In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis.

PURPOSE: To assess the in vitro larvicidal activity of ivermectin and povidone-iodine (PVP-I) against Oestrus ovis, the most frequent cause of external ophthalmomyiasis.
METHODS: L1 O. ovis larvae were collected from the nasal boots of sheep slaughtered in local abattoirs and transferred onto Petri dishes containing mucosal tissue (25 larvae/dish). The larvicidal activity of the following formulations was tested: 1% ivermectin suspension in balanced sterile saline solution (BSSS), 1% ivermectin solution in propylene glycol, propylene glycol, 0.6% PVP-I in hyaluronic acid vehicle (IODIM®), and combination of ivermectin 1% solution and 0.6% PVP-I. One mL of each formulation was added to different Petri dishes containing the larvae. The time needed to kill the larvae was recorded.
RESULTS: 893 larvae were tested. The median time needed to kill the larvae was 46, 44, 11, 6, and 10 minutes for Iodim®, ivermectin 1% suspension, propylene glycol, ivermectin 1% solution, and a combination of ivermectin 1% solution with 0.6% PVP-I, respectively. Kaplan-Meyer analysis disclosed that the survival curves were significantly lower in samples treated with ivermectin 1% solution, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol than in samples receiving other treatments or BSSS.
CONCLUSION: In this in vitro study, ivermectin 1% solution in propylene glycol, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol alone showed a good, relatively rapid larvicidal activity against O. ovis larvae. Further experimental and clinical studies are necessary to establish whether, or not, these formulations may be considered as potential candidates for the topical treatment for external ophthalmomyiasis caused by O. ovis.

Introduction

Ophthalmomyiasis is the infestation of the eye with maggots (larvae) of certain flies. Oestrus ovis, Diptera:Oestridae, is the most frequent cause of ocular myiasis, particularly in countries with tropical or mild climates [1, 2]. The presence of this pathogen is widely known in the Mediterranean area, where sheep farming is common [1-3].

Based on its location, ophthalmomyiasis is classified into external, internal, and orbital. In the external form, larvae are found on the conjunctiva and eyelid margins, leading to inflammation of the ocular surface. Typical symptoms include tearing, photophobia, foreign body sensation, and pain. Larvae may survive on the ocular surface for several days with consequent worsening of signs and symptoms, usually due to an IgE-mediated immune response to larval antigens.

Currently, mechanical removal of the larvae is the only available treatment for external ophthalmomyiasis. However, this procedure is time-consuming and often not completely effective, because the highly motile larvae can easily be missed. This usually results in persistence of ocular surface inflammation, with the need for repeated consultations.

There are sparse reports describing cases of ophthalmomyiasis caused by other Diptera species (Dermatobia hominis and Cochliomyia hominivorax) treated with ivermectin (MK-933, 22,23-Dihydroavermectin B1), a semisynthetic anthelmintic agent [4-7]. Oral ivermectin has also been successfully used in the treatment of one case of human conjunctival myiasis caused by O. ovis [8]. Furthermore, 0.6% povidone-iodine (PVP-I), an iodinated polyvinyl polymer, has recently been found to be effective against Demodex mites, a parasite colonizing the eyelid margins [9], thus offering new prospects for a possible use against other parasites, such as Diptera larvae.

The purpose of this study was to assess the in vitro larvicidal activity of ivermectin and PVP-I, alone and in combination, against L1 O. ovis larvae.

Materials and methods

Pharmacological preparations

Two different formulations of ivermectin were tested. The first formulation was 1% ivermectin suspension in balanced sterile saline solution (BSSS). One-hundred mg of ivermectin powder (Sigma-Aldrich; ID PubChem: 24278497) was suspended in 10 mL of BSSS and placed on a magnetic stirrer for 5 minutes. The pH of the suspension was 5.3. The second formulation was 1% ivermectin solution in propylene glycol (propane-1,2-diol, C3H8O2) vehicle. One-hundred mg of ivermectin powder (Sigma-Aldrich; ID PubChem: 24278497) was suspended in 5 ml of propylene glycol and placed on a magnetic stirrer for 5 minutes. After complete solubilization of ivermectin, the solution was brought to a volume of 10 mL by adding propylene glycol. The final pH was 5.4.

A combination of ivermectin 1% solution in propylene glycol and 0.6% PVP-I, obtained from 5% PVP-I ophthalmic solution (Oftasteril®, Alfa Intes, Casoria, Italy) was also tested. One-hundred mg of ivermectin powder was suspended in 5 mL of propylene glycol and placed on a magnetic stirrer for 5 minutes. Then, 1.2 mL of Oftasteril® was added and this solution was finally made up to 10 mL with propylene glycol. The final pH was 4.2.

Propylene glycol, the diluent used for ivermectin, was also tested alone, to avoid potential bias due to a possible larvicidal activity.

Finally, we assessed the larvicidal activity of a new commercial ophthalmic solution containing PVP-I 0.6% in hyaluronic acid vehicle (IODIM®, Medivis, Catania, Italy).

All the formulations were weighted on an analytical balance (Readability 0,1 mg, Mettler Toledo ab-204) and pH was measured with a pH-meter model 700 XS.

In vitro larvicidal tests

In vitro experiments were performed at the Section of Parasitology and Parasitic Diseases, Department of Veterinary Medicine, University of Sassari, Sassari, Italy.

Larvae were collected from the nasal boots of sheep slaughtered in local certified abattoirs. Each day, a maximum of 15 heads from freshly slaughtered animals were obtained and transported in specific containers to the autopsy room of the Department of Veterinary Medicine. The sheep heads were cut into halves on the sagittal axis and the nasal boots were removed. Although every effort was made to process all the specimens on the slaughter day, when this was not feasible, the surplus heads were maintained intact in specific containers at 39°C and processed one day later.

All the nasal boots were examined by stereomicroscope. All the viable L1 larvae found were collected and transferred onto separate Petri dishes containing mucosal tissue obtained from the nasal boots.

Each Petri dish was seeded with 25 larvae, filled with 1 mL of each study drug, and examined by light microscopy to assess larval viability. In order to quantify the time needed for each drug to kill the larvae, we defined time of death as the time necessary to obtain total larval immobility. When time of death was reached, the larvae were returned to drug-free culture medium and re-examined for a minimum of 5 minutes, in order to double-check their viability. If the absence of viability was confirmed, the time of death was accepted.

Control Petri dishes received 1 mL of BSSS.

All assays were performed in six replicates.

Statistical analysis

Data were tested for normality (Shapiro Wilk and Kolmogorov-Smirnov test) and homogeneity of variance (Levine test). Furthermore, we performed a logarithmic (base 10) transformation of data, to make them closer to normal distribution and reduce skewness.

Due to non-parametric distribution and heterogeneity of variance, Kruskal Wallis H test and Dunnett’s test with Sidák adjustment for multiple comparisons were performed on transformed data.

Kaplan-Meier survival analysis using the log-rank test was performed to compare the different drugs assessed.

A p value <0.05 was considered to be statistically significant. Statistical analysis was carried out using Stata software (Stata/MP 14.1 for Mac, StataCorp, College Station, TX).

Results

Only three sheep heads were held overnight in specific containers at 39°C and processed early in the morning the following day.

In total, 893 fully viable L1 O. ovis larvae were tested in 50 days. The median time needed to kill the larvae was 96, 46, 44, 11, 6, and 10 minutes for BSSS, Iodim®, ivermectin 1% suspension in BSSS, propylene glycol, ivermectin 1% solution in propylene glycol, and a combination of ivermectin 1% solution in propylene glycol with 0.6% PVP-I, respectively (Table 1).

Table 1

Median time required by Balanced Sterile Saline Solution (BSSS), Iodim®, ivermectin 1% suspension in BSSS, propylene glycol, ivermectin 1% solution in propylene glycol, and a combination of ivermectin 1% solution in propylene glycol with 0.6% povidone-iodine (PVP-I) to kill L1 O. ovis larvae.

Drug	Time	Median	Interquartile Range	Minimum	Maximum
BSSS	minutes	94.35	132.35	6	253.53
	Log10	-3.753876	-0.5485945	-2.556303	-4.182786
Iodim®	minutes	46.32	27.2	2.23	139.3
	Log10	-3.445915	-0.2815163	-2.155336	-3.922725
Ivermectin 1% Suspension	minutes	44.21	17.36	6.26	64.28
	Log10	-3.425034	-0.1974986	-2.586587	-3.587487
Propylene glycol	minutes	11.2	6.23	4.1	28.43
	Log10	-2.832188	-0.2144022	-2.39794	-3.236285
1% Ivermectin Solution	minutes	6.2	3.6	1.19	12.59
	Log10	-2.579202	-0.2913892	-1.897627	-2.891537
1% Ivermectin Solution + PVP-I 0.6%	minutes	10.13	4.21	2.8	16.18
	Log10	-2.787461	-0.1931484	-2.10721	-2.990339

Results of Kaplan-Meyer survival analysis using the log-rank test are reported in Fig 1.

Fig 1

Kaplan Meyer survival estimates.

The graph shows the survival curves of all the preparations tested.

The survival curves were significantly lower in the samples treated with ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol than in the samples receiving other treatments or BSSS.

Both original data and log10 transformations did not show a normal distribution; however, statistical analysis was performed using the latter. Kruskal-Wallis H test showed a statistically significant difference in time of death between the different drug treatments (χ2[2] = 668.8, with 5 d.f.; p = 0.0001). Dunnet test with Sidák adjustment for multiple comparisons showed that all drugs tested were significantly more effective than BSSS (p< 0.001).

Propylene glycol, the solvent chosen for ivermectin, showed a significantly higher larvicidal activity than Iodim® and ivermectin 1% suspension in BSSS (p< 0.001). However, ivermectin 1% solution in propylene glycol and the combination ivermectin 1% solution in propylene glycol + PVP-I 0.6% were found to be significantly more effective than propylene glycol alone in terms of time needed to kill L1 O. ovis larvae (p = 0.001 and p = 0.0422, respectively).

Discussion

O. ovis larvae usually colonize the frontal and nasal sinuses of sheep and goat, but occasionally they can infest the human eyes [1–3, 10, 11] and naso-pharyngeal tract [12, 13]. O. ovis life cycle starts when a gravid fly sprays up to 25 larvae on the target animal face. Then, L1 larvae migrate to the nasal boot using their hooks and feed on the mucus produced in response to the inflammatory process. Here, L1 larvae stay for several months during winter (hypobiosis). When weather conditions become more favorable, normally during spring/summer, they start their metamorphosis through the L2 and L3 stages. L3 larvae migrate out of the nasal boot and fall on the ground, where they develop into the adult form, thus starting a new life cycle.

Ivermectin, a macrocylic lactone, is a semisynthetic anthelmintic agent for oral administration in the treatment of ascariasis, filariasis, gnathostomiasis, and hookworm infections [14]. Ivermectin binds selectively and with high affinity to glutamate-gated chloride ion channels in invertebrate muscle and nerve cells. This binding causes an increased cell membrane permeability to chloride ions and results in cell hyperpolarization, which leads to paralysis and death of the parasite. Furthermore, ivermectin also acts as a gamma-aminobutyric acid (GABA) agonist, thereby disrupting GABA-mediated neurosynaptic transmission. In general, ivermectin does not cross the blood-brain barrier in most animal species, including humans, and does not interact with peripheral neurotransmitters, making it safe enough for human use [15].

Oral ivermectin has been reported to be effective in the treatment of head and neck myiasis in humans [4, 16]. Furthermore, topical ivermectin showed a beneficial effect in the treatment of rosacea and blepharitis, conditions in which the mite Demodex is believed to play a role [17].

Povidone–iodine (PVI) is a disinfectant and antiseptic agent used for preoperative preparation of the skin and mucous membranes, as well as for the treatment of contaminated wounds. Because of its broad-spectrum antimicrobial activity, 5% PVI has been widely used in ophthalmology. A peculiar chemical property of povidone is that the concentration of free iodine, the active antimicrobial element, increases with the dilution of PVI, due to a weakening of the chemical bonding between iodine and povidone. Recently, a new ophthalmic preparation containing 0.6% PVI has been shown to be bactericidal against several Gram-positive and Gram-negative isolates [18]. Furthermore, 0.6% PVI has also been found to be effective against Demodex mites [8], a parasite colonizing the eyelid margins, thus offering new prospects for a possible use against other parasites, such as Diptera larvae.

In this in vitro study, we found that ivermectin 1% solution in propylene glycol vehicle and a combination of ivermectin 1% solution in propylene glycol with 0.6% PVP-I showed a good, rapid larvicidal activity against L1 O. ovis larvae. Surprisingly enough, propylene glycol, the solubilizer chosen for ivermectin, had a similar larvicidal activity.

Ivermectin is insoluble and unstable in water but soluble in propylene glycol [19], a commonly used drug solubilizer in topical, oral, and injectable medications (e.g., intravenous diazepam, lorazepam, phenobarbital, phenytoin, and nitroglycerin). In Ophthalmology, propylene glycol is used in eye lubricants.

To the best of our knowledge, we are unaware of any former in vitro study assessing the larvicidal activity of ivermectin and PVP-I, alone and in combination, against L1 O. ovis larvae. Our results suggest that ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol might be potential candidates for the topical treatment, as ointment and/or eye-drops, for external ophthalmomyiasis caused by O. ovis.

Some authors have reported sporadic cases of human ophthalmomyiasis treated with a combination of petroleum ointment and ivermectin or ivermectin solution alone as adjuvant to the mechanical removal of the larvae. However, all these reports described cases of ophthalmomyiasis caused by other Diptera species (Cochliomyia hominivorax and Dermatobia hominis) [4-7].

There is already at least one paper indicating efficacy of oral ivermectin (12 mg in a single dose) in treating human conjunctival myiasis caused by O. ovis larvae [8]. However, the use of systemic ivermectin for the management of ocular surface myiasis is rather questionable, as a topical approach with ointment and/or eye-drops would be much more appropriate.

A clear limitation of our study is that we performed an in vitro experiment, which may not reflect exactly the real situation in vivo. Furthermore, we do not know how toxic ivermectin and propylene glycol can be for the corneal and conjunctival epithelium, at the concentrations used in this experiment. This topic will be the subject of future research. On the other hand, we feel strongly that our in vitro evaluation of different anthelmintic formulations on O. ovis viability represents a potentially impactful approach to addressing ophthalmomyiasis in the Mediterranean Basin or other sheep-rearing regions globally.

Conclusions

We found that ivermectin 1% solution in propylene glycol vehicle, 1% ivermectin solution in propylene glycol + 0.6% PVP-I, and propylene glycol alone showed a good, relatively rapid larvicidal activity against L1 O. ovis larvae. Further experimental and clinical studies are necessary to establish whether, or not, these formulations may be considered as potential candidates for the topical treatment for external ophthalmomyiasis caused by O. ovis.

6 Sep 2021

PONE-D-21-22404In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovisPLOS ONE

Dear Dr. Antonino Pinna,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: The results described in the manuscript are preliminary, however they are very interesting and provide the basis for clinical studies. The only negative point, but not an impediment to publication, is that the authors should have performed the in vitro assays with a greater number of repetitions.

Reviewer #2: Re: PLOS One Manuscript PONE-D-21-22404

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

By Giuseppe D’Amico Ricci

General:

The authors have submitted a well prepared manuscript describing laboratory effectiveness of five formulations, containing one or more of three main components, the parasiticide ivermectin, propylene glycol, used mainly as a carrier, but also tested as a sole ingredient as a control, and povidone-iodine in hyaluronic acid vehicle which is used primarily as an antiseptic but with a previous report of insecticidal activity against Demodex mites noted by the authors.

Usually laboratory testing, such as conducted here, is conducted as a preliminary study to in vivo trials to establish whether any insecticidal action against the target organism is present. If so in vivo effectiveness is validated later. Ivermectin often acts systemically and lack of a topical effect in laboratory studies does not necessarily mean that it would not be effective in vivo and provides very little information about optimal formulation.

However, in this instance there is already at least one paper indicating efficacy of ivermectin in treating human ocular myiasis caused by Oestrus ovis larvae (Kumar et al. ), some studies indicating efficacy of ivermectin against O oestrus in human nasal infestations and a significant number of papers indicating in vivo activity against O. ovis in sheep which draws into question the novelty of the work and need for new laboratory studies to demonstrating insecticidal effect against O. ovis. Furthermore, it could perhaps be argued from the results that the polypropylene glycol was more active in the laboratory studies than ivermectin per se, although whether this would be the case in a clinical setting is questionable.

Methods:

More detail is needed to for the reader to accurately interpret the design used for the study. It is noted that “Each Petri dish was seeded with 25 larvae, filled with 1 mL of each study drug” and that all assays were conducted in triplicate. I may have misunderstood, but to me this suggests that 5 treatments + saline control x 25 larvae x 3 replicates = 450 larvae were used. However, it is mentioned in the abstract and in the body of the paper that 893 larvae were tested. How were the other larvae used? This needs to be clarified

It is noted that larvae for the study were collected over a number of days and that in most cases the experiments were conducted on the day of collection, but in some instances when all heads could not be processed, they were held overnight and processed the following day. Often such closely associated parasites die quickly once separated from their host, or their host dies. Thus how the replicates/treatments were established with respect to time of collection needs to be specified to ensure there is no confounding between treatment effects and time of collection.

Results.

Figure 1.

As I understand it Figure 1, Graph A contains a plot of all treatments, Graph B is exactly the same as A but with 3 of the treatments from Graph A omitted and Graphs C and D just contain subsets of Graph A replotted with a different scale on the X axis?. That is, for example the same data 1% ivermectin in BSSS treatment is replotted 3 times in Graphs A, B, and C. It is unusual to replot the same data repeatedly and is potentially confusing for the reader. Consider just using Graph A with statements about which treatments were significantly different in the caption.

References.

As noted above there is at least one previous reported study of the use of ivermectin to treat human ocular myiasis (Kumar et al. 2013 Annals of Tropical Medicine and Public Health 6: 315-316) as well as a quite a number of previous reports of the effectiveness of ivermectin against Oestrus ovis larvae (in human hosts and in sheep). None of these papers have been cited in the reference list. These papers would seem to be very relevant to the current study and at least some of them should be noted.

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14 Sep 2021

Journal requirements

- Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

Reply: done

- In your Methods section, please provide additional details regarding the ivermectin used in your study and ensure you have described the source.

Reply: additional details regarding the ivermectin used in our study have been provided.

Reviewer #1

- The results described in the manuscript are preliminary, however they are very interesting and provide the basis for clinical studies. The only negative point, but not an impediment to publication, is that the authors should have performed the in vitro assays with a greater number of repetitions.

Reply: All assays were performed in six replicates, not in triplicate. Triplicate was a misprint

Reviewer #2

- More detail is needed to for the reader to accurately interpret the design used for the study. It is noted that “Each Petri dish was seeded with 25 larvae, filled with 1 mL of each study drug” and that all assays were conducted in triplicate. I may have misunderstood, but to me this suggests that 5 treatments + saline control x 25 larvae x 3 replicates = 450 larvae were used

Reply: We wish to thank Reviewer #2 for highlighting our mistake. Indeed, there was a misprint. All assays were performed in six replicates, not in triplicate (5 treatments + saline control x 25 larvae x 6 replicates = ~900 larvae were used)

- It is noted that larvae for the study were collected over a number of days and that in most cases the experiments were conducted on the day of collection, but in some instances when all heads could not be processed, they were held overnight and processed the following day. Often such closely associated parasites die quickly once separated from their host, or their host dies. Thus how the replicates/treatments were established with respect to time of collection needs to be specified to ensure there is no confounding between treatment effects and time of collection.

Reply: Only three sheep heads were held overnight in specific containers at 39°C and processed early in the morning the following day. This procedure did not significantly affect larval viability. All the nasal boots were examined by stereomicroscope. All the viable L1 larvae found were collected and transferred onto separate Petri dishes containing mucosal tissue obtained from the nasal boots.

- Figure 1.

As I understand it Figure 1, Graph A contains a plot of all treatments, Graph B is exactly the same as A but with 3 of the treatments from Graph A omitted and Graphs C and D just contain subsets of Graph A replotted with a different scale on the X axis?. That is, for example the same data 1% ivermectin in BSSS treatment is replotted 3 times in Graphs A, B, and C. It is unusual to replot the same data repeatedly and is potentially confusing for the reader. Consider just using Graph A with statements about which treatments were significantly different in the caption.

Reply: Figure 1 has been changed according to Reviewer #2 suggestions.

-References.

As noted above there is at least one previous reported study of the use of ivermectin to treat human ocular myiasis (Kumar et al. 2013 Annals of Tropical Medicine and Public Health 6: 315-316) as well as a quite a number of previous reports of the effectiveness of ivermectin against Oestrus ovis larvae (in human hosts and in sheep). None of these papers have been cited in the reference list. These papers would seem to be very relevant to the current study and at least some of them should be noted.

Reply: the study by Kumar et al. has been quoted (Reference 8) and additional references have been added (References 11, 12 and 13).

The following comment has been included in the Discussion: “There is already at least one paper indicating efficacy of oral ivermectin (12 mg in a single dose) in treating human conjunctival myiasis caused by O. ovis larvae.8 However, the use of systemic ivermectin for the management of ocular surface myiasis is rather questionable, as a topical approach with ointment and/or eye-drops would be much more appropriate.”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Oct 2021

PONE-D-21-22404R1In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovisPLOS ONE

Dear Dr. Antonio Pinna,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

Please improve the paper with the suggestion of the Reviewer #2.

==============================

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors respond to reviewers' comments in a satisfactory manner, in this way the manuscript can be accepted as is.

Reviewer #2: Re: PLOS One Manuscript PONE-D-21-22404-R1

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

The manuscript has been revised appropriately and with some minor suggested amendments noted below should be suitable for publication in PLOS 1.

Abstract

Lines 39-41 “Kaplan-Meyer analysis

…disclosed that the survival curves were significantly lower in the samples treated with ivermectin 41 1% solution, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol.” Please state lower than what. Lower than the RSSS control or lower than the other treatments

Introduction

Line 54. In entomological protocol it is most common, though not essential to state both the Order and the Family e.g. [Diptera:Oestridae]

Line 115- The BSS would usually be considered a negative control. A positive control would usually mean a treatment known to be effective in killing larvae. I suggest just delete “Positive”: ie Control Petri dishes received 1 mL of BSSS.

Line 124 – Log rank test, rather than “..long rank test”

Line 147 – Again, as above, significantly lower than what?

Lines 175-177. This is an important point which would benefit from a supporting reference.

Line 193 to 195 Delete “also”. That is: ‘Surprisingly enough, propylene glycol, the solubilizer chosen for ivermectin, had similar larvicidal activity.

Lines 203-204 Suggest “Our results suggest that ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol might be potential candidates for the topical treatment, as ointment and/or eye-drops, for external ophthalmomyiasis caused by O. ovis.”

Line 224 – closing sentence. Suggest ‘… to establish whether or not these formulations may be considered….’ Rather than “…they..”

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Oct 2021

Journal requirements

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reply: As far as we know, there are no retracted references in the list. Reference 8 (Kumar MA, Joseph NM, Srikanth K, Stephen S. Conjunctival Myiais caused by Oestrus ovis in a medical college student which responded to Ivermectin. Ann Trop Med Public Health 2013;6: 315-316), whose inclusion in the list was recommended by Reviewer #2, does not appear in PubMed, but can be retrieved in Scopus

Reviewer #2

Abstract

Lines 39-41 “Kaplan-Meyer analysis

…disclosed that the survival curves were significantly lower in the samples treated with ivermectin 41 1% solution, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol.” Please state lower than what. Lower than the RSSS control or lower than the other treatments

Reply: the sentence has been changed as follows: Kaplan-Meyer analysis disclosed that the survival curves were significantly lower in samples treated with ivermectin 1% solution, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol than in samples receiving other treatments or BSSS.

Introduction

Line 54. In entomological protocol it is most common, though not essential to state both the Order and the Family e.g. [Diptera:Oestridae]

Reply: changed according to Reviewer suggestion

Line 115- The BSS would usually be considered a negative control. A positive control would usually mean a treatment known to be effective in killing larvae. I suggest just delete “Positive”: ie Control Petri dishes received 1 mL of BSSS.

Reply: changed according to Reviewer suggestion

Line 124 – Log rank test, rather than “..long rank test”

Reply: changed according to Reviewer suggestion

Line 147 – Again, as above, significantly lower than what?

Reply: the sentence has been changed as follows: The survival curves were significantly lower in the samples treated with ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol than in the samples receiving other treatments or BSSS.

Lines 175-177. This is an important point which would benefit from a supporting reference.

Reply: as recommended the following reference (#15) has been included in the list: Laing R, Gillan V, Devaney E. Ivermectin - Old Drug, New Tricks? Trends Parasitol. 2017;33: 463-472.

Line 193 to 195 Delete “also”. That is: ‘Surprisingly enough, propylene glycol, the solubilizer chosen for ivermectin, had similar larvicidal activity.

Reply: changed according to Reviewer suggestion

Lines 203-204 Suggest “Our results suggest that ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol might be potential candidates for the topical treatment, as ointment and/or eye-drops, for external ophthalmomyiasis caused by O. ovis.”

Reply: changed according to Reviewer suggestion

Line 224 – closing sentence. Suggest ‘… to establish whether or not these formulations may be considered….’ Rather than “…they..”

Reply: changed according to Reviewer suggestion

Submitted filename: Response to Reviewers BIS.docx

Click here for additional data file.

12 Oct 2021

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

PONE-D-21-22404R2

Dear Dr. Antonio Pinna,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

14 Oct 2021

PONE-D-21-22404R2

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

Dear Dr. Pinna:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Filippo Giarratana

Academic Editor

PLOS ONE