In the present study, the flea infestation rate was high for both cats (28.1 ± 3.09%) and dogs (14.4 ± 2.67%), but both host species were almost equally likely to carry the cat flea, C. f. felis. Relatively few dog fleas were found, but these did occur more frequently on dogs. Relatively low numbers of rabbit, hedgehog and hen fleas were found, with no Pulex irritans identified. The protocol for recruitment to the study highlighted the need for veterinarians to seach pets brought into the practice for reasons other than flea infestation, suggesting that if known flea-infested animals were included, the prevalence would be even higher. Previous studies have shown that the flea species primarily associated with cats is C. f. felis; however, for dogs the results are more variable. Ctenocephalides felis felis was shown to be the most common species of flea on domestic dogs in the UK [30,31,32]. High levels of infestation of dogs by C. f. felis have also been reported in North America [33], Denmark [34] and Finland [35]. In contrast, C. canis was found to be more common on dogs in the UK than C. f. felis [36], as was the case in Ireland [37, 38]. In a further study in Ireland of 193 cases, 90% of all infestations on cats were with C. f. felis; only a single cat was found to be infested by C. canis. In contrast, in the dogs 17.5% were infested by C. f. felis and 75.7% by C. canis [39]. A preponderance of C. canis on dogs has also been reported in New Zealand [12], Denmark [40] and the Republic of Korea [41]. In a study of fleas infesting kennel dogs from two localities in Israel, a total of 355 fleas were collected from 107 dogs, of which 74.8% had C. canis, 63.6% had C.f. felis, 14.0% had P. irritans and one animal had Xenopsylla cheopis [42].

Flea borne pathogens in cats and dogs can result in significant levels of clinical disease and are of concern for veterinarians and pet owners [16]. In this study, 11% ± 2.85% of the pooled flea samples of different species and collected from both cats and dogs were found positive for Bartonella spp. Four different Bartonella species were found in the positive flea samples, and these samples were distributed mostly around central and southern UK. This could possibly be because fewer veterinary practices were recruited in northern parts of the UK, but equally may reflect a more southerly distribution of fleas since relatively few cases were submitted from northern England or Scotland; further spatial analysis is required to quantify this trend. The effects of infection by Bartonella spp. may range from asymptomatic to fatal. The most common zoonotic species is B. henselae, for which cats are the major natural reservoir. Fleas, C. f. felis in particular, are the known vectors for B. henselae, B. clarridgeiae and Bartonella koehlerae [17]. The results of this study highlight the anticipated strong association between Bartonella spp. and C. f. felis feeding on cats. Evidence of exposure to Bartonella spp. in cats has been found in many countries, particularly in regions with high humidity [43, 44]. In cats, bartonellosis can result in lymphadenopathy, endocarditis, myocarditis and hyperglobulinemia. However, most cats infected with a Bartonella spp. will show no clinical signs [45]. Since B. henselae survives at least nine days in flea faeces, flea control is imperative to attempt to reduce the risk of infection of other cats, dogs, or people [9, 46]. In this study Bartonella spp. were also found in fleas collected from dogs, which has also been noted in other studies [42] where 7.8% of pooled flea samples from kennel dogs from two localities in Israel were positive for Bartonella DNA. It should be noted that qPCR for Bartonella spp. detection alone may not be the most sensitive approach, which may be achieved more accurately using a combination of conventional and nested PCRs from blood and liquid culture samples [1]. Hence, the prevalence figures reported here may be an underestimate.

Dipylidium caninum is a common intestinal cestode parasite of dogs and cats. The onchospheres are contained in egg packets, each with about 20 eggs, and these are either expelled by the active segment or released by its disintegration. After ingestion by a larval flea intermediate host, the onchospheres travel to its haemocoel, where they develop into cysticercoids. The final host is infected by ingestion of the flea containing the cysticercoids. Occasionally humans have become infected following ingestion of the saliva of infected pets [12]. The potential zoonotic transmission and wide geographical range emphasize the importance of protecting dogs and cats from D. caninum. Routine treatment may be an effective approach to tapeworm management; however, the brief pre-patent period and lack of residual activity of most treatments means that reinfection may occur rapidly. Hence effective and persistent flea control is an important element of any tapeworm management regime. A relatively low prevalence of Dipylidium has been reported in studies using coproscopy. For example, in a study of 2775 dog faecal samples from the Lazio Region of central Italy (1156 from households and 1619 from shelter dogs) only 0.1% were found to be infected [16]. In Greece, in a study of the faeces from 1150 cats, a prevalence of 0.2% was detected [47]. However, the poor sensitivity of coproscopy means that faecal analysis is likely to significantly underestimate infection by Dipylidium. In PCR analyses of flea samples from 435 cats, 4.37% of fleas were found to be infected with D. caninum and in fleas from 396 dogs, 9.1% carried Dipylidium [48]. In the present study, the prevalence of D. caninum was similar, with 14 of the 470 (3 ± 1.53%) flea DNA samples found to be positive for D. caninum DNA but the majority of the D. caninum-positive flea samples were collected from cats and only one was collected from a dog. Of particular interest is the recent finding of two distinct genotypes of D. caninum in dogs and cats, suggesting that two distinct species may be present [49], but this was not investigated in the present study.

Like Bartonella spp., the DNA of haemoplasmas has been amplified from the blood of cats in many regions of the world [44], with M. haemofelis usually considered to be the most pathogenic species [22, 50]. The present study found only 3 flea samples positive for M. haemofelis or M. haemocanis DNA, and it was not possible to differentiate between these two haemoplasma species as their 16S rDNA sequences are near identical [51]. A similarly low prevalence of M. haemofelis DNA was found in ticks collected from pets in the UK [24] and no haemoplasma DNA was found in fleas collected from cats in southern Italy [44]. In contrast in a study involving 1585 cats, found 2.8% cats to be positive for M. haemofelis and 1.7% positive for “Ca. M. turicensis” [29]. The zoonotic importance of feline haemoplasmas is still being questioned [52]. Clinical signs of disease depend on the degree of anaemia, the stage of infection and the immune status of infected cats. Direct transmission may occur with the hemoplasmas, and studies have found some of the agents in saliva [53] and that subcutaneous inoculation of hemoplasma-containing blood resulted in infection transmission [54]. Infection does not necessarily result in clinical disease and in some cases healthy cats can also be positive for haemoplasma DNA in blood [29, 55, 56] and so PCR assay results do not always correlate well with clinical illness.

The methods used to evaluate the prevalence of flea-borne pathogens and the role of fleas as vectors vary in their sensitivity and each is subject to different biases. The use of host blood samples can be problematic. In a study of ectoparasites on cats and vector-borne pathogens in feline blood samples in southern Italy which used qPCR there was little agreement between serological and molecular results in individual cats and the presence of ectoparasites, with the exception of B. clarridgeiae and B. henselae [44]. The authors argued that the ability to detect pathogens in the blood depends strongly on the immunological sensitivity of the host; in addition, the bacteraemia of some pathogens is transient, lasting only a few hours. This makes it difficult to detect pathogens in the blood and requires samples to be taken at very specific time points and the use of highly sensitive molecular tools; the use of serology may therefore underestimate the prevalence of pathogens. The present study investigated the presence of pathogen DNA amplified from fleas but, even though fleas tend to remain on the same host once acquired, the blood in the gut could potentially have come from more than one host, particularly where pets live in close contact in the same household; the presence of pathogen DNA in the gut alone also does not demonstrate vectoral competence [24]. There is a possibility of carryover of the pathogen DNA from the host blood, especially where the Ct values of the qPCR is higher than 36 cycles. The detection of D. caninum DNA from adult fleas collected off a host also requires careful interpretation because, although it is highly indicative, it represents the potential for infection rather than infection itself, since the adult flea would need to be ingested by the grooming host to result in infection. As a result, ideally, a combination of epidemiological indicators is required to establish the true prevalence and the role of arthropod vectors in the transmission of pathogens [57].