Amblyomma mixtum (Acari: Ixodidae) Infestation in Humans in the Flooded Savanna Region of Colombia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 98 In the section on materials and methods the authors do not mention that all stages of ticks, except eggs were collected. It only becomes apparent in the results. 

Line 114 In the M&M where they mention the sequencing, which genus and species of ticks were used as the out group for the generation of the trees. This should be indicated and if not used indicate why not. 

Line 172 In Figure 5 caption there might be an error "different e body regions"

Line 187 Figure 6 lacks a caption. 

Line 196 Grammar should be checked. "...including accidentally human beings" 

In the human cases, bitten by A. mixtum the authors do not report on whether they were infected by Rickettsia spp. Why not? Is there any evidence of which Rickettsia spp. these A. mixtum ticks are vectors of? 

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The paper is fairly well written, however, there are parts of the paper where the authors should consider the way the information is related. 

I have mentioned some points in the feedback to the authors. 

Author Response

Dear

Reviewer

Wild

On behalf of the authors, we sincerely appreciate the time devoted to reviewing the manuscript, as well as the valuable comments and recommendations provided to improve it. All alterations are marked (word track changes).

With best regards

Sincerely

Arlex Rodríguez Durán

 

Q1

Comments:

Line 98 In the section on materials and methods the authors do not mention that all stages of ticks, except eggs were collected. It only becomes apparent in the results.

 

Answer: Thank you for your comment. In item 2.2. Tick Collection and Preservation, the tick stages have been added:

 

2.2. Tick Collection and Preservation

Ticks in larvae, nymphal, or adult stages were collected from accidentally infested individuals who voluntarily participated in the study. All participants were treated according to the ethical principles and procedures established in the Declaration of Helsinki. Voluntary participation was guaranteed by signing an informed consent form. Ticks were also collected at the larvae, nymphal, or adult stages, both in the non-parasitic stage on vegetation and at the parasitic stage infesting wild and domestic animals (see Table 1).

 

Q2

Comments:

Line 114 In the M&M where they mention the sequencing, which genus and species of ticks were used as the out group for the generation of the trees. This should be indicated and if not used indicate why not.

 

Answer: Thank you for your comment. For this analysis, the species Argas persicus (MK571448) was used as the outgroup. This information was clarified in section 2.3.3. Molecular Taxonomy: Sequencing and Phylogenetic Analyses of the methodology:

 

For the cox1 gene, the sequences of A. triste (OK576711 and OK576709), A. maculatum (OK576638 and OK576639) and A. mixtum (KY595136) were used. While the species Argas persicus (MK571448) was used as an outgroup of the genus Amblyomma.

 

Q3

Comments:

Line 172 In Figure 5 caption there might be an error "different e body regions"

 

Answer: Thank you for your comment. The title of Figure 5 has been modified to avoid confusion, and reads as follows:

 

Figure 5. Anatomical distribution of ticks and tick bite–associated skin lesions on different body parts of affected individuals from the flooded savanna of the department of Arauca, Colombia.

 

Q4

Comments:

Line 187 Figure 6 lacks a caption.

 

Answer: Thank you for your comment. The description for Figure 6 has been added, which reads as follows:

 

Figure 6. Maximum likelihood tree reconstruction using a: mitochondrial 16S rDNA gene sequences from Amblyomma mixtum and b: mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences from A. mixtum. Highlighted in black are the sequences from A. mixtum collected in this study. Additional sequences from GenBank (accession number in parentheses) and boot-strap values (1,000) are shown at each node. Argas persicus was used as the outgroup for the genus Amblyomma.

 

Q5

Comments:

Line 196 Grammar should be checked. "...including accidentally human beings"

 

Answer: Thanks for the comment. The wording has been improved, now reading as follows:

 

During the larvae and nymph life stages, it can parasitize a wide range of hosts with a preference for mammals, including humans [14].

 

Q6

Comments:

In the human cases, bitten by A. mixtum the authors do not report on whether they were infected by Rickettsia spp. Why not?

 

Answer: Thank you for your comment. Our study had a purely descriptive approach, focusing on the identification and distribution of A. mixtum in humans in this region of Colombia, with an emphasis on entomological aspects. Furthermore, ethical limitations prevented the collection of other types of samples from the individuals evaluated, as this would have required formal ethical approval.

 

Q7

Comments:

Is there any evidence of which Rickettsia spp. these A. mixtum ticks are vectors of?

 

Answer: Thank you for your comment. Several studies have reported Rickettsia species in A. mixtum (Ulloa-García et al., 2020; Chaparro-Gutiérrez et al., 2023). In the Floodplain Savanna region of Colombia, Chaparro-Gutiérrez et al. (2023) identified Rickettsia amblyommatis in A. mixtum. Meanwhile, in Mexico, Rickettsia typhi was identified in this tick species.

 

Chaparro-Gutiérrez, J.; Acevedo-Gutiérrez, L.; Mendell, N.; Robayo-Sánchez, L.; Rodríguez-Durán, A.; Cortés-Vecino, J.; Fernández, D.; Ramírez-Hernández, A.; Bouyer, D. First Isolation of Rickettsia amblyommatis from Amblyomma mixtum in Colombia. Parasit. Vectors 2023, 16(1), 332. https://doi.org/10.1186/s13071-023-05950-7.

 

Ulloa-García, A.; Dzul-Rosado, K.; Bermúdez-Castillero, S.; López-López, N.; Torres-Monzón, J. Detection of Rickettsia typhi in Rhipicephalus sanguineus s.l. and Amblyomma mixtum in southern Mexico. Public Health Mex. 2020, 62(4), 358-63. https://doi.org/10.21149/10160

 

Q7

Comments:

The English could be improved to more clearly express the research.

 

Answer: Thank you for the comment. The entire manuscript has been reviewed, and language modifications have been made. The changes are highlighted in yellow and tracked using the 'Track Changes' feature.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, 

In this paper, the authors describe the prevalence of Amblyomma mixtum in the flooded Savanna region of Colombia through collection of questing ticks from farms as well as voluntary case from humans and companion animals. The authors used molecular and taxonomic tools to identify the ticks collected from animals, vegetation, and humans. Upon completing both taxonomy and phylogenetic analysis, the authors confirm that majority of ticks were A. mixtum. In addition, the authors monitor human cases associated with accidental attachment of A. mixtum and in some cases record the presence of adverse conditions like rashes. This paper shows the abundance of this tick species in the region and highlights key public health implications associated with this species.

 

Major comments to be addressed:

 

• In the introduction, it would be useful to expand on mixtum species ecology and public health implications. Include the major zoonotic implications on animals and humans.

 

• In the results section, it is mentioned that in areas where human cases were identified, questing ticks were also collected from vegetation and domestic and wild animals (Table 1). However, in table 1, some areas did not have collection from vegetation. Is there is a reason why the area surrounding was not collected? If so, the authors need to clarify in the methods section whether the human associated with the case did not have a companion animal or whether they did not live or work in an area with access to tick habitat.

In addition, it would be beneficial to organize the table with location, this way readers can see the geographic location of the human case and the tick population surrounding the human.

 

• In the methods section, header 2.3.2, the authors use 12 ticks for molecular identification. Eight ticks were from humans and two of each animal and plants. Is there a rational for choosing this number of molecular ID? If so, it must be clarified in the section. Did the authors parse the ticks based on morphology and then pick representatives for molecular confirmation?

 

• Is pathogen testing on the collected ticks possible for Rickettsia sp? As this can help determine the secondary clinical symptoms associated with the two individuals.

 

Other comments:

• Lines 99 – 100 need to be revisited because of error in the sentence.
• Figure 6 is lacking a figure legend

Author Response

Dear

Reviewer

Wild

 

On behalf of the authors, we sincerely appreciate the time devoted to reviewing the manuscript, as well as the valuable comments and recommendations provided to improve it. All alterations are marked (word track changes).

With best regards

Sincerely

Arlex Rodríguez Durán

 

Q1

Comments:

In the introduction, it would be useful to expand on mixtum species ecology and public health implications. Include the major zoonotic implications on animals and humans.

 

Answer: Thank you for the comment. The ecology and pathogens identified in A. mixtum have been expanded upon.

 

In the Americas, the Amblyomma mixtum is widely distributed from southern Texas, USA, to western Ecuador [13,14]. It has been reported in both dry and humid regions of Mesoamerica, including the Mesoamerican Seasonal Highlands [13]. This tick species infests a variety of hosts, primarily cattle and equines, but it can also infested humans and birds [15,16]. Moreover, A. mixtum has been described as an important vector of zoonotic pathogens such as Anaplasma marginale, the causative agent of bovine anaplasmosis, and Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever [15,17-20]. It has also been associated with other pathogens, including Rickettsia amblyommatis and Rickettsia typhi [15,17-20].

 

Q2

Comments:

In the results section, it is mentioned that in areas where human cases were identified, questing ticks were also collected from vegetation and domestic and wild animals (Table 1). However, in table 1, some areas did not have collection from vegetation. Is there is a reason why the area surrounding was not collected? If so, the authors need to clarify in the methods section whether the human associated with the case did not have a companion animal or whether they did not live or work in an area with access to tick habitat.

 

Answer: Thank you for your comment. In Table 1, the numbers and stages of non-parasitic ticks collected were originally combined into a single item ('Farm Pastures and Forest'). Following the reviewer's recommendation, the table was modified to break down this information according to each area or property where the infested people reside (see items 14 to 19).

 

Q3

Comments:

In addition, it would be beneficial to organize the table with location, this way readers can see the geographic location of the human case and the tick population surrounding the human.

 

Answer: Thank you for your comment and recommendation. The geographic location of the individuals studied and the number of ticks identified in the non-parasitic stage have been added according to each stage (see items 14 to 19 in Table 1). See the answer to question Q2 for more details.

 

Q4

Comments:

In the methods section, header 2.3.2, the authors use 12 ticks for molecular identification. Eight ticks were from humans and two of each animal and plants. Is there a rational for choosing this number of molecular ID? If so, it must be clarified in the section.

 

Answer: Thank you for your comment. The number of ticks selected was random, and there were no inclusion or exclusion criteria to determine the number of samples to be used for DNA extraction.

 

Q5

Comments:

Did the authors parse the ticks based on morphology and then pick representatives for molecular confirmation?

 

Answer: Thank you for your comment. Initially, morphological identification was performed, and then representatives were selected for molecular confirmation, as described in sections 2.3.1. Classical Taxonomic Identification and 2.3.2. Molecular Taxonomy: DNA Extraction.

 

Q6

Comments:

Is pathogen testing on the collected ticks possible for Rickettsia sp? As this can help determine the secondary clinical symptoms associated with the two individuals.

 

Answer: Thank you for the comment and recommendation. Tick samples were preserved for the purpose of further studies on pathogens identified in A. mixtum, such as Rickettsia amblyommatis, previously reported in this region of Colombia. However, these analyses were not considered within the scope of this research.

 

Q7

Comments:

Lines 99 – 100 need to be revisited because of error in the sentence.

 

Answer: Thanks for the comment. The sentence has been modified.

 

Ticks were identified using an SZ61 binocular stereomicroscope (Olympus, USA) at magnifications of up to 100x, following the taxonomic keys and morphological descriptions provided by Strickland et al. [32], Barros-Battesti et al. [33, 34], Kleinjan and Lane [35], Estrada-Peña [36], and Nava et al. [37].

 

Q8

Comments:

Figure 6 is lacking a figure legend

 

Answer: Thank you for your comment. The description for Figure 6 has been added, which reads as follows:

 

Figure 6. Maximum likelihood tree reconstruction using a: mitochondrial 16S rDNA gene sequences from Amblyomma mixtum and b: mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences from A. mixtum. Highlighted in black are the sequences from A. mixtum collected in this study. Additional sequences from GenBank (accession number in parentheses) and boot-strap values (1,000) are shown at each node. Argas persicus was used as the outgroup for the genus Amblyomma.

Reviewer 3 Report

Comments and Suggestions for Authors

General comment

This manuscript presents novel and important observations of Amblyomma mixtum tick infestations in humans in the flooded savanna region of Colombia. The integration of classical and molecular taxonomy, coupled with clinical monitoring and ecological observations, contributes to the growing awareness of ticks as a One Health concern. The work is timely and relevant. However, several areas require clarification and revision to improve scientific rigor, reproducibility, and interpretation.

Major issues to address

1. Terminology – use of "Parasitosis"

The term "parasitosis" is used throughout the manuscript, including in the title, abstract, and introduction. This term typically refers to a pathological condition caused by parasitic organisms, often internal parasites like helminths or protozoa. Since ticks are ectoparasites and not all bites result in disease, the preferred term in this context is "infestation", or more specifically, "tick infestation" or "tick bite exposure."
Suggested revisions:

• Abstract: “...voluntary case reports of tick infestation in humans...”
• Introduction: “...documented and analysed a series of human and animal tick infestations...”
• Replace “parasitosis” throughout the manuscript unless a pathological condition is explicitly described.

2. Final sentence of the introduction

The last sentence of the introduction inappropriately presents results and lacks clear study rationale.
Current version:

“Through voluntary case reports and tick collections from animals... this study documented and analyzed... The findings showed...”

Suggested revision:

“This study aimed to document human tick infestations and compare them with tick collections from domestic and wild animals in the flooded savanna region of Arauca, Colombia. Our findings highlight the predominance of Amblyomma mixtum in this ecosystem and provide the first description of associated clinical skin manifestations in humans in this region.”

This version improves clarity, avoids premature result presentation, and better aligns with the study's goals.

3. Clarify technical terms

• Clearly define “classical and molecular taxonomy” for readers who are not entomologists or molecular biologists.
• Ensure tick identification processes are transparent and accessible to a broad readership.

4. Study area context

• Provide more detail on the flooded savanna environment and explain the rationale for its selection.
• Clarify whether the area is flooded year-round or seasonally, and how the timing of sampling relates to these conditions.
• For example: "During the rainy season (typically April to November), rivers such as the Meta, Arauca, and Guaviare flood surrounding regions."

Methodological clarifications

5. Tick Reporting by Humans

• Clarify how human participants reported infestations:
• Was there a prior awareness campaign?
• How did participants contact the research team (e.g., phone, field visits)?
• Specify who collected the ticks from participants, especially since some ticks were located in private body areas.

6. Sample size and attachment site inference

• The total number of 22 ticks from 6 individuals is too small to generalize life stage–body site associations.
• Rephrase statements in lines 149 and 153 to simply describe the distribution observed, rather than suggest a pattern.

 

7. Clinical monitoring

• Indicate exactly which individuals (from Table 1) were monitored clinically.
• Link the images shown in Figure 5 with the individuals listed in Table 1 for transparency.

8. Figure 4b label

• The label on the horizontal axis is misleading. Suggested change:
• Replace “Number of people infested by Amblyomma mixtum” with “Individuals infested by Amblyomma mixtum”.

 

9. Table 1 terminology

• “Wild pork” is unclear. Please confirm if this refers to wild boar or collared peccary (Dicotyles tajacu), and use the correct common name.

10. Sequence information

• Confirm that sequence accession numbers are deposited in GenBank and properly cited in the results section.

Discussion and conclusion

11. Unsubstantiated claim

• The statement in lines 206–207:

“...the tick collected from the patient was perhaps not infected by pathogens such as rickettsiae...”

• This is speculative and unjustified without diagnostic confirmation. It should be removed or rephrased as a hypothesis requiring further study.

12. Seasonality context missing

• Add a discussion of seasonality, especially in lines 213–217, where environmental risk is mentioned. When was the data collected, and how might seasonal flooding affect tick distribution?

13. Study limitations

• Explicitly state the small sample size as a limitation and clarify that findings should be interpreted cautiously.

14. Wording in conclusion

• Suggested revision:

“Our study documents cutaneous clinical manifestations associated with Amblyomma mixtum infestations in humans in the flooded savanna region and in Colombia generally.”

• Avoid “caused by” due to sample size limitations and absence of diagnostic confirmation.

Recommendation

Minor Revision
The manuscript is valuable and scientifically sound, but it requires clarifications, careful wording, and some methodological elaboration to strengthen its impact and reproducibility.

Author Response

Dear

Reviewer

Wild

 

 

On behalf of the authors, we sincerely appreciate the time devoted to reviewing the manuscript, as well as the valuable comments and recommendations provided to improve it. All alterations are marked (word track changes).

With best regards

Sincerely

Arlex Rodríguez Durán

 

Q1

Comments:

This manuscript presents novel and important observations of Amblyomma mixtum tick infestations in humans in the flooded savanna region of Colombia. The integration of classical and molecular taxonomy, coupled with clinical monitoring and ecological observations, contributes to the growing awareness of ticks as a One Health concern. The work is timely and relevant. However, several areas require clarification and revision to improve scientific rigor, reproducibility, and interpretation.

 

Answer: Thank you for your comments. The reviewer's clarifications and revisions are described below.

 

Q2

Comments:

Terminology – use of "Parasitosis"

The term "parasitosis" is used throughout the manuscript, including in the title, abstract, and introduction. This term typically refers to a pathological condition caused by parasitic organisms, often internal parasites like helminths or protozoa. Since ticks are ectoparasites and not all bites result in disease, the preferred term in this context is "infestation", or more specifically, "tick infestation" or "tick bite exposure."

 

Abstract: “...voluntary case reports of tick infestation in humans...”

Introduction: “...documented and analysed a series of human and animal tick infestations...”

Replace “parasitosis” throughout the manuscript unless a pathological condition is explicitly described.

 

Answer: Thanks for the clarification. The word "parasitosis" has been changed to "infestation".

 

Q3

Comments:

Final sentence of the introduction

The last sentence of the introduction inappropriately presents results and lacks clear study rationale.

Current version:

“Through voluntary case reports and tick collections from animals... this study documented and analyzed... The findings showed...”

Suggested revision:

“This study aimed to document human tick infestations and compare them with tick collections from domestic and wild animals in the flooded savanna region of Arauca, Colombia. Our findings highlight the predominance of Amblyomma mixtum in this ecosystem and provide the first description of associated clinical skin manifestations in humans in this region.”

This version improves clarity, avoids premature result presentation, and better aligns with the study's goals.

 

Answer: Thanks for the comment. The suggestion was made as the last paragraph of the introduction.

 

This study aimed to document tick infestations in humans, domestic animals, and wild animals in the flooded savanna region of the Arauca department, Colombia. Our findings highlight the predominance of Amblyomma mixtum in this ecosystem and provide the first description of associated cutaneous clinical manifestations in humans from this region.

 

Q4

Comments:

Clarify technical terms

Clearly define “classical and molecular taxonomy” for readers who are not entomologists or molecular biologists.

Ensure tick identification processes are transparent and accessible to a broad readership.

 

Answer: Thank you for the comment. A definition has been provided for each type of technique used to identify ticks:

 

2.3.1. Classical Taxonomic Identification

The external morphological characteristics of the collected ticks were examined using an SZ61 binocular stereomicroscope (Olympus, USA) at magnifications of up to 100x, following the taxonomic keys and morphological descriptions provided by Strickland et al. [32], Barros-Battesti et al. [33, 34], Kleinjan and Lane [35], Estrada-Peña [36], and Nava et al. [37].

 

2.3.2. Molecular Taxonomy: DNA Extraction

Genetic analysis was used to classify tick species. DNA was extracted from 12 ticks obtained from animals (n = 2), plants (n = 2), and humans (n = 8). For the extraction of nucleic acids, the reagent TRIzol® LS (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, US) was used, based on volumes and procedural steps indicated in the manufacturer's protocol.

 

Q5

Comments:

Study area context

Provide more detail on the flooded savanna environment and explain the rationale for its selection.

Clarify whether the area is flooded year-round or seasonally, and how the timing of sampling relates to these conditions.

For example: "During the rainy season (typically April to November), rivers such as the Meta, Arauca, and Guaviare flood surrounding regions."

 

Answer: Thank you for your comment. The item was modified taking into account the evaluator's recommendations:

 

2.1. Study Area

For this study, the municipality of Arauca, in the department of Arauca, Colombia, was selected because previous studies had reported tick infestations in animals, especially farm animals, suggesting the possibility of also recording infestations in humans by this arthropod [25-27]. Arauca is located in the lowlands of the western Orinoco River basin, Colombia [28]. The region's topography is predominantly flat, with open areas and wetlands covered with vegetation during the rainy season [29]. The climate is characterized by two distinct seasons: a dry season (typically from December to March) and a rainy season (from April to November), with an average annual rainfall of 1,477 mm, relative humidity of 90%, an elevation of 120 meters above sea level, and a temperature range reaching up to 30.9 °C [30].

 

25. Sánchez-Montes, S.; Ríos-Muñoz, C.; Espinosa-Martínez, D.; Guzmán-Cornejo, C.; Berzunza-Cruz, M.; Becker, I. First report of "Candidatus Rickettsia amblyommii" in west coast of Mexico. Ticks Tick Borne Dis 2016, 7(6), 1139-1145. https://doi.org/10.1016/j.ttbdis.2016.08.007

 

26. Rivera-Páez, F.; Labruna, M.; Martins, T.; Perez, J.; Castaño-Villa, G.; Ossa-López, P.; Camargo-Mathias, M. Contributions to the knowledge of hard ticks (Acari: Ixodidae) in Colombia. Ticks and tick-borne diseases 2018, 9(1), 57-66. https://doi.org/10.1016/j.ttbdis.2017.10.008

 

27. Rodríguez-Durán, A.; Chaparro-Gutiérrez, J.; Cortés-Vecino, J. Factors associated with tick infestation in cattle in the department of Arauca, Colombia. Rev Colomb Cienc Pecu 2024, 37(Suppl), 150-151.

 

28. Beck, H.; Zimmermann, N.; McVicar, T.; Vergopolan, N.; Berg, A.; Wood, E. Present and Future Köppen-Geiger Climate Classification Maps at 1-km Resolution. Sci. Data 2020, 7(1), 274. https://doi.org/10.1038/s41597-020-00616-w

 

29. Pinzón, C.; Rangel-Ch, J.; Minorta-Cely, O.; Aymard, G. Plant richness and diversity of flowering plants in the wetlands and floodplain savannas of the Arauca Department, Colombia. BioLlania 2017, 15, 470-532.

 

30. Rangel-Ch, J.; Gopar-Merino, L.; Minorta-Cely, V. Caracterización Climática de las Sabanas Inundables y los Humedales de Arauca, Colombia. BioLlania 2017, 15, 357-409.

 

Q6

Comments:

Tick Reporting by Humans

Clarify how human participants reported infestations:

Was there a prior awareness campaign?

 

Answer: Thank you for your comment. During tick collection, the owners of the animals studied stated that they had been infested or were infested at the time of the visit.

 

Q7

Comments:

Tick Reporting by Humans

How did participants contact the research team (e.g., phone, field visits)?

 

Answer: Thank you for the comment. It has been reviewed, clarifying the type of communication and monitoring of infested individuals:

 

2.4. Monitoring of Clinical Manifestations in Humans

Individual monitoring was conducted via telephone and field visits for up to five days to detect possible clinical manifestations related to tick bites, such as (i) skin lesions: red spots, swelling and itching at the bite site.

 

Q8

Comments:

Tick Reporting by Humans

Specify who collected the ticks from participants, especially since some ticks were located in private body areas.

 

Answer: Thank you for the comment. Sample collection was carried out by the author of this article, Diana Peña-Navarro, a physician by profession (see Figure 1. Professional diploma) with experience in tick research. She was responsible for collecting specimens from humans and analyzing the associated cutaneous clinical manifestations.

 

 

Figure 1. Professional title in Medicine from the author Diana Peña-Navarro.

 

Q9

Comments:

Sample size and attachment site inference

The total number of 22 ticks from 6 individuals is too small to generalize life stage–body site associations.

Rephrase statements in lines 149 and 153 to simply describe the distribution observed, rather than suggest a pattern.

 

Answer: Thanks for the comment. The sentence has been modified, taking into account your recommendation:

 

The anatomical distribution of A. mixtum attachments in humans varied, with ticks attaching to different regions of the body. For example, larvae were most frequently observed on the lower extremities of infested individuals (Figure 4), while nymphs were primarily found in the central body region. Adult ticks are predominantly attached to the torso and upper extremities (Table 1).

 

Q10

Comments:

Clinical monitoring

Indicate exactly which individuals (from Table 1) were monitored clinically.

Link the images shown in Figure 5 with the individuals listed in Table 1 for transparency.

 

Answer: Thank you for your comment. Table 1, in the "Monitoring time (days)" column, indicates the number of individuals who were monitored. In Figure 5, the location of the individuals has been added to the legend:

 

Figure 5. Anatomical distribution of ticks and tick bite–associated skin lesions on different body parts of affected individuals from the flooded savanna of the department of Arauca, Colombia. a. Infestation by an adult Amblyomma mixtum on the left ankle (Geographic location: Caño Colorado), b. Infestation by an adult A. mixtum on the right ankle (Geographic location: DNMI Cinaruco 1), c. Cutaneous manifestations (primary lesions) caused by the bite of an adult A. mixtum on the left ankle (Geographic location: Caño Colorado), d. Infestation of several larvae of A. mixtum on the left foot (Geographic location: Saya), e. Infestation of an adult A. mixtum on the thoracic dorsum (Geographic location: DNMI Cinaruco 2), f. Infestation by nymphs of A. mixtum in the lumbar region (Geographic location: Arrecifes), and g. Infestation by A. mixtum nymphs on the left arm (Geographic location: DNMI Cinaruco 2). Arrow indicates ticks and erythema. Circle indicates skin alterations following A. mixtum infestation.

 

Q11

Comments:

Figure 4b label

The label on the horizontal axis is misleading. Suggested change:

Replace “Number of people infested by Amblyomma mixtum” with “Individuals infested by Amblyomma mixtum”.

 

Answer: Thank you for your comment. The legend for Figure 4b has been modified according to the recommendation.

 

Q12

Comments:

Figure 4b label

The label on the horizontal axis is misleading. Suggested change:

Replace “Number of people infested by Amblyomma mixtum” with “Individuals infested by Amblyomma mixtum”.

 

Answer: Thank you for the comment. Collared peccary has been added to Table 1.

 

Q13

Comments:

Sequence information

Confirm that sequence accession numbers are deposited in GenBank and properly cited in the results section.

 

Answer: Thank you for your comment. The scheduled release date for these data is May 15, 2026, or upon publication of the article citing these accession numbers, whichever comes first.

 

Q14

Comments:

Unsubstantiated claim

The statement in lines 206–207:

“...the tick collected from the patient was perhaps not infected by pathogens such as rickettsiae...”

This is speculative and unjustified without diagnostic confirmation. It should be removed or rephrased as a hypothesis requiring further study.

 

Answer: Thanks for the comment. The sentence has been modified, taking into account the reviewer's recommendation:

 

Regarding the secondary manifestation on the skin observed in the second person studied, perhaps the presence of the pustules could be due to a bacterial infection caused by the same person when controlling the itching, having fingers or nails infected by some bacterial species [41]. However, specific diagnostic laboratory studies are required to verify these hypotheses.

 

Q15

Comments:

Seasonality context missing

Add a discussion of seasonality, especially in lines 213–217, where environmental risk is mentioned. When was the data collected, and how might seasonal flooding affect tick distribution?

 

Answer: Thanks for the comment. Changes have been made to the Results and Discussion sections to address the findings related to tick identification across the different climatic seasons.

 

Results

In the non-parasitic phase (from vegetation), 279 ticks were identified, consisting of 13 adults (3 males and 10 females), 9 nymphs, and 257 larvae. Ticks were identified as A. mixtum (38%) and Amblyomma spp. (62%). The highest tick frequency was recorded during the dry season, with 65% of the collected specimens (n = 181), compared to the rainy season, which accounted for 35% of the records (n = 84).

 

Discussion

The homes where the infested individuals lived or worked represent a high-risk factor, as they are located in a region of Colombia whose environmental conditions, such as relative humidity and temperature, can favor the development of A. mixtum [18, 21]. This observation is supported by our findings, which showed the highest frequency of ticks during the dry season. Additionally, the presence of a variety of domestic and wild animals may facilitate the establishment and persistence of this tick species in the region [18, 21, 27].

 

Q16

Comments:

Study limitations

Explicitly state the small sample size as a limitation and clarify that findings should be interpreted cautiously.

 

Answer: Thanks for the comment and recommendation. A clarification has been added to the discussion:

 

Although our results expand and compile information, for the first time, information on A. mixtum infestation in humans and the associated cutaneous clinical manifestations, the sample size was small, with only six individuals analyzed, and clinical monitoring was possible in just two cases. This limitation restricts the analysis to a localized population and reduces the representativeness of the data, which limits a comprehensive understanding of the clinical and epidemiological impact of A. mixtum on humans in this region of Colombia.

 

Q17

Comments:

Wording in conclusion

Suggested revision:

“Our study documents cutaneous clinical manifestations associated with Amblyomma mixtum infestations in humans in the flooded savanna region and in Colombia generally.”

Avoid “caused by” due to sample size limitations and absence of diagnostic confirmation.

 

Answer: Thanks for your comment. The modification has been made:

 

Our study expands and compiles for the first time information on the follow-up of cutaneous clinical manifestations associated by A. mixtum infesting humans in the flooded savanna region and in Colombia in general.

 

Q18

Comments:

The manuscript is valuable and scientifically sound, but it requires clarifications, careful wording, and some methodological elaboration to strengthen its impact and reproducibility.

 

Answer: We thank the reviewer for his various comments and recommendations, as they helped us improve the article.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

 

General assessment

The authors have done a commendable job in addressing the previous reviewer comments. Their revisions have notably improved the clarity and quality of the manuscript. The limitations of the study are now more clearly acknowledged, and the presentation of findings is better structured. However, a few methodological and presentation issues remain and should be clarified to ensure transparency and reproducibility.

Remaining observations and required clarifications

1. Clarity on Human Participant Selection:
• The manuscript still lacks clarity on how infested humans were identified and selected.
• Was tick infestation self-reported, or were individuals examined randomly as part of the study protocol?
• If people were randomly examined, how many individuals in total were screened, and how many were found to be infested?
• The current explanation ("the owners of the animals stated that they had been infested") is vague. Were individuals instructed to self-inspect, or were infestations discovered by the research team?
• These details are essential for understanding sampling bias, study reproducibility, and the potential scale of human infestation.

 

1. Selection of Households/Farms/sample sites:
• The manuscript should explain how farms or households were selected where the infested individuals were found.
• Was a random, convenience, or purposive sampling approach used?
• Clear information on the sampling strategy strengthens the scientific rigor of the methodology.

 

1. Human Tick Collection Procedure:
• The response indicates that a physician collected ticks from humans—this is an important methodological point and should be explicitly stated in the Methods section of the manuscript.

 

1. Introduction – Repetition of Findings:
• The final sentence of the Introduction reintroduces findings:

"Our findings highlight the predominance of A. mixtum in this ecosystem and provide the first description of associated cutaneous clinical manifestations in humans from this region."

• This sentence should be relocated to the Discussion or Conclusion sections. The Introduction should focus solely on background, rationale, and study objectives.

 

1. Tick Collection Section – Clarify Human Infestation Note:
• The phrase "Ticks in larvae, nymphal, or adult stages were collected from accidentally infested individuals..." is too vague. Amblyomma mixtum can and does infest humans, particularly in endemic rural and livestock-exposed settings. So, calling humans “accidentally infested” is not biologically wrong, but the phrase itself is vague from a scientific reporting perspective. Saying ticks were collected from “accidentally infested individuals” raises questions that need clearer answers. Was human infestation expected given the setting, or was it a rare, surprising event? Were the individuals systematically examined, or did they just report being infested? Did tick attachment happen during normal daily activity, livestock handling, or something unusual? Better alternatives might include: “Ticks at all life stages were collected from incidental human hosts encountered during the field investigation.”

 

 

1. Accession Numbers – Evidence for Scheduled Release:
• The manuscript includes the line:

"The scheduled release date for these data is May 15, 2026, or upon publication of the article citing these accession numbers, whichever comes first."

• It is advisable to provide evidence (e.g., GenBank submission confirmation or screenshot) to the editor, confirming this scheduled release for transparency.

Recommendation

Minor revisions required: Following the resolution of these small but important issues, particularly on human sampling methodology and clarity of tick collection procedures, the manuscript will be suitable for acceptance.

 

Author Response

Dear

Reviewer

Wild

 

On behalf of the authors, we sincerely appreciate the time devoted to reviewing the manuscript, as well as the valuable comments and recommendations provided to improve it. All alterations are marked (word track changes).

Clarifying note: The letter and manuscript are added in a single PDF, because the platform did not allow me to add the image of sequence evidence deposited in GenBank within this space of the platform.

With best regards

Sincerely

 

Arlex Rodríguez Durán

 

Author Response File: Author Response.pdf