Distribution and Abundance of the Tick-Borne Piroplasm Theileria cervi Parasitizing White-Tailed Deer (Odocoileus virginianus) in Massachusetts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study reports distribution of the tick-borne piroplasm Theileria cervi in white-tailed deer (Odocoileus virginianus) populations in Massachusetts, and assesses the possible usability of T. cervi detection for a sentinel marker for the establishment of the lone star tick Amblyomma americanum, the only putative vector of T. cervi, in their ever-expanding range. This hypothesis is interesting, and this study thoroughly verifies this hypothesis, particularly by use of Oxford nanopore sequencing techniques. The manuscript is well organized and written. Some minor corrections are required as suggested below.

1. L59, L82: “ixodid”
2. L77: “Infected individuals …”
3. L148–L151: Change the order of two sentences written here: “Ticks were collected … the head and neck. Ticks were placed in separate vials …”
4. L153–L155: “Yunker et al. [19], Cooley and Kohls [20], and Keirans and Clifford [21] were … identification.”
5. L166–L167: Replace “White blood cells” by “Leukocytes”, and “red cell” by “erythrocyte” to unify the terms.
6. L170–L171: At the second and repeated appearances of “(LGC Biosearch Technologies, Hoddesdon, UK)”, simply “(LGC Biosearch Technologies)” could be used. Insert “(LGC Biosearch Technologies)” after “MPC Precipitation Reagent” at L173.
7. L210–L416: “the small ribosomal subunit (18S)” The same entity is expressed by two or more different terms. It is better to unify the term.
8. L212–L213: “Berkley et al. [23] and Scott et al. [17]. Amplification of this gene …”
9. L254: “Ixodes” (?) If it is really “Ixodes sp.”, why did the authors not identify the species?
10. L278: “(Gene Codes Corporation, Ann Arbor, MI, USA)”
11. L312-L314: “Berkley et al. [23] found … for cervi.”
12. L320–L322: “Thompson et al. [25] reported …. Babesia-positive.”
13. L326–L327 : “… by Cauvin et al. [26], which …. Were infected with cervi.”
14. L356: “white-tailed deer”
15. L459–L521 [References]: Unify the format of references. Reduce excess capital letters in titles, for example, in [5], [6], [7], [8], [9], [11], and [16].

Author Response

This study reports distribution of the tick-borne piroplasm Theileria cervi in white-tailed deer (Odocoileus virginianus) populations in Massachusetts, and assesses the possible usability of T. cervi detection for a sentinel marker for the establishment of the lone star tick Amblyomma americanum, the only putative vector of T. cervi, in their ever-expanding range. This hypothesis is interesting, and this study thoroughly verifies this hypothesis, particularly by use of Oxford nanopore sequencing techniques. The manuscript is well organized and written. Some minor corrections are required as suggested below.

 

L59, L82: “ixodid”

The recommended revisions were implemented here.

L77: “Infected individuals …”

The recommended revisions were implemented here.

L148–L151: Change the order of two sentences written here: “Ticks were collected … the head and neck. Ticks were placed in separate vials …”

The recommended revisions were implemented here to improve paragraph structure.

L153–L155: “Yunker et al. [19], Cooley and Kohls [20], and Keirans and Clifford [21] were … identification.”

The recommended revisions were implemented here, placing in-text citations after each author(s) for all related instances.

L166–L167: Replace “White blood cells” by “Leukocytes”, and “red cell” by “erythrocyte” to unify the terms.

The recommended revisions were implemented here.

L170–L171: At the second and repeated appearances of “(LGC Biosearch Technologies, Hoddesdon, UK)”, simply “(LGC Biosearch Technologies)” could be used. Insert “(LGC Biosearch Technologies)” after “MPC Precipitation Reagent” at L173.

The recommended revisions were implemented here and applied to related instances.

L210–L416: “the small ribosomal subunit (18S)” The same entity is expressed by two or more different terms. It is better to unify the term.

The recommended revisions were implemented here.

L212–L213: “Berkley et al. [23] and Scott et al. [17]. Amplification of this gene …”

The recommended revisions were implemented here and all related instances.

L254: “Ixodes” (?) If it is really “Ixodes sp.”, why did the authors not identify the species?

Clarification was added to the methods for identifying tick species. Due to the large volume of Ixodescollected, these samples were identified based on the position of the anal groove being anterior to the anus. This method distinguished them from the collected non-Ixodes, but did not identify them at a species level. Ixodes scapularis is the predominant Ixodes species in the region, and while there are other Ixodes local to our chosen sampling regions, these species do not prefer white-tailed deer. This method was performed in an effort to efficiently sort the collected ticks.

L278: “(Gene Codes Corporation, Ann Arbor, MI, USA)”

The in-text citation including the address may be found in section 2.4. It immediately follows the first reference of Sequencher software.

L312-L314: “Berkley et al. [23] found … for cervi.”

The recommended revisions were implemented here.

L320–L322: “Thompson et al. [25] reported …. Babesia-positive.”

The recommended revisions were implemented here.

L326–L327 : “… by Cauvin et al. [26], which …. Were infected with cervi.”

The recommended revisions were implemented here.

L356: “white-tailed deer”

The recommended revisions were implemented here.

L459–L521 [References]: Unify the format of references. Reduce excess capital letters in titles, for example, in [5], [6], [7], [8], [9], [11], and [16].

The recommended revisions were implemented here.

We thank the reviewer for their suggestions to improve the accuracy, consistency, and structure of the language used throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The study addresses a topic of significant epidemiological and ecological interest, namely the monitoring of pathogen vector expansion (specifically Amblyomma americanum) through wildlife surveillance.

The authors acknowledge that the BJ1/BN2 primers (originally designed for Babesia) also amplify Theileria cervi and Babesia odocoilei. The use of Oxford Nanopore sequencing to obtain a consensus sequence entails the inherent limitation of masking low-concentration co-infections. Consequently, it would be appropriate to discuss in greater depth the impact of this limitation on the potential underestimation of Babesia odocoilei co-infections in areas where T. cervi is highly dominant.

Furthermore, blood and tick samples were collected during the autumn/winter hunting season from October to December. As correctly observed by the authors, this period does not coincide with the peak activity window of A. americanum nymphs and adults, which occurs between May and July. Although this approach is justified by the practical nature of hunter-based surveillance, it is recommended to emphasize with greater caution that the low number of A. americanum ticks recovered from deer in autumn reflects seasonal dynamics rather than necessarily representing true local vector density. 

Finally, although no inland samples tested positive for T. cervi following sequencing, initial PCR screening yielded positive bands due to off-target amplification, which were largely identified as Babesia odocoilei or Sarcocystis sp.. It is crucial that the Results and Discussion sections clearly distinguish the absolute absence of T. cervi inland from the presence of other hemoparasites, thereby avoiding ambiguity in the interpretation of raw agarose gel data

Author Response

The study addresses a topic of significant epidemiological and ecological interest, namely the monitoring of pathogen vector expansion (specifically Amblyomma americanum) through wildlife surveillance.

The authors acknowledge that the BJ1/BN2 primers (originally designed for Babesia) also amplify Theileria cervi and Babesia odocoilei. The use of Oxford Nanopore sequencing to obtain a consensus sequence entails the inherent limitation of masking low-concentration co-infections. Consequently, it would be appropriate to discuss in greater depth the impact of this limitation on the potential underestimation of Babesia odocoilei co-infections in areas where T. cervi is highly dominant.

An additional statement was added to the Discussion section to note that the prevalence of Babesia odocoilei may be underrepresented on Martha’s Vineyard due to detection methods.

Furthermore, blood and tick samples were collected during the autumn/winter hunting season from October to December. As correctly observed by the authors, this period does not coincide with the peak activity window of A. americanum nymphs and adults, which occurs between May and July. Although this approach is justified by the practical nature of hunter-based surveillance, it is recommended to emphasize with greater caution that the low number of A. americanum ticks recovered from deer in autumn reflects seasonal dynamics rather than necessarily representing true local vector density.

We added to the Discussion section to further express the low seasonal density of Amblyomma americanum at the time of sampling, which does not reflect the density observed during seasons with greater activity.

Finally, although no inland samples tested positive for T. cervi following sequencing, initial PCR screening yielded positive bands due to off-target amplification, which were largely identified as Babesia odocoilei or Sarcocystis sp.. It is crucial that the Results and Discussion sections clearly distinguish the absolute absence of T. cervi inland from the presence of other hemoparasites, thereby avoiding ambiguity in the interpretation of raw agarose gel data

Additional language was added to the Results and Discussion sections to emphasize the absence of Theileria cervi-positive samples from inland sampling sites

We thank the reviewers for their suggestions in clarifying and emphasizing important aspects of our Results and Discussion sections.