Presence of Porcine Circovirus Type 2 in the Environment of Farm Facilities without Pigs in Long Term-Vaccinated Farrow-to-Wean Farms

Round 1

Reviewer 1 Report

The authors investigated the contamination of porcine circovirus (PCV2) in four pig farms immunized with vaccine for a long time without pig breeding. From the analysis of the viral inspection of the internal and external environment in the pig farms, many links in the external environment without pigs have serious  problems of the virus pollution. Therefore, the authors suggest that the disinfection and sanitation of the external environment and personnel should be paid attention to when the pig farm is empty. The study also reminds that environmental disinfection in biosafety is a link that must be addressed. On the whole, this study is not deep and the data is not much, but it can be used as information for the farmers and managers. However, there are still some major issues to be clarified in this study. 

(1) The author found that the external environment of non pigs was seriously infected, but the detected nucleic acid could not represent the positive infectious substance. Is it capable of further infecting pigs? Additional experiments should be conducted to prove the infectivity of nucleic acids.

(2) Is the second sampling conducted in the same place of the pig farm? How long can nucleic acids remain in nature?

(3) Where is the source of positive nucleic acid for the staff and their offices? Is there a possibility of aerosol transmission? Is air sample collected for testing?

(4) In the table, there is no inspection showing the disinfection frequency and disinfection effect of the four pig farms? Is the method and frequency of disinfection consistent in the environment with and without pigs?

Author Response

Following the instructions of the editor, we have not addressed the comments of reviewer 1.

Reviewer 2 Report

Like in previous article Authors estimate presence of Porcine Circovirus Type 2 (PCV2). In 2019 analyze environmental distribution of Porcine Circovirus Type 2 in swine herds with natural infection, in 20022  via monitoring of porcine circovirus type 2 infection through air and surface samples. In all three articles methodology/laboratory analysis is completely the same.

 

Major comments:

Table 1 – piglets were vaccinated, what about sows? What vaccines were used? Type of cleaning methods and type of disinfection?

Line 99 – first environmental sampling was in spring. Lack sampling from piglets. Why? PCV2 infection was assessed in piglets at the end of the weaning stage, by PCR from blood. Blood/serum is good material in case acute viremia. In some cases or in stage with very low viremia PCR results might be false-negative. For screening samples of oral fluid will be better.

Table 3 - number of samples (33 – 36) is not enough.

Line 123 - for those samples which tested negative another isolation od DNA was perform. Why? How purity and concentration od DNA was estimated?

Line 147 - Regarding the environmental samples, the results of each sample from each farm are positive to PCV2. Without sequencing, it is impossible conclude that is potentially infective PCV2 or vaccine residues (e.g. form vaccine containing inactivated porcine circovirus type 2). Additionally, genotype determinations - PCV2a, PCV2b, PCV2d should be added.

Minor comments:

Line 107-108 - animals were clinically evaluated in order to detect clinical signs  compatible with PCV2 infections such as wasting, dyspnoea and/or skin pallor. What about other infection or disorder?

Line 127-129 - An exogenous internal control (EXOone EXOPOL S.L.) was added to each sample during the extraction in both protocols to identify possible qPCR inhibition. In case of EXOPOL test, and others, exogenous IC most of all check quality of isolation of DNA. Moreover, in EXOPOL EXOone IC i salso added to reaction mix, where check possible inhibition.

Line 131- 134 - repeating the method of isolation. Add blood samples to swab samples isolation methods.

Author Response

 Thank you for all your comments, which have allowed us to improve the quality of this paper. Please find our responses to your comments in the file attached in blue color.

Author Response File: Author Response.pdf

Reviewer 3 Report

First, I would like to thank you for the opportunity to review this scientific article.  The general the idea and suggested aim of the study is relevant to pig industry, specially to farms who have been vaccinating against PCV2 and they could consider cease vaccination. The study tried to identify possible reservoirs of PCV2 in the farm environment as possible source of contamination for pigs, which can contribute to establish better stewardships measures, and in my opinion this is the major strength of the article.

The article is easy to understand, however, the aim is not clear and concise, although the title is informative and relevant to the content of the manuscript. Overall, the article has some major points which need clarification, rewrites and/or additional information to improve the article. Authors could use as reference to improve this article, their previous peer-reviewed article (reference 12) as it is very similar but in non-vaccinated farms.

Therefore, I recommend that a revision. I explain my concerns in more detail below, and I ask that the authors to address them.

In addition to review the English language, could authors be consistent in terminology? For example: area or facility, farrow-to-wean or farrow-to-weaning (L 77), amount (L 19) and load

 

Simple summary: following author’s guidelines it should contain a clear statement of the problem addressed, the aims and objectives, pertinent results, conclusions from the study and how they will be valuable to society. However, it provides non relevant background information and redundant explanation of methodology and discussion. It does not state the aim of the study.

Abstract: The background does not place the question addressed in a broad context and does not state the purpose of the study; results broadly summarize the article's main findings but without providing values (for example: what the authors mean with “most of the samples”?) Adding some figures would provide clarity. Please remove the discussion sentences (L 35 - L 37).

Keywords: include biosecurity (as the article highlighted cleaning & disinfection and good management practices of staff), remove one the environment keywords, and vaccination keyword, as the paper is not discussing the effect of vaccination on the presence of PCV2 but possible routes of introduction and maintenance of PCV2 in the farm.

Introduction
According to the authors guidelines, the introduction should briefly place the study in a broad context and highlight why it is important. It should define the purpose of the work and its significance, including specific hypotheses being tested.

The first two paragraphs highlighted the importance of vaccination against PCV2, but there is not information about the disease, the relevance and importance of the disease in pig industry, its prevalence, etc. Then it relates the failure of vaccination with the occurrence of PCV2 in the environment, both in vaccinated and non-vaccinated farms, but there is not any background/state-of-art on the prevalence/occurrence and/or survival of this virus in the environment.

Finally, it is clearly outlined the research question (no information on the PCV2 distribution environment of vaccinated farms), and the aim to identify critical points for PCV2 contamination in the environment. (NB: it is not coincident with L 174 - L 175) However, this aim does not directly match with title, abstract, results and conclusion of the study.

Material and methods
This section has several flaws that should be addressed, as it should describe with sufficient detail to allow others to replicate and build on published results.

The following critical information related to the farms is missed:
How was the selection of farms performed? When was the study carried out? Where are farms located? What type of farms (intensive, extensive, outdoor, organic or conventional)?

Which is the definition of “small size”? Livestock unit (UGM or “unidad ganadera mayor” is not a standard measure and therefore it should be explained.

There are other important characteristics of the farms that should be described: do farms have a visitor register book? Which is the origin of sows/semen or gilts? Do they only check for workers/farm staff and not occasional visitors (veterinarians, lorry drivers, etc.)?

Overall, the article highlights the importance of vaccination, but what type of vaccine is used? Is it the same for all three farms?

Table 1. There is no need of such a big table, as only the number of sows, number of buildings, number of farrowing, weaning rooms, and years of PCV” vaccination are different between the three farms. Please modify accordingly and add the explanation to the text.

The following critical information related to the samples is missed: there is no information on the number of samples collected in each surface, in each farm or in total.

Reference 12 (L 99) is not appropriate, the reference does not describe the sampling but referred to another article of same authors. A briefly description of the swabbing protocol should be added: “Sterile cotton-tipped swabs of 11 mm in diameter were used. The protocol of sampling consisted of moistening each swab with PBS-T (phosphate buffer saline with 0.05% of Tween 20, pH = 7.4) and swabbing the entire area of sampling” as extracted from reference 16.

L 105: when the laboratory analyses was performed? Please specify time, temperature, and transport conditions.

L 107-L 112: which are the selection criteria for sampling piglets (location, age, weight, etc.)? Why 20 blood samples? Which is the robustness of this number?

For consistency, same classification of farm facilities and samples should be presented in table 2 and table 3.

L 131: Which are the criteria for the number of pool samples? For example, is it based on age/location of piglets? Which is the epidemiological unit?

Results
L 144-L 146: Do the authors examined every single animal in the farm/herd? Please change “herds” for “piglets” or “animals”.

L 151-L 153: Please remove “systematically”

Discussion/ conclusion
L 166: if animals in the studied farms are long-term clinically healthy it should be mentioned in material and methods and explained to which disease.

L 166-L 168: I would not say “remarkable result is the systematic presence” as there are quite a few farms areas negative.

L 168-L 170: this conclusion is not very accurate, as only offices and farm perimeter are contaminated in Farm B and farm staff clothes in farm D and farm A. Maybe it could be added in material and methods which is the threshold value to consider a sample with high or low viral load.

L 174- L 175: according to lines 85-86 this is not the aim of the study. Please review accordingly.

L 175 and L 178: statements seem to be different meaning. Please double check.

L 186-L 191: Did the authors consider this possible risk factor? If they do not, as showed in material and methods and results, authors are not discussed their results but providing background information. In such case, move to introduction.

L 209-L 211: Authors did not describe the origin of replacement animals in previous sections. Please see my previous comment (L 186-L 191).

L 224-L 228: As before, this is background information since authors have stated that animals where long term healthy and vaccination scheme in place for many years.

Conclusion only answers the aim of the study, if it was to describe the viral load of PCV2 in environmental samples of 4 farms

Author Response

 Thank you for all your comments, which have allowed us to improve the quality of this paper. Please find our responses to your comments in the file attached in blue color.

Author Response File: Author Response.pdf