The role of cofeeding arthropods in the transmission of Rickettsia felis

Rickettsia felis is an emerging etiological agent of rickettsioses worldwide. The cosmopolitan cat flea (Ctenocephalides felis) is the primary vector of R. felis, but R. felis has also been reported in other species of hematophagous arthropods including ticks and mosquitoes. Canines can serve as a bacteremic host to infect fleas under laboratory conditions, yet isolation of R. felis from the blood of a vertebrate host in nature has not been realized. Cofeeding transmission is an efficient mechanism for transmitting rickettsiae between infected and uninfected fleas; however, the mechanism of transmission among different orders and classes of arthropods is not known. The potential for R. felis transmission between infected fleas and tick (Dermacentor variabilis) and mosquito (Anopheles quadrimaculatus) hosts was examined via cofeeding bioassays. Donor cat fleas infected with R. felis transmitted the agent to naïve D. variabilis nymphs via cofeeding on a rat host. Subsequent transstadial transmission of R. felis from the engorged nymphs to the adult ticks was observed with reduced prevalence in adult ticks. Using an artificial host system, An. quadrimaculatus exposed to a R. felis-infected blood meal acquired rickettsiae and maintained infection over 12 days post-exposure (dpe). Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on rats infection persisting in the mosquito for up to 3 dpe. The results indicate R. felis-infected cat fleas can transmit rickettsiae to both ticks and mosquitoes via cofeeding on a vertebrate host, thus providing a potential avenue for the diversity of R. felis-infected arthropods in nature.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.     

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.     

Plasmodium berghei: Heat-treated sporozoite vaccination of mice

Repeated intravenous (IV) immunizing injections with 25,000 Plasmodium berghei heat-treated sporozoites gave an average protection of 13% in five experiments (0–53%). Four injections of 10⁵ sporozoites gave 50% protection, seven injections of 10⁵ gave 37% protection, and six injections of 10⁵ gave 11% protection. Viable spleen cells (1.2 × 10⁸) from twice challenged immune syngenic mice did not protect naïve mice against iv challenge. Six ip injections of the supernatant of Parr Bomb disintegrated sporozoites gave no protection against ip challenge, but 7 ip injections gave 40% protection. Centrifuged pellets from French Pressure Cell-disintegrated sporozoites gave almost no protection either iv, sc, or ip. The supernatant was disc-electrophoresed and compared to normal mosquito heads and salivary glands in order to select sporozoite bands for immunizing injections. Results were discussed with respect to uniformity of antigen batches.     

Cat fleas (Ctenocephalides felis) carrying Rickettsia felis and Bartonella species in Hong Kong

Fleas are commonly recorded on stray as well as domestic dogs and cats in Hong Kong. Fleas can be a major cause of pruritus in dogs and cats and also vectors of potentially zoonotic bacteria in the genera Rickettsia and Bartonella. Morphological examination of 174 fleas from dogs and cats living in Hong Kong revealed only cat fleas (Ctenocephalides felis). Cytochrome c oxidase subunit 1 gene (cox1) genotyping of 20 randomly selected specimens, revealed three cox1 haplotypes (HK-h1 to HK-h3). The most common haplotype was HK-h1 with 17 specimens (17/20, 85%). HK-h1 was identical to cox1 sequences of fleas in Thailand and Fiji. HK-h1 and HK-h2 form a distinct cat flea cox1 clade previously recognized as the Clade 3. HK-h3 forms a new Clade 6. A multiplex Bartonella and Rickettsia real-time PCR of DNA from 20 C. felis found Bartonella and Rickettsia DNA in three (15%) and ten (50%) C. felis, respectively. DNA sequencing confirmed the presence of R. felis, B. clarridgeiae and Bartonella henselae. This is the first reported study of that kind in Hong Kong, and further work is required to expand the survey of companion animals in the geographical region. The sampling of fleas on domestic cats and dogs in Hong Kong revealed them to be exclusively infested by the cat flea and to be harbouring pathogens of zoonotic potential.     

Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector Rhipicephalus appendiculatus

Young A. S., Leitch B. L. and Mutugi J. J. 1984. Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector, Rhipicephalus appendiculatus. International Journal for Parasitology14: 97–102. The effect of various temperature treatments on the sporogony cycle of Theileria parva in the salivary glands of unfed adult Rhipicephalus appendiculatus ticks of various ages was investigated. It was found that ticks incubated at 28 or 37°C would develop sporozoites infective to cattle but never in such large numbers as in ticks fed on rabbits. Heat stimulation of sporogony was possible for isolates of T. parva with minimal laboratory handling. The age of the ticks incubated at 28 or 37°C was important since sporozoites could only be induced at the earliest on day 27 or 28 p.repl. (post-repletion) and at the latest by day 41 p.repl. The age of ticks fed on rabbits was not as important for the production of sporozoites.     

Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

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.     

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 Findings

A 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/Significance

Examining 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.     

Genome‐wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q‐fever patient sera and naïve controls in order to discover C. burnetii‐specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q‐fever cases than naïve controls. The remaining eight antigens were cross‐reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q‐fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof‐of‐concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.     

A primary Corynebacterium pseudotuberculosis low dose infection in alpacas (Lama pacos) protects against a lethal challenge exposure

Corynebacterium pseudotuberculosis is the agent of alpaca's lymphadenitis. The present study was to demonstrate the effect of a primary infection with low (1.1 × 10³), moderate (1 × 10⁴), and high (1.2 × 10⁵) doses of C. pseudotuberculosis against a significant higher challenge dose of 9 × 10⁸ CFU of C. pseudotuberculosis. Three groups of 4 healthy male alpacas were inoculated subcutaneously (SC) in the left flank behind the costal arch with the above doses of bacteria. A fourth group of 4 alpacas was sham inoculated with phosphate buffered saline as control. After 5 weeks all animals were challenged with a dose of 9 × 10⁸ CFU of C. pseudotuberculosis inoculated SC in the right flank. The alpacas were clinically inspected for local and regional abscesses, body temperature and behavior changes. The primary infected alpacas had a febrile response, and abscesses at the inoculation point and regional lymph nodes. However, after challenge, the primary infected animals showed no superficial lesions or febrile response. In contrast, the immune naïve alpacas from group D developed a severe disease characterized by fever, abscesses in regional lymphnodes, and in one alpaca a subcutaneous edema and sudden death 2 weeks after exposure. In addition, primary infected alpacas had a robust antibody response against C. pseudotuberculosis cell wall antigen with significant differences with respect the naïve challenged alpacas. At necropsy, the primary infected alpacas had abscesses only in the regional or internal renal-lymph nodes from the left or primary inoculation side of the body, with no lesions in the right challenged side. In contrast, the primary sham inoculated alpacas had abscesses in the regional and internal lymph nodes from the right challenged side. This work showed that a primary infection with at least 1.1 × 10³ viable C. pseudotuberculosis induces protection against a second high dose exposure to this bacterium. These results will be useful for further study of prevention methods to control lymphadenitis in alpacas.     

Ecological host-range of Lilioceris cheni (Coleoptera: Chrysomelidae), a biological control agent of Dioscorea bulbifera

Open-field host-specificity testing assesses the host-range of a biological control agent in a setting that permits the agent to use its full complement of host-seeking behaviors. This form of testing, particularly when it includes a no-choice phase in which the target weed is killed, may provide the most accurate assessment of the ecological host-range of an agent. We conducted a two-phase field host-specificity test with experienced and naïve adults of Lilioceris cheni Gressitt and Kimoto (Coleoptera: Chrysomelidae), a biological control agent of Dioscorea bulbifera L. (Dioscoreales: Dioscoreaceae). We followed field tests with a no-choice laboratory consumption study with the congeneric plant species that received test feeding in the field, and an additional field evaluation of spillover risk. Both experienced and naïve adults strongly preferred D. bulbifera to non-targets in the field. Within 47 h post-release, 90% of the released beetles that remained in the plots were found on D. bulbifera. In the laboratory no-choice test, the beetles consumed significantly more D. bulbifera and survived longer on this plant than the non-targets. All naïve beetles in the Dioscorea sansibarensis and Dioscorea villosa treatments and 75% of naïve beetles on Dioscorea floridana died within 7 d. Potted plants of the native D. floridana experienced minor test feeding in the spillover experiment when surrounded by large populations of L. cheni in the field. At the end of this experiment, L. cheni eggs and/or larvae were present on 83% of D. bulbifera plants but none of the D. floridana plants. We conclude that L. cheni is host-specific to D. bulbifera and does not pose a spillover risk to the native D. floridana.     

Life Cycle of Cystoisospora felis (Coccidia: Apicomplexa) in Cats and Mice

Cystoisospora felis is a ubiquitous apicomplexan protozoon of cats. The endogenous development of C. felis was studied in cats after feeding them infected mice. For this, five newborn cats were killed at 24, 48, 72, 96, and 120 h after having been fed mesenteric lymph nodes and spleens of mice that were inoculated with C. felis sporulated sporocysts. Asexual and sexual development occurred in enterocytes throughout the villi of the small intestine. The number of asexual generations was not determined with certainty, but there were different sized merozoites. At 24 h, merogony was seen only in the duodenum and the jejunum. Beginning at 48 h, the entire small intestine was parasitized. At 24 h, meronts contained 1–4 zoites, and at 48 h up to 12 zoites. Beginning with 72 h, the ileum was more heavily parasitized than the jejunum. At 96 and 120 h, meronts contained many zoites in various stages of development; some divided by endodyogeny. The multiplication was asynchronous, thus both immature multinucleated meronts and mature merozoites were seen in the same parasitophorous vacuole. Gametogony occurred between 96 and 120 h, and oocysts were present at 120 h. For the study of the development of C. felis in murine tissues, mice were killed from day 1 to 720 d after having been fed 10⁵ sporocysts, and their tissues were examined for the parasites microscopically, and by bioassay in cats. The following conclusions were drawn. (1) Cystoisospora felis most frequently invaded the mesenteric lymph nodes of mice and remained there for at least 23 mo. (2) It also invaded the spleen, liver, brain, lung, and skeletal muscle of mice, but division was not seen based on microscopical examination. (3) This species could not be passed from mouse to mouse.     

Orientation behavior of Propylaea japonica toward visual and olfactory cues from its prey–host plant combination

The lady beetle Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae) is an important predator of aphids in agroecosystems. The inundative release of coccinellid beetles can be an effective biological control strategy. An understanding of how biological control agents perceive and use stimuli from host plants is the key to successfully implement commercially produced predators. Here, we studied the relative role of visual and volatile cues. Dual‐choice assays using foraging‐naïve and foraging‐experienced P. japonica adults were conducted using cotton plants [Gossypium hirsutum L. (Malvaceae)] with or without infestation by the cotton aphid, Aphis gossypii (Glover) (Hemiptera: Aphididae). Overall, experienced beetles were more attracted than naïve beetles toward cues associated with aphid‐infested plants. Experienced beetles were also more responsive to olfactory cues compared with naïve beetles. Both foraging‐naïve and ‐experienced lady beetles integrate olfactory and visual cues from plants infested with aphids, with an apparently greater reliance on olfactory cues. The results suggest that foraging experience may increase prey location in P. japonica.     

Plasmodium vivax infection induces expansion of activated naïve/memory T cells and differentiation into a central memory profile

Immunity to malaria is widely believed to wane in the absence of reinfection, but direct evidence for the presence or absence of durable immunological memory to malaria is limited. Here, we characterized the profile of circulating naïve and memory (including central and effector) CD4⁺ T cells responses of individuals naturally infected by Plasmodium vivax. In the current study, we demonstrated that acute P. vivax infection induces a significant increase in the absolute number of both naïve and memory cells, which were responsible for the production of anti-inflammatory (IL-10) and pro-inflammatory (IFN-γ) cytokines. Finally, we described the profile of memory cell subtypes (T_CM-CD45RO^highCCR7⁺ and T_EM-CD45RO^highCCR7⁻), as well as the pattern of cell migration based on CD62L selectin expression, demonstrating that P. vivax-infected donors presented with a predominantly central memory cell profile. Our results indicate that the expansion of both naïve and memory T cells, responsible for the production of both pro-inflammatory and regulatory cytokines, which might also contribute to the modulation of immune responses during P. vivax infection.     

Nasal peptide vaccination elicits CD8 responses and reduces viral burden after challenge with virulent murine cytomegalovirus

Infection of BALB/c mice with murine cytomegalovirus (MCMV) leads to CD8 cell responses to an immunodominant epitope YPHFMPTNL. We presented this epitope as a nasal peptide vaccine in combination with cholera toxin adjuvant, and evaluated immune responses and protection from MCMV challenge. Vaccination of naïve mice generated elevated numbers of peptide‐specific interferon‐7‐secreting splenocytes (median 80/million, range 60 to 490), compared to control mice (median 2/million, range —4.5 to 8; P=0.008, Mann‐Whitney test). Twelve days after challenge with virulent MCMV, vaccinated mice had a 1.1 log₁₀ reduction in salivary gland viral titer compared to unvaccinated controls (5.36±0.24 vs. 6.42±0.12, mean±SD log₁₀ plaque‐forming‐units; P<0.001, t‐test). Mice with chronic MCMV infection had consistent responses to the peptide (183±24/million interferon‐γ‐secreting splenocytes). Nasal peptide vaccination during chronic infection boosted peptide‐specific responses in two of four mice to >900/million interferon‐γ‐secreting splenocytes. Nasal peptide vaccination was immunogenic in naïve and MCMV‐infected mice, and reduced viral burden in naïve mice after virulent MCMV challenge. The nasal route may be useful for peptide presentation by novel human vaccines.