Individual Fleas Research Articles

Evidence for a specific host-endosymbiont relationship between 'Rickettsia sp. genotype RF2125' and Ctenocephalides felis orientis infesting dogs in India.

BackgroundFleas of the genus Ctenocephalides serve as vectors for a number of rickettsial zoonoses, including Rickettsia felis. There are currently no published reports of the presence and distribution of R. felis in India, however, the ubiquitous distribution of its vector Ctenocephalides felis, makes it possible that the pathogen is endemic to the region. This study investigates the occurrence of Rickettsia spp. infection in various subspecies of C. felis infesting dogs from urban areas of Mumbai, Delhi and Rajasthan in India.MethodsIndividual fleas collected off 77 stray dogs from Mumbai, Delhi and Rajasthan were screened for Rickettsia spp. by a conventional PCR targeting the ompB gene. Further genetic characterisation of Rickettsia-positive fleas was carried out using nested PCR and phylogenetic analysis of partial DNA sequences of the gltA and ompA genes. Ctenocephalides spp. were morphologically and genetically identified by PCR targeting a fragment of cox1 gene.ResultsOverall, 56/77 fleas (72.7%), including 22/24 (91.7%) from Delhi, 32/44 (72.7%) from Mumbai and 2/9 (22.2%) from Rajasthan were positive for Rickettsia DNA at the ompB gene. Sequences of gltA fragments confirmed the amplification of Rickettsia sp. genotype RF2125. The ompA gene of Rickettsia sp. genotype RF2125 was characterised for the first time and shown 96% identical to R. felis. Three species of Ctenocephalides were identified, with the Ctenocephalides felis orientis being the dominant flea species (69/77; 89.6%) in India, followed by Ctenocephalides felis felis (8/77; 10.4%).ConclusionsHigh occurrence of Rickettsia sp. genotype RF2125 in C. felis orientis and the absence of R. felis suggests a specific vector-endosymbiont adaptation and coevolution of the Rickettsia felis-like sp. within subspecies of C. felis.

Speed of kill efficacy and efficacy of flavored spinosad tablets administered orally to cats in a simulated home environment for the treatment and prevention of cat flea (Ctenocephalides felis) infestations

The efficacy of spinosad against adult fleas (Ctenocephalides felis) on cats was evaluated in two separate controlled, blinded studies—one to determine flea knockdown and speed of flea kill (SOFK) on experimentally infested cats, another to assess the ability of spinosad to prevent flea infestations in a simulated home environment (SHE) study design. In each study, pre-treatment live flea counts were used as a blocking factor to randomize cats to treatment, and treated in the fed state, with flavored tablets containing either no active ingredient (control) or spinosad (50–100mg/kg in the SOFK study; 50–75mg/kg body weight in the SHE study). In the SOFK study, 6 cats per group were infested with unfed adult fleas on Day −1. Groups 1–5 received control tablets; groups 6–10 received spinosad tablets. Flea counts were conducted at 0.5, 2, 4, 8 and 24h post-dosing. In the SHE study, 12 flea-free cats per group, treated on Days 0, 30 and 60, were maintained in solid-sided cages with solid carpeted floors. Each cat was infested on Days 1, 7 and 14 with 100 unfed adult fleas. Individual flea comb counts were performed on Days 3, 9, 16, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91 and 95. After each count, except Day 95, up to 300 live fleas were replaced on each cat. To augment flea challenge, the carpeted area in each cage was sprinkled weekly with larval flea growth media (dried blood, yeast). In the SOFK study, reductions in mean flea counts in the spinosad groups were observed at all post-treatment assessments, beginning at 0.5h post-infestation with significant differences (p<0.0001) from vehicle-treated cats from 2h post-treatment when efficacy was >90%, through the final flea counts 24h post-infestation when no fleas were found on spinosad treated cats. In the SHE study, GM post-treatment flea counts in the control group ranged between 38.9 and 107.0 (arithmetic means 58.8–118.1); no live fleas were combed from spinosad-treated cats (100% effectiveness) at any time point post-treatment. No adverse events that were attributable to the treatments were observed in either study. These studies demonstrated that spinosad administered orally to cats is safe and effective, providing >90% efficacy from 2h post-dosing and 100% knockdown at 24h, and preventing infestations over a 95 day study period from a flea-contaminated simulated home environment.

Prevalence of Rickettsia felis and the First Identification of Bartonella henselae Fizz/CAL-1 in Cat Fleas (Siphonaptera: Pulicidae) From Taiwan

Cat fleas (Ctenocephalides felis [Bouché]) are the primary ectoparasites of dog and cat populations. In this study, we report the monthly population dynamics of Rickettsia felis and Bartonella spp. (two zoonotic pathogens that can cause human disease) in cat fleas collected from dogs and cats in Taipei, Taiwan, from December 2006 to December 2007. Natural R. felis infection in individual cat fleas was assessed by polymerase chain reaction (PCR) using pRF-, ompB-, and gltA-specific primer pairs. Samples positive by PCR were confirmed with DNA sequencing. R. felis was detected in cat fleas year round, and the average infection rate was 21.4% (90 of 420) in 2007. Cat fleas also play an important role in the transmission of Bartonella between reservoirs and other mammalian hosts. In this study, we used primer pairs specific for the Bartonella gltA and rpoB genes to detect Bartonella infections. Of the 420 cat fleas tested, 38 were positive by PCR for Bartonella. Sequence similarities to Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were observed in 6.2% (26 of 420), 2.1% (9 of 420), and 0.7% (3 of 420) of the fleas, respectively. Based on the pap31 gene sequence, several amplicons of the B. henselae detected in the cat fleas could be subgrouped into three strains: Fizz/CAL-1 (n = 18), Marseille (n = 5), and Houston-1 (n = 3). These results demonstrate that cat fleas infected with R. felis are endemic to Taiwan, and highlight the role of C. felis in Bartonella transmission between reservoirs and other mammal hosts and demonstrate the genetic variability of B. henselae in Taiwan.

Discrimination of host sex by a haematophagous ectoparasite

Sex biases in parasite infestation are known for various host–parasite systems. In most cases, males of higher vertebrates are infested by more parasites than females. The mechanisms behind male-biased infestation may include not only more opportunities for males to encounter parasites and/or better performance of a parasite in male hosts but also active selection of these hosts by a parasite. To test this hypothesis, we studied behavioural responses of the flea Xenopsylla ramesis to males and females of its common rodent host Meriones crassus in Y-maze trials. We asked whether an individual flea is able to discriminate between male and female hosts and choose a male host, and whether previous experience of the fleas (never fed, fed once on a male host, fed once on a female host) affects the choice. Among 385 fleas, 62% made a choice. In general, proportions of female fleas making or not making a choice of a rodent did not differ significantly, whereas the majority of male fleas chose a rodent. Latency of rodent choice was similar between male and female fleas, but was strongly affected by experience: fleas that previously fed on a male rodent or were never fed made their choices significantly faster than fleas previously fed on a female rodent. Among fleas that made a choice, selection of a male versus a female rodent depended on flea sex only. Male fleas chose randomly between a male and a female rodent, whereas female fleas chose a male rodent significantly more often than a female rodent. Our results suggest that male-biased flea infestation may involve active choices of male hosts by fleas.

The effect of larval density on pre-imaginal development in two species of desert fleas

We studied the effect of density of larvae on pre-imaginal development in 2 flea species (Xenopsylla conformis and Xenopsylla ramesis) parasitic on 2 desert rodent species (Dipodillus dasyurus, adult body mass 20 g and Meriones crassus, 80 g). We predicted a decrease in duration of development with an increase in density of larvae. In general, in both flea species, duration of larva-to-pupa development decreased with an increasing larval density. In addition, this stage of development was longer in male fleas and in fleas from parents fed on D. dasyurus. The effect of larval density on larval development was manifested mainly when parent fleas fed on D. dasyurus. Duration of pupation decreased with increasing larval density only in offspring of fleas fed on G. dasyurus. In both fleas, pupation was longer in males. The effect of parent host on duration of pupation was found in X. ramesis only (longer if the host was M. crassus). Resistance of newly emerged fleas to starvation depended mainly on parent host species. Young X. conformis survived longer if their parents fed on D. dasyurus, whereas young X. ramesis survived longer if their parents fed on M. crassus. It was also found that (a) an individual flea that spent more time as a larva also spent more time as a pupa and (b) longer larval development resulted in a shorter time that a newly emerged flea was able to survive when starved.

Bacterial communities of disease vectors sampled across time, space, and species.

A common strategy of pathogenic bacteria is to form close associations with parasitic insects that feed on animals and to use these insects as vectors for their own transmission. Pathogens interact closely with other coexisting bacteria within the insect, and interactions between co-occurring bacteria may influence the vector competency of the parasite. Interactions between particular lineages can be explored through measures of alpha-diversity. Furthermore, general patterns of bacterial community assembly can be explored through measures of beta-diversity. Here, we use pyrosequencing (n=115,924 16S rRNA gene sequences) to describe the bacterial communities of 230 prairie dog fleas sampled across space and time. We use these communinty characterizations to assess interactions between dominant community members and to explore general patterns of bacterial community assembly in fleas. An analysis of co-occurrence patterns suggests non-neutral negative interactions between dominant community members (P<0.001). Furthermore, bacterial communities of fleas shift dramatically across years (phylotype-based: R=0.829, P<0.001; phylogenetic-based: R=0.612-0.753, P<0.001), but they also significantly differ across space (phylotype-based: R=0.418, P<0.001; phylogenetic-based: R=0.290-0.328, P<0.001) and between flea species (phylotype-based: R=0.160, P=0.011; phylogenetic-based: not significant). Collectively, our results show that flea-associated bacterial communities are not random assemblages; rather, an individual flea's bacterial community is governed by interactions between bacterial lineages and by the flea's place in space and time.

Rickettsia felisin Cat Fleas in Taiwan

We describe the first detection of Rickettsia felis in cat fleas (Ctenocephalides felis) in Taiwan. Natural infections of R. felis in cat fleas were isolated and confirmed using polymerase chain reaction (PCR) and an immunofluorescence assay. The infection rate in individual fleas and the minimum infection rate in pooled fleas detected by the PCR method were found to be 18.8% (13/69) and 8.2% (8/97), respectively. Partial sequences of the plasmid pRF, 17-kDa antigen, and outer membrane protein A genes obtained from the samples are identical to those of R. felis URRWXCal2. Serological studies confirmed R. felis infection in two stray cats, as demonstrated by the presence of serum IgG antibodies against R. felis with an immunofluorescence assay titer of 1:320.

Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas

Background Rickettsia felis is a flea-associated rickettsial pathogen recurrently identified in both colonized and wild-caught cat fleas, Ctenocephalides felis. We hypothesized that within colonized fleas, the intimate relationship between R. felis and C. felis allows for the coordination of rickettsial replication and metabolically active periods during flea bloodmeal acquisition and oogenesis.Methodology/Principal FindingsA quantitative real-time PCR assay was developed to quantify R. felis in actively feeding R. felis-infected fleas. In three separate trials, fleas were allowed to feed on cats, and a mean of 3.9×106 R. felis 17-kDa gene copies was detected for each flea. A distinct R. felis infection pattern was not observed in fleas during nine consecutive days of bloodfeeding. However, an inverse correlation between the prevalence of R. felis-infection, which ranged from 96% in Trial 1 to 35% in Trial 3, and the R. felis-infection load in individual fleas was identified. Expression of R. felis-infection load as a ratio of R. felis/C. felis genes confirmed that fleas in Trial 3 had significantly greater rickettsial loads than those in Trial 1.Conclusion/SignificanceExamining rickettsial infection dynamics in the flea vector will further elucidate the intimate relationship between R. felis and C. felis, and facilitate a more accurate understanding of the ecology and epidemiology of R. felis transmission in nature.

Asymmetry in the Expression of Bilateral Morphs as an Indicator Showing the Character of Interrelations Between Fleas and the Plague Pathogen

The level of fluctuating asymmetry (FA) of bilateral signs in fleas was shown to reflect a certain character of their interrelations with Yersinia pestis. Flea imagoes that had bacterial aggregations in their proventriculi demonstrated greater signs of FA chaetotaxy. Besides, in the experiments with groups of fleas originally differing in the intensity of FA expression, increased vector capacity was characteristic of individual fleas with higher levels of this index or those caught in the areas of the natural foci where epizootics had been registered. These peculiar interrelations manifested themselves both at intra- and inter-population levels in several species of Siphonaptera order, as well as in the imago hybrids originating from crosses between C.tesquorum altaicus and C.t.sungaris.

Bacterial Communities of Bartonella -Positive Fleas: Diversity and Community Assembly Patterns

We investigated the bacterial communities of nine Bartonella-positive fleas (n = 6 Oropsylla hirsuta fleas and n = 3 Oropsylla montana fleas), using universal primers, clone libraries, and DNA sequencing. DNA sequences were used to classify bacteria detected in a phylogenetic context, to explore community assembly patterns within individual fleas, and to survey diversity patterns in dominant lineages.

Interaction frequency across the geographical range as a determinant of host specialisation in generalist fleas

The strength of interspecific interactions varies over geographical scales, and can influence patterns of resource specialisation. Even with gene flow preventing local adaptation of a consumer to particular resources, we might expect that across its entire range, the consumer would show some specialisation for the resource types most likely to be encountered across the localities where it occurs. We tested the hypothesis that generalist fleas are more successful at exploiting small mammalian host species with which they co-occur frequently across their geographical range than host species that, though suitable, are encountered less frequently. This hypothesis was tested with data on 121 flea species compiled from field surveys across 35 regions of the Palaearctic. Using abundance (mean number of individual fleas per individual host) as a measure of flea success on a particular host species, positive correlations between flea abundance and the frequency of co-occurrence of a flea with each of its hosts amongst all regions surveyed were found in all but two of the flea species investigated, with one-fifth of these being significant. If overlap in geographical range between flea and host is used as a measure of frequency of encounters instead of the actual proportion of regions where they both occur, similar patterns are observed, though they are much weaker. In a comparative analysis across all flea species, there were significant relationships between the average abundance of fleas and average values of both measures of frequency of encounters (proportion of sites where they co-occur and range overlap), even when correcting for potential phylogenetic influences. The results suggest that for any given flea species, host species more commonly encountered throughout the spatial range of the flea are generally those on which the flea does best. Interaction frequency may be a key determinant of specialisation and abundance in host–parasite systems.

Rickettsia felisin Chile

To the Editor: Rickettsiosis due to Rickettsia felis is an emerging disease that has been reported worldwide (1). Fever, headache, myalgia, and macular rash have been attributed to R. felis infection in humans (1). In South America, R. felis infection in fleas (mostly Ctenocephalides spp.) has been reported only in Brazil, Peru, and Uruguay (2–3). Although a growing number of articles have reported that R. felis is transmitted by fleas, the acquisition mechanism of R. felis by vertebrates or uninfected fleas in nature remains unknown (4). Cats experimentally exposed to R. felis–infected fleas have been shown to become seropositive (5). However, neither serologic nor molecular evidence of R. felis infection has been reported in cats under natural conditions, despite the fact that most C. felis fleas are infected by R. felis (6,7). In November 2006, we investigated the presence of rickettsial DNA in 30 C. felis fleas randomly collected from 22 domestic cats privately owned and housed indoors in a single household in Santiago, Chile. To detect rickettsial DNA in each individual flea, PCRs were performed that targeted a 398-nt fragment of the rickettsial gltA gene and an 856-nt fragment of the rickettsial ompB gene (7,8). A total of 21 individual fleas (70%) yielded expected PCR products for both gltA and ompB genes. PCR gltA products from the 21 fleas and ompB products from 5 fleas were subjected to DNA sequencing as described (7). The gltA partial sequences obtained from 21 fleas were identical, as were the ompB partial sequences from 5 fleas. These sequences were 100% identical to corresponding sequences in the R. felis genome (GenBank accession no. {type:entrez-nucleotide,attrs:{text:C53,term_id:67003925,term_text:C53}}C53). Blood serum samples were collected from the 22 cats and tested by indirect immunofluorescence assay (IFA) with crude antigens derived from 6 Rickettsia isolates from Brazil: R. bellii, R. amblyommii, R. rhipicephali, R. rickettsii, R. parkeri, and R. felis (7,9). Serum was considered to contain antibodies against rickettsiae if it displayed a reaction at 1:64 dilution. End-point titers against each Rickettsia species were determined by testing serial 2-fold serum dilutions. Reactive serum specimens were tested in 2 or 3 replications by 2 readers before the end-point titer was determined. Serum showing a Rickettsia species titer at least 4-fold higher than those observed for the other Ricketttsia species was considered homologous to the first Rickettsia species or to a very closely related genotype (7,9). In each slide, a nonreactive cat serum specimen (negative control) and a known reactive cat serum specimen (positive control) were tested at the 1:64 dilution (7). IFA detected antibodies reactive with R. felis (titer >64) in 16 (72.7%) of 22 cats. Among those, 5 (22.7%) also reacted with R. rhipicephali, 4 (18.2%) with R. bellii, 3 (13.6%) with R. parkeri, 2 (9.1%) with R. rickettsii, and 1 (4.5%) with R. amblyommii. No serum reacted with any other Rickettsia species without reacting with R. felis (Table). Four cat serum specimens (cats 1, 3, 8, and 11) showed titers to R. felis at least 4-fold higher than those to any of the other 5 antigens. The antibody titers in these 4 animals were considered to have been stimulated by R. felis infection. For the remaining 12 seropositive cats, we could not discern whether R. felis had been the infection agent because the results displayed a single titer of 64 for R. felis or showed similar titers for other Rickettsia species. Table End-point titers of indirect immunofluorescence assay (IFA) for 6 Rickettsia species in cats from a household in Santiago, Chile* We report 70% R. felis–infected fleas in this study on the basis of the concordant results of 2 PCR amplifications (gltA and ompB) and DNA sequencing. This infection rate is within the range (13.5%–90%) that has been reported for R. felis infecting Ctenocephalides fleas in Brazil and Uruguay (2,3,7). Sixteen (72.7%) cats contained R. felis–reactive antibodies; 4 of them showed titers to R. felis at least 4-fold higher than those to the other 5 rickettsial strains, findings that enabled us to technically conclude that these cats were exposed to R. felis or a closely related organism (1,7,9). Our finding of 70% R. felis infection in fleas infesting the cats indicates that cats acquired the infection through infected fleas. However, the mechanism of R. felis transmission by fleas is yet to be demonstrated under experimental conditions. To our knowledge, the presence of R. felis, or a spotted fever group Rickettsia species, has not been reported in Chile. Recent investigations have provided clinical and serologic evidence of canine (10) and human (K. Abarca and J. Lopez, unpub. data) infection by spotted fever rickettsia in Chile, confirmed by IFA that used R. conorii commercial antigen. Since substantial serologic cross-reaction occurs between R. conorii and R. felis antigens (1), R. felis could be causing infection in dogs or humans in Chile.

Omnidirectional Electric Fish

Omnidirectional Electric Fish

Host condition and host immunity affect parasite fitness in a bird–ectoparasite system

SummaryParasites might preferentially feed on hosts in good nutritional condition as such hosts provide better resources for the parasites’ own growth, survival and reproduction. However, hosts in prime condition are also better able to develop costly immunological or physiological defence mechanisms, which in turn reduce the parasites’ reproductive success. The interplay between host condition, host defence and parasite fitness will thus play an important part in the dynamics of host–parasite systems.In a 2 × 2 design, we manipulated both the access to food in great titParus majorbroods and the exposure of the nestlings to hen fleasCeratophyllus gallinae, a common ectoparasite of hole‐breeding birds. We subsequently investigated the role of manipulated host condition, host immunocompetence, and experimentally induced host defence in nestlings on the reproductive success of individual hen flea females.The food supplementation of the nestlings significantly influenced the parasites’ reproductive success. Female fleas laid significantly more eggs when feeding on food‐supplemented hosts.Previous parasite exposure of the birds affected the reproductive success of fleas. However, the impact of this induced host response on flea reproduction depended on the birds’ natural level of immunocompetence, assessed by the phytohaemagglutinin (PHA) skin test. Flea fecundity significantly decreased with increasing PHA response of the nestlings in previously parasite‐exposed broods. No relationship between flea fitness and host immunocompetence was, however, found in previously unexposed broods. The PHA response thus correlates with the nestlings’ ability to mount immunological or physiological defence mechanisms against hen fleas. No significant interaction effect between early flea exposure and food supplementation on the parasites’ reproductive success was found.Our study shows that the reproductive success of hen fleas is linked to the hosts’ food supply early in life and their ability to mount induced immunological or physiological defence mechanisms. These interactions between host quality and parasite fitness are likely to influence host preference, host choice and parasite virulence and thus the evolutionary dynamics in host–parasite systems.

Rickettsia felis (Rickettsiales: Rickettsiaceae) in Ctenocephalides felis felis (Siphonaptera: Pulicidae) in the State of São Paulo, Brazil

Samples of 10 and 14 Ctenocephalides felis felis fleas were collected on dogs from Pedreira and Mogi das Cruzes municipalities, respectively, in the State of São Paulo, Brazil, for detection of Rickettsia spp. Individual fleas were submitted to Polymerase Chain Reaction targeting the 17-kDa and the 190-kDa (OmpA) genes of Rickettsiae. This later gene is specific for spotted fever group. Nine fleas from Pedreira (90%) and four fleas from Mogi das Cruzes (28%) were positive for the 17-kDa gene, and eight fleas from Pedreira (80%) and four from Mogi das Cruzes (28%) were positive for 190-kDa gene. The nucleotide sequence of the 190-kDa products of one flea from Pedreira and one flea from Mogi das Cruzes were 100% identical to each other, and when compared to the GenBank Data, they were 100% identical to the 190-kDa sequence of R. felis. This was the first report of its occurrence in the State of São Paulo.

Detection ofRickettsia felisin a New World Flea Species,Anomiopsyllus nudata(Siphonaptera: Ctenophthalmidae)

The flea and rodent samples studied in this project were collected from field study sites in New Mexico from winter 1998 to spring 2001. During this period, 155 small rodents (14 different species) were live-trapped and combed for the presence of fleas. A total of 253 fleas were collected, comprising 21 species. Two of the 253 fleas collected were polymerase chain reaction (PCR) positive for the Rickettsia 17-kDa protein gene. These two fleas were both Anomiopsyllus nudata Baker, each collected from an individual Neotoma albigula Hartley, on two occasions. Individual fleas positive for the Rickettsia 17-kDa protein gene were then tested with primers targeting the rickettsial genes for citrate synthase (gltA) and two major outer membrane proteins (ompA and ompB). The nucleotide sequences of the PCR products of these two fleas were identical to each other and were 100% (394/394), 100% (1150/1150), 99.8% (469/470), and 99.3% (818/824) similar to the corresponding sequences of the 17-kDa, gltA, ompA, and ompB genes of Rickettsia felis, respectively. Flea homogenates of individual PCR-positive fleas were inoculated into shell vials seeded with Vero cells, and the Gimenez stain technique was used to demonstrate the presence of Rickettsia-like organisms in detached cells found in aspirated medium 19 d after inoculation. These cells were harvested and tested by PCR, targeting portions of the 17-kDa and gltA genes, resulting in products 100% identical to R. felis. This work comprises the first report of R. felis detection in a flea species (A. nudata) endemic to the New World.

ミジンコ類急性遊泳阻害試験のための仔ミジンコ自動分取装置の開発

“Daphnia sp. acute immobilisation test” is a typical ecotoxicity test for chemicals. In the test procedures, the most troublesome operation is separating and collecting hundreds of water fleas in preparing the test. In this study, the automatic water flea separator and collector have been developed to simplify the test. Using the separator, 95% of the population of targeted individual Daphnia were separated from the untargeted Daphnia. Using the collector, the separated individual water fleas were collected in groups of 5 (average: 5.2, standard deviation: 0.8). The toxicity tests for potassium dichromate were performed and the efficacy of the device was evaluated. According to the results of the Daphnia sp., Acute Immobilisation Test, major damage was not observed in the individual Daphnia separated and collected automatically.

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.

Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations.

RV014 Q-fever — Epidemiology and control

Four hundred and sixty seven Ctenocephalides felis fleas removed from 185 feral cats living in residential areas of Jerusalem, Israel, were screened for bacterial infections of public health importance. The fleas were screened for bartonellae, rickettsiae and Coxiella burnetii by PCR and sequencing. Bartonella DNA was detected in 156 individual fleas collected from 91 of the 185 (49.2%) cats. DNA of Bartonella clarridgeiae, Bartonella henselae and Bartonella koehlerae was detected in 112/467 (24%), 29/467 (6.2%) and 15/467 (3.2%), respectively, indicating a significantly different distribution (P < 0.00001) of these Bartonella spp. among the fleas. However, no differences were observed between female and male fleas in their Bartonella-infection status (P > 0.05). Ninety one individual cats carried fleas infected with 1 to 3 Bartonella species. No differences were found between fleas collected from male and female, pregnant and non-pregnant or young, juvenile and adult cats. Interestingly, a significant association was observed between the clinical status of the cat hosts (apparently healthy versus sick) and the carriage of Bartonella-positive fleas. One of the 467 (0.2%) fleas was positive for Rickettsia felis DNA and no other Rickettsia spp. or C. burnetii DNA were detected. Our findings indicate a relatively high prevalence of Bartonella spp. known to be human pathogens, and low prevalence of R. felis in fleas from the Jerusalem district cats, highlighting the abundance and importance of bartonellae for public health in this urban region.