-->PONE-D-25-22875-->-->Comparison of thoracic ultrasonography (TUS), clinical respiratory scoring (CRS), and blood analysis to evaluate respiratory dysfunction in transported calves-->-->PLOS ONE

Dear Dr. van Dijk,

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Following a careful assessment by the editorial team and a group of expert reviewers, we have determined that the paper shows significant potential for publication. However, based on the reviewers' feedback, the manuscript requires MAJOR REVISIONS to meet the journal's standards. Please see the enclosed reviewer comments for detailed suggestions and specific actions required to improve the manuscript. We invite you to revise and resubmit the manuscript to us for further consideration. We look forward to receiving the revised manuscript.

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PLOS ONE

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-->1. When submitting your revision, we need you to address these additional requirements.-->--> -->-->Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at -->-->https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and -->-->https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf-->--> -->-->2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (a) methods of sacrifice, (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering.-->--> -->-->3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. “The blood data presented in this paper has previously been reported in two separate papers (attached as Paper 1 and Paper 2), and the CRS data has been reported in Paper 2 in a different format. However, the current paper is the first to incorporate TUS data, and it is the only one in which multiple methods are directly compared. This paper also uses different data analysis approaches and reporting structures, and therefore constitutes a distinct and independent publication.”  -->-->Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.-->--> -->-->4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.-->--> -->-->5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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Reviewers' comments:

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: TUS and CRS comparisons with blood are made despite blood being sampled on different days than clinical assessments (T2, T4, T5). This can lead to inconsistencies and should be addressed with caution or sensitivity analysis.

Clarify this limitation in the methods more explicitly and consider whether statistical correction or subgroup analysis might mitigate this mismatch.

Some values were excluded due to suspected lab errors (e.g., IgM, eosinophils). However, no mention is made of imputation or sensitivity analyses.

The kappa value (0.038) indicates no meaningful agreement, yet this is only discussed briefly. This result is central to the paper's conclusions

Grouping TUS score 4 into score 3:

• This collapsing may affect results significantly given the small number of TUS 4 observations. A sensitivity analysis keeping TUS 4 separate, or pooling with discussion of clinical relevance, would improve rigor.

Use more concise language in the abstract and results.

“...followed by a gradually decrease” → should be “a gradual decrease”

“...severe signs of respiratory disease” → could be simplified to “severe respiratory signs”

"The CRS system was based on the Wisconsin calf health scorer..." → should be “based on the Wisconsin Calf Health Scoring Chart”

Ensure past tense is used consistently in the methodology (e.g., "calves were scored" vs. "calves are scored")

Several sections (especially Discussion) contain overly long paragraphs. Breaking these into thematic blocks would improve clarity.

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This is a scientifically sound manuscript with good potential. However, I recommend a major revision focusing on:

• Statistical robustness and discussion of multiple comparisons.

• Clearer language and grammar corrections.

• Enhanced explanation of mismatched sample timing and potential biases.

• A clearer discussion of the CRS vs. TUS discordance and implications for field diagnosis.

Reviewer #2:  Comparison of thoracic 1 ultrasonography (TUS), clinical 2 respiratory scoring (CRS), and 3 blood analysis to evaluate 4 respiratory dysfunction in 5 transported calves

1 . L31 | "TUS scores (0: healthy to 3: severe consolidation)" – Incomplete scale; Table 2 shows scores up to 5. | Revise to: "TUS scores (0: healthy to 5: severe pneumonia)" and clarify in text that scores 4–5 were rare and collapsed into score 3 for analysis (L196–197). |

2 . L32 | "CRS (0: healthy to 3: severe clinical symptoms)" – Implies linear scale, but scoring is categorical. | Clarify: "CRS categories (0: healthy, 1: mild, 2: moderate, 3: severe)" to reflect ordinal nature.

3 . L35 "3 or 7 days post-arrival (T3)" – Introduces variability without justification. | State: "T3 was defined as 3 or 7 days post-arrival due to logistical constraints; analysis adjusted for time."

4 . L41 "Total immunoglobulins and IgG were lower for TUS of 3" – IgG is part of total Ig; cannot be lower unless others decrease. | Recheck calculation: Total Ig = IgA + IgG + IgM. If IgG ↓ and total Ig ↓, clarify if IgA/IgM compensate.

5 . L59 "Calves that are diseased pre-transport should not travel" – Assumes causality not tested. Temper: "Calves with clinical signs pre-transport may be at higher risk during transport."

6 . L65 "CRS has low sensitivity (0.27–0.64)" – No citation for these values in transport context. | Cite source specifically for transport or pre-transport settings (e.g., Donlon et al. 2023, Ref 21).

7 . L74 "TUS has sensitivities >0.85 and up to 0.94" – Cites [10,11], but [10] is subclinical, [11] lacks necropsy validation. Specify: "Sensitivities vary by population; in subclinical calves, Ollivett et al. [10] reported 0.85–0.94 vs. BAL, not necropsy."

8 . L89–94 Calves from farms and marts mixed without accounting for origin differences. Include "origin (farm vs. mart)" as a covariate in models or stratify analysis.

9 . L95–98 Describes cohort differences (sex, age, breed) but no statistical adjustment. Adjust models for cohort (C1 vs C2) or include as random effect.

10 . L105 "Calves from livestock mart assessed on day of transport" – Timing differs from farm calves (L104: one day pre). Standardize or acknowledge bias in pre-transport assessment timing.

11 . L122–124 All calves received antibiotics upon arrival – confounds disease progression. Acknowledge that metaphylaxis limits natural disease progression and affects TUS/CRS/blood interpretation.

12 . L130–133 TUS and CRS not performed on same day at T4/T5 (blood sampled 1–2 days apart). State: "Non-simultaneous sampling may reduce correlation; results interpreted with caution."

13 . L134–136 No blood at T3, but TUS/CRS done – limits temporal comparison. | Justify gap and avoid claiming integrated assessment at T3.

14 . L147 "Depth of 11cm" – Not validated for calf thorax; may miss deep lesions. Cite justification or note potential for missed consolidations.

15 . L153 "6-tier TUS" – Inconsistent with Table 2 (6-point scale) and text (0–3 used). Correct: "A 6-point TUS scale was used, but scores 4–5 were combined with 3 due to low frequency."

16 . L161–163 | Rectal temperature omitted at T1 and imputed at T4 – introduces bias. Exclude temperature from T1 CRS or use alternative scoring; do not impute without validation.

17 . L167–168 CRS categories based on sum 0–1=0, 2–3=1, etc. – unequal binning. Justify binning or cite Wisconsin system for this grouping.

18 . L177 "26ml blood" – excessive volume for young calves (~28d). Confirm volume is safe (<10% total blood volume); typical calf blood volume ~80 mL/kg. 26 mL from 55kg calf = ~0.5% – acceptable, but state safety.

19 . L182–183 Two hematology analyzers used (Advia 2120, XT-1800i) – may introduce batch effects. Perform cross-calibration or include analyzer as covariate in models.

20 . L187 Hb units converted (g/dL to mmol/L) – conversion factor not cited. Cite: "Hb converted using factor 0.6205 (mmol/L per g/dL) [Ref]."

21 . L195–196 One calf removed due to missing TUS – violates intention-to-treat. Report as missing data and use imputation or sensitivity analysis.

22 . L196–197 TUS 4 reclassified as 3 – alters disease severity. Report both ways: primary analysis with TUS 0–3, secondary with TUS 4 separate.

23 . L198–200 Outliers removed without statistical justification (e.g., >3 SD). Define outlier criteria (e.g., >3 IQR) and report number excluded.

24 . L207–212 "Minimal model" not defined; no random effects for repeated measures. Use mixed models with calf ID as random effect to account for repeated measures.

25 . L212 Residuals tested for normality – but no mention of transformation validation. Show Q-Q plots or Shapiro-Wilk results in supplement.

26 . L214–220 Different transformations per variable – but no consistency in back-transformation. Ensure all SEs and CIs are correctly back-transformed (L224–229 are correct; verify in tables).

27 . L222 Weighted kappa used – appropriate, but no confidence interval reported. Add 95% CI for kappa in text or figure.

28 . L241 "No calves died" – but mortality is relevant to BRD severity. Discuss survival bias: severe cases may have been treated early.

29 . L243 "67% had TUS ≥2" – implies disease, but no validation against gold standard. Use: "67% had ultrasound findings suggestive of lung pathology."

30 . L244–248 Location of lesions described, but not analyzed statistically. Report if right cranial lobe predominance is significant (e.g., chi-square).

31 . L257 "Chi-square; p=0.03" – Chi-square for transition frequencies? Inappropriate. Use McNemar’s test or generalized estimating equations (GEE) for paired ordinal data.

32 . L289 "Chi-square; p<0.01" – same error as L257. Replace with GEE or marginal model for longitudinal CRS changes.

33 . L307 "9 out of 14 variables changed" – cherry-picking; no adjustment for multiple testing. Apply FDR correction (e.g., Benjamini-Hochberg) and report q-values.

34 . L308 "Neutrophil/lymphocyte ratio decreased between T4 and T5" – contradicts Table 4 (T3 missing). Correct: "N/L ratio decreased from T1 to T5" or clarify trend.

35 . L315–316 Table 4: HGB different superscripts (a,b) but p= <0.01 – correct, but T5 lower than T2/T4? Verify post-hoc test: T5 is lowest, should be 'c' if significant.

36 . L322 "Haemoglobin did not show differences despite significant p=0.04" – contradiction. Recheck: p=0.04 is significant; either remove "did not show" or correct p-value.

37 . L323 "Lymphocyte count was lower for TUS of 3 than any lower TUS" – but TUS 0 vs 1 not different? Specify: "significantly lower than TUS 0, 1, and 2".

38 . L324 "N/L ratio higher for TUS 3 vs 0 or 2" – but p=0.03 and 0.01 – significant, but not vs 1? Clarify: "not significantly different from TUS 1".

39 . L325 "SAA higher for TUS 2 or 3 than TUS 1" – but Table 5 shows TUS 1=49.9, TUS 2=65.4, TUS 3=73.3. Correct: "higher for TUS ≥2 than TUS 0 or 1".

40 . L326 "Total Ig lower for TUS 3 than TUS 2" – but TUS 1=14.2, TUS 2=16.4 – TUS 1 not different? State: "significantly lower than TUS 2", not "any lower TUS".

41 . L331 "Haemoglobin did not show differences" – but p=0.04 – error. Correct: "Haemoglobin decreased with increasing TUS (p=0.04), though post-hoc differences were not significant."

42 . L341 "Haemoglobin lower for CRS 1 than 0" – but Table 6 shows CRS 2 and 3 also lower. Say: "lowest at CRS 1 and 2", or recheck pairwise tests.

43 . L344 "Eosinophil count did not show differences despite p=0.05" – p=0.05 is significant. Correct: "marginally significant" or adjust alpha.

44 . L355 "Severe TUS never correlated to severe CRS" – but n may be too small. Add: "though low frequency of severe cases limits inference."

45. L377 "almost 50% had some inflammation" – but TUS 1 may not be pathological. Use: "ultrasound abnormalities", not "inflammation", without histology.

46 . L380 "prevalence of severe lesions 4.8%" – based on observations, not calves. Report per calf: "4.8% of calves had at least one TUS=3 or 4 observation."

47 . L382 "72% of study calves displayed lung lesions" – at what time? T3? Specify: "at T3, 72% of calves had TUS ≥1."

48 . L388 "consolidation to fully recover" – assumes reversibility without histology. Say: "ultrasound lesions resolved", not "fully recover".

49 . L394 "failure of ultrasound technique" – contradicts claim of high sensitivity. Replace: "inter-observer variability or transient lesions may explain changes."

50 . L400 "rectal temperatures omitted... due to practical concerns" – weakens CRS validity. Acknowledge CRS is incomplete pre-transport and may underestimate disease.

51 . L416–418 "174 observations of healthy CRS, 104 not healthy by TUS" – but TUS may detect subclinical disease. Reframe: "TUS detected abnormalities in 60% of clinically healthy calves."

52 . L430 "haemoglobin usually increases" – incorrect; Hb often decreases in BRD due to anemia of inflammation. Correct: "In contrast to some reports, haemoglobin decreased, possibly due to acute phase response."

53 . L439 "Low total Ig and IgG... more at risk" – cites [16], but [16] is about arrival Ig predicting disease. Accurate: "As shown by Pardon et al. [16], low Ig at arrival predicts later BRD."

54 . L463 "TUS requires up to five minutes" – but no data in manuscript supports this. Add: "Based on observer logs, average scan time was X minutes."

55 . L465 "scanning right cranial lobe captures 61% of severe scores" – useful, but not validated. Recommend: "may be sufficient for screening, pending validation."

56 . L476–478 Blood and TUS/CRS not simultaneous – major limitation for correlation. State: "non-synchronous sampling likely attenuated correlations between blood and clinical scores."

57 . L481–482 "suboptimal situations" – but no description of how this affected blinding or consistency. Describe environmental constraints and their potential impact on scoring reliability.

58 . L484 "observers wear respiratory masks" – safety note, not research finding. Move to methods or supplementary.

59 . L485 "study increased stress" – confounder not measured or adjusted for. Measure cortisol or behavior, or acknowledge as limitation.

60 . L487–497 Conclusion: "TUS should be compared to other methods" – already done. Reframe: "TUS showed better alignment with immune markers than CRS, supporting its use in high-risk settings."

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Reviewer #1: Yes: Prof Dr Raheela Akhtar

Reviewer #2: Yes: Ali Hussein Aldujaily

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Comparison of thoracic ultrasonography (TUS), clinical respiratory scoring (CRS), and blood analysis to evaluate respiratory dysfunction in transported calves

PONE-D-25-22875R1

Dear Dr. Van Dijk,

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Mourad Mahmoud

Academic Editor

PLOS ONE

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