New records of pathogenic bacteria in different species of fleas collected from domestic and peridomestic animals in Spain. A potential zoonotic threat?

Climate change is causing many vectors of infectious diseases to expand their geographic distribution as well as the pathogens they transmit are also conditioned by temperature for their multiplication. Within this context, it is worth highlighting the significant role that fleas can play as vectors of important pathogenic bacteria. For this purpose, our efforts focused on detecting and identifying a total of 9 bacterial genera (Rickettsia sp.; Bartonella sp.; Yersinia sp.; Wolbachia sp., Mycobacterium sp., Leishmania sp., Borrelia sp., Francisella sp. and Coxiella sp.) within fleas isolated from domestic and peridomestic animals in the southwestern region of Spain (Andalusia). Over a 19-months period, we obtained flea samples from dogs, cats and hedgehogs. A total of 812 fleas was collected for this study. Five different species were morphologically identified, including C. felis, C. canis, S. cuniculi, P. irritans, and A. erinacei. Wolbachia sp. was detected in all five species identified in our study which a total prevalence of 86%. Within Rickettsia genus, two different species, R. felis and R. asembonensis were mainly identified in C. felis and A. erinacei, respectively. On the other hand, our results revealed a total of 131 fleas testing positive for the presence of Bartonella sp., representing a prevalence rate of 16% for this genus identifying two species B. henselae and B. clarridgeiae. Lastly, both Y. pestis and L. infantum were detected in DNA of P. irritans and C. felis, respectively isolated from dogs. With these data we update the list of bacterial zoonotic agents found in fleas in Spain, emphasizing the need to continue conducting future experimental studies to assess and confirm the potential vectorial role of certain synanthropic fleas.

Vector biology of the cat flea Ctenocephalides felis.

Ctenocephalides felis, the cat flea, is among the most prevalent and widely dispersed vectors worldwide. Unfortunately, research on C. felis and associated pathogens (Bartonella and Rickettsia spp.) lags behind that of other vectors and vector-borne pathogens. Therefore, we aimed to review fundamental aspects of C. felis as a vector (behavior, epidemiology, phylogenetics, immunology, and microbiome composition) with an emphasis on key techniques and research avenues employed in other vector species. Future laboratory C. felis experimental infections with Bartonella, Rickettsia, and Wolbachia species/strains should examine the vector-pathogen interface utilizing contemporary visualization, transcriptomic, and gene-editing techniques. Further environmental sampling will inform the range and prevalence of C. felis and associated pathogens, improving the accuracy of vector and pathogen modeling to improve infection/infestation risk assessment and diagnostic recommendations.

Cat Flea Coinfection with Rickettsia felis and Rickettsia typhi.

Purpose: Flea-borne rickettsioses, collectively referred to as a term for etiological agents Rickettsia felis, Rickettsia typhi, and RFLOs (R. felis-like organisms), has become a public health concern around the world, specifically in the United States. Due to a shared arthropod vector (the cat flea) and clinical signs, discriminating between Rickettsia species has proven difficult. While the effects of microbial coinfections in the vector can result in antagonistic or synergistic interrelationships, subsequently altering potential human exposure and disease, the impact of bacterial interactions within flea populations remains poorly defined. Methods: In this study, in vitro and in vivo systems were utilized to assess rickettsial interactions in arthropods. Results: Coinfection of both R. felis and R. typhi within a tick-derived cell line indicated that the two species could infect the same cell, but distinct growth kinetics led to reduced R. felis growth over time, regardless of infection order. Sequential flea coinfections revealed the vector could acquire both Rickettsia spp. and sustain coinfection for up to 2 weeks, but rickettsial loads in coinfected fleas and feces were altered during coinfection. Conclusion: Altered rickettsial loads during coinfection suggest R. felis and R. typhi interactions may enhance the transmission potential of either agent. Thus, this study provides a functional foundation to disentangle transmission events propelled by complex interspecies relationships during vector coinfections.

The presence of Rickettsia felis in communities in the central highlands of Vietnam.

Rickettsia felis is an emerging flea-borne spotted fever pathogen that causes febrile illness in humans. In Vietnam, R. felis was detected in hospitalized patients, but there is no information on its presence in the Vietnamese community. This cross-sectional study aimed to determine the presence of R. felis in humans of the Central Highlands of Vietnam. A total of 158 blood and 213 serum samples were subjected to PCR and IFAT, respectively, to detect the presence of R. felis DNA and antibodies against R. felis. PCR assays detected R. felis DNA in four out of 158 blood samples, accounting for a prevalence of 2.53 % (95 % CI: 0.81 %-6.76 %). Phylogenetic analysis indicated the presence of R. felis and R. felis genotype RF2125 in the communities in the Central Highlands of Vietnam. The result of IFAT identified seven out of 213 serum samples (3.29 %, 95 % CI: 1.45 %-6.93 %) positive for antibodies against R. felis. This study was the first to demonstrate the presence of active R. felis infections in the communities in the Central Highlands of Vietnam utilizing both molecular and serological methods.

A novel Rickettsia species closely related to Rickettsia felis in Anopheles mosquitoes from Yingkou City, Northeast China.

Mosquitoes are generally recognized as the most important vector of many zoonotic pathogens. In this study, seven mosquitoes species were identified (Anopheles pullus, Anopheles sinensis, Anopheles lesteri, Anopheles kleini, Ochlerotatus dorsalis, Aedes koreicus and Culex inatomii) in samples collected from Yingkou City, Liaoning Province, Northeastern China. A novel Rickettsia species was detected in Anopheles sinensis (two of 71, 2.82%) and Anopheles pullus (one of 106, 0.94%) mosquitoes. Genetic analysis indicated that the rrs and ompB genes have highest 99.60% and 97.88%-98.14% identities to Rickettsia felis, an emerging human pathogen of global concern mainly harboured by fleas, mosquitoes and booklice. The gltA sequences of these strains have 99.72% of nucleotide similarity with Rickettsia endosymbiont of Medetera jacula. The groEL sequences have 98.37% similarity to both Rickettsia tillamookensis and Rickettsia australis. The htrA sequences have 98.77% similarity to Rickettsia lusitaniae. In the phylogenetic tree based on concatenated nucleotide sequences of rrs, gltA, groEL, ompB and htrA genes, these strains are closely related to R. felis. Herein, we name it 'Candidatus Rickettsia yingkouensis'. Its human pathogenicity to humans and animals is still to be determined.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Complete bacterial profile and potential pathogens of cat fleas Ctenocephalides felis.

Fleas are important ectoparasites and vectors associated with a wide range of pathogenic diseases, posing threats to public health concerns, especially cat fleas that spread worldwide. Understanding the microbial components is essential due to cat fleas are capable of transmitting pathogens to humans, causing diseases like plague and murine typhus. In the present study, metagenomic next-generation sequencing was applied to obtain the complete microbiota and related functions in the gut of Ctenocephalides felis. A total of 1,870 species was taxonomically recognized including 1,407 bacteria, 365 eukaryotes, 69 viruses, and 29 archaea. Proteobacteria was the dominant phylum among the six samples. Pathogens Rickettsia felis, Acinetobacter baumannii, Coxiella burnetii, and Anaplasma phagocytophilum were taxonomically identified and had high abundances in all samples. The resistance gene MexD was predominant in microbial communities of all cat fleas. We also performed epidemiological surveys of pathogens R. felis, A. baumannii, C. burnetii, and A. phagocytophilum among 165 cat fleas collected from seven provinces in China, while only the DNAs of R. felis (38/165, 23.03%) and C. burnetii (2/165, 1.21%) were obtained. The data provide new insight and understanding of flea intestinal microbiota and support novel information for preventing and controlling fleas and their transmitted diseases.

Rickettsia felis DNA recovered from a child who lived in southern Africa 2000 years ago.

The Stone Age record of South Africa provides some of the earliest evidence for the biological and cultural origins of Homo sapiens. While there is extensive genomic evidence for the selection of polymorphisms in response to pathogen-pressure in sub-Saharan Africa, e.g., the sickle cell trait which provides protection against malaria, there is inadequate direct human genomic evidence for ancient human-pathogen infection in the region. Here, we analysed shotgun metagenome libraries derived from the sequencing of a Later Stone Age hunter-gatherer child who lived near Ballito Bay, South Africa, c. 2000 years ago. This resulted in the identification of ancient DNA sequence reads homologous to Rickettsia felis, the causative agent of typhus-like flea-borne rickettsioses, and the reconstruction of an ancient R. felis genome.

Revisiting the sialome of the cat flea Ctenocephalides felis.

The hematophagous behaviour emerged independently in several instances during arthropod evolution. Survey of salivary gland and saliva composition and its pharmacological activity led to the conclusion that blood-feeding arthropods evolved a distinct salivary mixture that can interfere with host defensive response, thus facilitating blood acquisition and pathogen transmission. The cat flea, Ctenocephalides felis, is the major vector of several pathogens, including Rickettsia typhi, Rickettsia felis and Bartonella spp. and therefore, represents an important insect species from the medical and veterinary perspectives. Previously, a Sanger-based sialome of adult C. felis female salivary glands was published and reported 1,840 expressing sequence tags (ESTs) which were assembled into 896 contigs. Here, we provide a deeper insight into C. felis salivary gland composition using an Illumina-based sequencing approach. In the current dataset, we report 8,892 coding sequences (CDS) classified into 27 functional classes, which were assembled from 42,754,615 reads. Moreover, we paired our RNAseq data with a mass spectrometry analysis using the translated transcripts as a reference, confirming the presence of several putative secreted protein families in the cat flea salivary gland homogenates. Both transcriptomic and proteomic approaches confirmed that FS-H-like proteins and acid phosphatases lacking their putative catalytic residues are the two most abundant salivary proteins families of C. felis and are potentially related to blood acquisition. We also report several novel sequences similar to apyrases, odorant binding proteins, antigen 5, cholinesterases, proteases, and proteases inhibitors, in addition to putative novel sequences that presented low or no sequence identity to previously deposited sequences. Together, the data represents an extended reference for the identification and characterization of the pharmacological activity present in C. felis salivary glands.

Molecular detection of Rickettsia felis in fleas and ticks collected from dogs and cats of Puebla, Mexico.

Fleas and ticks represent the two main groups of ectoparasites that infest companion animals. In particular, the flea Ctenocephalides felis felis and several members of the Rhipicephalus sanguineus complex are the main vectors of a wide range of pathogens on the American continent. They are competent vectors for several members of the genus Rickettsia, which encompass at least 15 pathogenic obligate intracellular bacteria that colonize the endothelial cells of vertebrates. In Mexico, 10 species of Rickettsia belonging to three groups have been detected in six species of ectoparasites from dogs in 9 of the 32 states of the country. However, in some larger regions of the country, active epidemiological surveillance has not been carried out. For this reason, the aim of this study was to identify the presence of members of the genus Rickettsia in fleas and ticks of dogs and cats in the state of Puebla, Mexico. A cross-sectional study was carried out to collect ectoparasites of dogs and cats during August to November 2019. Samples were fixed in 70% ethanol and examined to identify the presence of Rickettsia DNA by the amplification and sequencing of specific fragments of the gltA and ompB genes using conventional PCR. The recovered sequences were compared with those deposited in GenBank, and phylogenetic analyses were carried out to identify the position of the pathogens detected with respect to the valid species previously reported worldwide. Additionally, ecological parameters of the ectoparasite infestations were also calculated. We recovered 196 ectoparasites belonging to two species, 33 C. felis felis and 163 R. sanguineus s.l. (Rhipicephalus linnaei), parasitizing 46 hosts (42 dogs and 4 cats) in 11 localities of the state of Puebla. We detected the presence of Rickettsia felis in three pools of C. felis felis, and five from R. sanguineus s.l. Our work provides the first record of R. felis in hard ticks of Mexico and Central America, with new collection localities for this pathogen in central Mexico.

Rickettsia felis and species of fleas parasitizing on household dogs in the Central Highlands of Vietnam.

Rickettsia felis is an obligate intracellular gra m-negative bacterium that belongs to the family of Rickettsiaceae. Ctenocephalides felis, cat flea, is the primary vector of the bacteria. The flea is the most common ectoparasite in dogs and associated with flea-borne spotted fever in humans. Information on R. felis and flea species parasitizing on dogs in Vietnam is limited. This study aimed to identify the species of fleas collected from dogs in the Central Highlands of Vietnam and detected the existence of R. felis in these fleas utilizing molecular tools. Morphological identification of 1618 fleas and molecular confirmation revealed the predominance of C. felis orientis parasitizing on dogs in the Central Highlands of Vietnam. Sixty-eight out of 100 fleas collected from household dogs were positive for spotted fever group rickettsiae; whilst R. felis was detected in 97.06 % (66/68) of C. felis orientis and C. felis felis. The results of this study indicate the potentially high risk of R. felis infection to humans and animals.

Confirmation of the presence of Rickettsia felis infecting Ornithodoros puertoricensis in Mexico.

Soft ticks are neglected competent vectors of a wide range of pathogenic microorganisms, among which bacteria of the genera Rickettsia and Borrelia stand out. In Mexico, previous studies have shown the presence of a member of the Ornithodoros talaje complex in the Virginia opossum (Didelphis virginiana Didelphimorphia: Didelphidae Kerr) from southeastern Mexico. However, its specific identification has not been achieved. Two D. virginiana were treated in a private clinic during the period of April-May 2022. Tick larvae were manually removed, DNA extraction was performed, and some genes from various bacterial and parasitic pathogens were amplified and sequenced. A total of 96 larvae were recovered, which were morphologically identified as Ornithodoros puertoricensis (Ixodida: Argasidae Fox); the 16 S sequences showed a similarity of 96.79%-99.51% with sequences of O. puertoricensis from Panama and Colombia. The presence of Rickettsia felis (Rickettsiales: Rickettsiaceae Bouyer et al.) was detected in 15 specimens from one host. The soft tick O. puertoricensis is recorded for the first time as an ectoparasite of the Virginia opossum in America and represents the second report for this soft tick in Mexico since 1963. This represents the most northern record of this tick species in its geographic distribution and brings a new soft tick-Rickettsia association.

Rickettsia felis is an emerging human pathogen associated with cat fleas: A review of findings in Taiwan.

Rickettsia felis is an emerging rickettsial agent principally associated with cat fleas (Ctenocephalides felis), formerly discovered in 1990. Since then, clinical cases of R. felis infection have been identified globally by specific DNA sequences in patients with undifferentiated febrile illness, including in Taiwan, but such evidence is limited. R. felis rickettsiosis is self-limiting and easily treated with doxycycline, but its diagnosis remains a challenge. Environmental risk factors for R. felis rickettsiosis have yet to be clearly demonstrated, and its transmission biology is incompletely understood. Cat fleas are naturally infected with R. felis at varying rates, and vector competence in the transmission of R. felis has been demonstrated in animal models, including dogs, which may serve as reservoir hosts. In northern Taiwan, despite ∼20% of cat fleas infesting companion animals consistently found to be infected with R. felis, only a few cases of potential R. felis infection have been identified through a retrospective serological investigation, though without molecular confirmation. Ecological studies have identified divergent R. felis-like organisms in different arthropod hosts, but these strains appear to serve as nonpathogenic endosymbionts. Although its association with disease is limited, we believe cat flea-borne R. felis warrants increased recognition in an aging population due to immunosenescence and the proximity of companion animals to the elderly. Adopting a One Health approach involving collaboration and communication between clinicians, veterinarians, public health practitioners, and environmental scientists will improve our knowledge about this neglected pathogen and promote the prevention and control of vector-borne diseases.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

Human Rickettsia felis infections in Mainland China.

We identified four flea-borne spotted fever cases caused by Rickettsia felis in a retrospective survey of 182 patients with fever of unknown origin (FUO) in China between 2021 and 2022. The clinical signs and symptoms of the patients were similar to those of other rickettsioses, including fever, rash, and liver and kidney dysfunction. All four patients in the present study developed pneumonia or lung lesions after R. felis infection. The cases of R. felis infection, a neglected infectious disease, were sporadic in multiple provinces of the country. The high prevalence (2.14%, 4/187) of R. felis among patients with FUO highlights the risk posed by this pathogen to public health in China.

Prevalence of selected pathogens in ectoparasites from free-roaming domestic cats in the southern Great Plains of the United States.

Rickettsia sp. and Bartonella sp. were detected in ectoparasites of free-roaming domestic cats (Felis catus) from a trap-neuter-release program in central Oklahoma during January and February 2020. We used polymerase chain reaction and sequencing to identify fleas containing DNA of five different pathogens: Rickettsia felis (84%), 'Candidatus R. andeanae' (8%), Bartonella henselae (32%), Bartonella clarridgeiae (36%), and Bartonella sp. (8%). Co-infections with R. felis with three Bartonella species were identified. One tick was positive for R. felis, one flea was positive for 'Candidatus R. andeanae' and one ear mite was positive for a Bartonella species. These results highlight the need for more focus on free-roaming domestic cats and their ectoparasites in the Great Plains region.

Molecular Detection of Rickettsia felis in Fleas of Companion Animals in East Texas.

Flea-borne spotted fever is an emerging insect-borne rickettsial infection caused by Rickettsia felis and has been identified worldwide. This study sought to explore the prevalence of rickettsiae associated with fleas on companion dogs and cats from Walker and Montgomery Counties in East Texas. Fleas were collected from animals entering local veterinary clinics for routine checkups. Collected fleas were identified as Ctenocephalides felis or Pulex irritans and analyzed by polymerase chain reaction for the presence of rickettsiae and subsequent sequencing. An estimation of the bcMLE (bias-corrected maximum likelihood estimation) of pooled samples was calculated. Four hundred eighty-eight fleas (comprising C. felis and P. irritans) were collected from 16 cats and 77 dogs. Our results demonstrate R. felis in 21 pools of fleas from dogs (bcMLE 15.28%) and a bcMLE of 7.25% from flea samples collected from cats. Sequence analysis revealed R. felis as the only Rickettsia that could be amplified in our samples using the rickettsial citrate synthase gene and subsequent sequencing. In this study, the presence of R. felis in fleas from companion cats and dogs suggests a potential risk of flea-borne spotted fever in humans who encounter flea-infested animals.

Booklice Liposcelis bostrychophila Naturally Infected by Rickettsia felis Cause Fever and Experimental Pneumonia in Mammals.

BACKGROUND: Rickettsia felis is emergent in tropical areas. Despite its high morbidity, its natural history has not yet been fully determined. We investigated the role of the common household booklouse, Liposcelis bostrychophila, recently found to harbor R. felis. METHODS: Blood samples from 372 febrile patients from Senegalese villages, as well as nasal and skin samples from 264 asymptomatic individuals, were tested for cat flea-associated and booklice-associated strains of R. felis. Dust samples from beds were collected to isolate booklice and R. felis. Mice were infected with aerosol of R. felis strain from naturally infected booklice. RESULTS: Forty febrile patients (11%) were infected by R. felis, including 26 (7%) by the booklice-associated strain. Nine nasal samples (3.4%) and 28 skin samples (10.6%) contained R. felis, including 7 and 24, respectively, with the booklice-associated strain. The presence of live L. bostrychophila was observed in 32 dust samples (16.8%); R. felis was identified in 62 dust samples (32.5%). Several mice samples were positive for R. felis; interstitial lymphohistiocytic infiltrates were identified in lungs. CONCLUSIONS: Liposcelis bostrychophila may be a reservoir of R. felis. The booklice-associated strain is pathogenic in mammals, causing pneumonia. Human infection may be acquired via inhalation of infected booklice particles.

Is vertical transmission the only pathway for Rickettsia felis?

The genus Rickettsia encompasses several species grouped into two main clusters, Typhus and the Transitional groups. The latter group contains Rickettsia felis, an endosymbiont of several arthropods with an uncertain human pathogenicity and whose most efficient transmission mechanism described thus far is transovarial. The aim of this study was to evaluate whether this pathway exists using phylogenetic analysis and partial sequences of the 17kDa and gltA genes and comparing them with host phylogeny using the cytb region. This is the first study that evaluates the vertical transmission of R. felis. In general, both phylogenies of R. felis showed no polytomies, as suspected if this pathway was the only pathway occurring. When phylogenies of the invertebrates and the gltA of R. felis were compared for strong coevolutionary insight, intricate relationships were observed, suggesting that other transmission pathways must occur, such as horizontal transmission. Further studies are needed to determine which other transmission routes occur in hematophagous arthropods.