In-ovo and Early Probiotic Supplementation to Control Salmonella in Broilers

Progress report for LNE21-425R

Project Type: Research Only
Funds awarded in 2021: $150,000.00
Projected End Date: 08/01/2024
Grant Recipient: University of Connecticut
Region: Northeast
State: Connecticut
Project Leader:
Mary Anne Amalaradjou
University of Connecticut
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Project Information

Summary:

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 hypothesize that probiotic 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 will investigate the efficacy of in-ovo probiotic spray application and incorporation in water replacer to reduce Salmonella population in hatchlings. Further, to promote Salmonella exclusion, we will supplement probiotics as an on-farm supplement to chicks. Through these approaches we aim to promote probiotic establishment in the growing chick gut and prevent Salmonella colonization. This study is based on our previous data demonstrating the ability of probiotics [Lactobacillus rhamonsus (LR) and Lactobacillus paracasei (LP)] to reduce Salmonella population on eggs and reduce its cecal colonization. Additionally, our study demonstrated the growth promoting effect of these strains in chickens. Thus, it is expected that these probiotics will help control Salmonella in broiler chicks while improving their performance. In the current reporting period, our efforts were focussed around identifying candidate probiotic strains and determining their ability to reduce SE in-ovo.

For the study, fertile eggs were obtained from the UConn poultry farm from 25–30-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) or Brain Heart Infusion broth [BHI; Hafnia alvei (HA)] 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 five treatments including i) Control (only SE; no antimicrobial treatment), ii) LP (SE + LP application), iii) LR (SE + LR application), iv) HA (SE + HA application), v) LR-LP (SE + application of LP and LR cocktail) and vii) 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.

Farmer engagement and outreach:  To ensure the feasibility and adoptability of our proposed interventions, we will work with farmers throughout the research process to share our preliminary findings and obtain feedback. Further, based on the feedback received from the local farming community, we will develop and evaluate a probiotic-based multi-hurdle approach to controlling Salmonella from hatchery to farm.  In addition, working with our project advisory committee, we will reach out to poultry producers in the Northeast to assess their needs and perceptions towards integrating probiotics in their current management practices.  We will also conduct workshops and demonstrations for stakeholders to update them of the project outcomes. Ultimately, we expect to develop a probiotic-based comprehensive, effective and adoptable strategy to improve meat safety while promoting poultry performance. This in turn, is expected to help promote the sustainability, viability, competitiveness and economic efficiency of small producers in the region.

Project Objective:

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.

Introduction:

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). Further, although HA was effective in reducing SE populations, they also exhibited an inhibitory effect on embryonic development. Hence this treatment was removed from further trials. 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. In the next reporting period, we plan to carry out the hatchling trials and engage with the advisory board and stakeholders to share our results and obtain their input/feedback.      

Research

Materials and methods:

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.

Research results and discussion:

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.

Research conclusions:

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.

Participation Summary

Education & Outreach Activities and Participation Summary

Educational activities:

2 Webinars / talks / presentations

Participation Summary:

15 Farmers participated
5 Number of agricultural educator or service providers reached through education and outreach activities
Outreach description:

A poultry outreach program was organized in CT 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. A follow-up workshop is planned for April 2024. In addition, results of the study were presented to extension educators, regulators and academics at the Poultry Science Association Annual meeting in July, 2023.

 

Project Outcomes

1 New working collaboration
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.