Gastrointestinal Nematodes and Protozoa in Small and Large Ruminants from Rural Agro-Climatic Regions of Northern India

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article needs revision:

1. Why "The correlation between meteorological parameters and the number of eggs per gram (EPG) of 245 parasites of the genus GIN in domesticated ruminants." is given only from February to July (Fig. 6).

2. It is necessary to provide a list of helminth species and compare with data for the region. Thus (Jithendran, 2000) the survey "Table 2. Important parasites are helminths in cattle and buffaloes in Himachal Pradesh."

3. The authors note that Article 369-372 "In order to reduce the biological and economic impact of gastrointestinal parasites on the health and productivity of livestock, it is necessary to develop and implement a comprehensive strategy to combat gastrointestinal parasites based on effective regular deworming of animals, improving the quality of animal nutrition, safer methods of animal husbandry, sheds for keeping animals "pakka", etc."..". With that wound (Jitendran, 2000), specific measures for deworming in that region of India were considered. In particular, "In the absence of an alternative to chemotherapy, research activities are aimed at minimizing the use of anthelmintic drugs and optimizing their use to prevent the widespread spread of resistance. Recent studies have highlighted the role of improved nutrition in mitigating the effects of concomitant parasitic infections. The use of non-protein nitrogenous additives in the form of a carbamide-molasses block (UMB) alone or a medicamental UMB with fenbendazole led to even greater efficiency. The use of nematophagous fungi and anthelmintic vaccines represent control options that have significant potential, but have yet to be tested in local conditions. However, it should be emphasized that no single method is likely to be beneficial if applied in isolation, but that a combination of methods is likely to be most effective for a regime of control of susceptible parasites that meets the needs of individual farmers."

Author Response

Response to Reviewer 1 comments

Point 1: Why "The correlation between meteorological parameters and the number of eggs per gram (EPG) of 245 parasites of the genus GIN in domesticated ruminants." is given only from February to July (Fig. 6).

Response 1: We are extremely thankful to the esteemed reviewer for the valuable comment. The study was performed from February to July month only as it was part of Master’s dissertation work of the research student, that’s why only limited period data is presented. However, in our future studies, we shall endeavor to provide comprehensive and long-term meteorological data.

Point 2: It is necessary to provide a list of helminth species and compare with data for the region. Thus (Jithendran, 2000) the survey "Table 2. Important parasites are helminths in cattle and buffaloes in Himachal Pradesh."

Response 2: As kindly suggested by esteemed reviewer, the following paragraph has been included in discussion section where data reported from Himachal Pradesh by Jithendran (2000) and other researchers are compared with the findings of our study:

“Previous studies carried out in the mountainous regions of northern Indian states revealed occurrence of gastrointestinal nematode infestations in large and small ruminants. An extensive study by Jithendran (2000) found 87.2 % and 94.3 % gastrointestinal helminths infection levels in cattle and buffaloes from Himachal Pradesh with the predominance of the species belonging to genera Haemonchus, Bunostomum, Mecistocirrus, Strongyloides, Trichostrongylus, and Oesophagostomum. Our study also found high prevalence of these nematode genera except Bunostomum and Mecistocirrus which were not detected. In sheep and goats, we have found very high level of Haemonchus, Strongyloides and Trichostrongylus infestations. Similar prevalence rates and trends were reported previously by Jithendran (2000) except that Bunostomum and Chabertia were not found in fecal samples of small ruminants in the current study. In terms of season-wise intensity of gastrointestinal parasites, month of July witnessed peak levels of EPG counts in ruminants which is comparable to the data reported by Jithendran (2000) where highest parasite counts were observed in period during June to September. Sharma et al. (2013) also found 86.1% prevalence of gastrointestinal nematodes with highest incidence rainy season in Gaddi sheep from Palam valley of Himachal Pradesh. Among nematodes, H. contortus was the predominant followed by Ostertagia and Trichuris but the prevalence of Trichostrongylus and Strongyloides was found to be low as compared to our findings”

Point 3: The authors note that Article 369-372 "In order to reduce the biological and economic impact of gastrointestinal parasites on the health and productivity of livestock, …………………..of susceptible parasites that meets the needs of individual farmers."

Response 3: Conclusions section has been thoroughly revised as per your kind suggestions and also according to the comments of reviewer # 2 and reviewer # 3.

We would like to express our sincere appreciation to the revered reviewer for the keen interest in the manuscript and valuable suggestions which helped us immensely to improve the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The study present the new, interesting data on the occurrence of parasites (eggs and oocyst) in selected species of ruminants from India.

I am of an opinion that the article fits into scope of Diversity and could be published after major corrections.

 

Comments:

 1. Abstract and other chapters:

1. 1. Please resign from using the abbreviations "GIN" and "GIPP" as they are inconsistent. Why is it specified that they are parasites for protozoan and not for nematodes - although elsewhere in the text the authors use the phrase "gastrointestinal nematode parasites" that is GINP?.

 

2. Introduction:

2.1. The introduction is chaotic, contains a lot of unnecessary information, general, obvious information, truisms at the level of popular textbooks, such as. "Helminth parasites are complex multicellular worms which infest the gastrointestinal tracts and systemic organs of domestic ruminants and other animals", "Helminth parasites are broadly categorized into two phyla, namely platyhelminthes (cestodes or tapeworms and trematodes or flukes) and nemathelminthes which includes nematodes or roundworms".

 In addition, there is a lot of repetition here regarding pathogenicity.

 2.2. I completely don't understand why here the authors write about the species Eimeria oviodinalis and E. ninakohlyakimovae when they didn't find them and don't refer to them in the Discussion section.

 2.3. Platyhelminthes and Nemathelminthes should be capitalized.

 2.4. When using a species name for the first time, the full name should be given - including the author and date.

 2.5. Please explain what the term "agro-climatic" means and refer to it in the Discussion

  2.6. In conclusion, this chapter must be rewritten.

  

3. Material and methods:

 3.1. ”kaccha/pakka” – what is it, please explain.

 3.2. The authors write that "... and sheep (Ovis aries) of native and exotic breeds were randomly selected from the sampling sites listed in Table 1" - but there is no information on this (native and exotic breeds) in Table 1, in Results and in Discussion.

 3.3 “The study was of non-invasive nature and does not involved any harm or discomfort as neither blood nor flesh samples were collected from any of the experimental animals and hence the approval of Institutional Animal Ethics Committee was not required.” – for deletion; since coproscopic examinations were used, it is known that they were non-invasive; this type of information can be included in the cover letter at most.

 3.4. Unit missing, line 113 (2-4 C°).

 3.5. Unnecessary information, e.g. lines 119-120 (Himeda, India).

 3.6. Inconsistent, different wordings: „the suspension was strained through a strainer” and “was strained through a tea strainer” – did these strainers differ ?

 3.7. No information on average high and low temperature - it should be either in the Material and methods section or under some figure - maybe Figure 4 ?

 3.8. The authors write that they used statistical analyses, but there is no information about the tests used; please complete.

 3.9. Line 142: „ EPG count = Number of eggs counted  100” - ??

 3.10. The authors write that "Experiments were performed in triplicates"- not clear, were three repetitions used for each type of flotation i.e. there were 9 samples in total, or did they mean that three types of flotation were used ?

 3.11. The authors write that „Experiments were performed in triplicates and data were exppressed as mean ± SD.” – where exactly is the data on SD; under Figure 6 there is only information “Data represents mean ± SD of three independent experiments”.

 

4. Results:

4.1. The first two paragraphs, Table 1 and Figure 1 belong to Materials and methods.

 4.2. In table 1, column 3, remove "district Sirmaur" - as this information is in the title and is the same for all sites/localities.

 4.3. To Table 1 at the end, one more row should be added with summary data on the examined hosts.

 4.4. Table 1: families are written in normal font.

 4.5. Ascaris belongs to the Ascarididae! please check systematic affiliations of other genera !

 4.6. Figure 1 B and C to be merged into one including numbers and percentages.

 4.7. Figure 2: Photo 4 and perhaps H are deformed ! No scale - F, H, K .

 4.8. Title Figure 2: repetition – „The characteristic features of parasite eggs/cysts mentioned in standard manuals and parasitology keys were considered for the identification”.

 4.9. Lines 192-196 belongs to Material and methods; besides, again, a lot of information is repetition! In addition, why only selected features of only three genera of parasites (Trichuris, Strongyloides, Eimeria) are given ?, what about the others. Why do the authors list features that were used for identification and in the Table 1 for most parasites they do not mention them !??

 4.10. Figure 4: not clear – What does "heat map" mean, no explanation of what the squares of different colors mean !.

 4.11. I don't see information on overall infection regarding the Epg for individual hosts (without division into months).

 4.12. The Results is chaotic - nematodes, protozoan and Epg data must be clearly separated in separate paragraphs.

Please remove unnecessary, obvious information (repetition) like "with detected in 17 animals out 18 which", or "In total, 154 ruminants".

 4.13. Figures 3 and 6 – please add prevalences values (data labels) for individual parasites and hosts.

 4.14. Figure 6: „The meteorological data was retrieved from IMD Shimla http://weathershimla.nic.in/index.html) and Accuweather (https://www.accuweather.com/en/in/nahan/188526/weather-forecast/188526).” – repetition, This information is already in the Materials and Methods.

 4.15. “Quantitative estimation of EPG counts in fecal samples of study animals was performed in two sampling sites i.e Lana Machher and Bongli Kech from February, 2019 to July, 2019. The highest rainfall during the study period was recorded in the month of July, 2019 in monsoon season as compared to March, April, May and June months (data not shown).” – this belongs to Material and methods.

 Besides what the authors mean by "months (data not shown)".

 4.16. „As evident from the data presented in Fig. 6, there was an influence of meteorological parameters on the GIN parasites infestations in small and large ruminants” – this is a comment and belongs to the Discussion.

 4.17. Was there really only one Eimeria species found in all hosts? (also Discussion lines 326-327), this is rather unlikely - therefore the notation “Eimeria spp.” should be used for this taxon.

 

5. Discussion:

5.1. Please avoid the wordings "To the best of our knowledge,..." - either the authors know the literature on the subject or they don't .

 5.2. The discussion, like the Introduction, is chaotic and must be rewritten.

The discussion contains many repetitions from Results, e.g. „The present study was undertaken from February 2019 to July 2019 to determine the prevalence of GIN and GIPP parasites in domesticated ruminants (buffaloes, cattle, goats and sheep) inhabiting the rural areas in 26 villages in five tehsils of district Sirmaur”, “Fecal sample analyses  of domesticated ruminants (11 buffaloes, 86 cattle, 48 goats and 18 sheep) from 26 villages representing 5 tehsils of Sirmaur”, etc.

 5.3. There is a lot of imprecise information in the Discussion - it is not known whether the Authors write about India or other regions of the world - it must always be precise. Please choose examples for comparisons more precisely - comparing with other regions of the world, sometimes very distant, e.g. Egypt, does not make sense. For example, the authors write that “In another study, Mohamaden et al. (2018) identified more than 15 species of Eimeria from the ruminants. However, the cysts of Balantidium coli, Giardia duodenalis and Cryptosporidium sp. were undetectable in either of animal groups during this study.” – however studies by Mohamaden et al. (2018) refers to Egypt! – another region very distant from India, besides, were the same species of hosts studied at that time? – all this must be precisely defined.

 5.4. Lines 312-313: „The prevalence of GIN and GIPP parasites was recorded in both male and female animals.” - ??? not clear.

 5.5. The authors write that “Another explanation for the higher parasite infestation in the ruminants is the poor animal husbandry practices adopted by the livestock owners" or "Rainfall, humidity, temperature and animal husbandry conditions, grazing, deworming etc. were found to exert a considerable influence on GIH parasites prevalence in the ruminants" – however, there are no specifics here, there are no comments, e.g. to the aforementioned deworming, or grazing.

In addition in the Materials and methods chapter, the authors list additional parameters (age of animals, gender, breed, animal husbandry practices, farm size, physical state of animal sheds, feedstuffs, fodders, cleaning and sanitation), but the Results and Discussions lack any specific data and comments.

In addition, drawing conclusions is very risky with single samples, especially since the authors did not use any statistics to analyze the data obtained.

 5.6. The Discussion also needs to refer to the so-called "domesticated animals/ruminants" - since the authors use this term, it would be appropriate to refer to it, that is, to compare species described as domesticated with wild species.

 

6. References

6.1. The citation and references list are incorrect – please sea Instruction Diversity MDPI.

Comments on the Quality of English Language

Extensive editing of English language required.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments for author File: Comments.doc

Comments on the Quality of English Language

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In general, all the authors' comments were taken into account. Thank you for your attention to criticism. The authors have collected a lot of material, but the presented manuscript requires some revision. The study can be considered relevant, but it requires some improvement.

Note:

Lines 90-91: “Figure 1. Geographic location, sampling locations and animals studied. (A) Sampling sites (indicated by 90 pins) located in five tehsils of rural tropical Himalayan regions of Himachal Pradesh, India.” Enlarge the first drawing “India” 1.5 times. Drawing "Himachal Pradesh" make a designation of localities - geographical points with numbers in "Table 1". This makes your drawing more informative.

To relate (lines 96-98) to “Table 1. Sampling sites located in the Sirmaur district of the northern Indian state of Himachal Pradesh from where fecal samples from domestic animals were collected and analyzed to determine the prevalence of gastrointestinal nematodes and gastrointestinal protozoa. parasites."

Additions:

For a number of species indicated in the work, identified to genus, definitions to species from the study area are known, which must be indicated in the publication. It is necessary to note the presence of differences (their degree) for the species of the indicated genera. For example (Alok et al., 2020), “Five different species identified were Eimeria arlongi, E. ninakohlyakimovae, E. hirci, E. christenseni and E. caprina with infection of 42%, 26.5%, 17.0%, 9.75% and 4.75 %, respectively in children and 46.25%, 28.75%, 14.0%, 7.5% and 3.5%, respectively in adults."

At the same time, Eimeria arloingi, E. ninakohlyakimovae and (Alok et al., 2020) are more common. For example, E. ninakohlyakimovae has been found to be more pathogenic in goat populations (Chartier and Paraud, 2012), Eimeria arloingi causes focal mucosal hyperplasia and polyp formation, Eimeria caprina causes mucosal destruction in both the small and large intestines, and Eimeria christenseni and Eimeria hirci are also possibly pathogenic. (Andrews, 2013). E. ninakohlyakimovae, Eimeria arloingi and E. christenseni have caused abdominal pain, loss of appetite, bloody diarrhea and papilloma-like intestinal lesions in young children (Yvore et al., 1980). Thus, the clinical picture of the affected area may indicate the probable taxonomic status of the identified taxa of Eimeria sp. This analysis can be included in additional material, taking into account data for the study area.

Author Response

Point 1: In general, all the authors' comments were taken into account. Thank you for your attention to criticism. The authors have collected a lot of material, but the presented manuscript requires some revision. The study can be considered relevant, but it requires some improvement. Lines 90-91: “Figure 1. Geographic location, sampling locations and animals studied. (A) Sampling sites (indicated by 90 pins) located in five tehsils of rural tropical Himalayan regions of Himachal Pradesh, India.” Enlarge the first drawing “India” 1.5 times. Drawing "Himachal Pradesh" make a designation of localities - geographical points with numbers in "Table 1". This makes your drawing more informative. To relate (lines 96-98) to “Table 1. Sampling sites located in the Sirmaur district of the northern Indian state of Himachal Pradesh from where fecal samples from domestic animals were collected and analyzed to determine the prevalence of gastrointestinal nematodes and gastrointestinal protozoa. parasites."

Response 1: Sampling locations are now designated with number corresponding to list of location mentioned in Table 1. In addition, the size of figure has been appropriately changed as kindly suggested by the esteemed reviewer.

 

Point 2: For a number of species indicated in the work, identified to genus, definitions to species from the study area are known, which must be indicated in the publication. It is necessary to note the presence of differences (their degree) for the species of the indicated genera. For example (Alok et al., 2020), “Five different species identified were Eimeria arlongi, E. ninakohlyakimovae, E. hirci, E. christenseni and E. caprina with infection of 42%, 26.5%, 17.0%, 9.75% and 4.75 %, respectively in children and 46.25%, 28.75%, 14.0%, 7.5% and 3.5%, respectively in adults." At the same time, Eimeria arloingi, E. ninakohlyakimovae and (Alok et al., 2020) are more common. For example, E. ninakohlyakimovae has been found to be more pathogenic in goat populations (Chartier and Paraud, 2012), Eimeria arloingi causes focal mucosal hyperplasia and polyp formation, Eimeria caprina causes mucosal destruction in both the small and large intestines, and Eimeria christenseni and Eimeria hirci are also possibly pathogenic. (Andrews, 2013). E. ninakohlyakimovae, Eimeria arloingi and E. christenseni have caused abdominal pain, loss of appetite, bloody diarrhea and papilloma-like intestinal lesions in young children (Yvore et al., 1980). Thus, the clinical picture of the affected area may indicate the probable taxonomic status of the identified taxa of Eimeria sp. This analysis can be included in additional material, taking into account data for the study area.

Response 2: In consideration of the valuable suggestion of the revered reviewer, modifications have been incorporated in the manuscript.

We greatly appreciate the keen interest and valuable suggestions of the revered reviewer which greatly help us in revising the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

I have a few more comments:

1. Please do not provide authors and dates for genera, e.g. Ascaris Linnaeus, 1758; the author and date are customarily given for species. There are no full names for the species, e.g. Haemonchus contortus, or for the hosts - Bos taurus, Bubalus bubalis, Capra hircus, Ovis aries.

2. Table 2: Please do not write families (e.g. Strongyloididae) in italics; only genera and species should be in italics.

3. Fig. 6. In my opinion, the sentence "Data represents mean ± SD of three independent experiments" should remain in the title of this figure.

4. Figure 2: Trichuris egg (F) is still deformed!! – apparently the photo was deformed when copying into Word – it is too wide, Trichuris eggs are narrower.

5. Figure 4: Sorry authors, but the previous wording was correct; I didn't notice the units next to the legend/colored squares. In the current wording, the information regarding infection/prevalence has been repeated twice (in the legend/colored squares and in the title). So, please go back to the earlier version.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Point 1: Please do not provide authors and dates for genera, e.g. Ascaris Linnaeus, 1758; the author and date are customarily given for species. There are no full names for the species, e.g. Haemonchus contortus, or for the hosts - Bos taurus, Bubalus bubalis, Capra hircus, Ovis aries.

Response 1: Appropriate rectifications and amendments have been incorporated in the manuscript.

Point 2: Table 2: Please do not write families (e.g. Strongyloididae) in italics; only genera and species should be in italics.

Response 2: The necessary change has been incorporated.

 

Point 3: Fig. 6. In my opinion, the sentence "Data represents mean ± SD of three independent experiments" should remain in the title of this figure.

Response 3: As kindly suggested by the esteemed reviewer, the figure legend has been accordingly revised.

 

Point 4: Figure 2: Trichuris egg (F) is still deformed!! – apparently the photo was deformed when copying into Word – it is too wide, Trichuris eggs are narrower.

Response 4: The issue with Trichuris egg (F) in figure 2 has been rectified.

 

Point 5: Figure 4: Sorry authors, but the previous wording was correct; I didn't notice the units next to the legend/colored squares. In the current wording, the information regarding infection/prevalence has been repeated twice (in the legend/colored squares and in the title). So, please go back to the earlier version.

Response 5: As kindly suggested, the figure 4 legend has modified according to the first submitted version.

 

Point 6: Comments on the Quality of English Language: Minor editing of English language required.

Response 6: The manuscript has been rigorously and thorough checked for quality and correctness of the English language.

We greatly appreciate the keen interest and valuable suggestions of the revered reviewer which greatly help us in revising the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have substantially revised the text of the manuscript and added relevant details. In this form, the paper can be considered for publication.

Author Response

Dear Esteemed Reviewer, we greatly appreciate your valuable insights during the revision of the manuscript.