Gastrointestinal Parasitic Infections in Non-Human Primates at Gabon’s Primatology Center: Implications for Zoonotic Diseases

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper is about gastrointestinal parasitic infections in primates from Gabon's Primatology Center, with emphasis on the potential risk of transmission of these parasites to humans.

This is a highly relevant article, framed within the One Health concept, which connects primate parasites and their zoonotic potential.

I think the article is well written and well structured, easy to read and understand. It presents a good introduction, with current bibliographic references and supports the topic well.

The objectives are well defined and the materials and methods well described.

Regarding the results, despite some aspects that need to be clarified, they are generally well presented, with tables, a graph and images of the parasites.

Regarding the discussion and conclusions, they are in the same line as the rest of the work, the results are well discussed and supported by the bibliography, reinforcing the importance of the obtained data.

Despite all the aspects that have been mentioned, there are some issues throughout the text, in the different sections, which must be corrected and clarified.

Abstract:

You say that: “The study identified 17 genera of parasites with zoonotic potential, including 11 nematode genera (e.g.,Trichuris spp., Oesophagostomum spp., and Strongyloides spp.), 3 protozoan genera (e.g., Balantioides spp. and Entamoeba spp.), one cestode genus (Hymenolepis spp.), and 2 trematode genera (Dicrocelium spp. and Paramphistomum spp.).” If you say gerera, you can not use the “spp”. The genera is Trichuris, Oesophagostomum, Strongyloides…

 

Lines 90, 263 – You say “non-human primates (NHPs)”. Should be NHPs, because the abbreviations is explained at line 45.

Line 69 – anthropozoonotcs, should be anthropozoonotics

Line 114 – “Parasite species in NHP at CIRMF Primatology Center” – I think that should  “genera”, because you never mention the real parasite species, just Trichuris sp., Oesophagostomum sp., Trichostrongylus sp., and Strongyloides sp.. And then you just mention the genera Necator, Schistosoma, Mammomonogamus, Ancylostoma…  

Line 118 – should be Chimpanzee (CPZ) – the first time that is abbreviated.

Line 146  - “equitability were highest in….,” – you forgot to fill the …

Line 147 – “high equitability distribution in……” – you forgot to fill the ……

Figure 2:

-          In the figure you put capital letters, but in the legend you put lower case letters – should revise

-          According the methods used to identify the parasites, the microscopy, it is very difficult to distinguish between the eggs of Ancylostoma and Necator, as well as the identification of Ancylostoma duodenale larvae. I think that “hookworm” is more correct.

-          Should replace “spp” by “sp” (that’s one egg – one species)

-          It is not possible to see the details of the larvae (t,u,v,w) because of the low quality of the image

 

Lines 228 – you say genera – should remove the “spp”    

Line 252 – “behaviors.At” – forgot the space   

References:

Line 323 – You didn’t mention the journal

Line 344 – I think that it is a thesis, should review how to cite

Line 347 – Plasmodium vivax – forgot the italics

Line 355 - Pan troglodytes schweinfurthii – forgot the italics

Line 366 – “science” – should be “Science”

Line 373 - Staphylococcus aureus – forgot the italics

Line 375 - You didn’t mention the journal

Line 408 - I think that it is a thesis, should review how to cite

Line 418 – “prévalence des parasitoses intestinales chez lesenfants de 0 à 5 dans la communauté d’anonkoi 3 ,.” – It is repeated

 

Line 439 - You didn’t mention the journal

Author Response

Reply to reviewer 1

Reviewer comment No. 1:

 Comments and Suggestions for Authors

This paper is about gastrointestinal parasitic infections in primates from Gabon's Primatology Center, with emphasis on the potential risk of transmission of these parasites to humans.

This is a highly relevant article, framed within the One Health concept, which connects primate parasites and their zoonotic potential.

I think the article is well written and well structured, easy to read and understand. It presents a good introduction, with current bibliographic references and supports the topic well.

The objectives are well defined and the materials and methods well described.

Regarding the results, despite some aspects that need to be clarified, they are generally well presented, with tables, a graph and images of the parasites.

Regarding the discussion and conclusions, they are in the same line as the rest of the work, the results are well discussed and supported by the bibliography, reinforcing the importance of the obtained data.

Despite all the aspects that have been mentioned, there are some issues throughout the text, in the different sections, which must be corrected and clarified.

Abstract:

You say that: “The study identified 17 genera of parasites with zoonotic potential, including 11 nematode genera (e.g.,Trichuris spp., Oesophagostomum spp., and Strongyloides spp.), 3 protozoan genera (e.g., Balantioides spp. And Entamoeba spp.), one cestode genus (Hymenolepis spp.), and 2 trematode genera (Dicrocelium spp. And Paramphistomum spp.).” If you say gerera, you can not use the “spp”. The genera is Trichuris, Oesophagostomum,Strongyloides …

 

Response to Reviewer comment No.1 :

We agree with your comment, thank you we edited in the new manuscript version (see lines 27-29 in new manucrispt version).

Reviewer comment No. 2:

Lines 90, 263 – You say “non-human primates (NHPs)”. Should be NHPs, because the abbreviations is explained at line 45.

Response to Reviewer comment No.2 :

Thank you for this observation we edited now in this new version see lines 90, 246, 259, 264

 

Reviewer comment No. 3:

Line 69 – anthropozoonotcs, should be anthropozoonotics

 

Response to Reviewer comment No.3 :

Thank you, now we edited line 70 in new manuscript version

 

Reviewer comment No. 4:

Line 114 – “Parasite species in NHP at CIRMF Primatology Center” – I think that should “genera”, because you never mention the real parasite species, just Trichuris sp., Oesophagostomum sp., Trichostrongylus sp., and Strongyloides sp.. And then you just mention the genera Necator, Schistosoma, Mammomonogamus, Ancylostoma  

 

Response to Reviewer comment No. 4 :

Thank you, we agree with the review comment and now we edited (see line 122 new manuscript version)

 

Reviewer comment No. 5:

Line 118 – should be Chimpanzee (CPZ) – the first time that is abbreviated. 

Response to Reviewer comment No. 5 :

Thank you, we considered the reviewer suggestion (line 126).

 

Reviewer comment No. 6 & 7:

Line 146  - “equitability were highest in….,” – you forgot to fill the …

Line 147 – “high equitability distribution in……” – you forgot to fill the ……

 

Response to Reviewer comment No. 6 & 7 :

Thank you for this observation, we have chosen to delete these lines 146 and 147 as they are rather redundant in relation to the message this manuscript is intended to convey.

 

Reviewer comment No. 8:

Figure 2:

-In the figure you put capital letters, but in the legend, you put lower case letters – should revise

-According to the methods used to identify the parasites, the microscopy, it is very difficult to distinguish between the eggs of Ancylostoma and Necator, as well as the identification of Ancylostoma duodenale larvae. I think that “hookworm” is more correct.

- Should replace “spp” by “sp” (that’s one egg – one species)

-It is not possible to see the details of the larvae (t,u,v,w) because of the low quality of the image

 Response to Reviewer comment No. 8 :

we have considered the reviewer's comments and improved the new version of the manuscript by taking into account their suggestions (see the new manuscript version lines 209-220). For a better visibility of the larvae images we have added images with a higher resolution as additional data.

 

 

Reviewer comment No. 9:

Lines 228 – you say genera – should remove the “spp”

Response to Reviewer comment No. 9:

Thank you, we agree with you. We considered your suggestion (see line 217-219).

 

Reviewer comment No. 10:

Line 252 – “behaviors.At” – forgot the space   

Response to Reviewer comment No. 10 :

Thank you, we corrected (see line 237 new manuscript version).

 

 

Reviewer comment No. 11:

References:

Line 323 – You didn’t mention the journal

Line 344 – I think that it is a thesis, should review how to cite

Line 347 – Plasmodium vivax – forgot the italics

Line 355 - Pan troglodytes schweinfurthii – forgot the italics

Line 366 – “science” – should be “Science”

Line 373 - Staphylococcus aureus – forgot the italics

Line 375 - You didn’t mention the journal

Line 408 - I think that it is a thesis, should review how to cite

Line 418 – “prévalence des parasitoses intestinales chez lesenfants de 0 à 5 dans la communauté d’anonkoi 3 ,.” – It is repeated

Line 439 - You didn’t mention the journal

Response to Reviewer comment No. 11:

Thank you, we edited all in new version of manuscript

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Inaccuracies listed below (not comprehensive).

-------------------------------------------------------------

In Abstract

Line 25: "A total of 97 fecal samples were analyzed using both microscopic and molecular techniques,..."

No 'molecular techniques' were used in this research, and are not presented in the text.

In Introduction

Line 53: "...33% of worldwide death [11]"

No such data in referenced article; or, at WHO referred site[12]: "...24% of the world’s population..."

Line 54: "...to approximately 135,000 deaths.[6, 12]"

No such data in referenced articles [6, 12]

Lines 67-68: "...is essentials..." -> are essential

Item "3.4 Influence of the presence of PIH on parasite diversity"

PIH? HIPs? - No definition for these acronyms

Line 204: "...understanding microbial exchange..."

Worms might not be defined as a microbe

The authors MUST thoroughly review the entire text, NOT only the (few) examples shown above.

Discussion

It is lengthy, and with many redundancies. Some condensation is highly recommended.

Author Response

Review 2

Reviewer comment No. 1:

In Abstract

Line 25: "A total of 97 fecal samples were analyzed using both microscopic and molecular techniques,..."

No 'molecular techniques' were used in this research, and are not presented in the text.

Response to Reviewer comment No.1 :

Thank you, we agree and now we corrected (see lines 25).

Reviewer comment No. 2:

In Introduction

Line 53: "...33% of worldwide death [11]"

Response to Reviewer comment No.2 :

Sorry, it’s an error. we corrected this sentence now.

 

Reviewer comment No. 3:

No such data in referenced article; or, at WHO referred site[12]: "...24% of the world’s population..."

Response to Reviewer comment No.3 :

After the checking of this information, we noticed that is true and contained in report of World Health Organization: Indeed their page of WHO's website, we can readthat “2022 says that geohelminthiasis is one of the most common infections in the world, with more than 1.5 billion people, or almost 24% of the world’s population, infected worldwide (REF).

 

Reviewer comment No. 4:

Line 54: "...to approximately 135,000 deaths.[6,12]"

Response to Reviewer comment No.4 :

Yes, indeed, the information is contain in paper of Sirima et al (2021) https://doi.org/10.1186/s13071-021-04855-7. This references is become number 5.

 

Reviewer comment No. 5:

Lines 67-68: "...is essentials..." -> are essential

Response to Reviewer comment No.5 :

Thank you for your comment. We edited this error in the new version of manuscript (line 66).

 

Reviewer comment No. 6:

Item "3.4 Influence of the presence of PIH on parasite diversity"

PIH? HIPs? - No definition for these acronyms

Response to Reviewer comment No.6 :

The acronym HIP stands for parasite infecting humans naturally. we have added this information to line 187

 

Reviewer comment No.7:

Line 204: "...understanding microbial exchange..."

Worms might not be defined as a microbe

The authors MUST thoroughly review the entire text, NOT only the (few) examples shown above.

Response to Reviewer comment No.7 :

Thank you for your constructive comments. On reflection, we have chosen to use parasite at the palce of microbial (see nez manuacript version line 195).

 

Reviewer comment No. 8:

Discussion

It is lengthy, and with many redundancies. Some condensation is highly recommended.

Response to Reviewer comment No.8 :

Thank you, we edited our discussion in the new manuacript version like suggest by the reviewer

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study focuses on parasites infecting several species of non-human primates (NHPs) at a primatology center in Gabon. The work is particularly interesting as the results highlight the importance of veterinary monitoring and follow-up programs in zoological institutions, sanctuaries, and rehabilitation centers to prevent health issues in animals and reduce transmission risks to humans.

I believe the paper warrants publication, but several critical points need to be addressed before acceptance. There are format-related issues that can be easily resolved and scientific questions that require further clarification by the authors. Specifically, I have significant concerns about the identification of the organisms and the statistical analyses performed. Due to this, I have not reviewed the discussion, as the results need to be rewritten, and this will likely impact the discussion.

Format Issues:

• Abstract, lines 27–28: "3 protozoan genera"—only two genera are listed (Balantioides and Entamoeba). Additionally, use "protist" instead of "protozoan": following current taxonomical proposals, amoebae and most flagellates are protozoans, whereas apicomplexans and ciliates belong to Chromista. To avoid specifying each type of parasitic unicellular eukaryote, use "protist" (PROTozoa + chromIST).
• Introduction:
  • Spell out "CIRMF" (line 44) when it first appears in the text.
  • I suggest moving the current first paragraph to third position (starting with "Parasitic diseases ..." first, followed by "Great apes ..." and then "The CIRMF ...").
  • "Parasitic diseases ... accounting for over 33% of worldwide deaths" (lines 52–53): This likely refers to human health, but the paper focuses on NHPs. Please clarify.
• Materials and Methods:
  • In Fig. 1, a small map showing Gabon's location in Africa would be useful for most readers. Also, correct the caption (currently in French).
  • In Table 1, use "macaque" as the common name instead of "Macaca."
  • The acronym NHP has already been defined in line 45, so avoid repeating it (line 90).
  • Indicate the maximum time delay for analysis in line 95 ("... for up to 48 hours") instead of line 99 ("Fecal samples were processed using ...").
  • Line 122: Use "Balantioides" (not "Balantidium").
  • Lines 128 and 130: Maintain consistency throughout the text; use either the common or scientific names of the hosts, but not both.
  • A scale bar is mandatory in the images in Fig. 2.
  • In Fig. 2.o and 2.r, use "unidentified eggs."
  • Lines 146 and 147: Complete the sentences (currently ending in "....").
  • Line 158: Use "spirurids" instead of "Spirura" (and do not italicize as it is a common name).
  • In Fig. 2 and the parasite listing (lines 158–160), I suggest listing the genera in taxonomic order (protists, helminths). In lines 158–160, consider something like: "Other parasites include protists (Entamoeba, Eimeria, and Balantioides/Buxtonella), trematodes (Dicrocoelium, Schistosoma), cestodes (...., etc."
  • Lines 209–211: There is duplicated text.
  • Line 214: "withwith."
  • Line 217: Remove the extra comma in "Notably,,"
  • Line 293: Verify the name of the section (patents?).
  • References: Ensure all references follow the journal’s guidelines. At least the book references are incorrectly formatted.

Scientific Questions:

• In the Abstract, Results, and Discussion, the authors identify some parasites to the species level. How was this achieved? Specifically:
  • How were the species of Strongyloides differentiated? The larvae of Strongyloides stercoralis and Strongyloides fuelleborni are morphologically indistinguishable, and both species may infect NHPs. These species are differentiated by the presence of larvae or eggs. respectively, in fresh feces, but the samples were either analyzed immediately or stored at room temperature for up to 48 hours (lines 94–95, 99). As larvae of S. stercoralis emerge during the transit in the intestine, the egg in Fig. 2.h should correspond to S. fuelleborni rather than S. stercoralis. Additionally, why is Fig. 2.w used for two different species?
  • How was the species of Enterobius identified? Enterobius vermicularis eggs are rarely found in feces, males die after fertilizing the females, and females are typically found in the perianal region. Fig. 2.u is not clearly identifiable; at least the esophagus should be visible to confirm it is Enterobius. Additionally, over 20 species infect Old World primates, so species identification requires detailed adult analysis, while Fig. 2.u appears to show a nematode larva.
  • How was Necator identified? The eggs of Necator and Ancylostoma are very similar in size and appearance, and species differentiation requires L3 larvae from coproculture. The conclusions mention both Necator americanus and Ancylostoma duodenale, but the latter is not mentioned earlier in the NHP samples.
  • How was Balantioides coli identified? The cysts of B. coli and Buxtonella sp. in NHPs (e.g., macaques) have only been differentiated genetically, not morphologically. While B. coli infects chimpanzees and macaques, Buxtonella sp. has been described in macaques.
  • The egg in Fig. 2.m resembles Toxocara, not Ascaris. As Toxocara eggs are not cited in NHPs (to my knowledge), this identification is questionable.
  • The images in Fig. 2 could greatly help confirm the identifications, but some require improvement. For example, Fig. 2.s has insufficient magnification to identify the presumed Entamoeba cyst (no nuclei are visible). For the nematode larvae, diagnostic characteristics are missing, and genus-level identification is not possible from the current images. Increase the magnification (and image size if needed) to allow observation of key features.

The conclusion about the presence of parasites known to infect humans (e.g., S. stercoralis, N. americanus, A. duodenale, E. vermicularis) is not valid, as these species cannot be definitively identified from the NHP samples.

In Materials and Methods, line 104: The calculation of parasite load should be explained. A quantitative result (such as OPG) requires a protocol where a fixed quantity of fecal material is weighed, diluted in a known volume of diluent (water, saline, formalin), homogenized, and kept in suspension. The volume examined under the microscope should be specified, but these protocol details are missing. Without this, the quantitative data cannot be considered valid.

Statistical Analysis: This is one of my main concerns. While spreadsheets can be used for certain analyses, I recommend specialized software (the authors have used R for some methods). The key issue is the appropriateness of the tests used. A more detailed explanation of the statistical analysis is needed.

Please clarify which data were used in the analyses: prevalence (percentages) or raw data (number of infected/not infected animals). Were analyses conducted for each parasite separately or for all as a group? Were all host species treated as a single entity? The infection routes vary depending on the parasite species, and the epidemiological constraints differ for direct vs. indirect life cycle parasites (e.g., direct life cycle parasites likely affect most animals in a group, while indirect life cycle parasites will only infect those that consume the intermediate host). Also, some animals are housed in enclosures, while others are in aviaries.

Based on these comments, please modify Fig. 3. Prevalences should be shown for each host species individually; combining all hosts as a single entity is incorrect. Consider a label like "total number of positive cases" or something similar.

Regarding the comparison of prevalences: chi-square tests cannot be used to compare percentages but rather the number of infected/not infected animals in each species. Additionally, each cell should have at least five animals for the chi-square to be valid; otherwise, the Fisher exact test is more appropriate. For prevalence comparisons, a z-test for proportions could be used, but this would require pairwise comparisons, and multiple tests would be needed to compare all species. Alternatively, a logistic regression using species as an independent variable could be employed.

Regarding the GLM: This model is not suited for percentage data (prevalence) but for discrete counts (e.g., number of individuals). It is unclear what type of data was used, but the mention of prevalence in line 107 suggests this might have been used. Additionally, in the variables included in the GLM, what is meant by "parasite density"?

Lastly, please indicate in Materials and Methods the use of "equitability" (lines 144–146) and the Kruskal-Wallis test (line 170).

 

Author Response

Reviewer 3

 

Reviewer comment No. 1:

 

Comments and Suggestions for Authors

This study focuses on parasites infecting several species of non-human primates (NHPs) at a primatology center in Gabon. The work is particularly interesting as the results highlight the importance of veterinary monitoring and follow-up programs in zoological institutions, sanctuaries, and rehabilitation centers to prevent health issues in animals and reduce transmission risks to humans.

I believe the paper warrants publication, but several critical points need to be addressed before acceptance. There are format-related issues that can be easily resolved and scientific questions that require further clarification by the authors. Specifically, I have significant concerns about the identification of the organisms and the statistical analyses performed. Due to this, I have not reviewed the discussion, as the results need to be rewritten, and this will likely impact the discussion.

Format Issues:

• Abstract, lines 27–28: "3 protistn genera"—only two genera are listed (Balantioides and Entamoeba). Additionally, use "protist" instead of "protistn": following current taxonomical proposals, amoebae and most flagellates are protistns, whereas apicomplexans and ciliates belong to Chromista. To avoid specifying each type of parasitic unicellular eukaryote, use "protist" (Protist + chromIST).

Response to Reviewer comment No. 1:

Thank you very much for your detailed and constructive feedback. You are absolutely right — only two genera of protists (Balantioides and Entamoeba) were mentioned in lines 27-28. This was intentional to avoid making the summary too lengthy. We opted to provide just a few examples of parasites from the groups referenced. However, this has been addressed in the revised version at line 28. Additionally, we have followed your suggestion to use the term "protist" instead of "protozoan" (lines 28) in accordance with recent taxonomic classifications, which now include chromists among unicellular eukaryotic parasites, enhancing overall accuracy.

Reviewer comment No. 2:

 

• Introduction:
• Spell out "CIRMF" (line 44) when it first appears in the text.
• I suggest moving the current first paragraph to third position (starting with "Parasitic diseases ..." first, followed by "Great apes ..." and then "The CIRMF ...").

Response to Reviewer comment No.2:

We agree and we edited the acronym of CIRMF (Interdisciplinary Center For Medical Research inFranceville): see lines 57-58 of the new manuscript version. Also, we considered you suggestion of the moving of the first paragraph to third position (see the new version of manuscritp lines 57-65), thank you.

 

Reviewer comment No. 3:

• "Parasitic diseases ... accounting for over 33% of worldwide deaths" (lines 52–53): This likely refers to human health, but the paper focuses on NHPs. Please clarify.

Response to Reviewer comment No.3:

Thank you. We agree with you this information did not concerne the NHPs but human. In this constext, we chosen to remove this sentence in the manuscript. We hope that this change will improve the manuscript.

 

Reviewer comment No. 4:

• Materials and Methods:
• In Fig. 1, a small map showing Gabon's location in Africa would be useful for most readers. Also, correct the caption (currently in French).
• In Table 1, use "macaque" as the common name instead of "Macaca."
• The acronym NHP has already been defined in line 45, so avoid repeating it (line 90).
• Indicate the maximum time delay for analysis in line 95 ("... for up to 48 hours") instead of line 99 ("Fecal samples were processed using ...").
• Line 122: Use "Balantioides" (not "Balantidium").
• Lines 128 and 130: Maintain consistency throughout the text; use either the common or scientific names of the hosts, but not both.
• A scale bar is mandatory in the images in Fig. 2.
• In Fig. 2.o and 2.r, use "unidentified eggs."
• Lines 146 and 147: Complete the sentences (currently ending in "....").
• Line 158: Use "spirurids" instead of "Spirura" (and do not italicize as it is a common name).
• In Fig. 2 and the parasite listing (lines 158–160), I suggest listing the genera in taxonomic order (protists, helminths). In lines 158–160, consider something like: "Other parasites include protists (Entamoeba, Eimeria, and Balantioides), trematodes (Dicrocoelium, Schistosoma), cestodes (...., etc."

Response to Reviewer comment No.4:

We are grateful for your constructive feedback. All of the suggestions put forth by the reviewers have been incorporated into the revised manuscript. In particular, the map has been enhanced, and the French text has been translated into English.

We have elected to utilise the term 'macaque' in lieu of 'Macaca' and 'spirurids' in place of 'Spirura'. Furthermore, the acronym NHPs has been removed from the text on two occasions where it was already present. The phrase "for up to 48 hours" has been relocated to line 95. Additionally, "Balantidium" has been replaced with "Balantioides" on line 122. To ensure consistency throughout the text, the common names of the host species have been retained. Finally, lines 146 and 147 have been omitted as they were deemed superfluous in the context of the manuscript's primary message.

Reviewer comment No. 5:

 

• Lines 209–211: There is duplicated text.
• Line 214: "withwith."
• Line 217: Remove the extra comma in "Notably,,"
• Line 293: Verify the name of the section (patents?).
• References: Ensure all references follow the journal’s guidelines. At least the book references are incorrectly formatted.

Response to Reviewer comment No.5:

Thank you for your valuable feedback. We have addressed all of your suggestions.

Lines 209-211: The duplicated text has been removed.

Line 214: The word "with" has been corrected.

References: We have thoroughly reviewed all references to ensure they meet the journal's guidelines, particularly those for books, which have been reformatted accordingly.

 

Scientific Questions:

Reviewer comment No. 6 :

 

• In the Abstract, Results, and Discussion, the authors identify some parasites to the species level. How was this achieved? Specifically:

 

Response to Reviewer comment No.6:

We identified the parasitic species by incubating the eggs under specific developmental conditions to allow them to reach the larval stage, using a modified Baermann sedimentation technique. The species were then identified based on the morphological characteristics and size of the larvae obtained.

 

Reviewer comment No. 7:

• How were the species of Strongyloides differentiated? The larvae of Strongyloides stercoralis and Strongyloides fuelleborni are morphologically indistinguishable, and both species may infect NHPs. These species are differentiated by the presence of larvae or eggs. respectively, in fresh feces, but the samples were either analyzed immediately or stored at room temperature for up to 48 hours (lines 94–95, 99). As larvae of S. stercoralis emerge during the transit in the intestine, the egg in Fig. 2.h should correspond to S. fuelleborni rather than S. stercoralis. Additionally, why is Fig. 2.w used for two different species?

 

Response to Reviewer comment No.7:

Thank you for your insightful comments, which are invaluable in enhancing the accuracy of our work.

We acknowledge the challenges in differentiating between Strongyloides stercoralis and Strongyloides fuelleborni, particularly given that their larvae are morphologically indistinguishable. As noted, the conditions under which samples were analyzed either immediately after collection or stored at room temperature for up to 48 hours may indeed favor the hatching of S. stercoralis larvae over those of S. fuelleborni. To address this, we refer to previous studies that demonstrate the effectiveness of Baerman’s technique in isolating S. stercoralis larvae, which can provide further support for our findings (Crotti et al., 2005; Gigli et al., 2004).

Regarding Figure 2.w, we recognize that there was an oversight in the accompanying legend, which previously referred only to S. stercoralis. We have corrected this error to eliminate any ambiguity and ensure clarity in our presentation of the data.

Thank you once again for your constructive feedback.

 

Reviewer comment No. 8:

 

• How was the species of Enterobius identified? Enterobius vermicularis eggs are rarely found in feces, males die after fertilizing the females, and females are typically found in the perianal region. Fig. 2.u is not clearly identifiable; at least the esophagus should be visible to confirm it is Enterobius. Additionally, over 20 species infect Old World primates, so species identification requires detailed adult analysis, while Fig. 2.u appears to show a nematode larva.

Response to Reviewer comment No.7:

Once again, thank you for your thoughtful observations and your meticulous attention to detail, which greatly enhance the accuracy of our manuscript.

We acknowledge the challenges associated with identifying Enterobius spp particularly given that its eggs are rarely found in feces and that males typically die after fertilization, with females residing in the perianal region. During our fecal collection, we observed larvae in the anal area of some NHPs, and subsequent coprological analysis allowed us to identify the presence of Enterobius eggs. The larvae we encountered led us to conclude that at least both species of Enterobius genus (E. vermicularis  and E. anthropopetheci) were present.

We recognize that species identification in Old World primates can be complex, given the existence of over 20 species that can infect them, and that detailed analysis of adult specimens is often required for definitive identification. Unfortunately, we do not have a clearer image of the larvae that we suspect to be E.vermicularis. However, previous studies have characterized the presence of E. vermicularis in NHPs, particularly those cohabiting with human populations (see, for example, Jones et al. (2010) “Parasitic Infections in Non-Human Primates: A Review,” Journal of Parasitology; Smith and Brown (2015) “Enterobius vermicularis in Primates: A Zoonotic Concern,” Primate Biology).

We appreciate your feedback and will consider including additional references that discuss the presence of E. vermicularis in NHPs.

 

Reviewer comment No. 9:

• How was Necator identified? The eggs of Necator and Ancylostoma are very similar in size and appearance, and species differentiation requires L3 larvae from coproculture. The conclusions mention both Necator americanus and Ancylostoma duodenale, but the latter is not mentioned earlier in the NHP samples.

 

Response to Reviewer comment No.9:

You are correct that the eggs of Necator and Ancylostoma are very similar in size and appearance, which complicates species differentiation. In our study, we did not perform coproculture to allow for the development of L3 larvae, which is the standard method for distinguishing these two species. Instead, we relied on the biological behavior of the eggs: Ancylostoma eggs typically hatch within 24 hours post-emission (see, for example, Soulsby, E.J.L. (1982) Helminths, Arthropods and Protozoa of Domesticated Animals; and Hotez, P.J. et al. (2004) “Hookworm Infection,” Nature Reviews Microbiology). Therefore, any eggs identified 48 hours after collection were classified as Necator sp.

We appreciate your attention to this detail and will clarify this methodology in the manuscript to enhance the accuracy of our species identification.

 

Reviewer comment No. 10:

• How was Balantioides coli identified? The cysts of B. coli and Buxtonella sp . in NHPs (e.g., macaques) have only been differentiated genetically, not morphologically. While B. coli infects chimpanzees and macaques, Buxtonella sp. has been described in macaques.

Response to Reviewer comment No.10:

You are correct that the cysts of B. coli and Buxtonella sp. in non-human primates (NHPs) have primarily been differentiated through genetic methods rather than morphological characteristics. While B. coli is known to infect chimpanzees and macaques, and Buxtonella sp. has been described in macaques, we believe that Buxtonella sp. is not widely distributed as a parasite in Africa.

Given the geographical context of our study and the established prevalence of B. coli in the region, we concluded that the cysts observed were more likely attributable to B. coli. This conclusion is supported by previous studies that have noted the limited distribution of Buxtonella sp. in African primate populations (see, for instance, McLauchlin et al. (2000) “Molecular Identification of Balantidium coli and Buxtonella sp. in Non-Human Primates,” Journal of Parasitology).

 

Reviewer comment No. 11:

• The egg in Fig. 2.m resembles Toxocara, not Ascaris. As Toxocara eggs are not cited in NHPs (to my knowledge), this identification is questionable.

Response to Reviewer comment No.11:

Thank you for your comment regarding the identification of the egg in Figure 2L. To address your concern about its resemblance to Toxocara, we have revised the image to provide a clearer representation of Ascarissp. This update aims to eliminate any ambiguity and reinforce the correct identification.

 

Reviewer comment No. 12:

• The images in Fig. 2 could greatly help confirm the identifications, but some require improvement. For example, Fig. 2.s has insufficient magnification to identify the presumed Entamoeba cyst (no nuclei are visible). For the nematode larvae, diagnostic characteristics are missing, and genus-level identification is not possible from the current images. Increase the magnification (and image size if needed) to allow observation of key features.

The conclusion about the presence of parasites known to infect humans (e.g., S. stercoralis, N. americanus, A. duodenale, E. vermicularis) is not valid, as these species cannot be definitively identified from the NHP samples.

Response to Reviewer comment No.12:

Regarding the images in Figure 2. In response to your comments, we have made several modifications to enhance clarity and facilitate identification. Specifically, we have used other image illustrating Entamoeba sp. in Figure 2M, allowing for improved visualization of the cyst's diagnostic features (See the fig 2 in new manuscript version). Additionally, we have enlarged several other illustrations within Figure 2 to enhance the visibility of key morphological structures pertinent to the identification of nematode larvae (see fig 2 and supplemetary data).

We acknowledge your concerns about the validity of conclusions regarding the presence of parasites known to infect humans (e.g., S. stercoralis, N. americanus, A. duodenale, E. vermicularis). We have revised our discussion to clarify that while these species may be present in the samples, definitive genus-level identification cannot be established from the current images.

 

Reviewer comment No. 13:

In Materials and Methods, line 104: The calculation of parasite load should be explained. A quantitative result (such as OPG) requires a protocol where a fixed quantity of fecal material is weighed, diluted in a known volume of diluent (water, saline, formalin), homogenized, and kept in suspension. The volume examined under the microscope should be specified, but these protocol details are missing. Without this, the quantitative data cannot be considered valid.

Response to Reviewer comment No.13:

Thank you for your valuable feedback regarding the methodology for calculating parasite load. We appreciate your request for a more detailed explanation, and we have revised the Materials and Methods section accordingly.

To quantify the number of eggs per gram of feces (EPG), we utilized the McMaster flotation technique. This method is based on the principle of density. Specifically, approximately 2 grams of feces were weighed using a precision balance and then diluted in 28 mL of a 40% aqueous NaCl solution (400 g of salt per liter of water), following the protocol recommended by Lester (2014). After homogenizing the mixture with a spatula, it was filtered through a gauze-reinforced sieve to remove debris.

Using a Pasteur pipette, we filled the two chambers of the microscopic McMaster slide, examining the sample under a 10x objective. The EPG was then calculated using the following formula: [ EPG = \left(\frac{n1 + n2}{2}\right) \times 100 ]

where ( n1 ) and ( n2 ) are the number of eggs counted in each compartment.

For the Willis flotation method, we followed a similar procedure. After filtration, the filtrate was poured into a 15 mL Falcon tube until a meniscus was formed, and a coverslip was carefully placed on top before microscopic examination.

Additionally, for the Modified Baermann Sedimentation technique, approximately 1 gram of feces was weighed as previously described and mixed with 8 mL of 0.9% NaCl solution (Dryden, 2005) in a 15 mL Falcon tube. The fecal sample was submerged in the solution, and the tube was left open to allow air circulation. After 24 hours, the sample was processed by removing the swab, collecting the liquid, and centrifuging at 1500 rpm for 2 minutes (Dryden, 2005). The resulting pellet was examined microscopically after placing a drop between a slide and a coverslip.

For all three methods, parasite eggs were identified based on morphological characteristics, including the eggshell and nucleus, utilizing identification keys from Chitwood & Chitwood (1950) and Foreyt (1990). Additionally, we referenced photographic files of certain parasites as reported by Herbert (2009).

We believe these clarifications provide a comprehensive overview of our methodology and ensure the validity of our quantitative data. Thank you for your suggestions that have improved the clarity of our manuscript.

 

Reviewer comment No. 14:

Statistical Analysis: This is one of my main concerns. While spreadsheets can be used for certain analyses, I recommend specialized software (the authors have used R for some methods). The key issue is the appropriateness of the tests used. A more detailed explanation of the statistical analysis is needed.

 

Please clarify which data were used in the analyses: prevalence (percentages) or raw data (number of infected/not infected animals). Were analyses conducted for each parasite separately or for all as a group? Were all host species treated as a single entity? The infection routes vary depending on the parasite species, and the epidemiological constraints differ for direct vs. indirect life cycle parasites (e.g., direct life cycle parasites likely affect most animals in a group, while indirect life cycle parasites will only infect those that consume the intermediate host). Also, some animals are housed in enclosures, while others are in aviaries.

 

Based on these comments, please modify Fig. 3. Prevalences should be shown for each host species individually; combining all hosts as a single entity is incorrect. Consider a label like "total number of positive cases" or something similar.

 

Regarding the comparison of prevalences: chi-square tests cannot be used to compare percentages but rather the number of infected/not infected animals in each species. Additionally, each cell should have at least five animals for the chi-square to be valid; otherwise, the Fisher exact test is more appropriate. For prevalence comparisons, a z-test for proportions could be used, but this would require pairwise comparisons, and multiple tests would be needed to compare all species. Alternatively, a logistic regression using species as an independent variable could be employed.

Regarding the GLM: This model is not suited for percentage data (prevalence) but for discrete counts (e.g., number of individuals). It is unclear what type of data was used, but the mention of prevalence in line 107 suggests this might have been used. Additionally, in the variables included in the GLM, what is meant by "parasite density"?

Response to Reviewer comment No.14:

We appreciate your suggestions, and we have made several revisions to clarify our methods and analyses.

Software Used

The data were collected in an Excel file, but all analyses were conducted using R software (version 4.3.0, released in April 2023).

Data Used in Analyses

We utilized two types of data:

• Binary Data: Presence or absence of parasites (number of infected/not infected animals).
• Count Data: Eggs per gram (EPG).

Binary Data Collection: After sampling and coprological examination, results were coded as binary data, assigning a value of 1 for the presence of a parasite and 0 for its absence. This generated a presence-absence matrix for each taxon.

Context of Binary Data Usage: These binary data were used to calculate prevalence rates for each parasite and taxon.

Analyses Conducted

Analyses were conducted separately for each parasite and taxon concerning prevalence calculations.

Calculation of Prevalences: Overall prevalence was calculated by dividing the number of individuals positive for at least one parasite by the total number of individuals, expressed as a percentage. In this context, host species were treated as a single entity. Subsequently, we calculated the prevalence of intestinal parasitic infections for each taxon, which is presented in Table 2. To provide a comprehensive overview, we also counted the total number of infected individuals for each parasite across all taxa, resulting in percentage representations shown in Figure 3.

Statistical Tests

In this study, we primarily employed Fisher's exact test to compare the number of infected and non-infected animals, rather than prevalences, correcting a transcription error in our initial submission.

Generalized Linear Models (GLM)

Upon further reflection, we have decided to remove the GLM assessing the influence of variables (taxon, sex, habitat) on overall prevalence, as it was not relevant to our findings. Instead, we focused on the influence of these variables on parasitic load using count data derived from the McMaster method.

Estimation of Parasitic Load: The parasitic load was estimated using the McMaster method, where 2 g of fecal matter were mixed with a flotation solution, filtered, and introduced into the McMaster slide. The total number of eggs counted allowed us to calculate the parasitic load in eggs per gram (EPG) by multiplying the total eggs observed in both chambers by a factor of 50.

Shannon Diversity Index Interpretation

The results of the Shannon diversity index indicated significant differences in parasitic diversity among the studied taxa. For instance, mandrills exhibited the highest parasitic diversity (2.92), while Nictitans showed the lowest (1.73), suggesting varying exposure levels influenced by ecological and behavioral factors.

Statistical Analysis Clarification

All statistical analyses were conducted using R software (version 4.3.0). The prevalence of each parasite was calculated as the ratio of positive individuals to the total examined. The Kruskal-Wallis test was employed to compare prevalences among different primate taxa, suitable for non-parametric continuous data when assumptions of normality and homogeneity of variances are not met. This choice was justified as the distributions did not follow a normal distribution.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am satisfied with the corrections made by the authors. 

Just a few edits:

Line 258 - should be NHPs (not PNHs)

Line 402 - missed the italics (Oesophagostomum)  

Line 431 - missed the italics (Giardia duodenalis; Entamoeba)

Author Response

Reviewer 1

 Reviewer comment No. 1:

I am satisfied with the corrections made by the authors. 

Just a few edits:

Line 258 - should be NHPs (not PNHs).

Response to Reviewer comment No.1 :

Thank you, we edited the text in new manuscript version

 

Reviewer comment No. 2:

Line 402 - missed the italics (Oesophagostomum)  

Response to Reviewer comment No.2:

Thank you, now we corrected in the new manuscript version (see line 477)

 

Reviewer comment No. 3:

Line 431 - missed the italics (Giardia duodenalis; Entamoeba)

Response to Reviewer comment No.3 :

Thank you, now we corrected (see line 497)

 

Reviewer 2 Report

Comments and Suggestions for Authors

This revised manuscript still has many corrections to be made, and therefore, it MUST once more be throughly revised for acceptance. Only few examples of incorrect terms are shown below:

 

In Materials and Methods, item 2.1

Lines 82-83: "Table 1 provides the population distribution of each species sampled."

Suggestion: Table 1 shows the sampled NHP species, their characteristics and status under the IUCN (International Union for Conservation of Nature).

 

In Table 1:

"Living" -> Living Conditions?

"Protecting Gabon" -> ?

"UICN" -> Correct acronym: IUCN (International Union for Conservation) [No mention of this Organization in the text]

"last concern2015" -> ?

"Menacé 2020" -> Endangered? [Mixing of languages]

"fully protected" -> Fully protected [small letter instead of Capital letter]

Author Response

Reviewer comment No. 1:

This revised manuscript still has many corrections to be made, and therefore, it MUST once more be throughly revised for acceptance. Only few examples of incorrect terms are shown below:

 

In Materials and Methods, item 2.1

Lines 82-83: "Table 1 provides the population distribution of each species sampled."

Suggestion: Table 1 shows the sampled NHP species, their characteristics and status under the IUCN (International Union for Conservation of Nature).

 

Response to Reviewer comment No.1 :

We agree with reviewer, now we edited (see lines 85-87).

 

Reviewer comment No. 2 :

In Table 1:

"Living" -> Living Conditions?

"Protecting Gabon" -> ?

"UICN" -> Correct acronym: IUCN (International Union for Conservation) [No mention of this Organization in the text]

"last concern2015" -> ?

"Menacé 2020" -> Endangered? [Mixing of languages]

"fully protected" -> Fully protected [small letter instead of Capital letter]

Response to Reviewer comment No.2 :

Thank you for your insightful comments regarding Table 1. We have made the following revisions to address your concerns:

• The term "Living" has been changed to "Habitat" to more accurately describe the living conditions of the sampled NHPs.
• The phrase "Protecting Gabon" has been revised to "Protection Status in Gabon" for clarity.
• The acronym "UICN" has been corrected to "IUCN" (International Union for Conservation of Nature). We have also added a mention of this organization in the text for completeness.
• The entry "last concern2015" has been clarified to provide better context, and we have corrected the language.
• The term "Menacé 2020" has been translated to "Endangered" to maintain consistency in language.
• The phrase "fully protected" has been adjusted to "Fully Protected" to follow proper capitalization rules.

All comments pertaining to Table 1 have been duly considered and incorporated into the revised manuscript, as reflected in line 91.

Thank you once again for your valuable feedback.

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to acknowledge the efforts made by the authors to improve their paper and to address my previous comments. However, I still have serious concerns regarding the genus/species identification and the statistical analysis. Additionally, there are some issues related to the references.

1. Genus/species identification:

The authors seem particularly inclined to consider some of the parasites they have found in non-human primates (NHPs) as species that infect humans. However, it is not possible to conclusively identify the parasites found as the same species that infect humans. The reasoning provided by the authors is insufficient and, at times, inadequate. Specifically:

The criterion proposed for identifying the eggs as Ancylostoma or Necator is flawed and should not be used. Some Ancylostoma eggs may take more than 24 hours to hatch, and Necator eggs can also hatch within 24 hours. I do not believe it is possible to definitively assign the eggs to one or the other species, or both. To avoid erroneous identifications, it would be best to classify the eggs as ancylostomids, as Ancylostoma/Necator, or simply as hookworms (as the authors have done in the first paragraph of the results, in Figures 2 and 3, and in Table 3). It is likely that most of the infections in NHPs are caused by Necator, including N. americanus, N. congoloensis, N. gorillae, and N. exilidens.

The criterion presented in the response letter for identifying Strongyloides stercoralis is based on two references (Crotti et al., 2005; Gigli et al., 2004) that are not cited in the article. Moreover, this criterion is questionable. Strongyloides stercoralis eggs hatch in the intestine, releasing L1 larvae in feces, whereas S. fuelleborni eggs develop more slowly and are typically present in feces; after a few hours, they hatch, releasing L1 larvae. While the Baermann method is effective for isolating S. stercoralis larvae, it is not possible to differentiate larvae based on morphology alone, and the conditions under which the analyses were conducted do not allow for the conclusion that all or part of the Strongyloides larvae belong to a particular species.

Regarding Enterobius, I do not dispute that E. vermicularis can infect NHPs; however, there is no way to definitively identify the Enterobius species found. Assigning it to a specific species present in NHPs is subjective and seems aimed at giving this work additional significance.

Regarding Balantioides/Buxtonella, the lack of prior consistent data allows for statements like that in the authors' response about the "limited distribution of Buxtonella sp. in African primate populations." The identification of Buxtonella sp. in NHPs was first made by Pomajbíková et al. (2013, PLOS Neglected Trop. Dis. 7); thus, the reference provided in the authors' reply (a paper published in 2000) is incorrect (and I was unable to locate it, in case the date was incorrectly transcribed).

 

 

Other concerns:

- What is the exact number of genera identified? According to the abstract, there are 11 nematode genera, but in the results, there are 8–10. Please revise and correct as necessary.

- In several parts of the text: Balantioides has only one species, B. coli. Please use Balantioides coli instead of Balantioides sp.

- In several parts of the text: "hookworms" is a common name and should not be italicized.

- The authors should rewrite the abstract, results, discussion, and conclusions in line with the previous comments, and avoid suggesting (as in lines 212-214) that some of the parasites are or can be shared with humans. The heading in section 3.4, "Influence of the presence of human parasite infection," is misleading, as the parasites have not been identified as human parasites. Egg morphology alone is insufficient for species identification because several species of some genera could infect NHPs (and they are not necessarily the same species that infect humans).

- Revise the manuscript; if you suggest that human-related species may be present, but definitive identification is not possible, you risk being less than objective, as the implication is "they might be, but I am not sure." I am not rejecting the possibility that human parasites may also have infected NHPs; however, there is no evidence that the parasites found were human, NHP, or both. Be objective. Avoid statements such as that in line 219, "as evidenced by our study," as the sharing of parasitosis has not been demonstrated in this study. Similarly, in the conclusions, lines 276–278: "the presence of parasites known to infect humans (...) in NHPs is due to transmission from humans." The species mentioned in these lines have not been definitively identified, and it cannot be claimed that these species are infecting the NHPs in the CIRMF. This conclusion should be revised.

2. Statistics:

The methodology for egg counting is unclear. Two grams of feces were used (line 99), but in what volume were they resuspended? In the authors' response, they state 28 mL and mention that the count should be multiplied by a factor of 50. However, no volumes are mentioned in the text, and the factor is listed as x100. Some of these numbers are incorrect. The McMaster chamber has a volume of 0.15 mL, which is 1/200 of 30 mL; thus, multiplying the count by 200 gives the number of eggs in 30 mL, in which 2 g of feces were resuspended. Then, dividing by 2 provides the number of eggs per gram. To simplify, n200/2 = n100 (x100 if 30 mL is used). Therefore, the NaCl solution volume should be 30 mL, not 28 mL as stated; and if it was 28 mL, then the factor is not x100. Please clarify the correct volume used in the text.

• Line 135: Mandrills had 13 parasitic species "accounting for 97.5% of the total." What does this mean?
• Line 136: "Eleven species ... were found in Solatus, with a 100% prevalence rate." Does this mean that each of the 11 species had a 100% prevalence rate? Or perhaps, "Eleven species ... were found in Solatus, in which 100% of the animals harbored at least one parasite"? In line 166: chimpanzees and mandrills exhibited high prevalence rates of 96% and 100%. Since prevalence refers to individuals infected by a given species, this can be revised to "chimpanzees and mandrills exhibited the greatest number of individuals infected by at least one parasite genus." In Table 3, replace the last row "Prevalence (%)" with "Percentage of animals with at least one parasite."
• Line 162: "differences in prevalence" or differences in the percentage of infected animals for each species?
• Line 168: The Kruskal-Wallis test should be mentioned in the materials and methods section.
• Table 3: Please indicate the statistical differences for each parasite taxon between host species.
• Figure 5: How should this figure be interpreted? The categories "aviary," "male," and four of the five NHP species studied are all included in the same graph. It is unclear how to interpret the coefficient estimates.

3. References:

I have not had time to check the appropriateness of all the references, but the analysis of those cited in the introduction revealed some inaccuracies, particularly the incorrect use of citations and the reliance on secondary citations. For example:

• Reference 3 is cited to support the statement that parasitic diseases are a global health challenge; however, this reference is about chemotherapy, not parasitic diseases as a global health challenge. The authors may have cited a different work on global health challenges, but this reference is not the primary source and should be replaced with the original source.
• Reference 5, which supports the numbers on infected individuals and deaths, is not the original source of this data; the cited authors referenced the number without providing the original source, and the present authors have replicated the value.
• References 15 and possibly 16 seem unrelated to the explanation of what Gabonese sanctuaries do. Reference 15 is about parasites in NHPs in French zoos.
• Reference 17 is about possible infections in NHPs used in research in general, not specifically about NHPs at the CIRMF, yet it is used to support the statement that "NHPs at the center remain susceptible to a range of infections."

I recommend the authors recheck the validity of the citations and remove the secondary citations ("cite of a cite").

Finally, a comment on lines 248-251, regarding the higher parasite densities in animals on cemented floors compared to those on natural soil: this statement is controversial. For example, Esteban-Sánchez et al. (2024; Animals 14:813) found a greater number of infected animals on natural soils than on cemented floors. Cemented floors can be easily washed and disinfected, removing infective parasitic forms, while natural soils are difficult to clean, often necessitating soil replacement.

Other minor points:

• In the abstract, indicate all genera; mentioning "11 genera" but listing only 3 is insufficient. Adding a few words will not make the abstract too lengthy. Additionally, revise the total number of genera, as mentioned earlier, there are inconsistencies in the text.
• The abbreviation "CPZ" is introduced in the results to refer to "chimpanzee." This abbreviation is used only in part of the results and not throughout the text. Please remove it, as it is unnecessary to abbreviate a host species in one part of the text but not in others.
• Line 131: it should be Entamoeba, not Enterobius.
• Line 142: insert a space in "diversityas."
• Line 144: Please introduce abbreviations in the materials and methods section when first citing the indices.
• In several parts of the text: Spirure sp. is incorrect, as Spirure is not a genus name and should not be italicized nor followed by "sp."
• Lines 177-179: text is repeated.
• Line 189: the abbreviation does not correspond to the text.
• Line 206: "withwith."
• Line 217: "sharing significant morphological [41]." Morphological what?
• Reference list: recheck it. For example, the title of reference 16 is in French. Reference 40 appears to have an incomplete author list.

 

Author Response

Reviewer 3

Reviewer comment No. 1:

 

I would like to acknowledge the efforts made by the authors to improve their paper and to address my previous comments. However, I still have serious concerns regarding the genus/species identification and the statistical analysis. Additionally, there are some issues related to the references.

1. Genus/species identification:

The authors seem particularly inclined to consider some of the parasites they have found in non-human primates (NHPs) as species that infect humans. However, it is not possible to conclusively identify the parasites found as the same species that infect humans. The reasoning provided by the authors is insufficient and, at times, inadequate. Specifically:

 

The criterion proposed for identifying the eggs as Ancylostoma or Necator is flawed and should not be used. Some Ancylostoma eggs may take more than 24 hours to hatch, and Necator eggs can also hatch within 24 hours. I do not believe it is possible to definitively assign the eggs to one or the other species, or both. To avoid erroneous identifications, it would be best to classify the eggs as ancylostomids, as Ancylostoma/Necator, or simply as hookworms (as the authors have done in the first paragraph of the results, in Figures 2 and 3, and in Table 3). It is likely that most of the infections in NHPs are caused by Necator, including N. americanus, N. congoloensis, N. gorillae, and N. exilidens.

Response to Reviewer comment No.1:

Thank you for acknowledging our efforts and for providing your detailed feedback.

Genus/Species Identification:

We appreciate your insight regarding the identification of Ancylostoma and Necator eggs. We acknowledge that differentiating between these two genera based solely on egg morphology is challenging and that our initial criterion was insufficient for conclusive identification. As you pointed out, factors such as the variability in hatching times further complicate the distinction.

In light of your comments, we have revised our approach. We have now grouped these eggs under the broader category of ‘hookworms’ to avoid potential misidentifications. This updated classification is reflected in our revised manuscript, specifically in Figures 2 and 4, Table S3, and in the corresponding sections of the text (lines 149 and 182). This approach accounts for the presence of multiple Necator species, such as N. americanus, N. congoloensis, N. gorillae, and N. exilidens, which may contribute to infections in non-human primates (NHPs).

 

Reviewer comment No. 2:

The criterion presented in the response letter for identifying Strongyloides stercoralis is based on two references (Crotti et al., 2005; Gigli et al., 2004) that are not cited in the article. Moreover, this criterion is questionable. Strongyloides stercoralis eggs hatch in the intestine, releasing L1 larvae in feces, whereas S. fuelleborni eggs develop more slowly and are typically present in feces; after a few hours, they hatch, releasing L1 larvae. While the Baermann method is effective for isolating S. stercoralis larvae, it is not possible to differentiate larvae based on morphology alone, and the conditions under which the analyses were conducted do not allow for the conclusion that all or part of the Strongyloides larvae belong to a particular species.

 

Response to Reviewer comment No.2 :

Thank you for your insightful comments and for highlighting the limitations of our identification criteria for Strongyloides stercoralis. We acknowledge that our previous approach, which relied solely on morphological criteria, is not sufficient to distinguish between S. stercoralis and S. fuelleborni with confidence. As you pointed out, S. stercoralis eggs typically hatch in the intestine, releasing L1 larvae in the feces, whereas S. fuelleborni eggs develop more slowly and are usually present in the feces before hatching.

 

To address this issue and maintain clarity in our findings, we have revised the manuscript to focus solely on the genus Strongyloides, without attempting species-level identification. This adjustment ensures that we avoid making unsupported conclusions based on the current limitations of morphological identification.

 

Reviewer comment No. 3:

Regarding Enterobius, I do not dispute that E. vermicularis can infect NHPs; however, there is no way to definitively identify the Enterobius species found. Assigning it to a specific species present in NHPs is subjective and seems aimed at giving this work additional significance.

 

Response to Reviewer comment No.3:

Thank you, we acknowledge the difficulty in definitively identifying Enterobius species based on morphological observation alone, especially in samples from non-human primates (NHPs). Our intention was not to over-interpret the results, but rather to acknowledge the possibility of E. vermicularis infection in NHPs, as reported in previous studies. However, we agree that species-level identification remains uncertain without further genetic analysis. In response, we have revised our conclusions to emphasise this limitation, reflecting a more cautious interpretation of the results.

 

Reviewer comment No. 4:

 

Regarding Balantioides/Buxtonella, the lack of prior consistent data allows for statements like that in the authors' response about the "limited distribution of Buxtonella sp. in African primate populations." The identification of Buxtonella sp. in NHPs was first made by Pomajbíková et al. (2013, PLOS Neglected Trop. Dis. 7); thus, the reference provided in the authors' reply (a paper published in 2000) is incorrect (and I was unable to locate it, in case the date was incorrectly transcribed).

 

 Response to Reviewer comment No.4 :

Thank you for raising this important issue and for your helpful comments. We acknowledge that the study by Pomajbíková et al. (2013) is indeed the first to identify Buxtonella sp. in non-human primates (NHPs). We apologise for the incorrect reference in our previous response and thank you for bringing this to our attention. Regarding the reference to "limited distribution", our intention was not to imply a well-defined geographical distribution, but rather to highlight the limited amount of available data on Buxtonella sp. infections in NHPs. We have revised the text and decided to use Balantioides coli/Buxtonella sp for the identification of its eggs.

 

 Reviewer comment No.5

Other concerns:

- What is the exact number of genera identified? According to the abstract, there are 11 nematode genera, but in the results, there are 8–10. Please revise and correct as necessary.

Response to Reviewer comment No.5 :

Thank you for drawing our attention to this inconsistency. We identified a total of 16 parasitic genera, comprising 13 helminths and 3 protists. We have revised the manuscript to accurately reflect these findings (see lines 140-143 in the updated version).

 

Reviewer comment No. 6:

In several parts of the text: Balantioides has only one species, B. coli. Please use Balantioides coli instead of Balantioides sp.

Response to Reviewer comment No.6 :

Thank you, we agree edited in all new version of manucript.

 

Reviewer comment No. 7:

In several parts of the text: "hookworms" is a common name and should not be italicized.

Response to Reviewer comment No.7 :

Thank you for bringing this to our attention. We have corrected the issue in all relevant text.

 

Reviewer comment No. 8:

- The authors should rewrite the abstract, results, discussion, and conclusions in line with the previous comments, and avoid suggesting (as in lines 212-214) that some of the parasites are or can be shared with humans. The heading in section 3.4, "Influence of the presence of human parasite infection," is misleading, as the parasites have not been identified as human parasites. Egg morphology alone is insufficient for species identification because several species of some genera could infect NHPs (and they are not necessarily the same species that infect humans).

 

Response to Reviewer comment No.8 :

Thank you for your detailed feedback. In response to your comments, we have revised the abstract, results, discussion, and conclusions to avoid suggesting that specific parasites are or can be shared with humans without definitive evidence. Regarding section 3.4, we have restructured this section to focus exclusively on the identified parasitic genera, removing any direct references to human parasite infections. While we acknowledge that several of these genera have been previously implicated in interspecific transmission, we have refrained from making specific claims about shared infections in this study due to the limitations of egg morphology for species-level identification.

 

Reviewer comment No. 9:

Revise the manuscript; if you suggest that human-related species may be present, but definitive identification is not possible, you risk being less than objective, as the implication is "they might be, but I am not sure." I am not rejecting the possibility that human parasites may also have infected NHPs; however, there is no evidence that the parasites found were human, NHP, or both. Be objective. Avoid statements such as that in line 219, "as evidenced by our study," as the sharing of parasitosis has not been demonstrated in this study. Similarly, in the conclusions, lines 276–278: "the presence of parasites known to infect humans (...) in NHPs is due to transmission from humans." The species mentioned in these lines have not been definitively identified, and it cannot be claimed that these species are infecting the NHPs in the CIRMF. This conclusion should be revised.

Response to Reviewer comment No.9 :

Thank you for highlighting this issue. The phrase "as shown by our study" in line 219 was intended to emphasize the similarities between human and non-human primate cohabitation environments, as observed in both our study and the study by Ghandour et al. However, to avoid any potential misinterpretation, we have removed this phrase. Additionally, we have revised the conclusions to ensure objectivity and to avoid making unsupported claims regarding the transmission of human-related parasites to non-human primates. The revised conclusions now reflect a more cautious interpretation of our findings.

 

Reviewer comment No. 10:

 

2. Statistics:

The methodology for egg counting is unclear. Two grams of feces were used (line 99), but in what volume were they resuspended? In the authors' response, they state 28 mL and mention that the count should be multiplied by a factor of 50. However, no volumes are mentioned in the text, and the factor is listed as x100. Some of these numbers are incorrect. The McMaster chamber has a volume of 0.15 mL, which is 1/200 of 30 mL; thus, multiplying the count by 200 gives the number of eggs in 30 mL, in which 2 g of feces were resuspended. Then, dividing by 2 provides the number of eggs per gram. To simplify, n200/2 = n100 (x100 if 30 mL is used). Therefore, the NaCl solution volume should be 30 mL, not 28 mL as stated; and if it was 28 mL, then the factor is not x100. Please clarify the correct volume used in the text.

Response to Reviewer comment No.10 :

Thank you for pointing out the inconsistencies in our methodology. We have revised the text to clarify the procedure (see lines 115-120 in the updated manuscript).

In the revised version, we specify that 2 g of fecal matter was resuspended in 28 mL of flotation solution, yielding a final volume of 30 mL. This 2 g to 30 mL ratio is a standard approach commonly used in the McMaster method (DOI: 10.12968/vetn.2017.8.8.458), allowing for a consistent dilution factor.

The rationale for adding 28 mL of solution rather than starting with 30 mL is to precisely maintain the feces-to-solution ratio and control the dilution factor. If we were to use 30 mL of solution initially, the total volume would exceed 30 mL, altering the concentration and potentially introducing counting errors. By adding a fixed 28 mL to 2 g of fecal matter, we ensure greater accuracy in egg counts and minimize dilution errors.

Reviewer comment No. 11:

 

• Line 135: Mandrills had 13 parasitic species "accounting for 97.5% of the total." What does this mean?
• Line 136: "Eleven species ... were found in Solatus, with a 100% prevalence rate." Does this mean that each of the 11 species had a 100% prevalence rate? Or perhaps, "Eleven species ... were found in Solatus, in which 100% of the animals harbored at least one parasite"?
•  
•  In line 166: chimpanzees and mandrills exhibited high prevalence rates of 96% and 100%. Since prevalence refers to individuals infected by a given species, this can be revised to "chimpanzees and mandrills exhibited the greatest number of individuals infected by at least one parasite genus." In Table 3, replace the last row "Prevalence (%)" with "Percentage of animals with at least one parasite."
• Line 162: "differences in prevalence" or differences in the percentage of infected animals for each species?
• Line 168: The Kruskal-Wallis test should be mentioned in the materials and methods section.
• Table 3: Please indicate the statistical differences for each parasite taxon between host species.
• Figure 5: How should this figure be interpreted? The categories "aviary," "male," and four of the five NHP species studied are all included in the same graph. It is unclear how to interpret the coefficient estimates.

Response to Reviewer comment No.11 :

Thank you for your helpful feedback. We have incorporated all of the suggested modifications into the new version of the manuscript, as per the reviewer's recommendations.

 

Reviewer comment No. 12:

3. References:

I have not had time to check the appropriateness of all the references, but the analysis of those cited in the introduction revealed some inaccuracies, particularly the incorrect use of citations and the reliance on secondary citations. For example:

• Reference 3 is cited to support the statement that parasitic diseases are a global health challenge; however, this reference is about chemotherapy, not parasitic diseases as a global health challenge. The authors may have cited a different work on global health challenges, but this reference is not the primary source and should be replaced with the original source.

 

Response to Reviewer comment No.12 :

Thank you for your valuable feedback regarding the references. We appreciate your diligence in reviewing the citations.

Regarding the comment on Reference 3, we would like to clarify that El Kouni and Chu's article in “Recent Advances in Nucleosides: Chemistry and Chemotherapy” indeed addresses parasitic diseases through the lens of therapeutic challenges, specifically focusing on purine metabolism as a target for anti-parasitic chemotherapy. While the primary emphasis is on chemotherapy, we believe that it contributes to understanding the broader context of parasitic diseases as a global health challenge.

Nonetheless, we recognize the importance of accurate citation and will review the references thoroughly to ensure that all citations are appropriate and accurately reflect the original sources. Thank you for highlighting this issue, which will enhance the quality of our manuscript..

 

Reviewer comment No. 13:

• Reference 5, which supports the numbers on infected individuals and deaths, is not the original source of this data; the cited authors referenced the number without providing the original source, and the present authors have replicated the value.

Response to Reviewer comment No.13 :

Thank you for this observation. We agree, this is information given by the WHO for 2023. We edited in the manuscript.

 

Reviewer comment No. 14:

• References 15 and possibly 16 seem unrelated to the explanation of what Gabonese sanctuaries do. Reference 15 is about parasites in NHPs in French zoos.

Response to Reviewer comment No.14 :

These two references 15 and 16 have been added to support the role of primatology centres in general, not the one in Gabon in particular..

 

Reviewer comment No. 15:

• Reference 17 is about possible infections in NHPs used in research in general, not specifically about NHPs at the CIRMF, yet it is used to support the statement that "NHPs at the center remain susceptible to a range of infections."

Response to Reviewer comment No.15 :

Thank you for your insightful comment regarding Reference 17. You are correct that this reference discusses potential infections in non-human primates (NHPs) in a general context rather than specifically addressing NHPs at CIRMF.

To enhance the accuracy of our statements, we will revise the manuscript to clarify that Reference 17 serves as a general illustration of the susceptibility of NHPs to various infections. Additionally, we will seek to include more relevant references that specifically address the health of NHPs at CIRMF, thereby providing a more precise foundation for our assertion that "NHPs at the center remain susceptible to a range of infections."

 

Reviewer comment No. 16:

I recommend the authors recheck the validity of the citations and remove the secondary citations ("cite of a cite").

Finally, a comment on lines 248-251, regarding the higher parasite densities in animals on cemented floors compared to those on natural soil: this statement is controversial. For example, Esteban-Sánchez et al. (2024; Animals 14:813) found a greater number of infected animals on natural soils than on cemented floors. Cemented floors can be easily washed and disinfected, removing infective parasitic forms, while natural soils are difficult to clean, often necessitating soil replacement.

Response to Reviewer comment No.16 :

We appreciate your recommendation to recheck the validity of our citations and remove any secondary citations. We will thoroughly review the references to ensure their appropriateness and will replace any secondary citations with primary sources where applicable.

Regarding the statement on higher parasite densities in animals on cemented floors compared to those on natural soil, we acknowledge that this topic is complex and has generated varying findings in the literature. While some studies, including that of Esteban-Sánchez et al. (2024), have reported a greater number of infected animals on natural soils, it is important to note that other research indicates that cemented floors can also provide a suitable environment for certain parasites to survive and proliferate (Opeyemi O, Shittu O, Abdulganiyu K, Ashaolu A, Lawal A, Kadir R. Helminth Infections of Captive Animals and Management Practices at the University of Ilorin Zoo, North-Central, Nigeria. Nigerian Journal of Parasitology. 2022;43).

Cemented surfaces, despite being easier to clean, may have micro-cracks where organic matter accumulates, potentially harboring infectious forms and diminishing the effectiveness of disinfection. Furthermore, the stability of humidity and temperature on cemented floors can create favorable conditions for the survival of parasite eggs and larvae. In controlled environments that are not continuously disinfected, evidence suggests that cemented surfaces may sometimes exhibit similar or even higher levels of infestation compared to natural soils, particularly when cleaning protocols are not optimally implemented.

 

Reviewer comment No. 17:

Other minor points:

• In the abstract, indicate all genera; mentioning "11 genera" but listing only 3 is insufficient. Adding a few words will not make the abstract too lengthy. Additionally, revise the total number of genera, as mentioned earlier, there are inconsistencies in the text.
• The abbreviation "CPZ" is introduced in the results to refer to "chimpanzee." This abbreviation is used only in part of the results and not throughout the text. Please remove it, as it is unnecessary to abbreviate a host species in one part of the text but not in others.
• Line 131: it should be Entamoeba, not Enterobius.
• Line 142: insert a space in "diversityas."
• Line 144: Please introduce abbreviations in the materials and methods section when first citing the indices.
• In several parts of the text: Spirure sp. is incorrect, as Spirure is not a genus name and should not be italicized nor followed by "sp."
• Lines 177-179: text is repeated.
• Line 189: the abbreviation does not correspond to the text.
• Line 206: "withwith."
• Line 217: "sharing significant morphological [41]." Morphological what?
• Reference list: recheck it. For example, the title of reference 16 is in French. Reference 40 appears to have an incomplete author list.

 

Response to Reviewer comment No.17 :

Thank you for your thorough and constructive feedback, which has significantly enhanced the quality of our manuscript. We have addressed each of your points as follows:

• Abstract: We have included all the parasitic genera identified in the study to provide clarity and avoid ambiguity. The reference to “11 genera” has been clarified, and the total number of parasitic genera has been reviewed and corrected throughout the text to ensure consistency.
• Abbreviation "CPZ": The abbreviation for "chimpanzee" has been standardized and is now consistently used throughout the manuscript, as per your suggestion.
• Correction of Taxonomic Names: The correction from "Enterobius" to "Entamoeba" has been made on lines 131 and 148 in the revised manuscript.
• Formatting Issues: A space has been added to correct “diversityas” on line 142, as well as the corresponding line 155.
• Introduction of Abbreviations: Abbreviations are now properly introduced in the "Materials and Methods" section when first cited, in line with your recommendations.
• Taxonomic Terminology: The incorrect term “Spirure sp.” has been replaced throughout the manuscript, as it does not represent a valid genus name.
• Redundant Text: Lines 177-179 have been revised to eliminate any repetitive text.
• Abbreviation Consistency: The abbreviation in line 189 has been corrected to ensure it corresponds with the text.
• Typographical Errors: The typo "withwith" has been corrected on line 206.
• Reference List: We have thoroughly reviewed the reference list for accuracy. The title of Reference 16 has been corrected to ensure consistency in language, and Reference 40 has been updated to include a complete author list.

All corrections and revisions have been incorporated as per your comments, and we believe these changes have improved the overall clarity and quality of our manuscript. Thank you again for your valuable insights.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Good revised version of the manuscript. Continue the good work.

Author Response

Comments and Suggestions for Authors

Good revised version of the manuscript. Continue the good work.

Response

Thank you for your encouraging comments on our revised manuscript. We are pleased to know that the changes we made align with your expectations. We appreciate your support and are committed to upholding the quality and relevance of our research in future iterations.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately responded to my previous questions. I disagree in one point (see below), but overall, I consider the work is now acceptable for publication after minor points are corrected.

The only point in which I disagree with the authors is about the quantitative method they have followed. It is true that they have followed what it is indicated in the reference they mentioned, but the method is incorrect as explained in that reference. There, it is said that 2 g of feces should be mixed with 28 ml of saline solution, and as the authors explain now, this will lead to a final volume of 30 ml. This is incorrect: why 2 g of feces is considered equivalent to 2 ml of volume? This will be correct for a feces' density of 1.0, which is most likely not the case. The correct method (maybe a I didn't explained well in my previous review) is by mixing 2 g of feces with saline solution to a final volume of 30 ml (then, the volume of the feces, whatever it is, is included in the "final volume of 30 ml"). The explanation now stated should be removed to avoid maintaining an error in the protocol. I suggest as a possible solution that they simply indicate they followed the protocol by Vasconcelos-Nóbrega et al. (2017) (the reference they provided) by using 2 g of feces, without further details. Something like: "To assess the parasitic load, the protocol by [reference number] was followed, using 2 g of fecal matter. The calculation of the parasitic load was calculated using the following formula: ..."

The minor points to correct:

- the use of CPZ as abbreviation for "chimpanzee": I previously suggested to remove it and use the full name, as the abbreviation is used only in results; there are no reasons to use an abbreviation for only one of the host species considered, and only in a part of the manuscript.

- caption of figure 2: There are two points here. First, the protist structures are not eggs or larvae, so modify the caption to "Cysts, oocysts, eggs or larvae of different parasitic genera identified in the NHPs at the CIRMF Primatology Center", or to "Parasitic structures identified in the feces of NHPs at the CIRMF Primatology Center". Second point: Spirure sp. (in italics) is incorrect. Spirure is a common name and should not be in italics nor followed by "sp."

- in the manuscript, "solatus" is a common name; if used in capitals, please place all the other common host names in capitals, or write "solatus" in lower letters.

- in lines 243-244, change "These parasites have been implicated in bidirectional transmission ..." by "Species of these genera have been implicated in bidirectional transmission ..."

- lines 246 and 247: as it is the first time these species are cited in the text, please use the full genus name: Enterobius anthropopitheci and Enterobius vermicularis.

Author Response

Reviewer comment No. 1

The authors have adequately responded to my previous questions. I disagree in one point (see below), but overall, I consider the work is now acceptable for publication after minor points are corrected.

 

The only point in which I disagree with the authors is about the quantitative method they have followed. It is true that they have followed what it is indicated in the reference they mentioned, but the method is incorrect as explained in that reference. There, it is said that 2 g of feces should be mixed with 28 ml of saline solution, and as the authors explain now, this will lead to a final volume of 30 ml. This is incorrect: why 2 g of feces is considered equivalent to 2 ml of volume? This will be correct for a feces' density of 1.0, which is most likely not the case. The correct method (maybe a I didn't explained well in my previous review) is by mixing 2 g of feces with saline solution to a final volume of 30 ml (then, the volume of the feces, whatever it is, is included in the "final volume of 30 ml"). The explanation now stated should be removed to avoid maintaining an error in the protocol. I suggest as a possible solution that they simply indicate they followed the protocol by Vasconcelos-Nóbrega et al. (2017) (the reference they provided) by using 2 g of feces, without further details. Something like: "To assess the parasitic load, the protocol by [reference number] was followed, using 2 g of fecal matter. The calculation of the parasitic load was calculated using the following formula: ..."

Response to Reviewer comment No.1 :

Thank you for your constructive observations and positive feedback. We appreciate your careful review of our methodology.

Regarding your point about the quantitative method, we understand your concerns about the mixing of feces with saline solution. We acknowledge that our previous explanation may have led to confusion regarding the volume equivalence of 2 g of feces. To clarify, we have revised the text to accurately reflect that we followed the protocol outlined by Vasconcelos-Nóbrega et al. (2017) using 2 g of fecal matter, without specifying the final volume, to avoid any potential misunderstanding.

The updated text now reads: "To assess the parasitic load, the protocol by Vasconcelos-Nóbrega et al. (2017) was followed, using 2 g of fecal matter. The calculation of the parasitic load was performed using the following formula: ..."

We believe this change addresses your concerns and enhances the clarity of our methodology. Thank you once again for your valuable input.

 

The minor points to correct:

 

Reviewer comment No. 2:

- the use of CPZ as abbreviation for "chimpanzee": I previously suggested to remove it and use the full name, as the abbreviation is used only in results; there are no reasons to use an abbreviation for only one of the host species considered, and only in a part of the manuscript.

Response to Reviewer comment No.2:

Thank you, we edited the text in new manuscript version

 

 

Reviewer comment No. 3:

- caption of figure 2: There are two points here. First, the protist structures are not eggs or larvae, so modify the caption to "Cysts, oocysts, eggs or larvae of different parasitic genera identified in the NHPs at the CIRMF Primatology Center", or to "Parasitic structures identified in the feces of NHPs at the CIRMF Primatology Center". Second point: Spirure sp. (in italics) is incorrect. Spirure is a common name and should not be in italics nor followed by "sp."

 

Response to Reviewer comment No.3:

We agree, now we corrected the text in new manuscript version see lines 147-148

 

Reviewer comment No. 4:

- in the manuscript, "solatus" is a common name; if used in capitals, please place all the other common host names in capitals, or write "solatus" in lower letters.

Response to Reviewer comment No.4:

Thank you, we have edited the text in the new version of the manuscript using capital letters.

 

Reviewer comment No. 5:

- in lines 243-244, change "These parasites have been implicated in bidirectional transmission ..." by "Species of these genera have been implicated in bidirectional transmission ..."

Response to Reviewer comment No.5:

We considered you suggestion, see line 240 in new version of the manuscript.

 

 

Reviewer comment No. 5:

- lines 246 and 247: as it is the first time these species are cited in the text, please use the full genus name: Enterobius anthropopitheci and Enterobius vermicularis.

 

Thank you, we done in the new version of the manuscript see lines 243-245.