Review Reports

Round 1

Reviewer 1 Report

Very well-written and concise paper. The authors stated clearly what study found and how they did it. The variables are well defined and measured appropriately. The study methods are valid and reliable. There are enough details provided in order to replicate the study.

The data is presented in an appropriate way. Results are discussed from different angles and placed into context without being overinterpreted.

The conclusions answer the aim of the study. The conclusions are supported by references and own results.

 

Specific comments on weaknesses of the article and what could be improved:

Major points  - none

Minor points

1. Please, state the limitations of the study
2. Could you please discuss the implications of the results in real-world practice, what are your recommendations based on your study

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Fang et al. in this study titled, Co-infection of E. coli, E. faecalis, C. psittaci contributes to salpingitis of laying layers and breeder ducks have analyzed the three different bacterial species that are affecting the poultry industry. They have performed all the required experiments to prove that these micoorganisms are the aetiological agents behind the disease in layers and ducks.

Author Response

Dear respected reviewers,

 We are grateful for your spending time on our submission and we revised the whole manuscript to maintain the journal quality of Pathogens. To highlight the revisions, we described the revised points as follows:

1. Result section, we revised the description of Figure 2:

   Figure 2. Phylogenetic tree of ompA genes of C.psittaci was constructed using Neighbor-joining method.

2.M&M, we revised the protocol of q-PCR assay in lines 265-380:Firstly, Chlamydia 23S rRNA and incA gene were determined using real-time quantitative polymerase chain reaction (q-PCR)(Applied Biosystems^TM 7500,Thermofisher, Beijing, China) .The q‐PCR reagents included 10 μl of 2× AceQ Universal U, Probe Master Mix V2 (Vazyme, Nanjing, China), 2 μl of DNA template, 0.4 μM of specific primers, 0.2 μM TaqMan Probe, and ddH₂O. Thermal cycling parameters were performed as follows: 37 °C for 2 min; 95 °C for 5 min; 40 cycles of 95 °C for 10 sec, 60 °C for 30 sec. Secondly, the ompA gene of C.psittaci, approximately 1,209 bp, was amplified and its products were separated on agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen,Beijing, China).The PCR reagents contained 10 μl of 2× Premix Taq ( Ex Taq Version 2.0) (Takara, Beijing, China), 2 μl of DNA template, 1 μM of specific primers, ddH₂O up. The purified PCR products were cloned with pMD™19-T vector (Takara, Beijing, China) and then sequenced by commercial company (BGI Genomics Co.,Ltd, Shenzhen, China).The other 24 sequences of ompA genes C.psittaci were downloaded from GenBank, aligned by MEGA (version 7.0) ClustalW algorithm and the Test Neighbor-Joining Tree for designing the ompA phylogenetic tree.To evaluate evolution of ompA sequences, 500 bootstraps were calculated and the rooted tree was based on ompA sequence of Chlamydia caviae (GPIC).

3. We improved grammatic mistakes across the manuscript.

Thank you again.

All the best wishes,

 Cheng He

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.