Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae

ABSTRACT Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 – 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

identified in this MS as being in the genus Actinobacillus are clearly NOT in this genus. This continued allocation of these problematic organisms to the genus Actinobacillus when they are clearly not in the genus is only prolonging and intensifying the confusion. It would help all workers if those organisms that are clearly not members of the genus Actinobacillus be named at each and occasion with a clear flag that they are not members of the genus e.g. by placing the in-correct genus name in quotation marks ("Actinobacillus") or in square brackets ([Actinobacillus]). Based on the latest edition of Bergey's Manual (see Blackall, P.J., Turni, C. 2020. Actinobacillus. In Bergey's Manual of Systematics of Archaea and Bacteria, Whitman, W.B., Rainey, F., Kämpfer, P., Trujillo, M., Chun, J., DeVos, P., Hedlund, B., Dedysh, S., eds. (John Wiley & Sons), the following [Actinobacillus] species featured in this MS are clearly NOT members of the genus Actinobacillus and should always have the genus name clearly marked as either "Actinobacillus" or [Actinobacillus] with a similar approach for the abbreviated genus name "A." or [A.]:a. "A." delphinocola b. "A." indolicus c. "A." minor d. "A." porcinus e. "A." rossi f. "A." seminis g. "A." succinogenes For the moment, the final resolution of the position of "A." porcitonsillarum also remains unclear and that species should also be flagged as well. 3. Lack of technical details. At lines 164-172, the text describes the PCRs used in this work. I found the text to be far too light on detail. There are many Taq enzymes supplied by NEB -which one was used? What were the cycling conditions of the assays? 4. Field isolates. Based on Table 1, this study included 108 field isolates. The text could be more informative by indicating the number of isolates per serovar (simply place the number in brackets after the serovar and explain via a footnote). 5. Table 1. This Table needs considerable refinement. The column headed "No." is irrelevant and the column should be deleted. The column headed Species is actually Genus and Species. The column headed Source/Reference is mostly presenting strain name and reference (ie ATCC 27088 in the first cell in this column is the strain name -the Source is simply ATCC. As well, the columns headed "Strain Name" and "Source/Reference" are largely duplicating each other in terms of the strain name. The column headed "Strain name" should record the ATCC or CCUG strain name (readers can consult the ATCC and CCUG web sites for the alternative names of each strain) for all strains obtained from CCUG and ATCC. The Source/Reference column should just be showing ATCC or CCUG (and not the strain name). What was the source of the bacteria without a source name e.g. A. pleuropneumoniae serovar 7? The column headed "Number of strains/isolate" is wasting valuable space. Delete this column and simply use a footnote to record the rare exception where more than a single strain/isolate was used. Where the formal type strain of species has been used the T signifying that the strain is the formal species type strain should be in superscript. ATCC 27088{T} (where {} shows superscript) is the type strain for A. pleuropneumoniae. 6. Confusion on strain names. As I indicated above, there are problems with confusion over strain names in Table 1. This lack of completed understanding has spoiled over into the general text. At lines 280 -293, there is Discussion about the absence of xerD in a strain called NCTC 11383 (note the space -should be used with all NCTC and ATCC strains). The authors appear not to understand that NCTC 11383 is actually K17. Based on Tables 1 and 6, the pure culture of NCTC 11383/ATCC 33377/K17 was used in this study as a live culture and ewas tested by PCR and was positive for xerD. 7. Confusion over strains. Given the confusion highlighted above, the authors need to go back into their data and look for other duplications of strains. There amy weel be additional duplications of strains that are actually the same strain but simply obtained from ATCC or NCTC or other sources. 8. Table 3. Again, remove the first column -it is irrelevant. 9. Table 5. Again, a poorly laid out Table. The column headed "nucleotide sequence" is simply too bulky and makes this table very hard for the reader to assimilate. Move the details in this column to a Supplementary Table. The column headed "Among WGS of Pasteurellaceae" is very confusing. At a guess, the data being shown in the three sub-columns here is actually limited to the similarities in the available incomplete A. pleuropneumoniae genomes. The last column is simply a waste of space and can be replaced by a footnote that records whatever was meant to be shown in this column (it is just empty cells at the moment ). I think the Column headed "Gene" should read "Target". 10. Table 6. The column headed "Serovar/Name" is inappropriate. Replace with "Serovar/Strain". Then correctly record (using ATCC and NCTC numbers for those strains obtained from ATCC and NCTC and original strain names for others) that actual strain tested. Add an extra row for the field isolates of serovar 5 and ensure that the reference serovar 5 strain or strains that was used is named. 11. Table 6 -serovar 5 results. It is important that the reference serovar 5 strains be separately recorded as noted in the above point. I found it somewhat difficult to believe the number of field serovar 5 isolates that appear to be listed in Table 6. My problem is based on the data shown in Table 1. In Table 1, there are three reference serovar 5 strains listed and 108 field isolates of serovars 1, 2, 5, 12, 15 and non-typeable. I will assume that all three reference strains of serovar 5 are part of the 104 recorded in Table 6. This means that at least 101 field isolates of serovar 5 were tested according to Table 6. Given that Table 1 indicates that the field isolates represented 5 serovars and non-typeable, it means that almost all of the Thai isolates were serovar 5. Is this the case??? Providing the details of the serovars of the filed isolates in Table 1 and then separating the details of the reference strains and the filed isolates in Table 6 would resolve this problem.
1. App or A. pleuropneumoniae. In my view, an bacterium that is worthy of study is worthy of being formally and correctly named at each and every occasion in a scientific MS. While virologists are willing to reduce the names of "their" life forms, the same should not apply to bacteria in my view. Please use the normal conventions for all bacterial names -full genus and species at first use and then abbreviated genus name there after. The term App should be removed totally from this MS. 2. Line 26. The word "same" is, in my view, superfluous and should be deleted. 3. Line 120. What animal was the source of the blood cells used in the blood agar and what concentration was used. 4. Tables. Each Table should commence on a new page. 5. Line 219. Suggest inserting "only" ahead of "11 complete". 6. Line 256. Insert "the" ahead of "five new" 7. Lines 264-266. Is the sentence starting "Although not encoding intact" based on data generated in this study or is it based on published data? If based on published work, then that work has to be cited. If based on an analysis done in this work, then the text needs to make this clear.

Reviewer #2 (Comments for the Author):
The manuscript titled "Novel DNA markers for detecting Actinobacillus pleuropneumoniae" (#Spectrum01311-21) by Srijuntongsiri et al. describes the identification of novel DNA markers specific for Actinobacillus pleuropneumoniae using GWS methods and verification of these markers with PCR amplification. The bioinformatics analysis is well-organized and the paper is well-written. These DNA markers would be helpful for development of alternative methods for identification of A. pleuropneumoniae. My major concern is that this work is a preliminary study. Further efforts on the development of specific diagnostic methods and functions of target genes might contribute more to the understanding and control of this bacterium General comments: 1. Line 168, how about the PCR cycle conditions? 2. Line 267, what's the mean of "significantly similarity"? 3. Table 4, why primers for those 5 markers in apxIVA gene are not included? 4. Table 5, why fragment apxIVA-1 shows 51 matches in 23 incomplete A. pleuropneumoniae genomes? 5. Table 6, representative results from gel electrophoresis analysis should be displayed, or may be as supplementary material(s).

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We thank the Reviewers for their comments, and our response to each one is given below. Line numbers indicate where changes have been made in response to the Reviewer's comments, and these are additionally highlighted in yellow in the marked copy of the revised manuscript.

Reviewer #1:
Major Comments 1. In my view, the title should use the word "identification" in place of "detection".

Response:
The title has been changed to "Novel DNA markers for identification of Actinobacillus pleuropneumoniae." 2. Taxonomy. The authors appear not to be aware of the now widely accepted situation that many of the bacterial species identified in this MS as being in the genus Actinobacillus are clearly NOT in this genus. This continued allocation of these problematic organisms to the genus Actinobacillus when they are clearly not in the genus is only prolonging and intensifying the confusion. It would help all workers if those organisms that are clearly not members of the genus Actinobacillus be named at each and occasion with a clear flag that they are not members of the genus e.g. by placing the in-correct genus name in quotation marks ("Actinobacillus") or in square brackets ([Actinobacillus]). Based on the latest edition of Bergey's Manual (see Blackall, P.J., Turni, C. 2020. Actinobacillus. In Bergey's Manual of Systematics of Archaea and Bacteria, Whitman, W.B., Rainey, F., Kämpfer, P., Trujillo, M., Chun, J., DeVos, P., Hedlund, B., Dedysh, S., eds. (John Wiley & Sons), the following [Actinobacillus] species featured in this MS are clearly NOT members of the genus Actinobacillus and should always have the genus name clearly marked as either "Actinobacillus" or [Actinobacillus] with a similar approach for the abbreviated genus name "A." or [A.]:a. "A." delphinocola b. "A." indolicus c. "A." minor d. "A." porcinus e. "A." rossi f. "A." seminis g. "A." succinogenes For the moment, the final resolution of the position of "A." porcitonsillarum also remains unclear and that species should also be flagged as well. 3. Lack of technical details. At lines 164-172, the text describes the PCRs used in this work. I found the text to be far too light on detail. There are many Taq enzymes supplied by NEBwhich one was used? What were the cycling conditions of the assays?

Response
Response: More details on PCR parameters have been added as suggested.

"Genomic DNA purification and PCR amplification
Genomic DNA of various bacterial species was extracted using a standard DNA purification protocol (29). PCR was performed using Taq DNA polymerase with Standard Taq Buffer (M0273, New England Biolabs, Ipswich, MA, USA) according to the manufacturer's protocol. Briefly, PCR reactions were prepared to contain final concentrations of 200 µM dNTPs, 0.2 µM of each primer (Table 4), 0.025 U/µl Taq DNA polymerase, and 1 ng/µL of bacterial genomic DNA. Thirty cycles of 95 ºC for 30 seconds, 60 ºC for 1 minute, and 68 ºC for 1 minute were performed using a C1000 Touch PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. Alternatively, Luna qPCR Master Mix (M3003, New England Biolabs) was used according to the manufacturer's protocol. Briefly, qPCR reactions were prepared to contain final concentrations of 0.25 µM of each primer and 1 ng/µL of bacterial genomic DNA. Forty-five cycles of 95 ºC for 15 seconds and 60 ºC for 30 seconds were performed using a Bio-Rad CFX96 real-time PCR machine. Fluorescence signals indicative of the presence of PCR products were measured." 4. Field isolates. Based on Table 1, this study included 108 field isolates. The text could be more informative by indicating the number of isolates per serovar (simply place the number in brackets after the serovar and explain via a footnote). Table 1  The column headed "No." is irrelevant and the column should be deleted.

Response: The number of isolates per serovar has been added in brackets to
The column headed Species is actually Genus and Species.
The column headed Source/Reference is mostly presenting strain name and reference (ie ATCC 27088 in the first cell in this column is the strain name -the Source is simply ATCC. As well, the columns headed "Strain Name" and "Source/Reference" are largely duplicating each other in terms of the strain name. The column headed "Strain name" should record the ATCC or CCUG strain name (readers can consult the ATCC and CCUG web sites for the alternative names of each strain) for all strains obtained from CCUG and ATCC. The Source/Reference column should just be showing ATCC or CCUG (and not the strain name). What was the source of the bacteria without a source name e.g. A. pleuropneumoniae serovar 7?
The column headed "Number of strains/isolate" is wasting valuable space. Delete this column and simply use a footnote to record the rare exception where more than a single strain/isolate was used. Where the formal type strain of species has been used the T signifying that the strain is the formal species type strain should be in superscript. ATCC 27088{T} (where {} shows superscript) is the type strain for A. pleuropneumoniae. Table 1 has been modified as suggested and now reads: *The Langford laboratory was the source of bacteria (or gDNA) that were not purchased from ATCC or CCUG. The growth and preparation of derived gDNA from these strains was carried out as described previously (5).

Response:
6. Confusion on strain names. As I indicated above, there are problems with confusion over strain names in Table 1. This lack of completed understanding has spoiled over into the general text. At lines 280 -293, there is Discussion about the absence of xerD in a strain called NCTC 11383 (note the space -should be used with all NCTC and ATCC strains). The authors appear not to understand that NCTC 11383 is actually K17. Based on Tables 1 and 6, the pure culture of NCTC 11383/ATCC 33377/K17 was used in this study as a live culture and ewas tested by PCR and was positive for xerD.

Response:
The discussion on xerD has been modified accordingly. The manuscript (lines 307-309) now reads: "Nonetheless, xerD-specific PCR product was observed when genomic DNA from the ATCC 33377 strain was used as template (Table 1 and Table 6), indicating that xerD can also serve as a marker for A. pleuropneumoniae identification." 7. Confusion over strains. Given the confusion highlighted above, the authors need to go back into their data and look for other duplications of strains. There amy weel be additional duplications of strains that are actually the same strain but simply obtained from ATCC or NCTC or other sources.

Response:
In Table 2, strain name has been changed to ATCC or CCUG number when these are available. We found no other duplication.
8. Table 3. Again, remove the first column -it is irrelevant.

Response:
The first column has been removed as suggested.
9. Table 5. Again, a poorly laid out Table. The column headed "nucleotide sequence" is simply too bulky and makes this table very hard for the reader to assimilate. Move the details in this column to a Supplementary Table. The column headed "Among WGS of Pasteurellaceae" is very confusing. At a guess, the data being shown in the three sub-columns here is actually limited to the similarities in the available incomplete A. pleuropneumoniae genomes. The last column is simply a waste of space and can be replaced by a footnote that records whatever was meant to be shown in this column (it is just empty cells at the moment ). I think the Column headed "Gene" should read "Target".
Response: Table 5 has been modified as suggested. Table 5 now reads: 10. Table 6. The column headed "Serovar/Name" is inappropriate. Replace with "Serovar/Strain". Then correctly record (using ATCC and NCTC numbers for those strains obtained from ATCC and NCTC and original strain names for others) that actual strain tested. Add an extra row for the field isolates of serovar 5 and ensure that the reference serovar 5 strain or strains that was used is named.
Response: Table 6 has been modified as suggested.
11. Table 6 -serovar 5 results. It is important that the reference serovar 5 strains be separately recorded as noted in the above point. I found it somewhat difficult to believe the number of field serovar 5 isolates that appear to be listed in Table 6. My problem is based on the data shown in Table 1. In Table 1, there are three reference serovar 5 strains listed and 108 field isolates of serovars 1, 2, 5, 12, 15 and non-typeable. I will assume that all three reference strains of serovar 5 are part of the 104 recorded in Table 6. This means that at least 101 field isolates of serovar 5 were tested according to Table 6. Given that Table 1 indicates that the field isolates represented 5 serovars and non-typeable, it means that almost all of the Thai isolates were serovar 5. Is this the case??? Providing the details of the serovars of the filed isolates in Table 1 and then separating the details of the reference strains and the filed isolates in Table 6 would resolve this problem.

Response:
The number of strains and isolates have been listed in more detail in Table 6 as suggested. The majority of Thai A. pleuropneumoniae isolates recovered from samples sent to the Veterinary Diagnostic Laboratory, Livestock Animal Hospital, Chulalongkorn University are indeed of serovar 5. Table 6 now reads: