Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle
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Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries []. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.
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The work presented is clear and the arguments well formed. Both reviewer comments have been satisfied, and as such as are happy to accept this manuscript for publication.
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Comments to Author
This manuscript describes the development and validation of a Luminex assay for the detection of five endemic diseases of cattle. Whilst it is generally well written, there are a number of gaps in the manuscript that need to be revised before being considered acceptable for publication which include: 1. See list for minor amendments. 2. The authors need to describe the Luminex assay in more detail e.g. intro and methodology. 3. Consider condensing the introduction on viral diseases and repetitiveness throughout the document - the focus should be about the technology, test developed and application. 4. There are no acknowledgments of other assays developed in this area or compared/contrast or benefits of this platform compared to other multiplex platforms. 5. Present the order of antigen results in the …
Comments to Author
This manuscript describes the development and validation of a Luminex assay for the detection of five endemic diseases of cattle. Whilst it is generally well written, there are a number of gaps in the manuscript that need to be revised before being considered acceptable for publication which include: 1. See list for minor amendments. 2. The authors need to describe the Luminex assay in more detail e.g. intro and methodology. 3. Consider condensing the introduction on viral diseases and repetitiveness throughout the document - the focus should be about the technology, test developed and application. 4. There are no acknowledgments of other assays developed in this area or compared/contrast or benefits of this platform compared to other multiplex platforms. 5. Present the order of antigen results in the same order each time, currently different order in each figure/table. 6. There are a number of inconsistencies in the data presented in tables, figures and/or text which do not always correlate e.g. Figure 1 BVDV monoplex AUC 0.981 whereas table 3 AUC 0.885. Therefore, this needs to be amended or clarified before being considered suitable for publishing. Font - different fonts have been used throughout the manuscript, please change to single font recommended by journal. Title - To aid the reader please consider amending the word undiagnosed as misleading as many countries have eradicated or have eradication programmes in place for many of the diseases that you are describing. Line 31-33 Please rephrase sentence and add in punctuation. Line 34 - lower case h for Herpes Line 35 - lower case b & l Line 35 - the use of 'underdiagnosed' is too strong and as described above many countries have eradicate or have eradication/surveillance programmes in place for these diseases. Line 38 - omit the word 'serum' Line 39 - Add in the 'in serum' after EBLV Line 41 - lysates may be more appropriate Line 42 - change sera to serum Line 43 - change towards to 'for' Line 44 - consider including Luminex in this sentence Line 46 - ensure the word 'diagnostic' sensitivity and specificity is used throughout the document Line 49 - change diagnose to diagnostic Line 51 - omit serum Line 52 - add 'in serum' Line 54 - add in the potential application of this assay e.g. how it would be used or its benefits Line 67 - lowercase h for herpes Line 68 - lowercase b & l Line 69 - add in 'often' underdiagnosed Line 70-71 - merge sentences to help with flow Line 75 - PI3V Line 77 - in cattle Line 79 - lowercase h 84 - add in comma Line 91 - lowercase EBL Line 101 - change further to therefore Line 103 - state impact and why Line 107 - would also confirm vaccination success Line 111- or treatment regimens? Line 114 - omit causing endemic viral diseases Line 115 - where? Or do you mean in general? Line 119 - NADL strain? Line 120 - change to BHV1 Line 122 - MDBK change to 'Madin-Darby bovine kidney' cells Line 123 - MEM change to 'Minimum essential media eagle' Line 125 - Was trypsin used to remove cells from flasks? Line 131 - there is not much detail on how the monoplex assays were developed or do you have a reference that you can use? Line 133 - BVD Line 180 - change reaction to 'interaction' Line 185 - The papers you have reference do not refer to the chemistry or development of Luminex they are just applications of Luminex (like your study). Identify suitable references to illustrate the Luminex chemistry etc. Line 192 - remove gap after uL plus capital L for litres throughout manuscript Line 195-197 - does not state conditions e.g. temp, shaking Line 199 - No details of blocking buffer composition? Did you sonicate the beads to mix? Line 202 - add conditions above Line 214 - add value and full stop Line 223 - I do not understand what is meant by 'Selectivity was evaluated'? Please rephrase Line 224 - Neat rather than 0? Line 228 - You have referenced OIE for your own method that you have developed as part of this study? Line 237 - 1:2? You mentioned neat earlier? Please align Line 248 - change further to 'therefore' Line 394 - change to therefore Line 467 - EBL should be abbreviated
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data This paper was designed and conducted to test a pentaplex test's diagnostic acuity to detect antibodies simultaneously. BRSV, PI3V, BoHV-1, BVDV and EBLV in bovine serum. It is my judgment, first of all, that it is very well written and presented, with a lot of coherence and a remarkable structure. Secondly, I think it is a very well-designed and executed work with high rigor in the laboratory, using appropriate methods. I had doubts about the sample size, as it is not specified why the number of samples used is a desirable detail to have a better idea of the power of the results. Fortunately, the results were outstanding, which helped the authors not notice this absence. The statistical tests, moreover, are appropriate for …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data This paper was designed and conducted to test a pentaplex test's diagnostic acuity to detect antibodies simultaneously. BRSV, PI3V, BoHV-1, BVDV and EBLV in bovine serum. It is my judgment, first of all, that it is very well written and presented, with a lot of coherence and a remarkable structure. Secondly, I think it is a very well-designed and executed work with high rigor in the laboratory, using appropriate methods. I had doubts about the sample size, as it is not specified why the number of samples used is a desirable detail to have a better idea of the power of the results. Fortunately, the results were outstanding, which helped the authors not notice this absence. The statistical tests, moreover, are appropriate for this type of study. Unfortunately, the absence of animals entirely negative for the five diseases did not allow us to see the actual performance of this diagnostic test in serum from adult cattle naturally exposed to multiple infectious agents that could cause cross-reactions and possible false positives; however, the use of fetal bovine serum is the right thing to do when utterly seronegative adult cattle are not available. 2. Presentation of results The form in which the results are presented is appropriate for the most part. A couple of details are noted as observations in the draft document (attached). The tables and figures are well constructed, and the titles are appropriate. 3. How the style and organization of the paper communicates and represents key findings As I noted, the article's structure is adequate because it has absolute coherence, from the objective to the conclusion, including the methodology, results, and discussion. The authors do not get lost in the search for answers to their questions. The style is clear and facilitates the fluent reading of the work, leaving space to look carefully at the details, especially the methodological ones. 4. Literature analysis or discussion The literature review used for the different sections of the paper is adequate. Perhaps, I suggest including a couple more references on the economic effects of diseases, trying to give more weight to the relevance of the study and its results. The introduction presents the necessary contents to learn about the subject of the study. The discussion has a correct thread, in addition to analyzing and contextualizing the results. Perhaps the only thing I would add is a paragraph on the possible limitations in the development of the study, which in some way affect its scope. 5. Any other relevant comments This type of assay will help cattle producers in their goal to control or eradicate these diseases, even to reduce their economic impact.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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