Coprological Survey of Helminths in Reindeer (Rangifer tarandus) in 50 Selected Zoos and Menageries in Russia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study aimed to investigate, using qualitative and quantitative methods, the occurrence of helminth worms in faecal samples from reindeer (Rangifer tarandus) from zoos in Russia. Which highlights the importance of the study. Overall, the study is important as it maps the occurrence of intestinal worms in reindeer. However, there are some issues that must be clarified.

General comments:

I believe it would be interesting to compare the intensity and prevalence of these parasites between different zoos.

Is there any factor that affects the occurrence of these pathogens between locations?

What is the procedure for collecting and fixing parasites?

How were the prevalence and intensity of parasites calculated?

How were the parasites identified? Did the authors use any specific dye to visualize the internal structures?

Were the parasites mounted between the slide and coverslips?

What is the most abundant parasite or group of parasites in reindeer feces?

Lines 219-220: I suggest that this sentence be written in a direct way.

The discussion is too long, and, in some sections, the results are not discussed and/or compared with the results published in other articles.

My recommendation: major revisions.

Author Response

This study aimed to investigate, using qualitative and quantitative methods, the occurrence of helminth worms in faecal samples from reindeer (Rangifer tarandus) from zoos in Russia. Which highlights the importance of the study. Overall, the study is important as it maps the occurrence of intestinal worms in reindeer. However, there are some issues that must be clarified.

We are very grateful to the Reviewer for the careful review of our manuscript.

General comments:

I believe it would be interesting to compare the intensity and prevalence of these parasites between different zoos.

Prevalence of these parasites in different zoos is already present in the Table 2 (former Table 3) (lines 120-121 of initial paper). Intensity in different zoos was described verbally in lines 126-133. As we wrote, to save space we described only positive results for prevalence and indicated the zoos where Intensity was “medium”, unlike “low” in all the rest. These terms (“low intensity” and “medium intensity” derived from the National Standard of the Russian Federation (GOST R 54627-2011) “Ruminant animals—Methods of Laboratory Helminthological Diagnostics” which we also cited and provided with ref [29].

Also we added Table 4 to highlight the intensity comparison between the zoos.

Table 4. Intensity of infestation with helminths of zoo reindeer according to GOST R 54627-2011

Helminths	Intensity of infestation depending on the number of detected helminth eggs and larvae, specimens / 1 g of feces
	Low	Medium	High	Very high
Nematodes, cestodes	1–100	101–500*	501–1000	>1000
GINs (eggs)	1–56 (zoos #7, 12, 14, 22, 24, 30, 34, 39)	–	–	–
Nematodirus spp. (eggs)	1–4 (zoos #12, 36, 44)	–	–	–
Dictyocaulus sp. (L1)	1 (zoo #30)	–	–	–
E. rangiferi (L1)	1–98 (zoos #4, 5, 6, 7, 8, 12, 13, 25, 29, 30, 36, 37, 46)	15–537 (zoo #9)	–	–
Trichuris sp. (eggs)	1 (zoos #14, 22)	–	–	–
Capillaria sp. (eggs)	1–8 (zoos #2, 4, 7, 8, 9, 10, 12, 16, 18, 25, 26, 28, 30, 34, 47, 48)	–	–	–
Moniezia spp.** (eggs)	1–17 (zoos # 9,14, 24, 26, 34)	–	–	–
Trematodes	1–10	11–100	>100	–
F. hepatica (eggs)	1–3 (zoos #13, 21)	–	–	–
Paramphistomum sp. (eggs)	1–8 (zoos #4, 5, 9, 11, 13, 37, 38, 38)	9–56 (zoo #30)	–	–

* Low intensity in case of adult animals

**Including M. expansa

 

Is there any factor that affects the occurrence of these pathogens between locations?

It is a great question indeed! It is tempting to suggest that climate affects the occurrence. However, it is much more complicated due to constant exchange of animals between the zoos, different ways of zoo management, deworming programs, etc. It is worth studying and modeling, but requires much more resources than we had this time.

What is the procedure for collecting and fixing parasites?

It is unclear what collection and fixation the Reviewer was asking about. We have three suggestions, our comments are below:

1) for microscopic examination, eggs and larvae of helminths were obtained (“collected”) via standard fecal examination procedures. These eggs and larvae were not additionally fixated. As we wrote in lines 81-83, these procedures were described in the open-accessed MDPI article, ref. 21 of the original paper.

2) for DNA extraction, larvae of nematodes were collected from slides using a hand-made manipulator consisting of an acupuncture needle 0.16 × 25 mm (Beijing Zhongyan Taihe Medical Instrument Co., China) with a pencil like handle. For DNA extraction, Fasciola hepatica eggs were collected individually using eyelash handmade manipulator (as described in [24]). These eggs and larvae were not additionally fixated.

3) for vouchers, larvae of nematodes were collected using a hand-made manipulator consisting of an acupuncture needle 0.16 × 25 mm with a pencil like handle; Fasciola hepatica eggs were collected individually using eyelash handmade manipulator. These eggs and larvae were fixated with 70° alcohol.

We are ready to add any of this information to the manuscript if the Editor considers it is necessary.

How were the prevalence and intensity of parasites calculated?

In lines 81-83 we wrote: “Qualitative and quantitative fecal analyses, light microscopic study, and measurements of obtained eggs and larvae of helminths were performed as described by Loginova et al. [21]”. Prevalence and intensity are indices used in quantitative analyses. They were calculated according to the National Standard of the Russian Federation (GOST R 54627-2011) “Ruminant animals—Methods of Laboratory Helminthological Diagnostics” which we also cited and provided with ref [29]. That is, sample prevalence was calculated as the proportion of positive samples (=infected individuals) within the sample set (=host sample) and expressed as a percentage (0–100%). Intensity was calculated as a number of parasites (eggs or larvae) per 1 g of feces.

We added a subsection on this (2.5), in particular: “Therefore, to analyze the obtained data, the index method was used. Namely, it was Prevalence and Intensity. Sample prevalence was calculated as the proportion of positive samples (=infected individuals) within the sample set (=host sample) and expressed as a percentage (0–100%). Intensity for every reindeer was calculated as a number of parasites (eggs or larvae) per 1 g of their feces. The VIGIS chamber (analogue of the McMaster device) of the Diapar kit (VIGIS, Moscow, Russia) was used to calculate the Intensity.”

How were the parasites identified? Did the authors use any specific dye to visualize the internal structures?

As we wrote in lines 98-99: “Identification of helminths was based on their morphology, morphometric data, and (where possible) DNA analysis”.

We did not use any specific dye to visualize the internal structures, otherwise it would be mentioned.

However, we added a subsection (2.3) on Helminths identification:

“Primary identification of helminths at the stage of egg or larva was based on their morphology and morphometric data. Morphology of eggs and larvae derived from zoo reindeer feces was examined via light microscopy (LM) with the optical microscope Micmed-6 (LOMO-MA, St. Petersburg, Russia) using the lenses of 4× (to navigate the slide), 10×, 20×, 40× magnification. The parasites were mounted between the slide and coverslip (24 × 24 mm). Larvae were there in tap water. Eggs were either in tap water (once obtained via sedimentation) or in the Darling’s solution (once derived via Darling’s method). No specific dye to visualize the internal structures of parasites was used. Micrographs were taken using the digital photo camera 5D Mark II (Canon, Tokyo, Japan) connected to the microscope with the C-mount adapter (LOMO-MA, Russia). Morphometry was based on the obtained micrographs using Figi/ImageJ Version 1.2.4 RRID:SCR_003070 software (National Institutes of Health, Bethesda, MD, USA) set using the microscope calibration slide (transmitted light object micrometer) OMP (LOMO-MA, Russia). Straight Line mode was used to measure eggs, and Segmented Line mode was used to measure larvae. Reference literature was used to identify obtained helminths. Where possible, morphological identification was supported by DNA analysis.”

Were the parasites mounted between the slide and coverslips?

Yes, the parasites (eggs and larvae) were mounted between the slide and coverslips. We did not mention this for brevity, for it is a standard practice, but added it to the subsection above.

What is the most abundant parasite or group of parasites in reindeer feces?

As it is shown in Fig. 2 (line 124) and written in line 180: “Brain worm E. rangiferi was the most widespread helminth in our research”.

Lines 219-220: I suggest that this sentence be written in a direct way.

Initially it was as follows: “Apart from husbandry “heritage”, how can zoo reindeer become infected with helminths? In particular, through the birds”.

We changed it as follows: “Apart from husbandry “heritage”, zoo reindeer can become infected with helminths due to birds” as suggested by the Reviewer.

The discussion is too long, and, in some sections, the results are not discussed and/or compared with the results published in other articles.

We are sorry that the Reviewer has got such an impression. We discussed all the found helminths, and offered our suggestions on their occurrence. As to the comparison with other articles, it is a fair demand, but in our case we are, basically, the ones, studied helminths exactly in zoo reindeer. There are no other works on the subject to compare. Given heterogeneous origin of zoo reindeer it is incorrect to compare them with purely wild or domestic reindeer.

We deleted this: “Tactile contact between humans and animals in petting zoos may pose a risk of helminth infestation. For instance, red fox (Vulpes vulpes) and Arctic fox (V. lagopus) are recognized definitive hosts for Echinococcus spp. The latter produces numerous eggs which are shed with feces. Those eggs are very light and flying, and easily contaminate animal’s fur. Stroking such sick fox or even breathing closely to it may result in ingestion of Echinococcus eggs which are instantly invasive [37].” to make the discussion shorter.

My recommendation: major revisions.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Author Response

 L40-44. Describe, what parasites are common in reindeer? More details on the impact of parasites on reindeer, should be included in this segment. These details should include, for example, nutritional deficiency, decreased physical condition, reduced reproductive success, weakened immune system, anemia and blood loss, behavioral changes, damage to organs and tissues, secondary infections, impact on herd dynamics, economic consequences.

 

We are very grateful to the Reviewer for the careful review of our manuscript.

We also thank the Reviewer for such a detailed advice! However, parasites include: helminths, arthropods and protists (protozoa). This article is only about helminths, so we would prefer to avoid off tops.

 

We added this as suggested: “Helminths may cause nutritional deficiency in reindeer, decrease their physical condition, reduce their reproductive success, weaken immune system, cause anemia and blood loss, behavioral changes, damage to organs and tissues, and contribute to secondary infections [3-8, 16].” and a ref to:

 

Hoberg, E.P.; Kocan, A.A.; Rickard, L.G. Gastrointestinal strongyles in wild ruminants. In Parasitic Diseases of Wild Mammals; Samuel, W.M., Pybus, M.J., Kocan, A.A., Eds.;Wiley: New York, NY, USA, 2001; pp. 193–227.

 

L54. Not only helminths. The risk of infection with protozoa and ectoparasites also. Provide references for this statement.

 

Initial sentence was as follows: “It might pose a threat of picking up some new helminths.” We did not mention protozoa and ectoparasites, because we wanted to focus this paper on helminths. However, we agree with this comment. We replaced “helminths” with “parasites” (to broad the context without specification) and added refs:

 

Manninen, S.M.; Thamsborg, S.M.; Laaksonen, S.; Oksanen, A. The reindeer abomasal nematode (Ostertagia gruehneri) is naturally transmitted to sheep when sharing pastures. Parasitol. Res. 2014, 113, 4033–4038. https://doi.org/10.1007/s00436-014-4071-x.

 

Utaaker, K.S.; Ytrehus, B.; Davey, M.L.; Fossøy, F.; Davidson, R.K.; Miller, A.L.; Robertsen, P.-A.; Strand, O.; Rauset, G. R. Parasite spillover from domestic sheep to wild reindeer—the role of salt licks. Pathogens 2023, 12, 186. https://doi.org/10.3390/pathogens12020186.

 

L63-76. “2.1. Helminths recovery” is not appropriate subtitle for this section. For example. “Study area” is better.

 

We replaced “Helminth recovery” with “Study area” as suggested

 

L77. Table 1 is too long. Authors should consider to move this table in supplementary material. A figure with a map showing the locations where samples were taken would be recommended.

 

Initially, we followed an example of our article on similar subject, published with MDPI journal, where there was a Table in the Materials and Methods. Yet there were 25 lines, not 50.

 

We added a figure with a map showing the locations where samples were taken, as suggested by the Reviewer. We also removed the Table 1 from the Materials and Methods section, as suggested by the Reviewer.

 

We placed the table (now Table A1) and the figure (A1) into the Appendix A, and replaced the content of initial Appendix A to the Appendix B.

 

L81-83. Describe in detail what qualitative and quantitative faecal analyses have been used in research. This should be another segment of Material and Methods

 

Initially we wrote: “Qualitative and quantitative fecal analyses, light microscopic study, and measurements of obtained eggs and larvae of helminths were performed as described by Loginova et al. [21].” in order to save place.

 

Now we added few subsections: 2.2 Fecal analysis, 2.3 Helminth identification and 2.5 Statistical analysis (kindly see the revised manuscript) 

 

In segment of Material and Methods, statistical analyses are missing. The correct selection of statistical methods was not made. Authors must provide a 95% confidence interval (CI) to express the uncertainty of sample prevalence as an estimate of population prevalence. More information for this task is described in:

 

Reiczigel, J., Marozzi, M., Fábián, I. and Rózsa, L., 2019. Biostatistics for parasitologists–a primer to quantitative parasitology. Trends in parasitology, 35(4), pp.277-281. https://doi.org/10.1016/j.pt.2019.01.003

 

We thank the Reviewer for detailed advise and useful link. However, In this study statistical sample equals population, because all possible zoo reindeer were examined. A total of 233 zoo reindeer cannot represent some other bigger group of reindeer. There are four breeds of domestic reindeer and few ecotypes or subspecies of wild reindeer in Russia. All these kinds of reindeer differ between each other, in particular in terms of helminth resistance. Zoo reindeer are heterogeneous. Some of them are known to originate from wild forest ecotype reindeer, others originated from domestic animals (but it is hard to identify their breed), and some have no remained origin information. Regular reindeer exchanges between the zoos make it even more complicated. Besides, above-mentioned GOST R 54627-2011 prescribes to examine no less than 10% of animals. For approximately 2 mln reindeer in Russia the statistical sample must be around 200 thousand reindeer, it is almost 900 times bigger than in our case. Thus, a 95% confidence interval (CI) to express the uncertainty of sample prevalence as an estimate of population prevalence is not applicable here. Therefore, to analyze the obtained data, the index method was used. Namely, it was Prevalence and Intensity. Sample prevalence was calculated as the proportion of positive samples (=infected individuals) within the sample set (=host sample) and expressed as a percentage (0–100%). Intensity for every reindeer was calculated as a number of parasites (eggs or larvae) per 1 g of their feces. The VIGIS chamber (analogue of the McMaster device) of the Diapar kit (VIGIS, Moscow, Russia) was used to calculate the Intensity.

 

We added this consideration to the manuscript (Materials and Methods. 2.5. Statistical analysis)

 

It would be interesting to compare results from selected zoos and menageries; also comparing results different sex and age.

 

We understand this interest. However, it is our principled position not to oppose zoos and menageries. We believe it is enough that difference between them is shown in the Table 2 (former Table 3).

As to comparison of reindeer with different sex and age, it was out initial plan. Unfortunately, not all the zoos managed to provide this information: they sent the list of reindeer with their names, sex and age + fecal samples, and sometimes these samples were not labeled. Therefore, there was no way to link the samples and the info. Not to mention that it was challenging to make some of them participate, so there was no option of double sampling. We consider it pointless to try to analyze incomplete data.

Once this research is continued, we hope to fill this gap and also to get material for monitoring through years. And *that* will be a material for proper statistical analysis. 

 

L97-99. In which table or figure is this result presented? It is not visible.

 

Initially we wrote: “Flukes, tapeworms and round worms were found at the stage of egg or larva in feces of 106 reindeer (45%) kept in 31 zoos (62%)”. This information is not doubled in any table or figure, that is right, and that is how we intended. In this line we offer the general information on positive reindeer and zoos.   

 

L98-99. This must be described in Material and Methods section.

 

Initially we wrote: “Identification of helminths was based on their morphology, morphometric data, and (where possible) DNA analysis (Table 1, former Table 2)”.

 

As it was suggested by the Reviewer, we added to the Materials and Methods, 2.3. Helminths identification: “Identification of helminths at the stage of egg or larva was based on their morphology and morphometric data” <…> “Where possible, morphological identification was supported by DNA analysis” and replaced these lines in the Results with: “Voucher and GenBank accession numbers of helminths recovered from zoo reindeer feces are presented in the Table 2”

 

 

L120. Table 3 and 4, and Figure 2. As I said previously, in this epidemiological study 95% confidence interval (CI) must be provided. In my opinion results in Table 3 and 4, and Figure 2 are not clearly presented. Thise tables are more informative and not provide any statistical result. Authors should consider to move this tables in supplementary. I am giving you example how to present results with prevalences:

We are deeply grateful to the Reviewer for such an effort to improve our manuscript, but we cannot follow this particular advice. As we explained previously, we offer our results so that a Reader can trace both zoos and reindeer. The offered table is only about reindeer, which contradicts the wish of the Reviewer to compare results between the zoos. As to the mixed infections, they were all singular. We added this word (“singular”) to the manuscript.

L126-133. Provide a table with quantitative results.

Initially we wrote: ”Intensity of invasion was mostly low in terms of National Standard of the Russian Federation (GOST R 54627-2011) Ruminant animals—Methods of Laboratory Helminthological Diagnostics [29]. Nematode eggs / gram of feces ranged from 1 to 56 in all reindeer samples. Nematode larvae / gram varied from 1 to 98. Cestode eggs / gram ranged from 1 to 17. Trematode eggs / gram varied from 1 to 8. Exceptions were: fecal sample of reindeer from the zoo #9 that showed 537 E.rangiferi larvae / gram; fecal sample of reindeer from the zoo #30 that showed 56 Paramphistomum sp. eggs / gram. Those numbers are estimated as medium intensity (GOST R 54627-2011)” to avoid long tables and save space.

Following an advice of the Reviewer we replaced this text and expanded the data within a Table 4.

Table 4. Intensity of infestation with helminths of zoo reindeer according to GOST R 54627-2011

Helminths	Intensity of infestation depending on the number of detected helminth eggs and larvae, specimens / 1 g of feces
	Low	Medium	High	Very high
Nematodes, cestodes	1–100	101–500*	501–1000	>1000
GINs (eggs)	1–56 (zoos #7, 12, 14, 22, 24, 30, 34, 39)	–	–	–
Nematodirus spp. (eggs)	1–4 (zoos #12, 36, 44)	–	–	–
Dictyocaulus sp. (L1)	1 (zoo #30)	–	–	–
E. rangiferi (L1)	1–98 (zoos #4, 5, 6, 7, 8, 12, 13, 25, 29, 30, 36, 37, 46)	15–537 (zoo #9)	–	–
Trichuris sp. (eggs)	1 (zoos #14, 22)	–	–	–
Capillaria sp. (eggs)	1–8 (zoos #2, 4, 7, 8, 9, 10, 12, 16, 18, 25, 26, 28, 30, 34, 47, 48)	–	–	–
Moniezia spp.** (eggs)	1–17 (zoos # 9,14, 24, 26, 34)	–	–	–
Trematodes	1–10	11–100	>100	–
F. hepatica (eggs)	1–3 (zoos #13, 21)	–	–	–
Paramphistomum sp. (eggs)	1–8 (zoos #4, 5, 9, 11, 13, 37, 38, 38)	9–56 (zoo #30)	–	–

* Low intensity in case of adult animals

**Including M. expansa

 

Disscusion

In this section, results of prevalences of every found helminth must be discussed with findings of other researches.

It is a fair demand, but in our case we are, basically, the ones, studied helminths exactly in zoo reindeer. There are no other works on the subject to compare. One mentioned case of F. hepatica in zoo reindeer described by Mizkewitsch has no prevalence information. Given heterogeneous origin of zoo reindeer it is incorrect to compare them with purely wild or domestic reindeer.

L140. How many samples had one, two or three helminths? Discuss with other studies.

As we explained earlier, all the mixed infections were singular. We added this word (“singular”) to the manuscript.

L146-147. Rewrite this sentence.

Initially we wrote: “It is a rare finding for reindeer, and the latter is considered as an accidental host.”

It would be easier for us if the Reviewer explained what was wrong with this sentence. We reworded as follows: “This liver fluke seldom parasitize reindeer, and the latter is considered as an accidental host”

147-150. This statement is wrong. The more possible reasons for infection with Fasciola hepatica could be presence of intermediate hosts or eating food contaminated with metacercaria.

Lines 147-150 were as follows: “Infestation of reindeer with F. hepatica usually is associated with other ruminants in contact and captive facilities [3]. In two private zoos where F. hepatica was found in reindeer, there were sheep, goats and sika deer (Cervus nippon) also infected. Rumen fluke Paramphistomum sp. is widespread in Russian reindeer.”

Of course, F. hepatica has a complex life cycle involving fresh water mollusks as intermediate hosts and having metacercaria as an infective stage. But a presence of intermediate host themselves does nothing to infestation. It is exactly a presence of typical definitive hosts (infected ruminants) that makes reindeer infestation possible. Metacercaria do not appear out of the thin air.

We believe it is rather obvious, yet to follow the hint of the Reviewer we added “infected” to “ruminants in contact” and “where definitive hosts shed F. hepatica eggs, and they eventually develop into metacercaria”.

We recognize the chance to infest the host with contaminated food, but it this article we wanted to underline the danger of keeping reindeer with sheep and goats.

L153. What were the thresholds you used to determine the degree or level of infection? This also should be described in Material and methods section.

It was described in lines 126-133 of the initial manuscript. This time it is explained in the Table 4. Namely, we relied on numbers of the National Standard of the Russian Federation (GOST R 54627-2011) Ruminant animals—Methods of Laboratory Helminthological Diagnostics.

L155-156. Provide reference for this sentence.

Lines 155-156 were: “Tapeworms found were present with M. expansa (easily recognizable by its triangular eggs) and Moniezia sp.”

We added a ref to:

Verocai, G.G.; Chaudhry, U.N.; Lejeune, M. Diagnostic Methods for Detecting Internal Parasites of Livestock. Vet. Clin. Food. Anim. 2020, 36, 125–143. https://doi.org/10.1016/j.cvfa.2019.12.003.

L157-158. Moniezia have indirect life cycle. Presence of oribatid mites as intermediate hosts is also important factor for infection of reindeers. Is it higher or lower when you compare your results with results of other researches?

With all due respect to the reviewer, oribatid mites are very widespread. Their presence itself does nothing to infestation as well as in case of Fasciola. Oribatid mites are the means by which reindeer got infected, but infected ruminants are the source.

We doubt that somebody studied a presence of oribatid mites in the zoos, housing reindeer. At least we did not. So there is nothing to compare. Yet it is a possible subject for future researchers.

L193. Correct the grammatical error “Round worm”.

Replaced “Round worm” with “Roundworm” as suggested

L236-241. This text is out of topic.

These lines were: “Tactile contact between humans and animals in petting zoos may pose a risk of helminth infestation. For instance, red fox (Vulpes vulpes) and Arctic fox (V. lagopus) are recognized definitive hosts for Echinococcus spp. The latter produces numerous eggs which are shed with feces. Those eggs are very light and flying, and easily contaminate animal’s fur. Stroking such sick fox or even breathing closely to it may result in ingestion of Echinococcus eggs which are instantly invasive [37].”

We intended to underline the potential danger of petting zoos in general and thus move to the discussion of potential risk for humans in case of petting reindeer.

We deleted it as suggested by the Reviewer.

Reference number 3 is too much cited in manuscript. I advise authors to find other new references for the statements which are cited with this reference.

Reference number 3 is for the monograph solely on helminths of reindeer written by Dr. Vera Mizkewitsch in 1967. Mizkewitsch studied different aspects of this subject. We see no point in replacing these refs with more fresh publications if they only repeat her findings.

However, we are ready to follow this recommendation if the Editor insists.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear editor,

 

I have no additional comments. After review, my recommendation is: accept.

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors of the paper for addressing the comments.