Molecular detection of zoonotic bartonellae (B. henselae,B. elizabethae and B. rochalimae) in fleas collected from dogs in Israel

Fleas represent an acknowledged burden on dogs worldwide. The characterization of flea species infesting kennel dogs from two localities in Israel (Rehovot and Jerusalem) and their molecular screening for Bartonella species (Rhizobiales: Bartonellaceae) was investigated. A total of 355 fleas were collected from 107 dogs. The fleas were morphologically classified and molecularly screened targeting the Bartonella 16S–23S internal transcribed spacer (ITS). Of the 107 dogs examined, 80 (74.8%) were infested with Ctenocephalides canis (Siphonaptera: Pulicidae), 68 (63.6%) with Ctenocephalides felis, 15 (14.0%) with Pulex irritans (Siphonaptera: Pulicidae) and one (0.9%) with Xenopsylla cheopis (Siphonaptera: Pulicidae). Fleas were grouped into 166 pools (one to nine fleas per pool) according to species and host. Thirteen of the 166 flea pools (7.8%) were found to be positive for Bartonella DNA. Detected ITS sequences were 99–100% similar to those of four Bartonella species: Bartonella henselae (six pools); Bartonella elizabethae (five pools); Bartonella rochalimae (one pool), and Bartonella bovis (one pool). The present study indicates the occurrence of a variety of flea species in dogs in Israel; these flea species are, in turn, carriers of several zoonotic Bartonella species. Physicians, veterinarians and public health workers should be aware of the presence of these pathogens in dog fleas in Israel and preventive measures should be implemented.     

Survey of flea infestation in cats in Spain

Fleas are a common cause of feline skin disorders as well as vectors of zoonotic diseases. This study evaluated the flea species infesting domestic cats in Spain and assessed factors influencing their distribution. Fleas from 217 cats from 57 localities in Spain were identified and associations between abundance, and host‐dependent, host habitat and environmental factors were examined. Variations in infracommunity and component community structure were also explored. Three species were present, of which Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) was the most abundant (98.4%), followed by Ctenocephalides canis (Curtis) (1.1%) and Pulex irritans (L.) (Siphonaptera: Pulicidae) (0.5%). Overall abundance and abundances of both C. felis and C. canis were higher on farms than in apartments, but overall flea abundance and abundances of both C. felis and C. canis were lower in rural than urban environments. Overall abundance and C. felis abundance were lower during the warmest months, and mean annual rainfall was positively correlated with overall, C. felis and C. canis abundances. No relationship between the number of species per cat and any host, habitat or physiographical variable was found. Species richness was not correlated with mean annual temperature or rainfall. Flea abundance was mainly associated with host habitat and environmental factors.     

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a useful tool for the rapid identification of wild flea vectors preserved in alcohol

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re‐emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI‐TOF MS profiling. The goal of the present work was to assess the performance of MALDI‐TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI‐TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis.     

Evidence for the presence of Ctenocephalides orientis in livestock dwellings in northwest Iran

Fleas are important vectors of diseases such as murine typhus, tularaemia, hymenolepiasis and plague. The presence of active foci and history of human‐ and flea‐transmitted plague in northwest Iran prompted the present group to collect and identify fleas from human and livestock dwellings across West Azerbaijan Province. Adult fleas were collected and identified using routine taxonomic keys. Species designation was confirmed by sequencing the cytochrome oxidase subunit I (COI). Of the collected specimens (n = 989), 104 were collected off‐host (30 from human dwellings and 74 in light traps) and the rest were found on hosts (107 on animals and 778 by human bait). Of these fleas, 394 (40%) were male and 595 (60%) were female. The collected specimens belonged to the species Ctenocephalides canis, Ctenocephalides felis, Ctenocephalides orientis and Pulex irritans (all: Siphonaptera: Pulicidae). The amplified COI fragment, in addition to confirming the morphological identification of species, showed good efficacy in separating the different species in the phylogenetic analysis. In addition to the identification of fleas from human and livestock dwellings using morphological and molecular characteristics, the current paper represents the first report of the presence of C. orientis in northwest Iran. This finding suggests that changing climate conditions may have expanded the distribution of this species.     

High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers

The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché), is the most common flea species found on cats and dogs worldwide. We investigated the genetic identity of the cosmopolitan subspecies C. felis felis and evaluated diversity of cat fleas from Australia, Fiji, Thailand and Seychelles using mtDNA sequences from cytochrome c oxidase subunit I (cox1) and II (cox2) genes. Both cox1 and cox2 confirmed the high phylogenetic diversity and paraphyletic origin of C. felis felis. The African subspecies C. felis strongylus (Jordan) is nested within the paraphyletic C. felis felis. The south East Asian subspecies C. felis orientis (Jordan) is monophyletic and is supported by morphology. We confirm that Australian cat fleas belong to C. felis felis and show that in Australia they form two distinct phylogenetic clades, one common with fleas from Fiji. Using a barcoding approach, we recognize two putative species within C. felis (C. felis and C. orientis). Nucleotide diversity was higher in cox1 but COX2 outperformed COX1 in amino acid diversity. COX2 amino acid sequences resolve all phylogenetic clades and provide an additional phylogenetic signal. Both cox1 and cox2 resolved identical phylogeny and are suitable for population structure studies of Ctenocephalides species.     

Origin,evolution, phylogeny and taxonomy of Pulex irritans

The human flea Pulex irritans Linnaeus, 1758 (Siphonaptera: Pulicidae) is one of the most studied species together with the cat flea Ctenocephalides felis Bouché, 1835, because they have a cosmopolitan distribution and are closely related to humans. The present study aimed to carry out a comparative morphometric and molecular study of two different populations of P. irritans (Spain and Argentina). Accordingly, internal transcribed spacer (ITS)1 and ITS2 of rDNA and the partial cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) mtDNA genes of these taxa were sequenced. Furthermore, the taxonomy, origin, evolution and phylogeny of P. irritans was assessed. The morphometric data obtained did not show significant differences between P. irritans specimens from Spain and Argentina, even when these two populations were collected from different hosts; however, there was a considerable degree of molecular divergence between both populations based on nuclear and mitochondrial markers. Thus, it is proposed that P. irritans, in contrast with other generalist fleas, maintains a certain degree of morphological similarity, at least between Western Palearctic and Neotropical areas. Furthermore, two well defined geographical genetic lineages within the P. irritans species are indicated, suggesting the existence of two cryptic species that could be discriminated by a polymerase chain reaction‐linked restriction fragment length polymorphism.     

Fleas from domestic dogs and rodents in Rwanda carry Rickettsia asembonensis and Bartonella tribocorum

Fleas (Siphonaptera) are ubiquitous blood‐sucking parasites that transmit a range of vector‐borne pathogens. The present study examined rodents (n = 29) and domestic dogs (n = 7) living in the vicinity of the Volcanoes National Park, Rwanda, for fleas, identified flea species from these hosts, and detected Bartonella (Rhizobiales: Bartonellaceae) and Rickettsia (Rickettsiales: Rickettsiaceae) DNA. The most frequently encountered flea on rodents was Xenopsylla brasiliensis (Siphonaptera: Pulicidae). In addition, Ctenophthalmus (Ethioctenophthalmus) calceatus cabirus (Siphonaptera: Hystrichopsyllidae) and Ctenocephalides felis strongylus (Siphonaptera: Pulicidae) were determined using morphology and sequencing of the cytochrome c oxidase subunit I and cytochrome c oxidase subunit II genes (cox1 and cox2, respectively). Bartonella tribocorum DNA was detected in X. brasiliensis and Rickettsia asembonensis DNA (a Rickettsia felis‐like organism) was detected in C. felis strongylus. The present work complements studies that clarify the distributions of flea‐borne pathogens and potential role of fleas in disease transmission in sub‐Saharan Africa. In the context of high‐density housing in central sub‐Saharan Africa, the detection of B. tribocorum and R. asembonensis highlights the need for surveillance in both rural and urban areas to identify likely reservoirs.     

Detection and identification of Bartonella sp. in fleas from carnivorous mammals in Andalusia,Spain

A total of 559 fleas representing four species (Pulex irritans, Ctenocephalides felis, Ctenocephalides canis and Spilopsyllus cuniculi) collected on carnivores (five Iberian lynx Lynx pardinus, six European wildcat Felis silvestris, 10 common genet Genetta genetta, three Eurasian badger Meles meles, 22 red fox Vulpes vulpes, 87 dogs and 23 cats) in Andalusia, southern Spain, were distributed in 156 pools of monospecific flea from each carnivore, and tested for Bartonella infection in an assay based on polymerase chain reaction (PCR) amplification of the 16 S–23 S rRNA intergenic spacer region. Twenty‐one samples (13.5%) were positive and the sequence data showed the presence of four different Bartonella species. Bartonella henselae was detected in nine pools of Ctenocephalides felis from cats and dogs and in three pools of Ctenocephalides canis from cats; Bartonella clarridgeiae in Ctenocephalides felis from a cat, and Bartonella alsatica in Spilopsyllus cuniculi from a wildcat. DNA of Bartonella sp., closely related to Bartonella rochalimae, was found in seven pools of Pulex irritans from foxes. This is the first detection of B. alsatica and Bartonella sp. in the Iberian Peninsula. All of these Bartonella species have been implicated as agents of human diseases. The present survey confirms that carnivores are major reservoirs for Bartonella spp.     

Out-of-Africa,human-mediated dispersal of the common cat flea,Ctenocephalides felis: The hitchhiker’s guide to world domination

The cat flea (Ctenocephalides felis) is the most common parasite of domestic cats and dogs worldwide. Due to the morphological ambiguity of C. felis and a lack of — particularly largescale — phylogenetic data, we do not know whether global C. felis populations are morphologically and genetically conserved, or whether human-mediated migration of domestic cats and dogs has resulted in homogenous global populations. To determine the ancestral origin of the species and to understand the level of global pervasion of the cat flea and related taxa, our study aimed to document the distribution and phylogenetic relationships of Ctenocephalides fleas found on cats and dogs worldwide. We investigated the potential drivers behind the establishment of regional cat flea populations using a global collection of fleas from cats and dogs across six continents. We morphologically and molecularly evaluated six out of the 14 known taxa comprising genus Ctenocephalides, including the four original C. felis subspecies (Ctenocephalides felis felis, Ctenocephalides felis strongylus, Ctenocephalides felis orientis and Ctenocephalides felis damarensis), the cosmopolitan species Ctenocephalides canis and the African species Ctenocephalides connatus. We confirm the ubiquity of the cat flea, representing 85% of all fleas collected (4357/5123). Using a multigene approach combining two mitochondrial (cox1 and cox2) and two nuclear (Histone H3 and EF-1α) gene markers, as well as a cox1 survey of 516 fleas across 56 countries, we demonstrate out-of-Africa origins for the genus Ctenocephalides and high levels of genetic diversity within C. felis. We define four bioclimatically limited C. felis clusters (Temperate, Tropical I, Tropical II and African) using maximum entropy modelling. This study defines the global distribution, African origin and phylogenetic relationships of global Ctenocephalides fleas, whilst resolving the taxonomy of the C. felis subspecies and related taxa. We show that humans have inadvertently precipitated the expansion of C. felis throughout the world, promoting diverse population structure and bioclimatic plasticity. By demonstrating the link between the global cat flea communities and their affinity for specific bioclimatic niches, we reveal the drivers behind the establishment and success of the cat flea as a global parasite.     

Survey of Flea Infestation in Dogs in Different Geographical Regions of Iran

Medically important arthropods, including fleas, play an important role in causing clinical disorders and disease in man and domestic animals. This study was conducted to determine the seasonal flea infestations for domestic dogs from different geographic regions of Iran. A total of 407 fleas, belonging to 5 different species, were recovered from 83 domestic dogs from 3 regions. There was a distinctive pattern of species distribution and infestations with the highest infestation rates observed in a temperate climate and higher rainfall. Additionally, fleas were observed over all seasons, except February and March, with the highest infestation rate observed in August (24.7%) and the lowest rate in January (1.7%). They also parasitize dogs with a different spectrum of species. The cat flea, Ctenocephalides felis (67.5%), exhibited the highest prevalence among all flea species found on dogs. Thus, climatic conditions and seasonal patterns impact on flea infestation and must be considered in developing control programs.     

Responses of artificially reared cat fleas Ctenocephalides felis felis (Bouché, 1835) to different mammalian bloods

The cat flea, Ctenocephalides felis felis (Bouche, 1835) (Siphonaptera: Pulicidae), which is found worldwide and which parasitizes many species of wild and domestic animal, is a vector and/or reservoir of bacteria, protozoa and helminths. To aid in the study of the physiology and behaviour of fleas and of their transmission of pathogens, it would be of value to improve the laboratory rearing of pathogen‐free fleas. The conditions under which artificially reared fleas at the University of Bristol (U.K.) and the Rickettsial Diseases Institute (France) are maintained were studied, with different ratios of male to female fleas per chamber (25 : 50, 50 : 100, 100 : 100, 200 : 200). The fleas were fed with bovine, ovine, caprine, porcine or human blood containing the anticoagulants sodium citrate or EDTA. Egg production was highest when fleas were kept in chambers with a ratio of 25 males to 100 females. In addition, the use of EDTA as an anticoagulant rather than sodium citrate resulted in a large increase in the number of eggs produced per female; however, the low percentage of eggs developing through to adult fleas was lower with EDTA. The modifications described in our rearing methods will improve the rearing of cat fleas for research.     

Real‐time and multiplex real‐time polymerase chain reactions for the detection of Bartonella henselae within cat flea,Ctenocephalides felis,samples

Bartonella henselae (Rhizobiales: Bartonellacae), the agent of cat‐scratch disease, is an emerging bacterial pathogen which can be transmitted via infective faecal material of Ctenocephalides felis Bouché (Siphonaptera: Pulicidae). Worldwide, B. henselae has been identified in 1–53% of felines and 2.9–17.4% of fleas. Although culture is the routine method for detection, the procedure is time‐consuming and is rarely used for isolation directly from flea vectors. The current study reports the development of a quantitative real‐time polymerase chain reaction (qPCR) to detect and quantify B. henselae organisms from vector samples. The qPCR is specific and detects as few as 2.5 genome copies. To enable direct quantification of Bartonella organisms in different vector samples, we developed a qPCR to detect C. felis DNA that also acts as an extraction control. Combining both PCRs into a multiplex format validates B. henselae results when sampling flea populations, although there is a reduction in sensitivity. This reduction might be counteracted by a different combination of probe fluorophores.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

Assessment of Persistence of Bartonella henselae in Ctenocephalides felis

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.     

Abiotic factors influencing the ecology of wild rabbit fleas in north-eastern Spain

During 1992, the population dynamics of rabbit fleas were compared at two sites in north-eastern Spain. The sites differed mainly in terms of annual rainfall and soil type. All flea species showed seasonal cycles of abundance, although peaks in numbers occurred at different times, reflecting their specific adaptations for coping with climatic variables. Adult Spilopsyllus cuniculi (Dale) (Siphonaptera: Pulicidae) were found largely parasitizing rabbits in spring and adult Caenopsylla laptevi (Beaucournu etal.) (Siphonaptera: Ceratophyllidae) in the autumn. In contrast, monthly flea indices of Xenopsylla cunicularis (Smit) (Siphonaptera: Pulicidae) and Echidnophaga iberica (Ribeiro et al.) (Siphonaptera: Pulicidae) peaked in summer. Spilopsyllus cuniculi was present at both sites, but was less common on the drier site, where monthly mean temperature and annual rainfall approached the flea's physiological limits. By contrast, E. iberica, X. cunicularis and C. laptevi, known to be better adapted for dryness, showed the opposite patterns of abundance. Nevertheless, even these arid-adapted species took advantage of the milder and wetter spring (X. cunicularis and E. iberica) or autumn (C. laptevi) for breeding and larval development. Although environmental temperature, rainfall and soil texture will influence the microclimate of the burrows where the flea larvae develop, burrow humidity seems to be more dependent on soil characteristics and past rainfall rather than the humidity of the external air.     

Indicators of sexual maturation and regression in the female cat flea, Ctenocephalides felis

Differences in the salivary glands, mesenteron epithelium and reproductive organs of female cat fleas, Ctenocephalides felis Bouché (Siphonaptera: Pulicidae), are related to the degree of reproductive maturation or regression. Contrary to previous ideas, blue bodies in the ovarioles are degenerate oocyte nuclei and their presence denotes failure of ripening oocytes to reach full maturity. A distinction between true corpora lutea and pseudo-corpora lutea is established, the presence of the former indicates successful oviposition, and of the latter, failure to complete maturation of eggs. Accurate indicators of sexual maturation and reproductive success are of potential value in assessing relative suitability of various hosts for a given flea species and therefore in assessing the degree of host specificity among fleas.     

Accumulation and persistence of flea larvicidal activity in the immediate environment of cats treated with imidacloprid

To investigate the persistence of flea larvicidal activity in the immediate environment of cats treated with imidacloprid, eggs of the cat flea Ctenocephalides felis felis Bouché (Siphonaptera: Pulicidae), from untreated donor cats, were incubated on samples of fleece blanket taken from the floor of cages used by treated or untreated cats for a total of 10 or 20 6-h periods over 2-4 weeks, respectively. Sufficient imidacloprid accumulated during these periods to reduce the emergence of adult fleas by 94.7-97.6% when the blankets were tested after 18 weeks' storage at room temperature. A typical laundry procedure (washing with detergent at 50 degrees C and low temperature tumble drying) removed this biological activity. Unwashed control blankets did not support the flea life-cycle as effectively as washed blankets or a sand substrate.     

Bartonella species in fleas from Palestinian territories: Prevalence and genetic diversity

Bartonellosis is an infectious bacterial disease. The prevalence and genetic characteristics of Bartonella spp. in fleas of wild and domestic animals from Palestinian territories are described. Flea samples (n=289) were collected from 121 cats, 135 dogs, 26 hyraxes and seven rats from northern (n=165), central (n=113), and southern Palestinian territories (n=11). The prevalent flea species were: Ctenocephalides felis (n=119/289; 41.2%), Ctenocephalides canis (n=159/289; 55%), and Xenopsylla sp. (n=7/289; 2.4%). Targeting the Intergenic Transcribed Spacer (ITS) locus, DNA of Bartonella was detected in 22% (64/289) of all fleas. Fifty percent of the C. felis and 57% of the Xenopsylla sp. contained Bartonella DNA. DNA sequencing showed the presence of Bartonella clarridgeiae (50%), Bartonella henselae (27%), and Bartonella koehlerae (3%) in C. felis. Xenopsylla sp. collected from Rattus rattus rats were infected with Bartonella tribocorum, Bartonella elizabethae, and Bartonella rochalimae. Phylogenetic sequence analysis using the 16S ribosomal RNA gene obtained four genetic clusters, B. henselae and B. koehlerae as subcluster 1, B. clarridgeiae as cluster 2, while the rat Bartonella species (B. tribocorum and B. elizabethae) were an outgroup cluster. These findings showed the important role of cat and rat fleas as vectors of zoonotic Bartonella species in Palestinian territories. It is hoped that this publication will raise awareness among physicians, veterinarians, and other health workers of the high prevalence of Bartonella spp. in fleas in Palestinian territories and the potential risk of these pathogens to humans and animals in this region.     

Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI‐TOF MS

Ticks and fleas are vectors for numerous human and animal pathogens. Controlling them, which is important in combating such diseases, requires accurate identification, to distinguish between vector and non‐vector species. Recently, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to the rapid identification of arthropods. The growth of this promising tool, however, requires guidelines to be established. To this end, standardization protocols were applied to species of Rhipicephalus sanguineus (Ixodida: Ixodidae) Latreille and Ctenocephalides felis felis (Siphonaptera: Pulicidae) Bouché, including the automation of sample homogenization using two homogenizer devices, and varied sample preservation modes for a period of 1–6 months. The MS spectra were then compared with those obtained from manual pestle grinding, the standard homogenization method. Both automated methods generated intense, reproducible MS spectra from fresh specimens. Frozen storage methods appeared to represent the best preservation mode, for up to 6 months, while storage in ethanol is also possible, with some caveats for tick specimens. Carnoy's buffer, however, was shown to be less compatible with MS analysis for the purpose of identifying ticks or fleas. These standard protocols for MALDI‐TOF MS arthropod identification should be complemented by additional MS spectrum quality controls, to generalize their use in monitoring arthropods of medical interest.     

Predictive factors for flea occurrence in red foxes (Vulpes vulpes) from semi-arid Mediterranean environments

The role of red fox as host for a wide range of parasites, particularly fleas and other arthropods causing vector-borne diseases, in combination with its capability to adapt to anthropized environments, makes this wild canid an epidemiologically remarkable species at the wildlife–domestic–human interface, especially in the present time of rise of emerging and re-emerging diseases. This study evaluated the prevalence and parasite intensity of fleas in 88 foxes from Murcia Region (Southeastern Spain) and determined the geographic distribution of areas with the highest potential risk of flea presence. Pulex irritans, Ctenocephalides felis, Spilopsyllus cuniculi and Nosopsyllus fasciatus were identified. The overall prevalence was 76.13%. This is the first time that N. fasciatus has been reported in foxes from Murcia Region. The predictive model established a certain pattern to determine the areas with the highest risk of acquiring fleas. Positive correlation of daily potential evapotranspiration (ET₀) in winter and the opposite effect occurring for ET₀ in summer were obtained, as well as positive correlations for mean daily temperature (T_mean) in summer and mean precipitation (P_mean) in winter and summer. The model was also found positively correlated in the forest habitat ecotone areas and the anthropized areas.