High Prevalence of Novel Beak and Feather Disease Virus in Sympatric Invasive Parakeets Introduced to Spain From Asia and South America
Round 1
Reviewer 1 Report
This manuscript describes a robust study examining BFDV distribution and diversity in introduced parrot populations in southern Spain. The findings are novel and important to our understanding of this significant pathogen, which is a key threatening process to a number of threatened parrot species, and are likely to be of wide interest to avian disease and conservation researchers and managers. My recommendations for the manuscript are minor and I commend the authors for the excellent presentation of both the text and figures.
Minor edits are below:
Line 36: remove ‘s’ from ‘Feathers’
Line 38: move ‘highly infectious and mutable’ before ‘single-stranded…’
Line 72: change ‘mandatory’ to ‘important’
Line 95: 5uM is a very high primer concentration, please check that this is correct
Line 101: correct to ‘by Sanger sequencing’
Line 177: ‘reported’ instead of ‘reporter’
Line 183: ‘other’ instead of ‘another’
Line 189: ‘infectiveness’ isn’t right here, perhaps you mean ‘infectiousness to native species…’?
Line 189: “Therefore, the novel and their closest viral variants can colonize psittacids from all continents.” This sentence doesn’t really make sense. Do you mean “The novel genotype in this study and its closest variants can colonize psittacids from all continents” or something else?
Line 197: ‘bred’ instead of ‘breed’
Methods section: Were two primer sets used on all samples? Please also describe how you defined a sample as positive (i.e. positive if at least one primer pair PCR was positive or positive only if both primer pair PCRs were positive). I see that you have touched on this in your discussion, but a sentence in the methods would be valuable to interpreting the results presented immediately after.
Author Response
Line 36: remove ‘s’ from ‘Feathers’
REPLY: Done.
Line 38: move ‘highly infectious and mutable’ before ‘single-stranded…’
REPLY: Done.
Line 72: change ‘mandatory’ to ‘important’
REPLY: Done.
Line 95: 5uM is a very high primer concentration, please check that this is correct
REPLY: This is correct. 5 uM was the primer quantity added to the reaction. Nevertheless, we have updated this sentence in order to make it clearer to the reader: PCR was performed in a reaction mixture of 20 μl containing 10 μl of 2x MyTaq HS Mix (Bioline), 250 nM of each primer (final concentration) and ~20 ng of template DNA. (lines 96-97)
Line 101: correct to ‘by Sanger sequencing’
REPLY: Done.
Line 177: ‘reported’ instead of ‘reporter’
REPLY: Done.
Line 183: ‘other’ instead of ‘another’
REPLY: Done.
Line 189: ‘infectiveness’ isn’t right here, perhaps you mean ‘infectiousness to native species…’?
REPLY: We appreciate and followed the reviewer’s suggestion: “…which deserves further research on the potential infectiousness to native species in the invasive range of these parakeet species.” (lines 192-193)
Line 189: “Therefore, the novel and their closest viral variants can colonize psittacids from all continents.” This sentence doesn’t really make sense. Do you mean “The novel genotype in this study and its closest variants can colonize psittacids from all continents” or something else?
REPLY: We appreciate and followed the reviewer’s recommendation and have revised this sentence, which now reads as follows: “The novel genotype characterized in this study and its closest viral variants can colonize psittacids from all continents.” (lines 193-194)
Line 197: ‘bred’ instead of ‘breed’
REPLY: Done.
Methods section: Were two primer sets used on all samples? Please also describe how you defined a sample as positive (i.e. positive if at least one primer pair PCR was positive or positive only if both primer pair PCRs were positive). I see that you have touched on this in your discussion, but a sentence in the methods would be valuable to interpreting the results presented immediately after.
REPLY: We appreciate and followed the reviewer’s recommendation. We have updated one sentence and introduced a new sentence in the “Materials and Methods” section:
“The screening of BFDV was performed on all samples using two primer sets…” (lines 92-93)
“Samples were considered BFDV positive if at least one primer pair PCR had an accurate amplification of the expected size fragment.” (lines 103-104)
Reviewer 2 Report
The manuscript from Morinha et al outline detection and sequence analysis of BFDV from 2 species of parakeets in Spain. The results found a relatively high prevalence of BFDV in both parakeet species and phylogenetic analysis showed similarity to BFDV isolates from other birds and other rose-ringed parakeets. The analysis is limited, but also of interest because of the novel genetic information of BFDV from monk parakeets.
Comments:
1) Line 101: Can the authors elaborate on this? Why 5 from each? Were they chosen at random or was there a reason each sample was chosen for sequencing?
2) Lines 125-126: Have the Genbank sequences been deposited yet? I can't find them. Also looking to confirm that the sequences from rose-ringed and monk parakeets were exactly the same.
3) I believe the difference between figure 1 and figure 2 is that fig 1 is comparing all BFDV sequences to those sequenced in this study, fig 2 is only comparing P. krameri in this study to other BFDV sequences from P. krameri. Is this correct? Can this be made more clear in lines 131-135?
4) "Positive samples for the virus only amplified with the primers reporter by Ypelaar et al.": This is really interesting and valuable to know.
5) Line 103: "GenBank thought a BLAST search". Should be "through a BLAST search"
6) Line 179: "thus may had underestimated the actual prevalence". Should be "thus may have underestimated..."
Author Response
1) Line 101: Can the authors elaborate on this? Why 5 from each? Were they chosen at random or was there a reason each sample was chosen for sequencing?
REPLY: We appreciate and followed the reviewer’s recommendation and have revised this sentence, which now reads as follows: “Five random positive samples of each species were bi-directionally sequenced by Sanger sequencing to assess genotype diversity.” (104-105)
2) Lines 125-126: Have the Genbank sequences been deposited yet? I can't find them. Also looking to confirm that the sequences from rose-ringed and monk parakeets were exactly the same.
REPLY: The sequences were submitted to GenBank but “they will not be released to the public database until the data or accession numbers appear in print”. This is an option available for the user to select during GenBank submission.
3) I believe the difference between figure 1 and figure 2 is that fig 1 is comparing all BFDV sequences to those sequenced in this study, fig 2 is only comparing P. krameri in this study to other BFDV sequences from P. krameri. Is this correct? Can this be made more clear in lines 131-135?
REPLY: The reviewer's statements are correct. This is now clear described in “Materials and Methods” and “Results” sections:
“Then, our sequences were compared with data available in GenBank through a BLAST search [34]. The program BEAST v.2.6.0 [35] was used to construct a Maximum Clade Credibility (MCC) tree using the 100 sequences most related with the genotype characterized in this study.” (lines 106-109)
“Our analysis indicates that the novel genotype differed by 9 to 13 nucleotide substitutions with the most similar genotypes among all BFDV sequences described to date for different psittacine species (Fig. 1).” (lines 130-132)
“… were used to construct a MCC tree using only P. krameri rep gene sequences with a 100% coverage of the query sequence in the BLAST search.” (lines 119-120)
“The phylogeny of the rep sequences isolated from P. krameri in various countries supports the proximity of the BFDV genotypes isolated from wild individuals in an invasive population in Spain with captive individuals in Saudi Arabia, which share an ancestor with the two genotypes also detected in captive individuals in Poland (Fig. 2).” (lines 136-139)
4) "Positive samples for the virus only amplified with the primers reporter by Ypelaar et al.": This is really interesting and valuable to know.
REPLY: This was discussed in the manuscript: “Positive samples for the virus only amplified with the primers reported by Ritchie et al. [32], likely due to variations in viral copy numbers or mutations in the primer binding sites in some BFDV genotypes. However, several studies only focused on one primer set for BFDV screening [10,12,17], and thus may had underestimated the actual prevalence in these populations. Further research using different molecular markers is thus needed to increase the robustness of the BFDV diagnosis test.” (lines 180-185)
We changed Ypelaar to Ritchie in the revised version. We put the wrong name by a mistake when formatting the reference numbers.
5) Line 103: "GenBank thought a BLAST search". Should be "through a BLAST search"
REPLY: Done.
6) Line 179: "thus may had underestimated the actual prevalence". Should be "thus may have underestimated..."
REPLY: Done.
