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In JoVE (1)
Other Publications (24)
- Journal of Vector Ecology : Journal of the Society for Vector Ecology
- Journal of Medical Entomology
- Journal of Medical Entomology
- Journal of Vector Ecology : Journal of the Society for Vector Ecology
- Applied and Environmental Microbiology
- Journal of Medical Entomology
- Veterinary Parasitology
- The ISME Journal
- Journal of Medical Entomology
- Vector Borne and Zoonotic Diseases (Larchmont, N.Y.)
- Journal of the American Mosquito Control Association
- Veterinary Parasitology
- Journal of Economic Entomology
- PloS One
- International Journal for Parasitology
- Journal of Medical Entomology
- Journal of the American Mosquito Control Association
- Journal of Medical Entomology
- Journal of Vector Ecology : Journal of the Society for Vector Ecology
- Journal of Medical Entomology
- Journal of Medical Entomology
- Vector Borne and Zoonotic Diseases (Larchmont, N.Y.)
- PloS One
- Molecular Ecology
Articles by Lane Foil in JoVE
JoVE General
Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)
Department of Entomology, Louisiana State University Agricultural Center
We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).
Other articles by Lane Foil on PubMed
Vertical Transmission of Rickettsia Felis in the Cat Flea (Ctenocephalides Felis Bouché)
Rickettsia felis can be maintained in cat fleas by vertical transmission for up to 12 generations without the benefit of an infected host. Horizontal transmission or the acquisition of R. felis by fleas feeding on cats or artificially infected meals were not demonstrated in this study. Horizontal transmission of R. felis by the ingestion of feces or eggs by flea larvae was not detected. We also tested for and found no evidence to support horizontal transmission by contact among positive fleas and negative fleas.
Use of the Polymerase Chain Reaction to Investigate the Dynamics of Pyrethroid Resistance in Haematobia Irritans Irritans (Diptera: Muscidae)
A field study was conducted from 1991 through 1997 to evaluate the use of pyrethroid and organophosphate (OP) ear tags, alternated yearly, for the control of a pyrethroid resistant horn fly, Haematobia irritans (L.), population in Louisiana. Fly resistance was monitored by weekly fly counts, filter paper bioassays and diagnostic polymerase chain reaction (PCR) assays for the presence of pyrethroid resistance-associated mutations in the sodium channel gene coding region. Fly control in the first study year was poor, as pyrethroid ear tags were effective for only 7 wk. The following year, OP ear tags provided 15 wk of fly control. However, in all subsequent years, fly control was poor with both types of ear tags. The PCR assays showed that there were very few female flies homozygous for the pyrethroid susceptible sodium channel allele, never rising above 10% of the total females in the population. A fitness cost appeared to be associated with the pyrethroid resistant allele, as the resistant form was selected against in the absence of the pyrethroid ear tags. Despite this selection in favor of the susceptible allele and the annual alternation of pyrethroid and OP ear tags, the percentage of homozygous susceptible flies never reached over 19% of the population, resistant alleles of the sodium channel remained at high levels in the population, and horn fly control on cattle with either type of tag quickly became minimal.
Studies on the Growth of Bartonella Henselae in the Cat Flea (Siphonaptera: Pulicidae)
Two out of three pools of cat fleas, Ctenocephalides felis (Bouche), that were fed Bartonella henselae-positive cat blood for 3 d and then bovine blood for 3 d, were polymerase chain reaction (PCR) positive for B. henselae. In a second experiment, three cats were inoculated with a streptomycin-resistant strain of B. henselae. After the cats were inoculated, caged cat fleas were fed on the cats during three different periods, and then pooled and transferred to noninfected recipient cats. In the first trial, the bacteria in the flea feces were below level of detection when the fleas were transferred from the infected cats to the recipient cat. After the fleas had fed on the recipient cat for 6 d, a bacteria level of 4.00 x 10(3) CFU/ mg was detected in the flea feces. Subsequently, the bacteria level increased for 4 d and then declined. In another experiment, the bacteria level in the flea feces was 1.80 x 10(3) CFU/mg at 2 h after collection and 3.33 x 10(2) CFU/mg at 72 h after collection. These data indicated that this strain of B. henselae can persist in flea feces in the environment for at least 3 d, and that B. henselae can multiply in the cat flea.
Seasonal and Geographical Distribution of Adult Ixodes Scapularis Say (Acari: Ixodidae) in Louisiana
The distribution and seasonality of adult black-legged ticks (Ixodes scapularis Say) in Louisiana was measured. The presence of adult ticks was determined by flagging at 106 sites throughout Louisiana. It was concluded that Ixodes scapularis is widely distributed throughout Louisiana. Ticks were also collected twice per month at one site over a 15-month period by flagging and use of CO2 traps to establish the relative seasonal abundance pattern of free-living adult ticks. Host-seeking, black-legged adult ticks were collected from October to May. Peak adult abundance was observed in December. More ticks were collected by the use of CO2 traps compared to flagging in October, November, and February. No black-legged tick larvae or nymphs were collected in this study using either collection method.
Rickettsia Felis from Cat Fleas: Isolation and Culture in a Tick-derived Cell Line
Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.
Detection of West Nile Virus RNA in Pools of Three Species of Ceratopogonids (Diptera: Ceratopogonidae) Collected in Louisiana
Light traps were used to collect ceratopogonids in East Baton Rouge parish, Louisiana. In total, 46,496 ceratopogonids were sorted from 4,968 light trap collections from 20 November 2002 through 25 November 2004. Two hundred and nine pools containing specimens of 18 species of Culicoides Latreille, seven pools containing specimens of Atrichopogon Kieffer, and five pools containing specimens of Forcipomyia Meigen were tested for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) RNA using real-time reverse transcriptase polymerase chain reaction. Five out of the 209 pools of Culicoides specimens were positive for WNV RNA.
Development of Treated Targets for Controlling Stable Flies (Diptera: Muscidae)
Electrocution techniques were used to determine if treated targets similar to those used for tsetse control could be developed for stable fly control. In a series of two experiments, a half blue and half black (UK) 1 m2 target constructed of trigger cotton poplin was determined to be acceptable for development studies. In the first experiment, an average of 350 stable flies per hour (maximum 794 flies in 1 h) was collected using the UK target. A time-delayed circuit trial using untreated UK targets demonstrated that stable flies remained on or around the targets for at least 30 s. Two experiments were conducted with time-delayed circuits and UK targets treated with 0.1% lambda-cyhalothrin. In the first experiment, the number of flies collected using the 30 s on/off treated target treatment was not different from the number of flies collected using the other treatments. In the second experiment, the number of flies collected using the 30 s on/off treated target treatment was not different than the untreated target continuous or 30 s on/off treatments, but was significantly lower than the treated target continuous treatment. The number of flies collected with UK trigger targets was significantly higher than that for alsynite cylinder traps in two experiments. The mean number of flies collected during 22 1h assays using targets was 6.1-fold higher than that for alsynite traps, and the mean number of flies collected during 40 3 h using the targets also was 6.1-fold higher than that for alsynite traps. The results of this study indicate that treated cloth targets may be a viable addition for stable fly control programs.
Comparative Microbiota of Rickettsia Felis-uninfected and -infected Colonized Cat Fleas, Ctenocephalides Felis
Fleas serve as arthropod vectors for several emerging and re-emerging infectious disease causing agents including, Rickettsia felis. Although the prevalence of R. felis infection in colonies of fleas has been examined, the influence of the R. felis infection on flea microbiota has not been investigated. We identified three colonies of cat fleas, Ctenocephalides felis, with varying prevalence of R. felis infection (Louisiana State University (LSU), 93.8%; Professional Laboratory and Research Services Inc. (PLRS), 16.4%; Elward II (EL), 0%) and subsequently utilized polymerase chain reaction amplification, restriction fragment length polymorphism analysis and sequencing of the 1.4-kb portions of 16S rRNA genes to examine the diversity of bacteria in the flea populations. A total of 17 different bacterial 16S rRNA gene sequences were identified among the C. felis colonies. The prevalence of two Wolbachia species that were identified in each flea colony differed between colonies and R. felis-uninfected and -infected fleas. Species richness was unchanged among the R. felis-uninfected (LSU, PLRS and EL colonies) and -infected (LSU and PLRS colonies) fleas; however, between R. felis-uninfected and -infected fleas within both the LSU and PLRS colonies, R. felis-uninfected fleas have greater species richness. Diversity indices did not identify a difference in diversity between any of the flea samples. The interaction of endosymbionts within arthropods can widely impact the dissemination of vertically transmitted pathogenic bacteria; and the reciprocal may be true. These results suggest that carriage of R. felis has an impact on the richness of flea microbiota.
Comparison of the Efficiency of Biological Transmission of Anaplasma Marginale (Rickettsiales: Anaplasmataceae) by Dermacentor Andersoni Stiles (Acari: Ixodidae) with Mechanical Transmission by the Horse Fly, Tabanus Fuscicostatus Hine (Diptera: Muscidae)
Mechanical transmission ofAnaplasma marginale by horse flies (Tabanidae) is thought to be epidemiologically significant in some areas of the United States. We compared the relative efficiencies of mechanical transmission of Anaplasma marginale by the horse fly, Tabanus fuscicostatus Hine, during acute infection (approximately 10(7) to approximately 10(9) infected erythrocytes [IE]/ml blood) with biological transmission by Dermacentor andersoni Stiles in the persistent phase of infection (approximately 10(2.5) to approximately 10(6) IE/ml). Transmission of A. marginale was not observed when horse flies were partially fed on an acutely infected donor calf and immediately transferred to susceptible calves to complete their blood meal. Ticks that were acquisition fed on the same donor host after it reached the persistent phase of infection successfully transmitted A. marginale when transferred to the same recipient calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >240-fold higher than that from which ticks transmitted with 100% efficiency shows that tick-borne biological transmission is at least two orders of magnitude more efficient than mechanical transmission by horse flies.
Identification of Rickettsia Felis in the Salivary Glands of Cat Fleas
Rickettsia felis, a flea-associated rickettsial pathogen, has been identified in many tissues, including the digestive and reproductive tissues, within the cat flea, Ctenocephalides felis. We utilized transmission electron microscopy and polymerase chain reaction to identify R. felis in the salivary glands of fed fleas and further define the distribution of R. felis within the arthropod host. We identified Rickettsia-like organisms in salivary glands using electron microscopy. Sequence analysis of portions of the Rickettsia genus-specific 17-kDa antigen gene and R. felis plasmid confirmed the morphological identification of R. felis in cat flea salivary glands. This is the first report of R. felis in tissues critical for horizontal transmission of rickettsiae.
West Nile Virus Detection in Mosquitoes in East Baton Rouge Parish, Louisiana, from November 2002 to October 2004
The prevalence of West Nile virus (WNV) was determined in mosquitoes between November 2002 and October 2004 in East Baton Rouge Parish, LA. A total of 244,374 female mosquitoes were collected and tested by viral isolation. Additionally, 131,896 female mosquitoes were collected in 2003 and tested by VecTest and 167,175 female mosquitoes were collected in 2004 and tested by reverse transcriptase-polymerase chain reaction (RT-PCR). West Nile virus was isolated by cell culture from 17 (47.2%) out of 36 mosquito species collected over the study period. In 2003, WNV was detected in 9 (33.3%) out of 27 species tested by VecTest. In 2004, 14 (50%) out of the 28 mosquito species tested by RT-PCR were positive for WNV. The species with the greatest number of WNV-positive pools detected by all 3 testing methods was Culex quinquefasciatus. A significantly greater proportion of Cx. salinarius pools collected in light traps placed at a 3-m height were positive for WNV by viral isolation than in pools collected in light traps placed at a 1.5-m height.
Acetylcholinesterase Mutation in Diazinon-resistant Haematobia Irritans (L.) (Diptera: Muscidae)
Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.
Evaluation of Different Insecticides and Fabric Types for Development of Treated Targets for Stable Fly (Diptera: Muscidae) Control
Stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), once only a pest of pastured cattle, has become a serious pest of range cattle in the United States. Because of the difficulties associated with stable fly management under range conditions, a pesticide-impregnated cloth target is being developed as a management tool. We conducted studies to determine the influence of weather, time, fabric type, insecticide type, and insecticide concentration on the mortality of stable flies from a susceptible laboratory colony exposed for 30 s to treated cloth targets. We found that 100% of the flies exposed to trigger (Trigger-Royal Box, 65% polyester and 35% cotton) fabric targets that were treated with 0.1% h-cyhalothrin or 0.1% zeta-cypermethrin and weathered outdoors in Gainesville, FL., for up to 3 mo, were dead within 20 min after a 30-s exposure. The results of this study support the concept that treated targets can be developed for integration into stable fly control programs.
Prevalence and Infection Load Dynamics of Rickettsia Felis in Actively Feeding Cat Fleas
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.
Development of a Mathematical Model for Mechanical Transmission of Trypanosomes and Other Pathogens of Cattle Transmitted by Tabanids
Mechanical transmission of pathogens by biting insects is a non-specific phenomenon in which pathogens are transmitted from the blood of an infected host to another host during interrupted feeding of the insects. A large range of pathogens can be mechanically transmitted, e.g. hemoparasites, bacteria and viruses. Some pathogens are almost exclusively mechanically transmitted, while others are also cyclically transmitted. For agents transmitted both cyclically and mechanically (mixed transmission), such as certain African pathogenic trypanosomes, the relative impact of mechanical versus cyclical transmission is essentially unknown. We have developed a mathematical model of pathogen transmission by a defined insect population to evaluate the importance of mechanical transmission. Based on a series of experiments aimed at demonstrating mechanical transmission of African trypanosomes by tabanids, the main parameters of the model were either quantified (host parasitaemia, mean individual insect burden, initial prevalence of infection) or estimated (unknown parameters). This model allows us to simulate the evolution of pathogen prevalence under various predictive circumstances, including control measures and could be used to assess the risk of mechanical transmission under field conditions. If adjustments of parameters are provided, this model could be generalized to other pathogenic agents present in the blood of their hosts (Bovine Leukemia virus, Anaplasma, etc.) or other biting insects such as biting muscids (stomoxyines) and hippoboscids.
Microarray Analysis of Female- and Larval-specific Gene Expression in the Horn Fly (Diptera: Muscidae)
The horn fly, Haematobia irritans L., is an obligate blood-feeding parasite of cattle, and control of this pest is a continuing problem because the fly is becoming resistant to pesticides. Dominant conditional lethal gene systems are being studied as population control technologies against agricultural pests. One of the components of these systems is a female-specific gene promoter that drives expression of a lethality-inducing gene. To identify candidate genes to supply this promoter, microarrays were designed from a horn fly expressed sequence tag (EST) database and probed to identify female-specific and larval-specific gene expression. Analysis of dye swap experiments found 432 and 417 transcripts whose expression levels were higher or lower in adult female flies, respectively, compared with adult male flies. Additionally, 419 and 871 transcripts were identified whose expression levels were higher or lower in first-instar larvae compared with adult flies, respectively. Three transcripts were expressed more highly in adult females flies compared with adult males and also higher in the first-instar larval lifestage compared with adult flies. One of these transcripts, a putative nanos ortholog, has a high female-to-male expression ratio, a moderate expression level in first-instar larvae, and has been well characterized in Drosophila. melanogaster (Meigen). In conclusion, we used microarray technology, verified by reverse transcriptase-polymerase chain reaction and massively parallel pyrosequencing, to study life stage- and sex-specific gene expression in the horn fly and identified three gene candidates for detailed evaluation as a gene promoter source for the development of a female-specific conditional lethality system.
Evaluation of Surveillance Methods for Detection of West Nile Virus Activity in East Baton Rouge Parish, Louisiana, 2004-2006
A 3-year study was conducted to determine if testing mosquitoes collected in modified sentinel chicken boxes for West Nile virus (WNV) or testing sentinel chickens for WNV antibody would detect WNV activity before onset of human cases in East Baton Rouge Parish, LA. In each year mosquitoes tested positive for WNV before the onset of human cases were detected, but seroconversions of sentinel chickens were detected after the human cases occurred. In 1 year we also compared the effectiveness of Centers for Disease Control and Prevention (CDC) light traps, gravid traps, and sentinel chicken box traps for collecting WNV-positive mosquitoes. Gravid traps collected more WNV-positive mosquitoes than CDC light traps or sentinel chicken box traps. However, WNV was detected earlier in mosquitoes collected from sentinel chicken box traps than in mosquitoes collected with gravid traps or CDC light traps. In total, 1,222 pools containing 19,353 mosquito specimens representing 18 species were tested for WNV. West Nile virus was detected in 59 mosquito pools from 4 species; 87% of the positive pools were detected from Culex quinquefasciatus, which was the most abundant species collected in all 3 years.
Host Feeding Patterns of Culex Mosquitoes (Diptera: Culicidae) in East Baton Rouge Parish, Louisiana
Host feeding patterns were examined for four species of Culex mosquitoes collected from 18 sites in or adjacent to East Baton Rouge Parish, LA, from November 2002 to October 2004. Host DNA from 37 bloodfed Culex coronator Dyar and Knab, 67 bloodfed Cx. salinarius Coquillett, 112 bloodfed Cx. nigripalpus Theobald, and 684 bloodfed Cx. quinquefasciatus Say were identified. The percentages of bloodmeals containing mammalian DNA were 94.6% for Cx. coronator, 82.1% for Cx. salinarius, 66.1% for Cx. nigripalpus, and 40.1% for Cx. quinquefasciatus. Human DNA was detected in 7% of the bloodmeals from Cx. quinquefasciatus and 2.7% of the bloodmeals from Cx. nigripalpus. The northern cardinal was the most frequent avian host of Cx. quinquefasciatus and Cx. nigripalpus. In 2003 and 2004, there was no significant relationship from May through October between the proportion of Cx. quinquefasciatus feeding on mammalian hosts and the date of collection. Of the six avian species most frequently fed on by Cx. quinquefasciatus, the northern cardinal, northern mockingbird, common grackle, and brown thrasher were fed on more frequently than expected based on their abundance. House sparrows were fed on less frequently than expected based on their abundance. These data support the conclusions of previous studies that Cx. quinquefasciatus is the most important vector for both the enzootic amplification and transmission of West Nile virus to humans in southern Louisiana.
Evidence of Vertical Transmission of West Nile Virus in Field-collected Mosquitoes
Male and nulliparous female mosquitoes were surveyed for evidence of vertical WNV infection in East Baton Rouge Parish, Louisiana. Adult male mosquitoes collected by trapping and aspiration, and adult male and nulliparous female mosquitoes reared from field-collected larvae were tested. Adult male Culex spp., female Aedes albopictus (Skuse), and female Culex quinquifasciatus Say mosquitoes that were collected as larvae were test-positive for WNV RNA. Infectious WNV was detected using virus isolation in field-collected male Aedes triseriatus Say and Culex salinarius Coquillett; these data represent the first field evidence of vertical transmission of WNV in Ae. triseriatus and Cx. salinarius.
Detection of West Nile Virus RNA in Mosquitoes and Identification of Mosquito Blood Meals Collected at Alligator Farms in Louisiana
Since 2001, alligator farms in the United States have sustained substantial economic losses because of West Nile virus (WNV) outbreaks in American alligators (Alligator mississippiensis). Once an initial infection is introduced into captive alligators, WNV can spread among animals by contaminative transmission. Some outbreaks have been linked to feeding on infected meat or the introduction of infected hatchlings, but the initial source of WNV infection has been uncertain in other outbreaks. We conducted a study to identify species composition and presence of WNV in mosquito populations associated with alligator farms in Louisiana. A second objective of this study was to identify the origin of mosquito blood meals collected at commercial alligator farms. Mosquitoes were collected from 2004 to 2006, using Centers for Disease Control light traps, gravid traps, backpack aspirators, and resting boxes. We collected a total of 58,975 mosquitoes representing 24 species. WNV was detected in 41 pools of females from 11 mosquito species: Anopheles crucians, Anopheles quadrimaculatus, Coquillettidia perturbans, Culex coronator, Culex erraticus, Culex nigripalpus, Culex quinquefasciatus, Mansonia titillans, Aedes sollicitans, Psorophora columbiae, and Uranotaenia lowii. The blood meal origins of 213 field-collected mosquitoes were identified based on cytochrome B sequence identity. Alligator blood was detected in 21 mosquitoes representing six species of mosquitoes, including Cx. quinquefasciatus and Cx. nigripalpus. Our results showed that mosquitoes of species that are known to be competent vectors of WNV fed regularly on captive alligators. Therefore, mosquitoes probably are important in the role of transmission of WNV at alligator farms.
Development of Microsatellites for Genetic Analyses and Population Assignment of the Cat Flea (Siphonaptera: Pulicidae)
Cat fleas, Ctenocephalidesfelis (Bouché) (Siphonaptera: Pulicidae), are common ectoparasites of companion animals that negatively impact their hosts directly by causing dermatitis and blood loss during feeding and indirectly through the potential transmission of disease causing agents. We isolated and characterized seven novel microsatellite loci from a partial genomic library of the cat flea enriched for di-, tri-, and tetranucleotide repeats. We screened these loci in cat fleas from two laboratory colonies and one wild-caught population collected at a temporary animal shelter (Parker coliseum) in Baton Rouge, LA. Six loci were polymorphic, with two to 15 alleles per locus and an average observed heterozygosity of 0.21 across populations. Although the two laboratory cat flea colonies were isolated from each other for many years, they did not significantly differ in their genotypic composition. The cat flea population from Parker coliseum was genetically different from the laboratory colonies, but also showed high degrees of inbreeding. Multilocus genotypes of the polymorphic loci were sufficient to assign over 85% of cat fleas to their population of origin. Genetic markers for flea population identity will allow further studies to examine the origins and movement of cat fleas with important genetic traits such as insecticide resistance or pathogen susceptibility. The use of microsatellites also could determine if there are host-specific strains of cat fleas and add insight into the development of the different subspecies of C. felis.
Acquisition of Rickettsia Felis by Cat Fleas During Feeding
Evidence for horizontal routes of transmission for Rickettsia felis has come from detection of R. felis infection in vertebrates and multiple blood-feeding arthropods; however, infection of cat fleas, Ctenocephalides felis, during blood feeding has not been demonstrated. In this study, the ability of cat fleas to acquire R. felis through an infectious blood meal with subsequent vertical transmission was examined. Utilizing an artificial feeding system, Rickettsia-naive fleas were exposed to R. felis-infected blood meals and monitored for subsequent infection at weekly intervals for 4 weeks. At 7 days postexposure (dpe) ~52% of fleas successfully acquired rickettsiae and R. felis DNA; rickettsial transcript and DNA was detected in cat flea feces. Quantitative real-time polymerase chain reaction determined that both the R. felis infection load and R. felis infection density was significantly greater in fleas assessed at later time points. Although a persistent R. felis infection was detected in adult fleas, R. felis infection was not observed in F(1) progeny. This study demonstrates that cat fleas are able to acquire R. felis infection from an infectious blood meal and will serve as a model to examine R. felis transmission between arthropod and vertebrate hosts.
Isolation of a Rickettsial Pathogen from a Non-hematophagous Arthropod
Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.
Horizontal Transmission of Rickettsia Felis Between Cat Fleas, Ctenocephalides Felis
Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.
