Review Reports - Serological Survey of SARS-CoV-2 in Wild Canids in Serbia: First Report in Red Foxes and Golden Jackals

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Lupulović and colleagues performed serosurveillance study on red foxes and golden jackals in Serbia. Commercially available and in-house ELISA kits were applied. This study is appreciated but several major concerns must be addressed.

Major concerns:

Line 202: What does “S/P” stand for?
In section 2.2.2, the specificity of anti-dog IgG has to be validated for red foxes and golden jackals. Negative and positive controls have to be included. What is “PBST”? What is the rationale or reference to define the cut-off value? Any false positive or negative results in this assay? Since this assay is one of the most critical aims in this paper, clear and convincing presentation is required.
It is highly recommended to present all the ELISA results in figures. For example, the readout of ELISA needs to be shown.
In figure 1, higher resolution is required because the name of region is not clear.
In discussion section, what is the implication of SARS-CoV-2 infection in Jackal and fox? How can the virus be transmitted to these animals? What is the conservation of human ACE2 and the ACE2 found in these animals?

Author Response

Dear Reviewer 1,

Thank you for taking the time to read our paper. We thank you for your critical comments and we hope that we have been able to respond to all your remarks and suggestions. Our answers are in a separate file attached. In the manuscript itself, all changes are marked in red.

Best regards,

Diana Lupulović

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Although the work presents original data for the reference geographical area, the methodological structure and analysis of the results are overly simplified. Therefore, in my opinion, substantial revision is necessary to make it suitable for publication. The manuscript's most obvious weakness is the complete lack of risk factor analysis, which reduces what should be an analytical epidemiological study to a simple descriptive report. No investigation was conducted into environmental or biological variables that could influence seropositivity, such as the proximity of sampling sites to high-density urban centres, the presence of intensive livestock farming of susceptible species, or the demographic characteristics of the animals. It would be appropriate to enhance the data analysis with a multivariate logistic regression model to calculate odds ratios, thereby identifying the drivers that favour the spillover of the virus from the human population to wildlife. Another weakness concerns the data presentation, which is excessively fragmented in the text and lacks adequate tabular support. The results should be summarised in contingency tables that cross-reference species, districts of origin, and sampling periods, allowing the reader to assess the distribution of positive results at a glance. From a diagnostic perspective, the use of a multispecies ELISA test, although practical, raises doubts about the specificity of the results in the absence of a confirmatory test. In veterinary epidemiology, cross-reactivity with other animal coronaviruses is a real risk that can lead to overestimation of prevalence. It would therefore be appropriate to critically discuss the validity of the test used and indicate the sensitivity and specificity values provided by the manufacturer. Finally, the discussion lacks a robust "One Health" perspective and does not adequately explore the ecological implications of the golden jackal's expansion in Europe and its role as a potential viral reservoir. The text should develop a more complex interpretation, comparing Serbian data with those from other European contexts and analysing the risk of spillback to humans or other domestic animal species. These additions would enable the manuscript to provide management or concrete health recommendations, whereas in its current form it merely reports the presence of antibodies.

Author Response

Dear Reviewer 2,

Best regards,

Diana Lupulović

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my concerns. However, further clarification is required on the following points:

Line288, 310: What do "(IDVet ELISA)" and "(in-house ELISA)" mean?
There is a discrepancy between the data presented in Table S1 and the statement on line 298. Table S1 indicates that 10 samples were positive in both the commercial IDVet ELISA and the in-house ELISA. However, on line 298, the authors state that 12 out of 165 animals were positive for SARS-CoV-2 infection. The authors should provide a clearer explanation and discussion to reconcile these results.

Author Response

Response to Reviewer 1

We thank the reviewer for the constructive comments. The manuscript has been revised in accordance with your suggestions. Detailed responses are provided below, and the revised manuscript is attached.

Comments:

Point 1: Line 288, 310: What do "(IDVet ELISA)" and "(in-house ELISA)" mean?

Response 1: We thank the reviewer for this comment. The terms “IDVet ELISA” and “in-house ELISA”, used in the titles of Tables 2 and 3 (lines 288 and 310), refer to the commercial multi-species ELISA (ID Screen® SARS-CoV-2, IDVet, France) and the adapted assay developed in this study, respectively. To improve clarity, this has now been explicitly defined in the Materials and Methods section. We added the following sentence in line 183: “For clarity, the commercial ELISA (ID Screen® SARS-CoV-2 Double Antigen Multi-Species, IDVet, France) is hereafter referred to as “IDVet ELISA”, while the adapted assay developed in this study is referred to as “in-house ELISA”.

Point 2: There is a discrepancy between the data presented in Table S1 and the statement on line 298. Table S1 indicates that 10 samples were positive in both the commercial IDVet ELISA and the in-house ELISA. However, on line 298, the authors state that 12 out of 165 animals were positive for SARS-CoV-2 infection. The authors should provide a clearer explanation and discussion to reconcile these results.

Response 2: Thank you for this important comment. We agree that the difference between the number of seropositive animals detected by the commercial ELISA (17/165) and the in-house ELISA (12/165) required clearer explanation.

The apparent discrepancy arises because the two assays did not fully concord in all samples. As shown in Supplementary Table S1, 10 samples were positive in both assays (true positives), while 2 samples were positive only in the in-house ELISA (false positives) and 7 samples were positive only in the commercial IDVet ELISA (false negatives in the in-house assay).

Therefore, the total number of seropositive animals differs depending on the assay used (17 vs. 12), while the number of concordant positive results between the two methods is 10.

To clarify this, we have revised the manuscript and explicitly explained the discordant results and their impact on the total counts. A reference to Supplementary Table S1 has been added in the Results section (in line 314): “The comparison between the commercial IDVet ELISA and the in-house ELISA revealed a small number of discordant results, including false positive and false negative samples (Supplementary Table S1). Out of the 17 samples identified as positive by the commercial ELISA, 10 were also positive by the in-house ELISA, while 7 were not detected by the adapted assay. In addition, 2 samples were positive only by the in-house ELISA. Scatter plots showing the distribution of ELISA readout values for both assays are presented in Supplementary Figure S1.”

We hope that the revisions have adequately addressed your comments and improved the quality of the manuscript. Thank you for your valuable and constructive feedback.

Best regards,

Diana Lupulović

Corresponding author

Author Response File: Author Response.pdf