Trends in

Parasitology
Review

Vector biology of the cat flea

Ctenocephalides felis
Charlotte O. Moore , 1 Marcos Rogério André , 2 Jan Šlapeta , 3 and Edward B. Breitschwerdt 1,
*

Ctenocephalides felis, the cat flea, is among the most prevalent and widely dis- Highlights
persed vectors worldwide. Unfortunately, research on C. felis and associated Ctenocephalides felis is among the
pathogens (Bartonella and Rickettsia spp.) lags behind that of other vectors most prevalent vector species and
transmits multiple zoonotic Bartonella
and vector-borne pathogens. Therefore, we aimed to review fundamental as-
and Rickettsia spp.
pects of C. felis as a vector (behavior, epidemiology, phylogenetics, immunology,
and microbiome composition) with an emphasis on key techniques and research Research regarding C. felis and path-
avenues employed in other vector species. Future laboratory C. felis experimen- ogen biology, including the response
to environmental conditions, is nec-
tal infections with Bartonella, Rickettsia, and Wolbachia species/strains should essary to understand disease risk
examine the vector–pathogen interface utilizing contemporary visualization, and epidemiology.
transcriptomic, and gene-editing techniques. Further environmental sampling
will inform the range and prevalence of C. felis and associated pathogens, The diversity of C. felis and associated
bacterial species is high and the impact
improving the accuracy of vector and pathogen modeling to improve infection/ on pathogen transmission and C. felis
infestation risk assessment and diagnostic recommendations. survival is unknown.

Implementing contemporary visualiza-

tion, DNA and RNA sequencing, and ge-
C. felis and associated bacteria nomic/transcriptomic manipulation is
As of 14 November 2023, a PubMed search for the term ‘flea’ returned only 6167 results com- necessary to expand our understanding
pared with ‘tick’ (42 916 results) and ‘mosquito’ (69 994 results). This almost sevenfold disparity of the C. felis–pathogen interaction.
between flea and tick research reflects a lack of funding from the National Institutes of Health (NIH)
dedicated to flea and flea-borne pathogen research (Figure 1), despite fleas infesting more cats
and dogs worldwide and serving as the vector for multiple zoonotic pathogens [1]. C. felis is
the most common flea infesting domestic cats and dogs around the world [2,3], and is well
appreciated as the cause of flea allergy dermatitis (FAD), yet it remains underappreciated as the
vector of multiple zoonotic Bartonella and Rickettsia species [4–7]. Therefore, we aim to review
the behavior, anatomy, immunology, and microbiome of C. felis as well as C. felis and pathogen
epidemiology and phylogenetic diversity to identify future directions in C. felis research, highlight-
ing key findings and techniques utilized in other vector species that may be adapted to C. felis and 1
Intracellular Pathogens Research
associated pathogen research efforts. Laboratory, Department of Clinical
Science, North Carolina State University,
NC, USA
The C. felis life cycle begins with egg laying, followed by larval development, pupation, and finally adult 2
Vector-Borne Bioagents Laboratory
emergence. Adult C. felis of both sexes are hematophagous (see Glossary) and reside on the host. (VBBL), Department of Pathology,
The larval C. felis diet consists primarily of adult flea feces. C. felis is known to transmit bacterial path- Reproduction, and One Health, Faculty
of Agrarian and Veterinary Sciences, São
ogens from two genera: Bartonella and Rickettsia. Three Bartonella spp. are transmitted by C. felis Paulo State University (FCAV/UNESP),
(Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae), all of which utilize the cat as Jaboticabal, SP 14884-900, Brazil
3
a reservoir host [5,6,8]. Horizontal transmission from an infected cat to a naive flea is the only Sydney School of Veterinary Science,
Faculty of Science, The University of
confirmed source of C. felis infection with Bartonella [4]. Medically, B. henselae is the best described Sydney, New South Wales, Australia
of these species, typically causing self-limiting cat scratch disease. Atypical manifestations in humans
and other mammals have been reviewed previously (Figure 2) [9–11]. Stercorarian transmission
via inoculation with flea feces is likely the primary mode of infection for mammals, as transmission
by C. felis saliva or regurgitation during feeding are unconfirmed [12,13]. Laboratory C. felis has *Correspondence:
been successfully colonized by Bartonella birtlesii, Bartonella tribocorum, and Bartonella quintana ebbreits@ncsu.edu (E.B. Breitschwerdt).

324 Trends in Parasitology, April 2024, Vol. 40, No. 4 https://doi.org/10.1016/j.pt.2024.02.006

© 2024 Elsevier Ltd. All rights reserved.
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Figure 1. National Institutes of

Health (NIH) funding awarded to
flea, mosquito, and tick research
from 2013 to 2022. This bar graph
displays the NIH funding awarded to
three vector groups in millions USD (y-
axis). This includes all research tagged
with ‘flea’, ‘mosquito’, and ‘tick’, and is
not discriminated by vector or pathogen
species of interest. Data were retrieved
from the NIH RePORTER database
(https://reporter.nih.gov/) and visualized
in R utilizing ggplot2.
Trends in Parasitology
[13]. Therefore, C. felis may be able to transmit additional Bartonella species, but typically do not due
to the bacteria’s mammalian host specificity.
The Rickettsia typically associated with C. felis are classified within the Rickettsia felis-like organism
(RFLO) group, including R. felis, Rickettsia asembonensis, and ‘Candidatus Rickettsia senegalensis’.
Limited studies and case reports identify R. felis as a cause of acute febrile illness in humans (Figure 2)
[14–16]. Even fewer publications have investigated the potential pathogenicity of other RFLO species
[17]. C. felis and the domestic dog serve as the reservoirs for R. felis [7,18]. C. felis maintains R. felis via
transovarial transmission, while the domestic dog supports long-term rickettsemia in the absence of
hematological abnormalities or disease manifestations [7,18].
Glossary
Cofeeding transmission: a mode of
vector-borne pathogen transmission
that does not require an infected host,
but instead occurs when infected and
uninfected vectors feed in physical
proximity on the same host at a similar
time.
Hematophagous: refers to animals or
insects that feed on blood.
Horizontal transmission:
transmission of a pathogen through
nonhereditary means.
Pathogen transmission: the transfer
of a pathogen from an infected individual
to an uninfected individual.
Reservoir host: an organism or
species in which a given pathogen can
develop infection, proliferate, and be
transmitted to a new host or vector,
often in the absence of clinical
manifestations of infection.
Stercorarian transmission: pathogen
transmission via inoculation of a wound
or insect bite with vector feces.
Vertical nontransovarial
transmission: pathogen transmission
to developing vector life stages
(e.g., larva or nymph) after egg laying.
Vertical transovarial transmission:
pathogen transmission from mother to
egg.

Bartonella spp. Rickettsia spp.

Eye Central n ervous system Central nervous
Retinitis Meningitis system
Parinaud's oculoglandular Encephalitis Meningitis
syndrome Myelitis Encephalitis
Psychiatric disorders
Muscle
Myopathy Peripheral nervous system
Neuritis
Heart
Endocarditis em
Lymphatic syste
Myocarditis Regional lymphadenitis

Liver Spleen
Peliosis hepatis Granulomas/abscesses Skin
Granulomas/abscesses Maculopapular rash
Muscle
Aches
Bone Skin Chills
Osteomyelitis Bacillary angiomatosis
Bartonella associated
cutaneous lesions

Systemic
Fever
Weight loss Systemic
Night sweats Fever

Trends in Parasitology

Figure 2. Manifestations of Ctenocephalides felis-associated pathogens. This figure displays manifestations known
to be associated with Bartonella and Rickettsia spp. associated with C. felis by the organ system. The figure was generated
with BioRender.com.

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Rickettsia typhi and Yersinia pestis are not considered in this review as they are primarily associ-
ated with Xenopsylla cheopis, not C. felis. R. typhi is primarily maintained in flea and rodent spe-
cies and causes murine typhus, a rickettsiosis of severity similar to that caused by the RFLO
group. C. felis can acquire R. typhi, and therefore may be an occasional vector [19]. Y. pestis is
the causative agent of bubonic and pneumonic plague, a re-emerging infectious disease [20].
Due to its more frequent feeding behavior, C. felis does not form a Y. pestis biofilm and therefore
displays poor transmission efficacy [21].

The final genus of interest is Wolbachia [22,23]. While not pathogenic to mammals, Wolbachia
infects a majority of insect species and influences reproduction and vector capacity on a strain-
specific basis [24–26]. Within C. felis, Wolbachia is believed to be maternally inherited yet displays
divergent tissue localization based on strain [22]. The effect of various Wolbachia strains on
C. felis, or on the pathogens it transmits, is largely unknown.

C. felis behavior and husbandry

C. felis adults reside on the host and are resistant to host switching until flea population numbers are
high [27]. Feeding occurs rapidly and frequently; feces from the blood meal are observed 8–9 min
after infestation [21,28]. C. felis is considered minimally host specific, infesting cats, dogs, opos-
sums, cows, sheep, and goats [28,29–32]. Human infestation is typically reported when preferred
hosts (i.e., cats and dogs) are not available [33].

C. felis reproduce sexually and mates on the host following male flea feeding [34]. Female C. felis
emerges 2.7 days before males and displays increased egg production after mating with multiple
males, perhaps due to the ejaculate containing oviposition stimulants or nutrients [34,35].
This behavior negates genetic control strategies used for vector species that mate only once
(e.g., Anopheles gambiae) [36]. Interestingly, virgin females do lay eggs; however, the eggs are
not viable [35,37].

Experimental C. felis colonies are classically maintained on a mammalian host, usually a cat; how-
ever, the suitability of C. felis for artificial feeding systems has been documented repeatedly [38].
Further development of C. felis artificial feeding systems is critical to assess the factors influencing
pathogen acquisition and transmission. Larval C. felis is typically raised on a diet containing yeast
and spray-dried bovine blood, although alternative protein sources may be suitable [39]. Fleas
fed whole dog blood display the greatest blood ingestion, egg production, and larval hatching,
followed by bovine, cat, and human blood [40]. Wild-caught C. felis requires a period of adaptation
to artificial feeding systems that may be accompanied by divergence from the wild-caught C. felis
genotype, transcriptome, and protein expression. While additional control of the C. felis environ-
ment and decreasing mammalian use in research make artificial feeding an appealing model sys-
tem, findings must be interpreted in the context of low flea and microbiome diversity, the lack of
mammal–flea immune interactions, and the absence of other natural selection pressures.

Flea–bacteria interaction
During blood feeding, C. felis pierces the skin with sharp paired laciniae [41]. The blood then
passes through a groove on the median surface of the labrum and into the digestive tract
consisting of the pharynx, esophagus, proventriculus, midgut, hindgut, Malpighian tubules, and
rectum. Pathogens must resist passage in the feces, evidenced by the fast clearance of killed
B. henselae and the inability of Y. pestis to colonize the C. felis digestive tract [13,21].

B. henselae loads in experimentally infected C. felis decrease in the 2 days following ingestion then
remain constant [42,43]. B. henselae has been visualized in the C. felis gut via immunofluorescence

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imaging; however, the authors did not investigate other tissues [42]. Transmission to mammals via
C. felis feeding (salivary or regurgitation) has not been experimentally documented. Salivary trans-
mission would require dissemination to the C. felis salivary glands (Figure 3) and injection with saliva
during feeding via the salivary canal; however, the presence of Bartonella within the C. felis salivary
glands or secretion in the saliva has not been confirmed. Inoculation with C. felis feces is a more
established mechanism of infection [12].

Current evidence only documents Bartonella maintenance in C. felis populations via acquisition
from a mammalian host (i.e., the domestic cat). However, alternative methods of transmission
have been proposed. Vertical transovarial transmission of Bartonella by C. felis appears un-
likely, not occurring in early infection (<5 days post-infection), or in Xenopsylla ramesis fleas in-
fected with Bartonella kosoyi [13,44]. By contrast, acquisition of Bartonella by larval ingestion of
infected adult feces (often referred to as vertical nontransovarial transmission) is considered
likely based on findings in other flea species [44].

Following exposure of larval X. ramesis to Bartonella-infected adult flea feces, 16% of adult
X. ramesis display Bartonella infection [44]. Documenting larval C. felis infection with Bartonella
via ingestion of adult flea feces would reorient our understanding of Bartonella maintenance in
the environment, removing the need for an infected reservoir host and placing emphasis on flea
feces as a contaminate responsible for mammalian and flea infection.

Figure 3. Reproductive, neurological,

salivary, and digestive anatomy
of Ctenocephalides felis. This figure
displays (A) hematoxylin and eosin
staining and a pictorial depiction (B) of the
major reproductive (pink), neurological
(gray), salivary (blue), and digestive (red)
organs of the female hard tick and C. felis.
Abbreviations: DG, dorsal ganglion; ES,
esophagus; HG, hindgut; MG, midgut;
MT, Malpighian tubules; OV, ovary; OVD,
oviduct; PV, proventriculus; RA, rectal
ampulla; SEG, subesophageal ganglion;
SG, salivary glands; TG, thoracic ganglion;
V, vagina; and VN, ventral ganglion. The
flea hematoxylin and eosin micrograph is
courtesy of Cynthia Robveille.

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The reproductive and development effects of Bartonella on C. felis are unknown. Experimental in-
fection of X. ramesis with B. kosoyi results in no reproductive or developmental effects except for
a reduction in the size of males produced by Bartonella-infected females [45]. On an artificial feed-
ing system, Ixodes ricinus infected with B. henselae display increased nymphal feeding and
molting, while infection with Bartonella grahamii reduced the rate of female engorgement and
egg laying [46]. These studies suggest a variable influence of Bartonella infection on vector devel-
opment based on Bartonella and vector species, clearly indicating the need for C. felis experimen-
tal infection with multiple Bartonella species to document C. felis reproductive and developmental
outcomes. Artificial membrane feeding is ideal for such applications, removing the need for in-
fected mammalian hosts and providing greater control over the C. felis environment.

Rickettsia species range from highly pathogenic to symbiotic for their arthropod host [47]. Follow-
ing ingestion of an infected R. felis bloodmeal, rickettsia disseminate to the C. felis midgut, ova-
ries, salivary glands, and muscles as early as 7 days post-exposure [48,49]. Primary
proliferation of R. felis and R. typhi occurs within the epithelial cells of the flea gut [49,50]. In the
9 days following R. felis ingestion, no consistent pattern is observed in bacterial loads; however,
females carry significantly higher bacterial loads [51]. PCR detection of R. felis on a membrane
probed by C. felis, prior to R. felis dissemination to the salivary glands, suggests mechanical
transmission on the mouthparts or regurgitation [52]. Maintenance of R. felis within the flea pop-
ulation is believed to rely primarily on vertical transmission, based on observations in C. felis col-
onies [18]. The relative importance of horizontal transmission from the mammalian host is
unknown as C. felis is able to maintain R. felis by vertical transmission for at least 12 generations
[18,52]. Studies have observed R. felis transmission via cofeeding transmission in both artificial
and murine models, increasing the relative importance of cofeeding on nonreservoir hosts
(e.g., cats) [7,52,53].

Current research suggests that Wolbachia is maintained purely through transovarial transmission
in the flea, evidenced by maintenance in laboratory flea populations without access to infected
bloodmeals and detection by PCR in the C. felis ovary [22,54]. Wolbachia can be detected in
dogs via PCR, most often due to concurrent Dirofilaria immitis infection [26]. The effect of
Wolbachia infection in other arthropod species is often profound, ranging from the induction of
cytoplasmic incompatibility (CI) and parthenogenesis to influencing vector competence and
male killing [24]. Genomic comparison of two C. felis-associated Wolbachia strains identified bi-
otin synthesis operons and CI genes [22]. Therefore, it is believed that C. felis Wolbachia strains
have historically walked the line between parasite and mutualist.

Techniques and observations in other vector species can guide further investigation of the inter-
action between C. felis and Bartonella, Rickettsia, and Wolbachia. Salivary transmission is critical
for multiple tick-borne pathogens (Borrelia burgdorferi, Ehrlichia ewingii), and B. henselae is
known to disseminate to the salivary glands of I. ricinus and Rhipicephalus sanguineus following
ingestion of an infected bloodmeal [55–57]. Therefore, determining the tissue localization of
Bartonella, Rickettsia, and Wolbachia, as has been done with R. felis, is critical to understanding
the impact on multiple organ systems (i.e., salivary and reproductive tissues). Investigation should
employ both in situ visualization and RNA/DNA quantification techniques to visualize localization
within specific cell types as well as bacterial load over time. In the absence of commercially
available Bartonella or Wolbachia monoclonal antibodies, RNA in situ hybridization (RNAscope®)
technologies are a promising alternative for species or strain specific visualization. After
documenting tissue tropism, next-generation sequencing (NGS) and RNA-seq can facilitate
investigation of the microbiome and transcriptomic changes associated with infection in
specific organs.

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Epidemiology and bioclimatic considerations

Laboratory and epidemiological studies and niche modeling consistently associate C. felis
survival and reproduction with warmer, more humid environments (Figure 4) [58–60]. Uncovering
the effect of geography and climate on infestation and flea-borne pathogen infection risk and
severity will assist diagnosticians and facilitate improved vector or pathogen control measures.

Environmental conditions
The primary influence of environmental factors (temperature, humidity, etc.) on C. felis is not adult
fatalities, but instead changes in generation interval, population density, and survival of egg and
larval life stages that take place off the host. Optimal conditions for development off host are a
function of temperature, relative humidity, and soil moisture. Within the laboratory, development
is possible at 13–35°C and 50–92% relative humidity, and is optimal at 32°C and 92% relative hu-
midity [59]. Mating behavior increases at higher temperatures, with the most mating at 38°C and
up to temperatures of 42°C [34]. C. felis feeding and defecation increases at higher temperatures,
while increases in relative humidity (50–92%) result in increased adult body size [50,59]. These
factors all contribute to the general association of C. felis with warmer, more humid locations
where desiccation or freezing is less likely. This is confirmed by ecological niche modeling that in-
dicate minimum temperature, mean temperature of the coldest quarter, and precipitation are the
best predictors of C. felis distribution [58].

Considering the flea-associated pathogen response to environmental conditions, a single study

investigated the response of R. typhi infecting C. felis and reported significantly greater pathogen
loads at 24–30°C than at 18°C [50]. The effect of temperature and relative humidity, and the be-
haviors which they influence on other flea-borne pathogens, remain to be elucidated and likely
have considerable effects on pathogen prevalence and transmission.

Studies from Australia and Spain suggest that the C. felis range will expand and contract in a
location-specific manner [61,62]. In Australia, it is predicted that climate change will reduce

Trends in Parasitology

Figure 4. Predicted distribution of suitable habitat zones for Ctenocephalides felis. Prediction is based on maximum
entropy modeling of C. felis collection sites and 19 variables forming a bioclimatic envelope. Red indicates high predicted
probability of habitat suitability, green indicates medium predicted probability of habitat suitability, and light blue indicates low
probability of suitable conditions. Habitat suitability is defined as the probability that the bioclimatic conditions in the area are
suitable for the species or cox1 clades noted. The figure is adapted from Lawrence et al. [58].

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C. felis habitat and shift phylogenetic distribution, resulting in an overall southerly shift to more
temperate climates [61], while a model of C. felis in Spain predicted an overall increase in
C. felis prevalence [62]. Gálvez et al. also identified increased C. felis prevalence
in anthropogenic locations, those associated with higher human density [62]. Researchers
hypothesized that anthropogenic environments help to maintain fleas during ideal conditions
due to the movement and concentration of many suitable hosts. C. felis presence has also
been associated with human wealth, its presence being decreased in wealthier areas of the
UK, potentially due to the ability to afford ectoparasite-prevention products [60]. These findings
reinforce the association of C. felis with human factors and indicate the need to include human-
associated variables (presence, wealth, urbanization) in C. felis epidemiological modeling.

Future modeling of C. felis distribution will benefit from additional consideration of the sampling
approach, explanatory variables, and computational techniques selected. Active, quantitative
surveillance by trained personnel and species identification by a professional scientist is
ideal given the potential bias in sample collection, misclassification, and imprecise location data
in passive/opportunistic sampling [63]. Leveraging historic climate data, such as the WorldClim
database (https://www.worldclim.org/), in combination with climate change prediction data,
is critical to model changing host and vector range. Beyond climatic data, C. felis modeling
specifically is expected to benefit from the inclusion of human development and wealth data
due to the association of C. felis with human presence and wealth, as well as success of
C. felis within climate-controlled environments [60,62].

C. felis phylogenetics
C. felis is a highly diverse species originating from Africa [58]. In a survey of over 500 cat and dog
fleas from 57 countries (six continents) around the globe, Lawrence et al. utilized mitochondrial
gene sequencing (cox1 gene) to identify four bioclimatically limited clusters that composed
eight clades including the temperate (Clades 1–2), tropical (Clades 3–6), and African clades
(Clades 7–8) (Figure 4) [58].

Based on limited studies, there appears to be no association between C. felis clade and
Bartonella infection. Of the fleas surveyed in Clades 1, 3, 4, and 6, B. clarridgeiae and
B. henselae have been detected in fleas from Clades 1, 3, 4, and 6, while B. koehlerae has
been detected in Clades 1 and 4 [64–66]. Azrizal-Wahid et al. analyzed fleas on an individual
haplotype basis (cox1 and cox2 genes) and also found no associations between Bartonella
infection and flea phylogenetics; however, the relative diversity of these fleas in the context of
previously analyzed populations is unknown [67]. Although flea–Bartonella infection does not
appear to be clade-limited, the effect of C. felis clade on pathogen acquisition, transmission,
and epidemiology remains to be explored.

By contrast, Rickettsia and Wolbachia spp. infection in fleas does appear to be limited by clade.
R. felis has been detected in Clades 1 and 6, while R. asembonensis infection has been detected
in Clades 3 and 4 [64–66]. Furthermore, clade specificity held true when strains of C. felis of
different clades were co-infesting the same cat: in cats infested with both Clade 1 and 4 fleas,
Clade 1 and 4 fleas were infected with only R. felis and R. asembonensis, respectively [66].
Considering Wolbachia strains, in a study of C. felis Clades 1, 4, and 6, Wolbachia strain
wCfeT was found in all clades, while other strains (wCfeF, wCfeJ, and an undescribed strain)
were exclusively found in Clade 6 [66]. The strict association of Rickettsia/Wolbachia with
C. felis clade, even when fleas are infesting the same cat, necessitates further exploration in the
laboratory to determine if C. felis of various clades can acquire nontypical species/strains, and
if not, the mechanisms by which resistance is conferred.

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Understanding the response of phylogenetically diverse C. felis clades to bacterial infection and
climatic conditions is critical to understand the mediators of C. felis and associated pathogen ep-
idemiology. I. ricinus displays phylogenetic divergence across Europe with three main lineages
based on location: UK, western Norway, and continental Europe [68]. Comparing I. ricinus
from the UK with I. ricinus from continental Europe (France), I. ricinus from the UK display in-
creased host questing behavior at lower temperatures, a finding which the authors suggested
is due to genetic selection [69]. Similar experimentation in C. felis is critical to improving epidemi-
ological modeling and climate-change predictions.

While cox1 and cox2 sequencing is suitable to assess C. felis population structure, the implemen-
tation of shotgun sequencing would allow the concurrent sequencing of numerous vector and
bacterial genes, as well as the detection of a greater diversity of pathogenic and symbiotic
bacteria [70,71]. Utilizing this approach on seven tick genera and 74 individual ticks from
China, researchers employed the Mantel test to reveal a significant association between tick
and Rickettsia phylogeny [71]. Pairing wild-caught C. felis shotgun sequencing and phylogenetic
analysis with experimental infection of phylogenetically diverse C. felis will help to reveal the
influence of C. felis and bacterial diversity on pathogen acquisition, proliferation, and transmis-
sion, informing niche modeling and clinician risk assessment.

Pathogen epidemiology and phylogenetics

C. felis-associated Bartonella displays a geographic distribution similar to that of C. felis, with
increased flea and bartonellosis prevalence in areas of higher temperature and humidity
[72–75] (reviewed in [73]). Phylogenetic analysis historically utilizes multiple locus sequence typing
(MLST), the sequencing of multiple molecular markers [e.g., rpoB, gltA, and 16S-23S intergenic
spacer (ITS)] from pure culture, vector, or host samples for comparison with known sequences
[75]. B. henselae strain diversity in gene expression and phenotype has been documented in
the laboratory; however, the epidemiology or virulence of these strains in fleas and mammals is
relatively unexplored [76].

RFLOs have been reported on all continents except Antarctica (reviewed in [77]). To date, there
has been little investigation of RFLO epidemiology [45,78,79]. Two geographically limited studies
surveying C. felis from the USA report greater R. asembonensis prevalence in the Southeast com-
pared with more common detection of R. felis in the Mid-Atlantic and Mid-West, though these
findings may be due to flea phylogenetic diversity [65,66]. RFLOs are assigned to the Rickettsia
transitional group, as they share genotypic and phenotypic traits of both the spotted fever and ty-
phus groups [47,80]. Multiple R. felis strains have been identified and are reported to influence the
C. felis response to infection [81]. Specifically, Liposcelis bostrychophila-associated R. felis re-
sults in significantly greater infection loads, density, and prevalence than infection with C. felis-
associated R. felis [81].

C. felis is infected with the largest diversity of Wolbachia strains of any parasite, with representa-
tives of six supergroups (A, B, F, I, V, and W) [22,23,82–85]. Wolbachia infection in C. felis has
been documented around the world without reported differences in prevalence by location
[22,23,66]. Strains other than wCfeT are considered rare in wild-caught fleas, a finding that
may be explained by associations with C. felis clade [22,66]. The effect of Wolbachia strains on
C. felis is informed by genomic and not experimental studies. Strain wCfeT is known to harbor
a ‘biotin synthesis operon of obligate intracellular microbes’ (BOOM), suggesting a mutualistic
role in the flea, while wCfeJ contains a cinB-like operon implicated in CI induction in flies [22]. In-
terestingly, the C. felis genome harbors CI-like antidote genes suggesting resistance to induction
of a CI phenotype [22]. Experimental infection studies are limited to Synosternus cleopatrae fleas

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in which the Wolbachia burden increased with age in laboratory-origin fleas, but not in wild-
caught fleas, reinforcing the influence of host strain or origin on response to bacterial infection
[85]. Continued surveillance for Wolbachia diversity and uncovering their mutualistic and parasitic
roles in C. felis may hold a key for disrupting pathogen transmission. Given the ability of
Wolbachia to alter host (reproduction, behavior) and pathogen (colonization, proliferation) suc-
cess, understanding the mechanism of interference utilizing single-cell sequencing and gene-
editing technologies (plasmid transfection or genome editing) may uncover pathways to prevent
pathogen transmission by altering colonization or proliferation within the vector.

Movement to more descriptive whole-genome sequencing techniques and experimental flea and
mammalian infections with diverse Bartonella, Rickettsia, and Wolbachia species and strains is
critical to further our understanding of pathogen and symbiont transmission; however, it presents
technical and financial challenges. Due to the intracellular nature of these genera, low host bac-
teremia, and slow bacterial growth, bacterial isolation for whole-genome sequencing is difficult.
Therefore, shotgun sequencing and metagenome-assembled genome construction may be a
more practical approach to genome analysis and comparison [71]. Within the laboratory, ex-
perimental infection of C. felis and reservoir hosts with differing Bartonella, Rickettsia, and
Wolbachia species and strains should be paired with host and pathogen genotyping. Infection
with two or more bacterial strains and comparative genome analysis may uncover genes or
single-nucleotide polymorphisms (SNPs) associated with variable bacterial proliferation or
issue localization.

The immune system

The flea immune response consists of cellular and humoral innate immune functions triggered by
pattern-recognition receptors (PRRs) that bind pathogen-associated molecular patterns
(PAMPs). The triggered immune response relies on cellular immunity, signaling pathways, and an-
timicrobial molecules. Three primary immune pathways (Toll, Imd, and Jak/Stat pathways) dom-
inate the insect immune system and result in pathogen death via multiple mechanisms (Box 1).
Homologs for all Drosophila Imd and Toll pathway components have been identified in the
C. felis transcriptome, although research characterizing these pathways is limited [86]. Rennoll
et al. reported an increased R. typhi burden in C. felis following knockout of Relish and Imd
(two components of the Imd pathway) [86]. Hemocytes are the major arm of the C. felis cellular
immune response within the hemocoel (insect body cavity) and have been divided based on func-
tion or location (circulating and sessile hemocytes) [87].

Following ingestion, pathogens must first avoid excretion or death in the flea digestive tract. Ser-
ine proteases (e.g., chymotrypsin and trypsin) play a primary role in bloodmeal digestion in C. felis
and are increased during infection with B. henselae, Y. pestis, and R. typhi, suggesting an im-
mune protective role [88–90]. This may be due to their importance in activating the melanization
cascade that facilitates pathogen encapsulation and clearing, or in directly digesting Bartonella/
Rickettsia-associated or required proteins [91]. Serine protease activity is regulated by serine pro-
tease inhibitors (SPIs). Multiple SPIs are expressed by C. felis that localize in the digestive tract, fat
body, and pericardial cells [92].

Reactive oxygen species (ROS) and antimicrobial peptide (AMP) secretion, triggered by the Toll,
Imd, and Jak/Stat pathways, is critical to the immune response in the digestive tract. The
X. cheopis genome contains numerous ROS-regulating genes found to influence Y. pestis bac-
terial loads but they are not upregulated in response to systemic infection with Escherichia coli
or Micrococcus luteus [87,93]. Similarly, diverse AMPs are known to comprise the C. felis im-
mune response but are poorly described. Defensins are generally small, cationic AMPs that

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Box 1. Major insect immune pathways and pathogen-killing mechanisms

Immune pathways

• The Toll pathway is initiated by pathogen-recognition receptor (PRR) binding of Spatzle which binds the hemocyte
transmembrane Toll receptor. An intracellular cascade then results in the nuclear translocation of NF-κB transcription
factors and the transcription of antimicrobial peptides (AMPs) typically targeting Gram-positive bacteria, viruses, fungi,
and plasmodia.
• The Imd pathway is initiated by pathogen-associated molecular pattern (PAMP) binding of the PGRP-LC receptor and
inducing the intracellular cascade leading to nuclear translocation of NF-κB transcription factors and the transcription
of AMPs typically targeting Gram-negative bacteria, viruses, and plasmodia.
• The Jak/Stat pathway is activated by binding of the extracellular cytokine Unpaired to the Domeless receptor, trigger-
ing an intracellular cascade resulting in the dimerization and translocation of Stat into the nucleus for transcription of
antimicrobial genes targeting bacteria, viruses, and plasmodia.

Pathogen-killing mechanisms

• Phagocytosis is initiated by hemocyte receptor binding to pathogen proteins and results in internalization into a
phagosome, phagosome fusion with a lysosome, and hydrolytic enzyme digestion. In other insects, this process
begins seconds after pathogen introduction.
• Melanization is initiated by the recognition of PRRs and results in the encapsulation of the pathogen in a dark protein-
aceous capsule, resulting in death by either starvation or oxidative damage. Following melanization, small pathogens
are often phagocytosed. Although not characterized in the flea, X. cheopis has pro-phenoloxidase activating factor
genes necessary for the production of melanin.
• Encapsulation occurs in pathogens too large to be phagocytized and results in the formation of multiple cell layers
surrounding the pathogen. These multiple layers often require multiple cell types.
• Nodulation is an immune mechanism for killing bacterial aggregates. Nodulation frequently occurs prior to melanization
and involves the adherence of multiple hemocyte layers.
• Autophagy is an immune process for the degradation of intracellular materials (e.g., bacteria, viruses) by membrane
elongation for formation of an intracellular autophagosome which fuses with a lysosome.
• RNAi functions in protection from viruses by ribonuclease cleavage of viral double stranded (ds)RNA to form viral-
derived small interfering RNAs.
• Apoptosis, the triggering of programmed cell death, is utilized in the insect response to viruses and baculoviruses.
• Lysis, the killing of pathogens by disruption of the pathogen membrane, is typically triggered by AMPs. Insect-associated
AMPs are typically grouped into four categories: defensin and defensin-like peptides, cecropin and cecropin-like
peptides, attacins and gloverins, and lebocins. Additional effectors involved in lysis are lysozyme proteins and reactive
oxygen species (ROS).

cause the lysis of bacteria, fungi, viruses, and protozoa via membrane disruption [94]. Defensin
expression has been identified in C. felis; however, their diversity and effects remain unknown
[95,96]. As expected, B. henselae and Y. pestis have acquired genes for AMP and ROS immune
evasion [97–99].

Additional investigation of the C. felis immune system is necessary to understand pathogen trans-
mission and the C. felis–pathogen interaction. Analysis of the recently published C. felis genome
presents the opportunity to identify immune effector molecules, including defensins and SPIs.
With a high degree of phylogenetic diversity, tick defensins are considered candidates for tick-
borne pathogen control [94]. In I. ricinus artificially infected with B. henselae, four defensins
were identified by high-throughput RNA sequencing, all of which were downregulated in re-
sponse to infection [100]. The potential to exploit defensins for vector-borne pathogen control
or as an explanatory factor separating vector-competent and non-vector-competent species
should be explored in C. felis. In the same study, a BPTI/Kunitz family SPI (IrSPI) was upregulated
in B. henselae-infected I. ricinus [100]. Implementing RNA interference (RNAi) technology, the au-
thors silenced IrSPI, significantly decreasing B. henselae loads [99]. Utilizing RNA-seq and RNAi
on artificially infected C. felis or C. felis embryonic cell lines will allow researchers to identify differ-
ential transcription during bacterial infection and document the influence of silencing specific RNA
transcripts on bacterial infection. As flea-embryonic cell lines are not currently available, investiga-
tion may currently be assisted by better characterized Drosophila and tick cell lines, although

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these systems present their own drawbacks (e.g., reduced cell diversity, lack of blood-feeding
behavior).

The microbiome
Multiple studies have attempted to characterize the microbiome of wild-caught C. felis
[33,66,101]. These studies consistently identify three major genera infecting C. felis: Bartonella,
Rickettsia, and Wolbachia. However, due to the low biomass nature of C. felis, it is difficult to dif-
ferentiate the true C. felis microbiome from bacterial DNA contamination derived from laboratory
reagents and plastics [65,102]. Current studies should employ contaminant-removal methods
with negative controls such as the ‘decontam’ prevalence method [103,104].

The relationship between Bartonella, Rickettsia, and Wolbachia abundance appears to vary by
flea species. These three genera regularly coinfect C. felis at fluctuating relative abundance
[65]. In Oropsylla spp., a flea of rodents, Bartonella abundance is negatively correlated with
Rickettsia abundance [105]. NGS of C. felis fed on B. henselae experimentally infected and un-
infected cats identified increased microbial diversity in C. felis fed on B. henselae-infected cats
at 24 h, decreasing to baseline by 9 days [54]. The cause of increased diversity was hypothesized
to be cat or flea immunological changes associated with B. henselae infection.

Similarly, the ability of Rickettsia spp. to induce microbiome changes or influence pathogen trans-
mission is of interest. Utilizing amplification and ligation into a plasmid, Pornwiroon et al. identified
decreased species richness in naturally R. felis-infected fleas from two flea colonies [106]. Further
investigation in experimentally infected C. felis with more powerful sequencing technologies
(NGS) is necessary to confirm these findings. 16S rRNA NGS has identified widespread coinfec-
tion with multiple Rickettsia in individual C. felis; however, many of these sequences did not align
with a known strain of Rickettsia. Therefore, the application to our current understanding of
microbiome-induced resistance to acquisition or transovarial transmission is unknown [65]. The
inability of ticks to acquire and transovarially transmit multiple Rickettsia spp. is generally consid-
ered tick biology dogma, as studies have indicated that ticks infected with a single Rickettsia spp.
are resistant to acquisition and transovarial transmission of a second Rickettsia spp. [107]. How-
ever, high coinfection rates in Haemaphysalis montgomeryi question this belief, and suggests that
vector species and Rickettsia spp. relatedness may influence this phenomenon [108]. Further
Rickettsia experimental infection and coinfection in C. felis is necessary to elucidate the role of co-
infection on Rickettsia survival and transmission.

Across vector species, the ability of Wolbachia to influence pathogen transmission is of great in-
terest. This effect was first reported in RNA viruses of Drosophila with a majority of research fo-
cusing on mosquito-transmitted arboviruses [109]. The ability of certain Wolbachia strains to
block mosquito-borne pathogen transmission is attributed primarily to host innate immune acti-
vation via ROS, resource competition, and vector fitness costs [110,111]. Deployment of
Wolbachia strain wMel-colonized mosquitoes has resulted in significant changes to dengue
transmission in Townsville, Australia [112]. In ticks, the ability of Wolbachia to disrupt pathogen
transmission is less clear, and instead other known tick endosymbionts may be of greater interest
for tick-borne pathogen control [113]. Given the pathogen, vector, and Wolbachia strain specific-
ity for pathogen transmission blocking, further investigation of C. felis and non-C. felis-associated
Wolbachia infection in diverse C. felis clades may provide insights into vector and pathogen
biology, epidemiology, and control.

While 16S rRNA NGS is a valuable tool to sequence the bacterial microbiome, movement to
metagenomic sequencing is critical to appreciate the diverse nonbacterial microbiome of

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C. felis, including viruses and protozoa. This approach has been implemented in Ixodes Outstanding questions
persulcatus ticks from China with comparison to the Kyoto Encyclopedia of Genes and Genomes What immune pathways does C. felis
(KEGG) to identify known or predicted pathogen gene function [114]. Researchers concluded employ to combat Bartonella and
Rickettsia infection?
that viruses were a principal component of the microbiome with a large diversity of previously
uncharacterized viruses [114]. Such analyses in C. felis would broaden our appreciation for the How do environmental conditions
complex microbiome shifts associated with pathogen ingestion and identify organisms or influence C. felis and its associated
genes as candidates for C. felis pathogen control. pathogens?

What is the effect of C. felis genetic

Concluding remarks diversity on Bartonella, Rickettsia, and
Our understanding of C. felis vector biology continues to evolve with an increasing number of Wolbachia infection and transmission?
publications addressing flea and pathogen species. These efforts should leverage our knowledge
What is the role of C. felis-associated
of C. felis behavior and reinforce the need for an updated examination of the C. felis response to Wolbachia strains in influencing C. felis
infection (see Outstanding questions). The use of C. felis colonies and establishment of C. felis cell biology and pathogen transmission?
lines will be invaluable tools in exploring these research objectives. Leveraging contemporary
techniques (e.g., metagenomic sequencing, RNAi, single-cell sequencing) to investigate the
C. felis–pathogen interface will uncover the influence of C. felis phylogenetic diversity and the im-
mune and microbiome response to infection. These efforts should utilize our basic understanding
of the insect immune response to contextualize findings in C. felis, including the pathogen-killing
pathways, mechanisms, and effectors. Finally, epidemiological modeling of vector and pathogen
species should seek to employ our improved knowledge of C. felis and pathogen biology, and the
C. felis–pathogen interaction.

Acknowledgments
The authors would like to thank the many individuals who have contributed to our understanding of fleas and flea-borne path-
ogens, as well as thanking their various funding sources, including CNPq (National Council for Scientific and Technological
Development) for Productivity Grant conceived to M.R.A. (CNPq Process #303701/2021-8) and the NC State Molecular Bio-
technology Training Program of the National Institutes of Health under award number 1T32GM133366 to C.O.M.

Declaration of interests
In conjunction with Sushama Sontakke and North Carolina State University, E.B.B. DVM holds US Patent No. 7,115,385:
‘Media and Methods for Cultivation of Microorganisms’, issued 3 October 2006. He is a co-founder, shareholder, and Chief
Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella
species infections as well as a scientific consultant to IDEXX Laboratories (Westbrook, ME, USA). All other authors declare no
potential conflicts of interest.

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