Chemical Treatment to Remove or Prevent Salmonella Contamination of Poultry Feed

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Chemical treatment to remove or prevent Salmonella contamination of poultry feed" by Cawthraw, S. et al., is a good study looking at the efficacy of formaldehyde vs organic feed acids on reducing Salmonella contamination in livestock feed.

The manuscript is well laid out and the authors have nicely described their study along with the observed results. The introduction is nicely written with adequate references. The authors have done an excellent job describing the methods. As well, the results are concisely described and statistical significance has been indicated where applicable. Additionally, the discussion compares the authors findings with what has been described in the literature.

I have a few minor comments which I would like the authors to address:

1. I would like the authors to review bacterial nomenclature. For example, on L110, "S. Enteritidis" should be written out in full the first time as "Salmonella enterica subspecies enterica serovar Enteritidis" the first time and all subsequent references can remain as "S. Enteritidis" or "S. Infantis". Similarly, on L171, "Salmonella" should be italicized. Please review the entire manuscript to ensure consistency with bacterial nomenclature.
2. The Materials and Methods are very nicely described. I would like to suggest that the authors include references where applicable to further strengthen this section using statements like "as described previously [reference]".
3. There are various references to tables throughout the manuscript but most of them do not have a number and this makes it very difficult for the reader to determine which table the authors are referring to. For example, on L144, the authors reference a "table" without an actual number. There are several instances where this is the case. Please review to ensure that all table numbers are included where applicable.
4. On L165 and L173, there are some reference errors (this appears to be a technical error). Please review this and the entire manuscript to ensure that all references are included where appropriate. 

Author Response

Thank you for taking the time to review our paper and for providing your constructive comments. We have hopefully addressed each of the concerns in an acceptable manner. Our responses are detailed below.

All reviewers made comments that relate to errors that seem to have occurred post-upload, possibly in the conversion to pdf (comments relating to formatting, references and in-text table numbering). I have now uploaded a revised word doc with changes showing and a pdf version that seems to be correct.

1. I would like the authors to review bacterial nomenclature. For example, on L110, "S. Enteritidis" should be written out in full the first time as "Salmonella enterica subspecies enterica serovar Enteritidis" the first time and all subsequent references can remain as "S. Enteritidis" or "S. Infantis". Similarly, on L171, "Salmonella" should be italicized. Please review the entire manuscript to ensure consistency with bacterial nomenclature. See above
2. The Materials and Methods are very nicely described. I would like to suggest that the authors include references where applicable to further strengthen this section using statements like "as described previously [reference]". Added a statement (l 115)
3. There are various references to tables throughout the manuscript but most of them do not have a number and this makes it very difficult for the reader to determine which table the authors are referring to. For example, on L144, the authors reference a "table" without an actual number. There are several instances where this is the case. Please review to ensure that all table numbers are included where applicable. See above
4. On L165 and L173, there are some reference errors (this appears to be a technical error). Please review this and the entire manuscript to ensure that all references are included where appropriate. See above

Reviewer 2 Report

Comments and Suggestions for Authors

Chemical treatment to remove or prevent Salmonella contamination of poultry feed

Summary

Salmonella contamination is a challenge for livestock production and food safety which effects the health of livestock and the eventually the human beings in the food chain. Feed used in poultry livestock is contaminated at various stages such as sourcing, processing , storage and temperature, equipment used and environmental reservoirs. Thermal processing can reduce the contamination but cannot eradicate it completely .Formaldehyde based treatments have been widely used because of its antimicrobial property but in UK, regulatory bans have compelled the industry towards alternatives such as organic acids (OA).Previous studies showed OA as less efficient compared to formaldehyde at standard inclusion rates. The present study evaluates the anti-Salmonellae efficacy of commercially used organic acids and formaldehyde products applied to poultry feed. Unpelleted, heat –treated poultry feed is used with three field isolates of Salmonella. With low and moderate decontamination, the study compares prevention (treatment of feed before contamination with Salmonella) and decontamination (treatment after contamination with Salmonella) modes. Neutralizers such as histidine were added to prevent masking effects of formaldehyde and both OA and formaldehyde products were tested according to the manufacturer-recommended levels.

At moderate contamination levels of 10²-10³ CFU/25g, none of the OA products reduced or prevented Salmonella recovery. Among all the OA products tested, OA1 exhibited closer performance to formaldehyde at lower inclusion rates, although complete elimination occurred only in decontamination mode .These results suggests OA products may have limited application at naturally low contamination conditions. With formaldehyde, at higher inclusion rate of 3.0 ml/Kg, prevention was more effective than decontamination whereas at lower inclusion rates of 1.0 ml/Kg decontamination outperformed the prevention mode slightly better. OA products showed a similar trend at lower contamination, though overall efficacy is modest. Masking effect in formaldehyde treated samples was reduced by incorporating histidine during pre-enrichment to increase the Salmonellae recovery. All the three Salmonella isolates survived well in feed but at low moderate dose of 3X10²CFU, S. 13, 23:i:- was more susceptible than other two isolates. At low dose, no difference was observed.

In conclusion, formaldehyde based treatment is more effective than OA –based products at recommended inclusion rates. Some OA products reduce salmonella at low contamination levels which are natural levels in poultry farms. The authors stated that OA products may be still relevant in the real field but their performance is inferior to formaldehyde based products.

 

Major comments

1. Although the feed samples were obtained from the commercial feed manufacturer and the experiments were carried out in controlled conditions, it would be ideal if the study can replicate real field conditions. Because the authors stated, naturally low contamination levels are often observed in poultry farms. However, the authors did not replicate real environmental parameters such as fluctuating temperatures, humidity, percent of organic matter and biofilms. The contamination may also occur from equipment, conveyors and augers or wildlife contamination. Additionally, most broiler feeds are pelleted and testing only unpelleted feeds limit applicability.
2. The feed was only heat treated but not pelleted. Pelleting is one of the most important steps affecting the Salmonella survival, moisture redistribution, penetration of chemicals, and risk of recontamination. It is unclear why did the authors not incorporate pelleted feed into the experimental design.
3. Throughout the manuscript the authors were referring to heat–treated feed yet no temperature or time duration is provided. This is important because pelleting temperatures vary widely and also effects Salmonella survival.  Residual heat also impacts the volatility of formaldehyde. Given that different mills operate at different temperatures, the absence of specific heat –treatment parameters limit the reproducibility.
4. All experiments were carried out at room temperatures but in reality, the feed bins can reach to temperatures of 40-50 ºC in summer and can freeze in winter. Again, these temperatures effect the Salmonella survival, organic acid dissociation, moisture migration and formaldehyde evaporation.
5. The authors mentioned natural contamination differs in stress adaptation, clustering of cells, microenvironments, ingredient specific survival patterns and focal “hot spots” of contamination. Whereas the in-vitro inoculation produces uniform, freshly cultured cells, which behave differently from naturally stressed Salmonella in real feed mills.
6. Recontamination of feed can happen in coolers, conveyors, trucks, farm bins or wild birds or rodents. However, the study only tests initial contamination and treatment but not the recontamination dynamics.
7. Moisture is a major determinant for Salmonella survival, but the study uses a single feed moisturizer level, did not teat humidity conditions
8. Only qualitative culture rather than CFU enumeration was used in this study. This means 90% of reduction and 1% of reduction looks same if the sample is positive. Subtle differences between OA products are masked because of qualitative results and log- reduction comparisons cannot be accurately made.
9. Only one turkey grower feed formulation was used in this study yet the feed from different sources differ in ingredient type, fat content and particle size. Comparing different feeds will allow for more evaluation of feed matrix effects across different feed types.
10. All the experiments were limited to 24hrs or 72hrs, but the feed usually sits in the mills for weeks or months sometimes. Chemical efficacy and Salmonella survival may change over time.
11. Only three serovars were tested despite the fact that many additional serovars are commonly encountered in most of the feed mills such as Salmonella typhimurium, S. kentucky, S. heidelberg How representative is these three serovars to natural contamination patterns?

Minor comments

1. The citations referenced in lines 165 and 173 could not be referenced Please verify and update.
2. When referring to tables in the text, please include the corresponding table number. For example, see the line 217.

Author Response

Thank you for taking the time to review our paper and for providing your constructive comments. We have hopefully addressed each of the concerns in an acceptable manner. Our responses are detailed below.

Thank you for these constructive comments. Nos. 1, 2, 4, 5, 6, 7, 9, 10 and 11 are all valid criticisms but resources did not permit a study incorporating more variables. As presented, the work involved almost 2000 separate tests. In light of the comments, a couple of lines have been added to the discussion to highlight the limitations of the work (l275-279). Other comments are addressed below 

1. Although the feed samples were obtained from the commercial feed manufacturer and the experiments were carried out in controlled conditions, it would be ideal if the study can replicate real field conditions. Because the authors stated, naturally low contamination levels are often observed in poultry farms. However, the authors did not replicate real environmental parameters such as fluctuating temperatures, humidity, percent of organic matter and biofilms. The contamination may also occur from equipment, conveyors and augers or wildlife contamination. Additionally, most broiler feeds are pelleted and testing only unpelleted feeds limit applicability.
2. The feed was only heat treated but not pelleted. Pelleting is one of the most important steps affecting the Salmonella survival, moisture redistribution, penetration of chemicals, and risk of recontamination. It is unclear why did the authors not incorporate pelleted feed into the experimental design.
3. Throughout the manuscript the authors were referring to heat–treated feed yet no temperature or time duration is provided. This is important because pelleting temperatures vary widely and also effects Salmonella survival.  Residual heat also impacts the volatility of formaldehyde. Given that different mills operate at different temperatures, the absence of specific heat –treatment parameters limit the reproducibility. Our work was done with post-heat treatment product therefore we do not feel this information is relevant to our laboratory-based study. All our tests were run at ambient/room temperature (barring media incubations), so would be reproducible.
4. All experiments were carried out at room temperatures but in reality, the feed bins can reach to temperatures of 40-50 ºC in summer and can freeze in winter. Again, these temperatures effect the Salmonella survival, organic acid dissociation, moisture migration and formaldehyde evaporation.
5. The authors mentioned natural contamination differs in stress adaptation, clustering of cells, microenvironments, ingredient specific survival patterns and focal “hot spots” of contamination. Whereas the in-vitro inoculation produces uniform, freshly cultured cells, which behave differently from naturally stressed Salmonella in real feed mills.
6. Recontamination of feed can happen in coolers, conveyors, trucks, farm bins or wild birds or rodents. However, the study only tests initial contamination and treatment but not the recontamination dynamics.
7. Moisture is a major determinant for Salmonella survival, but the study uses a single feed moisturizer level, did not teat humidity conditions
8. Only qualitative culture rather than CFU enumeration was used in this study. This means 90% of reduction and 1% of reduction looks same if the sample is positive. Subtle differences between OA products are masked because of qualitative results and log- reduction comparisons cannot be accurately made. This is a valid comment, but we chose a qualitative model because the contamination levels are lower than are typically used in decontamination/disinfection studies. In particular, Study 2 in our paper uses only 20-30cfu per 25 g of feed so log reduction comparisons would not be possible. It’s not unreasonable to assume that if a treatment is going to have an effect, it would be able to ‘deal with’ such a low number so one would expect no recovery even from enriched samples (as was observed for some products)
9. Only one turkey grower feed formulation was used in this study yet the feed from different sources differ in ingredient type, fat content and particle size. Comparing different feeds will allow for more evaluation of feed matrix effects across different feed types.
10. All the experiments were limited to 24hrs or 72hrs, but the feed usually sits in the mills for weeks or months sometimes. Chemical efficacy and Salmonella survival may change over time.
11. Only three serovars were tested despite the fact that many additional serovars are commonly encountered in most of the feed mills such as Salmonella typhimurium, S. kentucky, S. heidelberg How representative is these three serovars to natural contamination patterns? See above re testing extra strains. Also, 2 of the strains we used were feed/mill isolates and the other from a feed-pan system on farm (where feed can become contaminated even if clean when leaving the mill).

Minor comments

1. The citations referenced in lines 165 and 173 could not be referenced Please verify and update. See above – these aren’t actually citations, they refer to the paper’s tables.
2. When referring to tables in the text, please include the corresponding table number. For example, see the line 217. See general response at the beginning

 

Reviewer 3 Report

Comments and Suggestions for Authors

Lines 15 – 18:  The “Gap Statement” is useful but could more clearly specify why current evidence on organic acid efficacy is insufficient, particularly under realistic contamination levels.

Lines 19 – 23: The methodology description is generally clear; however, specifying the number of replicates would improve transparency.

Lines 35 – 41: The impact statement clearly highlights the relevance of the work. However, the claim that some alternatives may be “as efficacious as formaldehyde products” should be phrased cautiously since this appears to apply only under very low contamination conditions.

Lines 56 – 68: Consider shortening this paragraph to maintain focus on chemical treatments.

Lines 76 – 84: consider adding a short sentence clarifying the practical implications for feed manufacturers.

Lines 147 – 153: The description of prevention tests could be clarified by including a short schematic summary of the experimental timeline.

Table 2: Provide the commercial names or manufacturers of the tested products. Clarify whether the inclusion rates correspond exactly to manufacturer recommendations or represent a range within those recommendations.

Lines 159 – 162: clarify whether each replication used independently prepared bacterial cultures.

Lines 164 – 170: Consider simplifying this section or including a summary table of experimental combinations.

Lines 171 – 175: The description of neutraliser testing could be clarified by briefly explaining how the three neutralisation conditions differ conceptually.

Lines 195 – 203: Consider reporting percentages alongside counts to improve readability.

Table 3: Consider including percent recovery values to facilitate comparison across treatments.

Lines 360 – 368: The suggestion that some OA products may still reduce contamination risk in practice is reasonable but should be expressed cautiously.

Author Response

Thank you for taking the time to review our paper and for providing your constructive comments. We have hopefully addressed each of the concerns in an acceptable manner. Our responses are detailed below.

All reviewers made comments that relate to errors that seem to have occurred post-upload, possibly in the conversion to pdf (comments relating to formatting, references and in-text table numbering). I have now uploaded a revised word doc with changes showing and a pdf version that seems to be correct.

Lines 15 – 18:  The “Gap Statement” is useful but could more clearly specify why current evidence on organic acid efficacy is insufficient, particularly under realistic contamination levels. Line added

Lines 19 – 23: The methodology description is generally clear; however, specifying the number of replicates would improve transparency. Stated in the 1^st para of the ‘Assays’ section

Lines 35 – 41: The impact statement clearly highlights the relevance of the work. However, the claim that some alternatives may be “as efficacious as formaldehyde products” should be phrased cautiously since this appears to apply only under very low contamination conditions. Agreed, but we think our phrasing, including specifying low contamination levels and ‘may be…’ is suitably cautious.

Lines 56 – 68: Consider shortening this paragraph to maintain focus on chemical treatments. Edited a little, but we feel some discussion of heat-treatment is merited.

Lines 76 – 84: consider adding a short sentence clarifying the practical implications for feed manufacturers. Added a line (l 72-80)

Lines 147 – 153: The description of prevention tests could be clarified by including a short schematic summary of the experimental timeline. Fig 1a shows timelines (reference to the figure was missing – see comment at the top of response).

Table 2: Provide the commercial names or manufacturers of the tested products. Clarify whether the inclusion rates correspond exactly to manufacturer recommendations or represent a range within those recommendations. Added an extra column to the table

Lines 159 – 162: clarify whether each replication used independently prepared bacterial cultures. Added

Lines 164 – 170: Consider simplifying this section or including a summary table of experimental combinations. Workflow depicted in Fig 1.

Lines 171 – 175: The description of neutraliser testing could be clarified by briefly explaining how the three neutralisation conditions differ conceptually. Included in Fig 1 b

Lines 195 – 203: Consider reporting percentages alongside counts to improve readability. Added to the table

Table 3: Consider including percent recovery values to facilitate comparison across treatments. Added

Lines 360 – 368: The suggestion that some OA products may still reduce contamination risk in practice is reasonable but should be expressed cautiously. Agreed, but we don’t think there are any extravagant claims made in this paragraph.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made changes in accordance with my comments. I do not have any further comments. The paper can be accepted in the current form.