The role of cofeeding arthropods in the transmission of Rickettsia felis.

Rickettsia felis is an emerging etiological agent of rickettsioses worldwide. The cosmopolitan cat flea (Ctenocephalides felis) is the primary vector of R. felis, but R. felis has also been reported in other species of hematophagous arthropods including ticks and mosquitoes. Canines can serve as a bacteremic host to infect fleas under laboratory conditions, yet isolation of R. felis from the blood of a vertebrate host in nature has not been realized. Cofeeding transmission is an efficient mechanism for transmitting rickettsiae between infected and uninfected fleas; however, the mechanism of transmission among different orders and classes of arthropods is not known. The potential for R. felis transmission between infected fleas and tick (Dermacentor variabilis) and mosquito (Anopheles quadrimaculatus) hosts was examined via cofeeding bioassays. Donor cat fleas infected with R. felis transmitted the agent to naïve D. variabilis nymphs via cofeeding on a rat host. Subsequent transstadial transmission of R. felis from the engorged nymphs to the adult ticks was observed with reduced prevalence in adult ticks. Using an artificial host system, An. quadrimaculatus exposed to a R. felis-infected blood meal acquired rickettsiae and maintained infection over 12 days post-exposure (dpe). Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on rats infection persisting in the mosquito for up to 3 dpe. The results indicate R. felis-infected cat fleas can transmit rickettsiae to both ticks and mosquitoes via cofeeding on a vertebrate host, thus providing a potential avenue for the diversity of R. felis-infected arthropods in nature.

Introduction

Rickettsia felis is an emerging pathogen typically associated with clinical symptoms similar to other rickettsioses including headache, chills, fever, myalgia and rash followed by complications involving neurological systems [1]. The first human infection with R. felis was described nearly three decades ago in the United States [2]; subsequently, case reports have increased throughout Central and South America, sub-Saharan Africa, Europe, and Asia over the last 25 years [3]. The cat flea, Ctenocephalides felis, functions as a primary reservoir host and vector, transmitting the agent both vertically and horizontally [4]. Transovarial transmission directly from female to progeny has been reported in laboratory-reared R. felis-infected cat flea colonies but with high variability in transmission rates, suggesting that vertical transmission alone is likely inadequate to maintain R. felis within flea populations [5-7]. Thus, horizontal transmission of R. felis from infected to naïve fleas via cofeeding was determined to be sufficient to perpetuate the agent within populations of cat fleas [8,9]. Given the success of cofeeding as a means of flea-to-flea transmission, it is plausible that cofeeding may also be an effective transmission strategy between infected fleas and naïve non-flea, hematophagous arthropods.

Nucleic acid from R. felis and related R. felis-like organisms (RFLOs) has been detected in other hematophagous arthropods, including more than 40 species of fleas, ticks, and mosquitoes [4,10]. Field surveys have identified R. felis in wild-caught ticks [11-13] and laboratory studies have demonstrated R. felis was vertically transmitted by the American dog tick, Dermacentor variabilis, when adult ticks were exposed via capillary feeding [14]. More recently, in sub-Saharan African countries where R. felis has been reported as an emerging cause of unknown fever, R. felis has been detected in mosquitoes [3,15,16]. Detection of R. felis in male mosquitoes in China and unfed adult mosquitoes in the United States suggests vertical transmission in nature [17,18]. Furthermore, horizontal transmission of R. felis by Anopheles gambiae was reported using a laboratory model of infection [19]. Thus, while field and laboratory studies support the transmission potential of R. felis by alternate vectors, conduits for introduction of the agent into arthropod populations remain undefined.

Cat fleas have a broad host range, infesting feral hosts as well as commonly being identified on companion animals [20]. Within domestic or peri-domestic environments, vertebrate hosts are exposed to fleas, mosquitoes, and ticks. Thus, concurrent feeding by infected fleas and other arthropods on the same host could contribute to transmission of pathogens. Indeed, transmission of R. felis from infected cat fleas to naïve rat fleas (Xenopsylla cheopis) cofeeding on vertebrate hosts was demonstrated [9]; however, potential for flea transmission of R. felis to other orders and classes of arthropods is not known. To test the hypothesis that R. felis-infected cat fleas may contribute to dissemination of the agent to other hematophagous vectors, cofeeding bioassays were employed to examine rickettsial transmission from infected cat fleas (C. felis) to ticks (D. variabilis) and mosquitoes (Anopheles quadrimaculatus) on a vertebrate host. The data suggest R. felis-infected cat fleas transmit rickettsiae to other hematophagous vectors via cofeeding on a vertebrate host.

Materials and methods

Ethics statement

All animals use in this research was performed under the approval of the Louisiana State University (LSU) Division of Laboratory and Animal Medicine (DLAM) with LSU IACUC 17–115.

Rickettsial propagation

Rickettsia felis str. LSU, originally isolated from cat fleas, was propagated in the Ixodes scapularis-derived embryonic cell line (ISE6), which was maintained in modified L15B growth medium as previously described [21]. Rickettsiae were semi-purified from ISE6 cells and enumerated by the LIVE/DEAD BacLight viability stain kit (Invitrogen, Eugene, OR), as described previously [22]. To generate infected bloodmeals, concentrations of R. felis (passage 3) were adjusted to 3 × 1010 rickettsiae in 700 μl (cat fleas) or 2 ml (mosquitoes) of heat-inactivated (HI) bovine blood.

Fleas, ticks, and mosquitoes

Newly emerged cat fleas were obtained from Elward II (Soquel, CA) and maintained on defibrinated bovine blood (HemoStat Laboratories, Dixon, CA) within an artificial dog feeding unit [23]. Prior to use in bioassays, genomic DNA (gDNA) was extracted from a portion of fleas and confirmed Rickettsia-negative by quantitative real-time PCR (qPCR) [24,25]. In order to generate R. felis-infected donor cat fleas, newly emerged cat fleas were pre-fed HI bovine blood for 24 hrs, starved for 5–6 hrs, then exposed to the R. felis-infected bloodmeal (3 × 1010 rickettsiae/700 μl) for 24 hrs [8]. Rickettsia-exposed cat fleas were fed on an uninfected bloodmeal for 48 hrs, and 20 fleas of random sex were collected to assess R. felis prevalence in the donor population by qPCR. For cofeeding bioassays, cat fleas treated in the same manner, except being exposed to a bacteria-free bloodmeal for the 24 hrs exposure, served as the control, uninfected ‘donor’ fleas.

Rickettsia-free D. variabilis (originally provided by Dr. Daniel Sonenshine) were maintained in a controlled environmental chamber at 27°C, with 92% relative humidity, and a 12:12 (light:dark) cycle [26,27].

Anopheles quadrimaculatus were provided by Dustin Miller (Centers for Disease Control and Prevention for distribution by BEI Resources, NIAID, NIH) and maintained in a controlled environmental chamber at 27°C, with 92% relative humidity, and a 12:12 (light:dark) cycle. Adults were fed with a 10% sucrose solution [28].

Flea to tick cofeeding transmission bioassay

To assess acquisition of R. felis by D. variabilis cofeeding with R. felis-infected cat fleas, 30 nymphal ticks were encapsulated on Sprague Dawley (SD) rats in the top portion of a 50 ml conical tube and allowed to attach and feed as described [14]. After 2 days, unattached ticks were removed from the feeding capsule and R. felis-infected donor cat fleas (10 male/25 female) were placed into the same feeding capsule with D. variabilis experimental group. The control group of ticks were cofed with 20 (10 male/10 female) Rickettsia-free cat fleas. After 24 hrs of cofeeding, 10 nymphal ticks were collected from each rat and subjected to gDNA extraction. Likewise, a portion of the donor cat fleas were subsampled for gDNA extraction. The remaining nymphs were left in the feeding capsule and the remaining viable cat fleas were added back to the capsule daily until tick engorgement (~5 days). After natural detachment, a portion of ticks and cat fleas were used for gDNA extraction, with the remaining ticks being allowed to molt to adult before assessment of rickettsial infection. Each experiment was conducted twice independently.

Mosquito exposure to R. felis by membrane feeding assay

To assess the ability of An. quadrimaculatus to harbor R. felis at various time points after exposure, female mosquitoes were starved for 5 hrs, before being allowed to feed on a glass membrane feeder containing either a R. felis-infected HI bovine blood meal (3 × 1010 rickettsiae/2 ml) or an uninfected blood meal. Mosquitoes fed through a Parafilm membrane and across a glass feeding chamber containing recirculating warm water to keep the blood at 37° C [29]. After the blood meal, engorged female mosquitoes were transferred to a new cage and maintained on a 10% sucrose feeding solution in the chamber. Mosquitoes were sampled and individually processed at 1, 3, 5, 7, 9, and 12 day post-exposure (dpe). Each experiment was conducted twice independently.

Flea to mosquito cofeeding transmission bioassay

To assess acquisition of R. felis by An. quadrimaculatus cofeeding with R. felis-infected cat fleas, R. felis-infected donor cat fleas (10 male/25 female) were encapsulated on SD rats in the top portion of a 50 ml conical tube and allowed to feed for 24 hrs. Subsequently, 10 uninfected mosquitoes per day were placed into the same feeding capsule and allowed to cofeed with fleas for 20 mins once per day for 3 days. Fed female mosquitoes were collected and transferred to new cage and provided access to a cotton ball soaked in 10% sucrose for 24 hrs before being assessed for rickettsial infection. Ten female R. felis-infected donor cat fleas were collected daily for immediate gDNA extraction. On separate vertebrate hosts, groups of Rickettsia-free donor fleas were cofed with mosquitoes in the same manner and served as the negative infection control. Each experiment was conducted twice independently.

Sample preparation and rickettsial quantification by PCR

All flea, tick, and mosquito samples were washed with 10% bleach for 5 mins, 70% ethanol for 5 mins, and three times with sterile distilled H2O for 5 mins to remove environmental DNA. Each sample was transferred to a 1.7 ml microcentrifuge tube and ground with sterile plastic pestles in liquid nitrogen. Extraction of gDNA was performed using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD) following the manufacturer’s instructions with a final elution in 35 μl of UltraPure DNAse/RNAse free distilled H2O (Invitrogen, Grand Island, NY). A negative environmental control, a 1.7 ml microcentrifuge tube containing buffer(s) but no sample, was included in each DNA extraction procedure as a negative environmental control for qPCR.

Assessment of rickettsial infection by qPCR was performed with a LightCycler 480 Real-Time PCR system (Roche, Indianapolis, IN). The plasmid pCR4-TOPO-RfelB was used as a standard template to create serial 10-fold dilutions and matched with RfelB primers and probe as previously described [24,25]. For An. quadrimaculatus gene expression measurements, mitochondrial DNA (mtDNA) and ribosomal protein S7 gene were used as reference gene [30,31].

Statistical analyses

All statistical analyses were performed in R Studio (version 1.3.1093) with base R (version 3.6.3). To compare concentration of R. felis in each arthropod, the concentration of each sample from three technical replicates was averaged. Two experimental trials (biological replicates) were conducted and data were aggregated over both trials after determination of no significant difference between trials. The rickettsial load was compared using the Kruskal-Wallis non-parametric analysis of variance (kruskal.test). When warranted, post-hoc test for pairwise differences was performed using the Dunn Test (dunnTest, package FSA). To test for differences in proportions of infected arthropods, the prop.test function was performed, where appropriate. In all analyses, statistical significance was assessed at the 95% confidence level.

Results

Transmission of R. felis from fleas to cofeeding ticks

To determine the potential for R. felis transmission from infected cat fleas to other arthropods, we first examined cofeeding transmission from cat fleas to D. variabilis ticks on a vertebrate host, SD rats. Infection status of the donor cat fleas was assessed following the experimental infection and determined to be 100% (23/23) infected with R. felis. Rickettsia-free nymphal D. variabilis actively feeding on a vertebrate host were cofed with either R. felis-infected cat fleas or uninfected cat fleas as a control and transmission was assessed in individual ticks that cofed for 24 hrs, at tick engorgement (4–5 days), and after molting to the adult stage. R. felis was detected in 75% (15/20) D. variabilis nymphs that were forcibly removed from the host 24 hrs after cofeeding. Comparably, 81.2% (13/16) of 4–5 days fed, engorged ticks were positive for R. felis; and, after molting, 56.2% (9/16) of adult ticks were positive. None of the ticks that cofed with uninfected fleas were positive for R. felis (). While a reduction in the overall proportion of adult ticks that were R. felis-positive compared to nymphal stages was observed, there were not statistically significant differences in the proportions of infected ticks among cofeeding duration and life cycle stages of exposed ticks. Further, there was no statistical difference in the rickettsial load from ticks among life cycle stages. These observations demonstrate that transmission of R. felis from infected cat fleas to immature ticks via cofeeding allows for a mechanism to generate R. felis-infected adult ticks.

Susceptibility of mosquitoes to R. felis infection

To assess the susceptibility of An. quadrimaculatus to acquire R. felis from oral exposure, individual mosquitoes were offered an R. felis-infected bloodmeal and infection was assessed at 1, 3, 5, 7, 9, and 12 dpe. At each time point, 100% of blood fed mosquitoes had detectable R. felis in whole body samples, compared to 0% of the control (mock-exposed) individuals. Due to the lack of variance in either group, statistical analysis could not be performed. Mosquitoes tested at day 1 post-exposure had the highest median rickettsial load (5.54 x 105) per mosquito, which decreased significantly by day 3 post-exposure (3.57 x 102). The significant changes when comparing rickettsial loads 1 dpe to the later time points is likely an indication that the R. felis detected was remnants of the bolus in the offered blood meal. After day 3, the rickettsial load in mosquitoes increased at days 5 (2.21 x 104), 7 (2.36 x 104), 9 (4.04 x 104), and 12 (1.44 x 104) post-exposure (Fig 1). Days 5–12 were not statistically significant from one another, while day 12 was not statistically different from any time points, likely due to the small sample size at that day (n = 7).

Fig 1

Concentration (log10) of rickettsiae in individual whole mosquitoes per time point after exposure to an R. felis-infected bloodmeal.

Mosquitoes ingested R. felis and maintained infection over the course of 12 days. Lines represent the median and letters indicate significance grouping according to Dunn’s post-hoc test.

Transmission of R. felis from fleas to cofeeding mosquitoes

Naïve An. quadrimaculatus were able to acquire infection through cofeeding with R. felis-infected cat fleas. Donor R. felis-infected cat fleas were assessed for infection on the same days of cofeeding as An. quadrimaculatus. At days 1, 2, and 3 post-cofeeding, 60% (12/20), 65% (13/20), and 70% (14/20), respectively, of cat fleas were positive for R. felis. There was no significant difference in the proportions of R. felis-infected cat fleas in cofed populations among the time points assessed. Of the cofeeding An. quadrimaculatus, 31.6% (6/19), 40% (8/20), and 60% (12/20) were R. felis-positive on days 1, 2, and 3 post-exposure, respectively. Similar to cat fleas, no statistical difference was observed in the proportion of R. felis-infected mosquitoes among the time points assessed. Comparing the proportions of infected between the two feeding arthropod populations, no significance difference was identified between the two vectors on any of the time points assessed. Combined, the data indicate that An. quadrimaculatus are susceptible to rickettsial infection via cofeeding with infected cat fleas.

Rickettsial load was compared among arthropod species and time points and no statistically significant difference across time for either mosquitoes or cat fleas was observed (). However, when comparing cat fleas to mosquitoes at each day of cofeeding, donor cat fleas exposed to R. felis prior to cofeeding consistently had significantly higher rickettsial loads than the mosquitoes with which they were cofed ().

Concentration (log10) of rickettsiae from individual mosquitoes (black circles) who were infected via cofeeding with cat fleas (blue circles) at 1, 2, or 3 days of cofeeding (lines represent medians).

There were significant differences in rickettsial load between the two arthropods (An = mosquitoes and Ct = cat fleas) at each timepoint, assessed by the Kruskal-Wallis test.

Discussion

The biology of R. felis is associated with varying degrees of disease, a distinct transmission cycle within the flea, and identification in a number of arthropods [1]. First coupled with human infection in the United States, R. felis and related RFLOs including Rickettsia asembonensis and Candidatus Rickettsia senegalensis have been associated with human disease worldwide [3]. Reported clinical signs in humans range from non-distinct flu-like symptoms to ulceration and more complex manifestations including neurological involvement [32-34]. Placed in the transitional group of Rickettsia, R. felis contains genetic and biological characteristics of both tick-associated spotted fever group (SFG) and insect-associated typhus group Rickettsia [35]. As obligate intracellular bacteria, arthropods often serve as both reservoirs and vectors of Rickettsia [36]. The most well-studied vector for R. felis is the common cat flea, which supports both vertical and horizontal transmission mechanisms [1]. In addition to cat fleas, R. felis has been described in a number of arthropods [4,37]. Furthermore, molecular detection of R. felis and RFLOs in field collected ticks and mosquitoes suggests that additional arthropod hosts, and possible transmission cycles, exist [11-15,17-19,38]. However, the route by which R. felis disseminates to infect such a large range of arthropod hosts is unknown.

Cofeeding transmission occurs when a pathogen is transmitted between two arthropods feeding in proximity on a vertebrate host, negating the intrinsic incubation period required for some pathogens during transmission events on a vertebrate host [39]. The cofeeding mechanism has been described for both flea- and tick- borne rickettsial pathogens, and occurs in the absence of a rickettsemic host [9,40]. Fleas deposit rickettsiae in the dermis at the vector-host skin interface resulting in dissemination across the skin [9], presumably where other arthropods, including ticks that have mouthparts in the same tissue, can acquire the agent during feeding events; thus, the transmission of R. felis from infected fleas to naïve ticks would be expected. In contrast, mosquitoes are capillary feeders and the mechanism by which rickettsiae transit between the dermis and associated capillaries targeted by mosquitoes remains to be examined. Although untested, the diversity of clinical symptoms associated with R. felis-rickettsioses, including skin presentation as well as systemic infection, may be related to the arthropod feeding duration (slow versus fast) or route of arthropod delivery (intradermal versus direct inoculation into the bloodstream). Further studies are needed to examine the potential for arthropod-dependent influence on rickettsial dissemination, elucidation of the host response to infection, and subsequent pathology in the vertebrate host.

The expansive distribution of R. felis is related to the cosmopolitan nature of cat fleas. There are several pieces of evidence supporting the potential for horizontal transmission of R. felis to vertebrate hosts, such as identification of R. felis in the salivary glands of cat fleas [41] and detection of R. felis in the blood of vertebrate hosts exposed to R. felis-infected fleas [5,42-44]. More recently, domestic canines were determined to be susceptible to R. felis infection and may serve as an infectious source of R. felis to arthropods other than fleas [45]. However, the mechanisms driving transmission of R. felis by arthropod vectors to vertebrate hosts or transmission between arthropod hosts are only in the initial stages of characterization. Intra- and inter-specific transmission of R. felis between cofeeding R. felis-infected cat fleas and naïve cat fleas and rat fleas via flea bite on vertebrate hosts has been demonstrated [9]. Similarly, ticks are known to transmit SFG Rickettsia via cofeeding [40]. The current study demonstrates cofeeding transmission by R. felis-infected donor fleas to naïve ticks. After only 24 hrs of cofeeding, uninfected recipient nymphal D. variabilis acquired R. felis with subsequent transstadial transmission to the adult stage. Under laboratory conditions, adult D. variabilis are susceptible to infection and subsequent transovarial transmission of R. felis for at least one generation [14]. However, the transovarial maintenance of R. felis infection in F1 larvae was not assessed in the current study. Although not tested in the current study, the tick lifecycle stage and rickettsial load after exposure may contribute to the efficiency of transovarial transmission. As both cat fleas and ticks can be recovered from an individual vertebrate host, further studies are needed to assess the role of horizontal acquisition and vertical transmission routes in dissemination of R. felis in tick populations.

Mosquitoes are important vectors of pathogens that cause disease in humans and animals, but little is known about rickettsiae infection and transmission in mosquitoes. In the current study, R. felis acquired by An. quadrimaculatus can be maintained, although at a reduced rickettsial load, within the mosquito host for up to 12 days. Twelve days is comparable to the time required for productive virus infection in mosquitoes after initial exposure [46,47]. Further study is required to examine the dissemination of R. felis to mosquito legs and saliva as an estimate of transmission potential. Laboratory and field studies postulate that mosquitoes may serve as vectors of R. felis. Indeed, there is evidence of transmission potential for Anopheles mosquitoes which in the laboratory are able to induce transient rickettsemia in mice [19]. Additionally, the detection of rickettsial DNA in field collected male and unfed females Anopheles mosquitoes suggests that vertical transmission may occur [17,18]. The detection of R. felis in molecular surveys of mosquitoes do not address the origin of rickettsial infection. Thus, the current study examined the potential for mosquitoes to acquire R. felis via cofeeding. Transmission of R. felis from infected cat fleas to naïve mosquitoes occurred after only 20 mins of cofeeding and acquisition efficiency increased corresponding to the duration infected fleas were fed on the vertebrate host. Additional studies, similar to ones demonstrating successive transmission from R. felis-infected fleas to uninfected fleas [9], are required to further characterize the role of mosquitoes in the ecology of R. felis distribution.

In summary, the current study demonstrated that R. felis-infected donor cat fleas can transmit the agent to naïve vectors via cofeeding on vertebrate hosts. The acquisition of rickettsiae by D. variabilis and An. quadrimaculatus suggests a new model for inter-class and inter-order transmission of R. felis possibly contributing to the wide-spread identification of this Rickettsia species in hematophagous arthropods. The arthropod species examined in this study were selected based on laboratory studies and species availability. Consistent with laboratory and field studies [17-19], the current results support the assertion that mosquitoes may have an active role in the ecoepidemiology of R. felis rickettsioses. The susceptibility of other species of ticks and mosquitoes is not known, but the diversity of arthropods associated with R. felis suggests widespread susceptibility of other species to R. felis. Additional studies are needed to elucidate the vector competency of arthropods other than fleas for R. felis, as defining potential alternative transmission mechanisms for rickettsial pathogens is necessary for designing sufficient intervention strategies to control the incidence of disease.

24 May 2022

Dear Dr. Macaluso,

Thank you very much for submitting your manuscript "The role of cofeeding arthropods in the transmission of an emerging rickettsial pathogen" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The method section was well written and described the methods used well.

Reviewer #2: I have read through the manuscript with these questions in mind. The objectives are clearly articulated and the study design was appropriate. The sample size and number of replicated experiments were appropriate to test the hypothesis and the statistical analyses were correct. I am fine with the overall design of the study.

Reviewer #3: I am happy with the well-described methodology in this article, Please refer to General Comments to the Authors.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are clearly and completely presented including a table and figures.

Reviewer #2: The results from the study were clearly presented with the overall objectives in mind and the tables and figures were clear.

Reviewer #3: The results in this work are clearly presented, and I am happy with the quality of the Table and Figures in this manuscript.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are supported well be the data presented. In addition the authors discuss how the data presented can be helpful to advance our understanding of R. felis transmission.

Reviewer #2: The conclusions were supported by the data and the limitations were described. While the authors could work on clarifying the discussion in one of the main paragraphs, overall, this study clearly advances our understanding of the topic.

Reviewer #3: The conclusions of this manuscript are well supported by the performed assays. The work is highly related to the public health.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Title:

Would add to or replace emerging rickettsial pathogen the name of the pathogen Rickettsia felis.

Abstract:

Lines 44 and 48: Would suggest that you include the name of the vertebrate host e.g. rat.

Author summary:

Introduction:

Line 70: remove the word “by”.

Line 78: suggest removing the word “vectors” and making the last word plural, i.e. arthropods.

Line 80: suggest replacing “vectors” with arthropods.

Materials and Methods:

Line 106: scapularis in italics i.e. scapularis-derived

Line 116: suggest defining qPCR as quantitative real-time PCR

Line 138: what was the portion of the donor cat fleas e.g. 10 or 10%?

Line 140: a portion, maybe this is written in the results?

Results:

Line 195: add Sprague Dawley or SD rat after vertebrate host.

Line 200: add number of positive adult ticks/number evaluated

Table 1:

Define ND

Discussion:

Line 252: Rickettsia senegalensis should be written as Candidatus Rickettsia senegalensis.

Reviewer #2: Line 43: transmit should be past-tense – transmitted

Line 45: There is no comma needed between observed and with.

Line 48: No comma needed between host and with

Line 53: The way this is written it seems to say that R felis is the common cat flea - take out the comma after felis

Line 60: Can take out 'other arthropods including' as ADT and mosquitoes were the only ones tested

Line 65: ‘with’ normally does not follow a semi-colon (;) so you can remove it. Might be better to phrase it: 'rash followed by complications involving neurological systems (1).'

Line 68: Better to change the tense: ‘have been increasing’ to ‘have increased’

Line 106: scapularis needs to be italicized

Lines 193-209: The data here is good but I struggled while I read with the question: how do they differentiate between live R. felis and DNA from dead R. felis in D variabilis nymphs? I realize the qPCR measures that - but I feel it could be stated more clearly in this section of the results so the reader isn't wondering what kind of DNA is being detected. For example, this aspect was taken care of in the mosquito section by presenting the linear changes in rickettsial concentration over the different time points.

Line 292: No need for semi-colon between ‘felis’ and ‘with’

Lines 301-321: There are several instances in this paragraph where there is a need to clarify ideas. Please re-work the whole paragraph so the ideas read more smoothly.

Lines 302-305: There are quite a few comma phrases here in this four line sentence. I would suggest making two sentences out of it so the ideas flow better.

Lines 308: no comma needed after mosquitoes. Could even change it to ‘which, in the lab, are able...’

Lines 309-311: sentence is a bit awkward - please smooth - two very different thoughts connected by an 'and'

Lines 311-315: Again, these are confusing sentences. Please work to smooth out the storyline as ideas feel like they're jumping around.

Lines 326-329: Key to highlight here is the need for transmission studies. I realize it is mentioned in the last sentence but it needs to be brought out more because only one aspect of competency has been demonstrated here (acquisition by the non-flea host) – transmission by the non-flea host still needs to be demonstrated.

Reviewer #3: I do not have advice to modify the existing data in this manuscript.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Overall the manuscript is well and clearly written. The introduction of R. felis transmission by co-feeding with potential vectors/hosts will initiate further investigations to clarify the diversity and breath of vectors of this unusual rickettsial pathogen.

Reviewer #2: The nidality of arthropod-borne rickettsial disease systems are fascinating and, at times, can boggle the mind. The Rickettsia felis system is particularly interesting with publications constantly popping up, reporting its presence in all kinds of arthropods. This study takes it one step further and moves into the question ‘how does this rickettsial pathogen get around so freely?’ I really enjoyed reading this paper and commend the research team for thinking through the feeding trials in such a way that produced some pretty clear results. In summary, with some minor revisions and clarifications, I feel this paper will be ready - and will help to fill in some gaps in our knowledge regarding how R. felis moves between different hosts.

Reviewer #3: The project in this manuscript used several bioassays to explore the role of cofeeding arthropods in the transmission of Rickettsia felis, an important emerging rickettsial pathogen. This reviewer enjoyed reading this manuscript which is well-written. The methods related to this project are described in the details, and the conclusion of this manuscript is well supported by the performed assays. The authors are encouraged to address the below comments:

The authors may consider including the keyword- the organism (Rickettsia felis) as part of the manuscript title.

R. felis has been identified in multiple vectors, including different species of ticks and mosquitoes. The authors shall provide the rationale regarding the choice of the tick and mosquito species used in this project. In addition, please discuss if the conclusions drawn by the use of the tick and mosquito species in this work will apply well to other tick species and mosquito species.

The starting copy number of R. felis is very high ((3 × 10~10 rickettsiae/700 μl). Then, the detected rickessial copies in ticks and mosquitoes are much lower, particularly in mosquitoes. Could the R. felis positivity in mosquitoes be due to the carry-over of R. felis from the original organism? The qPCR, not the culture, was used to determine the copy number of R. felis, not the viability of the pathogen.

Regarding the transmission of R. felis from fleas to cofeeding mosquitoes, lines 48-50 reads: Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on a vertebrate host, with infection persisting in the mosquito for up to 3 dpe. In this experiment, the R. felis identified in mosquitoes can possibly acquire the organism from cat flea and/or the host? Or, more likely from the host?

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7 Jun 2022

Submitted filename: Response Letter PNTD-D-22-00511_6-7-22 final.docx

Click here for additional data file.

11 Jun 2022

Dear Dr. Macaluso,

We are pleased to inform you that your manuscript 'The role of cofeeding arthropods in the transmission of Rickettsia felis' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Job Lopez

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

21 Jun 2022

Dear Dr. Macaluso,

We are delighted to inform you that your manuscript, "The role of cofeeding arthropods in the transmission of Rickettsia felis," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Table 1

Detection of rickettsiae by qPCR in D. variabilis nymphs cofed with uninfected or R. felis-infected cat fleas for 24 hours on a vertebrate host.

Rickettsial infection was also assessed in ticks after completion of blood meal acquisition (4–5 days) and as newly molted adults.

Life Stage	Cat fleas cofed with	% Positive (n)	Median Log10 Concentration (Range)
Nymph
(24 hours fed)	R. felis-infected	75% (20)	1.72 (ND, 3.66)
	Uninfected	0% (20)	ND
(4–5 days fed)	R. felis-infected	81.2% (16)	1.37 (ND, 3.06)
	Uninfected	0% (10)	ND
Adults (post-molt)
	R. felis-infected	56.2% (16)	1.10 (ND, 2.71)
	Uninfected	0% (10)	ND

ND, not detected