Hypobiosis and Development of Haemonchus contortus and Trichostrongylus colubriformis Infection in Lambs under Different Levels of Nutrition

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It's been a pleasure to read an estimulating manuscript such as this and I can appreciate the amount of work involved to complete the study.

Before the manuscript is published the authors should make the requested changes and answer all the questions raised. 

Figure 1 could be improved by assigning solid colours to the bars (please mind not to use green and red as contrast colours as they are difficult to read for colour-blinded people).

All other comments and questions are included in the attached file.

I look forward to the final version of this manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English language is of high quality. Regarding language, only some minor editing is required.

Author Response

Dear Reviewer,

Thank you very much for your valuable comments that resulted in considerable improvement of the manuscript. We have embedded specific responses to these below and made corresponding changes to the manuscript.

Reviewer 1: “Before the manuscript is published the authors should make the requested changes and answer all the questions raised”. 

RESPONSE: As far as possible, we made all changes according to your comments.

Reviewer 1: “Figure 1 could be improved by assigning solid colours to the bars (please mind not to use green and red as contrast colours as they are difficult to read for colour-blinded people)”.

RESPONSE: Patterns and colours changed as suggested

Reviewer 1: “Please explain why there was only four days between the last infection and the slaughter”

RESPONSE: In discussion section we addressed the problem related to the fact that several L4 would be developing and not hypobiotic, as you suggested (third paragraph of discussion). The present experiment was carried out also with the aim of determining, under controlled conditions of infection, what influence four levels of dietary supplementation would have on the performance of Dorper lambs. The serial infections aimed to reproduce the continuous infections that occur in the field.

Reviewer 2 Report

Comments and Suggestions for Authors

Submitted paper “Hypobiosis and development of Haemonchus contortus and Trichostrongylus colubriformis infection in lambs under different levels of nutrition” is a valuable contribution to the understanding of trichostrongylid life cycle in the host. In the described experiment the lambs were fed with four different diets, which contained different levels of metabolized protein and metabolizable energy. Each three days these animals were inoculated with 1000 of infective larvae of two widely distributed trichostrongylid species: Haemonchus contortus and Trichostrongylus columbriformis. After the end of the experiment (28 days) the animals were slaughtered and examined for intestinal parasitic nematodes.  Periodically during the experiment, the blood samples were taken from lambs and the level of eosinophils and IgC antibodies against these two nematode species were estimated. The main conclusion from the accurate count of nematodes is that the majority of H. contortus were on the stage of fourth stage juveniles. In concordance with the observations of other authors such a fact was considered as an indication of their hypobiosis – i.e. arrested stage of development. The most plausible explanation for H. contortus is that the established nematode population of this species increase the defense mechanisms of the host. As authors indicated in the Discussion “immune response to infection …………manifested by eosinophilia and production of anti-L3 immunoglobulins in comparison with the non-infected controls” was evident in all the diet groups. Remarkably, no such effect was reported for T. colubriformis. Concerning the quality of nutrition, authors revealed that ‘better’ diet decrease the ability of H. contortus to establish themselves in the host organism. In the same time the diet is not influencing directly the numbers of early-L4 hypobiotic larvae in the abomasal population of H. contortus.  No influence of diet on T. colubriformis ‘performance’ in the host was observed.

The submission is well prepared: the graphs are self-explanative, demonstrating the main statements of the paper. I consider this submission as ready for publication, but, in the same time, I would like to invite the attention of authors to several stylistic moments.

1.   In the Abstract authors mention ‘fifth stage larvae’. As a nematologist, I am more accustomed to the understanding of nematode life cycle as fourth juvenile (larval) stages and final stage - adult nematodes. Surely early fifth stage specimens are still looking as juvenile stage, but for me the expression “fifth stage larvae” sounds as contradiction. Probably just replace it with “nematodes after fourth molt” or “immature adult stage”?

2.   On the line 44 authors use the abbreviation FECs, but this first use of is not accompanied with explanation (Fecal Egg Count?), which can be found only in the line 111. Probably it is reasonable to do it on the line 44? E.g. first mention of MP and ME are accompanied with such explanation (line 120).

3.   On the line 214, 218, 220, 221 the Latin binomial H. contortus is not italicized.  

Author Response

Dear Reviewer,

Thank you very much for your valuable comments. Responses to your comments are below:

Reviewer  2:   In the Abstract authors mention ‘fifth stage larvae’. As a nematologist, I am more accustomed to the understanding of nematode life cycle as fourth juvenile (larval) stages and final stage - adult nematodes. Surely early fifth stage specimens are still looking as juvenile stage, but for me the expression “fifth stage larvae” sounds as contradiction. Probably just replace it with “nematodes after fourth molt” or “immature adult stage”?

RESPONSE: We replaced by “immature adult stage” as you suggested.

Reviewer  2:      On the line 44 authors use the abbreviation FECs, but this first use of is not accompanied with explanation (Fecal Egg Count?), which can be found only in the line 111. Probably it is reasonable to do it on the line 44? E.g. first mention of MP and ME are accompanied with such explanation (line 120).

RESPONSE: Correction made.

Reviewer  2:   On the line 214, 218, 220, 221 the Latin binomial H. contortus is not italicized.  

RESPONSE: Corrections made.

Reviewer 3 Report

Comments and Suggestions for Authors

The information shown in this study is relevant to veterinary parasitology studies. This study was well done.  

Minor suggestions and comments

Lines 95, 214, 218 and 221. Please, write the scientific names in italics. 

Line: 145. What diagnostic technique was used to confirm the monospecific infection (T. colubriformis?). Please, add another reference that supports the monospecific infection of the lambs with T. colubriformis.

Author Response

Dear Reviewer,

Thank you very much for your valuable comments. Responses to your comments are below:

Reviewer  3:   Lines 95, 214, 218 and 221. Please, write the scientific names in italics. 

RESPONSE: Corrections made.

Reviewer  3:   Line: 145. What diagnostic technique was used to confirm the monospecific infection (T. colubriformis?). Please, add another reference that supports the monospecific infection of the lambs with T. colubriformis.

RESPONSE: The information was added in Material and Methods, as suggested.

Reviewer 4 Report

Comments and Suggestions for Authors

In the 1980s and 1990s, many studies on hypobiosis in ruminants were carried out. The authors re-examine hypobiosis in ruminants infected with Heamonchus contortus and Trichostrongylus colubriformis. The current study investigated this phenomenon in sheep under local conditions in Brazil. The manuscript is clear and concise. The results were presented in a transparent manner and the conclusions were adequate to the results.

Minor comments

1. Since sheep were infected with both species of nematodes simultaneously, I suggest using the term "co-infection" or "mixed infection" in the manuscript.

2. lines 174-182

The antibody used in the ELISA requires specification.

3. lines 214, 218, 221/222

Use italics in the nematode name.

4. The graphs in Figure 2 require better analysis in the "results" section. Based on the average number of nematodes as a function of metabolized protein and the slope of the regression curve, it can be concluded that different stages are sensitive to diet. Consider explaining.

5. line 231

Please insert a space at P > 0.05

Author Response

Dear Reviewer,

Thank you very much for your valuable comments. Responses to your comments are below:

Reviewer  4:   Since sheep were infected with both species of nematodes simultaneously, I suggest using the term "co-infection" or "mixed infection" in the manuscript.

RESPONSE: We replaced by “mixed infection” as you suggested.

Reviewer  4:   lines 174-182; The antibody used in the ELISA requires specification.

RESPONSE: The information was added in Material and Methods (item 2.5) as suggested.

Reviewer  4:  lines 214, 218, 221/222; Use italics in the nematode name.

RESPONSE: Corrections made.

Reviewer  4:   The graphs in Figure 2 require better analysis in the "results" section. Based on the average number of nematodes as a function of metabolized protein and the slope of the regression curve, it can be concluded that different stages are sensitive to diet. Consider explaining.

RESPONSE: We added information about the slopes in the results section (last line of page 6).

Reviewer  4:   line 23; Please insert a space at P > 0.05

 RESPONSE: Corrections made.

Reviewer 5 Report

Comments and Suggestions for Authors

The authors aimed of determining, under controlled conditions of infection and nutrition, what influence four levels of dietary supplementation would have on the development of the immune response and on the biology of H. contortus and T. colubriformis, in Dorper lambs under serial artificial infections.

Major issues.

Please describe the experimental protocol in more clearly. Possibly include a table and a timeline figure to visualize the study protocol.

The statistical analysis may not be correct. The authors do not indicate if the data were parametric. Please carry out this assessment and if needed, please redo the analysis with non-parametric techniques.

The Discussion can be a bit longer to cover in greater depth all aspects of the findings.

Please include in the Conclusion a new paragraph to underline the novelty of the study.

Minor issues.

The objectives of the study must be expressed plainly and clearly.

Please include some tables with summary presentation of the findings.

Overall.

Can be published after correction as indicated and re-evaluation.

Author Response

Dear Reviewer,

Thank you very much for your valuable comments. Responses to your comments are below:

Reviewer  5:   Please describe the experimental protocol in more clearly. Possibly include a table and a timeline figure to visualize the study protocol.

RESPONSE: As you suggested, a figure with the timeline was included (Figure 1).

Reviewer  5:   The statistical analysis may not be correct. The authors do not indicate if the data were parametric. Please carry out this assessment and if needed, please redo the analysis with non-parametric techniques.

RESPONSE: Information about normality test was added. Data without normal distribution were analysed under log transformation (item 2.6).

Reviewer  5:   Please include in the Conclusion a new paragraph to underline the novelty of the study.

RESPONSE: A new line was added in the first paragraph calling attention to occurrence of hypobiosis involving H. contortus. According to journal guidelines “Conclusion section may contain only one or two paragraphs summarizing the main conclusions”. Therefore, it was not possible to insert a new paragraph.

Reviewer  5:   The objectives of the study must be expressed plainly and clearly.

RESPONSE: Done.

Reviewer  5:   Please include some tables with summary presentation of the findings.

RESPONSE: We opted to present the results only in figures.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been immensely improved.

A few things remain to be amended, corrected and/or clarify before publication.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Dear Reviewer,

We greatly appreciate your thoughtful review of our article. We corrected the manuscript according to your suggestions.

Reviewer 5 Report

Comments and Suggestions for Authors

The authors have made significant changes and have improved the manuscript. In relation to the presentation of the results: whilst I understand the point of the authors, I still consider that inclusion of tables presenting the results in summarised form, will greatly benefit the manuscript.
In any case, I let the editor decide on this matter.

Author Response

Dear Reviewer,

Thank you for your comments and suggestions regarding our manuscript.