching (day 18 – Day 21): As seen during initial incubation, SE counts in the probiotic treated groups were consistently reduced on day 20 when compared to control and PAA. On day 20, we sampled the embryonic intestine and recovered ~ 3.5 log SE in the control while we only less than 1.5 log was recovered in the probiotic groups. Similarly in the hatchlings, chicks from the LR and LP group had significantly lower SE counts in the liver, spleen and ceca when compared to control and PAA. Specifically, we recovered ~2.4, 3.0 and 3.5 log of SE from the LR, LP and PAA/Control group, respectively. Similarly, we only recovered 2.5 log of SE from the ceca of the LR group, while we recovered 2.7 log in the ceca of the control and PAA birds. This clearly demonstrates that application of probiotics to the fertile eggs significantly reduce pathogen populations at hatch.
Shipping simulation and grow-out period: Once hatched, birds were placed in transport boxes to simulate shipping and provided with water replacer with (LR/LP) or without probiotics (control/PAA). The treatment assignment followed the in ovo treatment application. These birds were sampled every twelve hours to determine SE populations. As seen previously, probiotic treatments led to a significant reduction in pathogen numbers in the liver, spleen and ceca when compared to control and PAA. For instance, in the spleen, pathogen counts were below the detection limit in the LP group while ~2.22 log of SE was recovered from the control and PAA groups. In addition, we did not observe any significant difference between the control and PAA groups through out the grow-out period. However, LP and LR consistently reduced SE populations in the chicks during the grow-out period. For instance, while SE counts were below the detection limit in the liver of the grow-out birds in the LP and LR groups, ~1.5 log of SE was retrieved from the control and PAA groups. Moreover, we also observed significant reduction in cecal colonization of SE in the probiotic groups. On day 10, post-hatch, supplementation with LP and LR resulted in a greater than 1.5 log reduction in cecal SE populations when compared to control and PAA.
Conclusions: Overall, our results demonstrate the ability of the select probiotic strains to control SE in developing embryos, hatchlings, chicks during transport and during grow-out. Hence this could serve as a comprehensive approach to reduce SE colonization in broilers, thereby resulting in decreased pathogen shedding and controlling SE in the pre-harvest environment. In summary, application of these probiotics could serve as a multi-hurdle, user-friendly approach that can be integrated with current management practices to control SE along the pre-harvest production pipeline while supporting broiler performance.
Cooperators
Research
We hypothesize that probiotic supplementation along the hatchery to farm continuum will significantly reduce i) delivery of Salmonella-positive chicks to the grow-out farms and ii) subsequent Salmonella dissemination in the flock, thereby improving poultry meat safety. Further, we hypothesize that in addition to their direct antimicrobial effect, probiotic supplementation in-ovo and in water replacer will help initiate the establishment of desirable microbes in the neonatal gut thereby promoting pathogen exclusion.
For the study, fertile eggs were obtained from Aviagen from 40-45-week-old layers. Following receipt, any damaged eggs were removed. The eggs were then weighed, and spot inoculated with a five-strain mix of SE. These SE strains were pre-induced for resistance to nalidixic acid (NA; 50 µg/ml) to enable selective enumeration. To prepare the inoculum, the individual SE strains were grown in tryptic soy broth containing nalidixic acid. The overnight cultures were washed twice in phosphate buffered saline (PBS; pH 7.4) and the approximate bacterial count in each culture was determined spectrophotometrically. Equal portions from each of the five strains were then combined to make the pathogen cocktail. The bacterial population in the five-strain mixture was determined by plating 0.1-ml portions of appropriate dilutions on Xylose Lysine Deoxycholate Agar (XLD) with NA (XLD-NA) followed by incubation at 37°C for 48 h. Appropriate dilutions of the five-strain mixture in PBS was used to obtain the desired level of inoculum (~8 log CFU/ml). The fertile eggs were then spot inoculated with 100 µl of SE cocktail on the broad end. Following inoculation, the eggs were held at room temperature for an hour to allow for the inoculum to dry. Eggs (n = 8-10) were randomly sampled to determine SE population on eggs following inoculation. In addition to sampling eggshell, the internal contents were also sampled to determine SE counts.
For the antimicrobial treatment, probiotic cultures [Lactobacillus rhamnosus (LR) and Lactobacillus paracasei (LP)] were grown in de Mann Rogosa Sharpe broth (MRS) overnight and washed in PBS as previously described. The washed probiotic cultures were used to treat the eggs (8 log CFU/egg) and the bacterial count in the inoculum was enumerated following plating. In addition to the different probiotics, we also tested the application of 0.3% peracetic acid (PAA) to simulate commercial egg disinfection. For egg treatment, inoculated eggs were randomly assigned to one of four treatments including i) Control (only SE; no antimicrobial treatment), ii) LP (SE + LP application), iii) LR (SE + LR application), iv) PAA (SE + 0.3% PAA). For treatment application, eggs in each group were placed on egg trays and manually sprayed with the respective treatments. Following spray application, eggs (n = 6/group) were sampled to determine SE and probiotic populations on eggs surface and internal contents. The remaining eggs in each group were then placed in separate hovabators and held at 37.8°C and 55% RH for 18 days. At each sampling time (Day 0, 1, 3, 10, 14 and 18), six eggs from each group were randomly sampled to enumerate surviving SE and probiotic populations.
On day 18, the eggs were transferred from the incubator to the hatcher and held until hatch. Eggs were randomly sampled on day 20 to determine SE translocation into the developing intestine. Further, once hatched, a sub-set of hatchlings were sampled to enumerate pathogen and probiotic populations in the ceca, liver, and spleen. The remaining birds were placed in shipping boxes and held at held at 28-30°C for 24 h in the dark in a climate-controlled room to simulate shipping. Fifteen chicks were placed in each box containing water replacer with the different probiotic treatments (8 log CFU/ml). Hatchlings from the control and PAA group received water replacer without any probiotics. During this holding time, birds were sampled every 12h to assess the effect of probiotics in water replacer on SE population in the chicks. Following the 24 h shipping simulation, remaining chicks were transferred to floor pens. Based on the in-ovo and water replacer treatment regimen, chicks from the control and PAA group were provided regular feed. While chicks from the LP and LR group were provided with feed supplemented with the respective probiotics (8 log CFU/kg of feed). The chicks were sacrificed on day 1, 4, 7 and 10 to determine SE populations in the liver, spleen, and cecum.
During incubation (day 0 – day 18): The results of our study demonstrated that approximately 3 log CFU/ml of SE was recovered from egg surface following inoculation and spray treatment in all treatment samples on Day 0. However, on all following sampling days, SE population in the treatment samples was significantly reduced compared to control (P<0.05). Specifically, with the surface counts, spray application of LR, LP, LR-LP and PAA reduced SE populations to below detection limit (< 1 log CFU/ml) by day 3 of incubation. A similar reduction was observed with the internal shell membrane and internal contents. It is significant to mention that although 0.3% PAA was able reduce SE populations on the shell surface, it was less effective in reducing SE population inside the egg when compared to LR and LP. This is significant since internalization of SE is an important source for contamination of the growing embryo and eventual colonization in the hatchling. Further, on day 18, ~80% of the eggs (internal contents) were positive for SE in the control group whereas SE was detected in only 20% of the eggs in the probiotic treated groups. Besides SE counts, our study also revealed that the LR and LP could survive in significant numbers on the eggs throughout the study. Overall, these data demonstrate that spray application of LR and LP could potentially be used to reduce SE in embryonated eggs. Moreover, they also exert an inhibitory effect on SE translocation and survival in the internal contents.
Late incubation period and hatching (day 18 – Day 21): As seen during initial incubation, SE counts in the probiotic treated groups were consistently reduced on day 20 when compared to control and PAA. On day 20, we sampled the embryonic intestine and recovered ~ 3.5 log SE in the control while we only less than 1.5 log was recovered in the probiotic groups. Similarly in the hatchlings, chicks from the LR and LP group had significantly lower SE counts in the liver, spleen and ceca when compared to control and PAA. Specifically, we recovered ~2.4, 3.0 and 3.5 log of SE from the LR, LP and PAA/Control group, respectively. Similarly, we only recovered 2.5 log of SE from the ceca of the LR group, while we recovered 2.7 log in the ceca of the control and PAA birds. This clearly demonstrates that application of probiotics to the fertile eggs significantly reduce pathogen populations at hatch.
Shipping simulation and grow-out period: Once hatched, birds were placed in transport boxes to simulate shipping and provided with water replacer with (LR/LP) or without probiotics (control/PAA). The treatment assignment followed the in ovo treatment application. These birds were sampled every twelve hours to determine SE populations. As seen previously, probiotic treatments led to a significant reduction in pathogen numbers in the liver, spleen and ceca when compared to control and PAA. For instance, in the spleen, we did not recover any countable colonies in the LP group while ~2.22 log of SE was recovered from the control and PAA groups. In addition, we did not observe any significant difference between the control and PAA groups through out the grow-out period. However, LP and LR consistently reduced SE populations in the chicks during the grow-out period. For instance, while we did not recover any countable SE colonies in the liver of the grow-out birds in the LP and LR group, ~1.5 log of SE was retrieved from the control and PAA groups. Moreover, we also observed significant reduction in cecal colonization of SE in the probiotic groups. On day 10, post-hatch, supplementation with LP and LR resulted in a greater than 1.5 log reduction in cecal SE populations when compared to control and PAA.
Conclusions: Overall, our results demonstrate the ability of the select probiotic strains to control SE in developing embryos, hatchlings, chicks during transport and during grow-out. Hence this could serve as a comprehensive approach to reduce SE colonization in broilers, thereby resulting in decreased pathogen shedding and controlling SE in the pre-harvest environment. In summary, application of these probiotics could serve as a multi-hurdle, user-friendly approach that can be integrated with current management practices to control SE along the pre-harvest production pipeline.
Education & Outreach Activities and Participation Summary
Educational activities:
Participation Summary:
With assistance from Dr. Upadhyaya, we engaged farmers to identify their needs (needs assessments) and interests in participating in poultry workshops. Based on this we organized a poultry outreach program on April 26, 2023 to share project results and obtain feedback from poultry producers in the state. Topics related to sustainable poultry production, poultry diseases, beneficial bacteria in poultry production and future of sustainable poultry production were discussed. In addition, results of the study were presented to extension educators, regulators and academics at the Poultry Science Association Annual meeting in July, 2023.
The results of the study were presented to extension educators, regulators, industry and academics at the
- Kosuri P, Reddyvari R, Kanike E, Muttathukonam SH, Amalaradjou MA. From hatching eggs to grow-out birds: Probiotic interventions to control Salmonella Enteritidis along the broiler production continuum. 2024 PSA Annual Meeting, July 15-8, 2024.
- Kosuri P, Muttathukonam SH, Reddyvari R, Gao M, Ren Y, Amalaradjou MA. 2023. Hatching egg sanitation using probiotics to control Salmonella Enteritidis. 2023 PSA Annual Meeting, July 10-13, 2023.
Learning Outcomes
sustainable poultry production, poultry diseases, beneficial bacteria in poultry production, the future of sustainable poultry production