Flea Infection Research Articles

Opossums and Cat Fleas: New Insights in the Ecology of Murine Typhus in Galveston, Texas.

Murine typhus is an acute undifferentiated febrile illness caused by Rickettsia typhi The classic reservoir (Rattus spp.) and flea vector (Xenopsylla cheopis) were once culprits of murine typhus in the United States. Vector and rodent control efforts have drastically decreased the prevalence of disease, except in a few endemic foci where opossums and cat fleas play a role in transmission. Since 2012, there has been a reemergence of murine typhus in Galveston, TX. We hypothesize that opossums and cat fleas are involved in the transmission of R. typhi in Galveston. To explore this, we sought to find the seroprevalence of typhus group antibodies from opossums. We also sought to find the prevalence of R. typhi in fleas parasitizing these animals. We collected blood from 12 opossums and found that eight (66.7%) had the presence of anti-R. typhi antibodies. All opossums were infested with fleas; a total of 250 Ctenocephalides felis fleas were collected from these animals. Seven opossums (53.8%) were infested with fleas that had molecular evidence of R. typhi infection, while six (46.2%) were infested with fleas that contained Candidatus Rickettsia senegalensis, an organism closely related to R. felis The minimum flea infection rate for R. typhi was 7.0%. The minimum infection rate for Candidatus R. senegalensis was 6.1%. Our study demonstrates that fleas infected with R. typhi parasitize opossums in Galveston. It is therefore likely that opossums and their fleas play a role in the city's recent reemergence of murine typhus.

A LysR-Type Transcriptional Regulator, RovM, Senses Nutritional Cues Suggesting that It Is Involved in Metabolic Adaptation of Yersinia pestis to the Flea Gut

Yersinia pestis has evolved as a clonal variant of Yersinia pseudotuberculosis to cause flea-borne biofilm–mediated transmission of the bubonic plague. The LysR-type transcriptional regulator, RovM, is highly induced only during Y. pestis infection of the flea host. RovM homologs in other pathogens regulate biofilm formation, nutrient sensing, and virulence; including in Y. pseudotuberculosis, where RovM represses the major virulence factor, RovA. Here the role that RovM plays during flea infection was investigated using a Y. pestis KIM6+ strain deleted of rovM, ΔrovM. The ΔrovM mutant strain was not affected in characteristic biofilm gut blockage, growth, or survival during single infection of fleas. Nonetheless, during a co-infection of fleas, the ΔrovM mutant exhibited a significant competitive fitness defect relative to the wild type strain. This competitive fitness defect was restored as a fitness advantage relative to the wild type in a ΔrovM mutant complemented in trans to over-express rovM. Consistent with this, Y. pestis strains, producing elevated transcriptional levels of rovM, displayed higher growth rates, and differential ability to form biofilm in response to specific nutrients in comparison to the wild type. In addition, we demonstrated that rovA was not repressed by RovM in fleas, but that elevated transcriptional levels of rovM in vitro correlated with repression of rovA under specific nutritional conditions. Collectively, these findings suggest that RovM likely senses specific nutrient cues in the flea gut environment, and accordingly directs metabolic adaptation to enhance flea gut colonization by Y. pestis.

Resistance to Innate Immunity Contributes to Colonization of the Insect Gut by Yersinia pestis.

Yersinia pestis, the causative agent of bubonic and pneumonic plague, is typically a zoonotic vector-borne disease of wild rodents. Bacterial biofilm formation in the proventriculus of the flea contributes to chronic infection of fleas and facilitates efficient disease transmission. However prior to biofilm formation, ingested bacteria must survive within the flea midgut, and yet little is known about vector-pathogen interactions that are required for flea gut colonization. Here we establish a Drosophila melanogaster model system to gain insight into Y. pestis colonization of the insect vector. We show that Y. pestis establishes a stable infection in the anterior midgut of fly larvae, and we used this model system to study the roles of genes involved in biofilm production and/or resistance to gut immunity stressors. We find that PhoP and GmhA both contribute to colonization and resistance to antimicrobial peptides in flies, and furthermore, the data suggest biofilm formation may afford protection against antimicrobial peptides. Production of reactive oxygen species in the fly gut, as in fleas, also serves to limit bacterial infection, and OxyR mediates Y. pestis survival in both insect models. Overall, our data establish the fruit fly as an informative model to elucidate the relationship between Y. pestis and its flea vector.

Role of the PhoP-PhoQ gene regulatory system in adaptation of Yersinia pestis to environmental stress in the flea digestive tract.

The Yersinia pestis PhoPQ gene regulatory system is induced during infection of the flea digestive tract and is required to produce adherent biofilm in the foregut, which greatly enhances bacterial transmission during a flea bite. To understand the in vivo context of PhoPQ induction and to determine PhoP-regulated targets in the flea, we undertook whole-genome comparative transcriptional profiling of Y. pestis WT and ΔphoP strains isolated from infected fleas and from temperature-matched in vitro planktonic and flow-cell biofilm cultures. In the absence of PhoP regulation, the gene expression program indicated that the bacteria experienced diverse physiological stresses and were in a metabolically less active state. Multiple stress response genes, including several toxin–antitoxin loci and YhcN family genes responsible for increased acid tolerance, were upregulated in the phoP mutant during flea infection. The data implied that PhoPQ was induced by low pH in the flea gut, and that PhoP modulated physiological adaptation to acid and other stresses encountered during infection of the flea. This adaptive response, together with PhoP-dependent modification of the bacterial outer surface that includes repression of pH 6 antigen fimbriae, supports stable biofilm development in the flea foregut.

LPS modification promotes maintenance of Yersinia pestis in fleas.

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

Factors associated with flea infestation among the different rodent species in Mbulu and Karatu districts, northern Tanzania

Flea infection with the bacterium, Yersinia pestis is acquired from reservoirs which include several rodents and other small mammals. In areas that are endemic of plague, reservoirs of Y. pestis and various flea vectors are responsible for perpetuating existence of the disease. The objective of this cross sectional study was to investigate the magnitude and factors associated with flea infestation among different rodent species of northern Tanzania, where outbreaks of plague have been recently reported. House rodents were trapped with box traps, while field and forest rodents were trapped with Sherman live traps. Fleas were removed from the rodents by using shoe-shining brush and were identified to genus level. Among the captured rodents, Rattus rattus (26.5%), Lophuromys flavopunctatus (16.5%), Praomys delectorum (16.2%) and Mastomys natalensis (32.3%) were most abundant rodent species, accounting for 91% of all species. Altogether, 805 fleas belonging to nine species were collected from 61% of the captured rodents. The most common fleas were Xenopsylla spp.; Dinopsyllus spp and Ctenophthalmus spp. Fleas were found to be highly abundant in M. natalensis, R. rattus, P. delectorum and L .flavopunctatus. Most of rodents were heavily infested with various flea species. These flea species probably play an important role in the transmission of plague in these two districts. We conclude that rodent species was the most important risk factor associating with flea infestation among the rodent population. Therefore, measures for control and prevention of plague in this area should particularly target rodents associated with high intensity of flea infestation.

Yersinia pestis insecticidal-like toxin complex (Tc) family proteins: characterization of expression, subcellular localization, and potential role in infection of the flea vector

BackgroundToxin complex (Tc) family proteins were first identified as insecticidal toxins in Photorhabdus luminescens and have since been found in a wide range of bacteria. The genome of Yersinia pestis, the causative agent of bubonic plague, contains a locus that encodes the Tc protein homologues YitA, YitB, YitC, and YipA and YipB. Previous microarray data indicate that the Tc genes are highly upregulated by Y. pestis while in the flea vector; however, their role in the infection of fleas and pathogenesis in the mammalian host is unclear.ResultsWe show that the Tc proteins YitA and YipA are highly produced by Y. pestis while in the flea but not during growth in brain heart infusion (BHI) broth at the same temperature. Over-production of the LysR-type regulator YitR from an exogenous plasmid increased YitA and YipA synthesis in broth culture. The increase in production of YitA and YipA correlated with the yitR copy number and was temperature-dependent. Although highly synthesized in fleas, deletion of the Tc proteins did not alter survival of Y. pestis in the flea or prevent blockage of the proventriculus. Furthermore, YipA was found to undergo post-translational processing and YipA and YitA are localized to the outer membrane of Y. pestis. YitA was also detected by immunofluorescence microscopy on the surface of Y. pestis. Both YitA and YipA are produced maximally at low temperature but persist for several hours after transfer to 37°C.ConclusionsY. pestis Tc proteins are highly expressed in the flea but are not essential for Y. pestis to stably infect or produce a transmissible infection in the flea. However, YitA and YipA localize to the outer membrane and YitA is exposed on the surface, indicating that at least YitA is present on the surface when Y. pestis is transmitted into the mammalian host from the flea.

Bartonella and Rickettsia in arthropods from the Lao PDR and from Borneo, Malaysia.

Rickettsioses and bartonelloses are arthropod-borne diseases of mammals with widespread geographical distributions. Yet their occurrence in specific regions, their association with different vectors and hosts and the infection rate of arthropod-vectors with these agents remain poorly studied in South-east Asia. We conducted entomological field surveys in the Lao PDR (Laos) and Borneo, Malaysia by surveying fleas, ticks, and lice from domestic dogs and collected additional samples from domestic cows and pigs in Laos. Rickettsia felis was detected by real-time PCR with similar overall flea infection rate in Laos (76.6%, 69/90) and Borneo (74.4%, 268/360). Both of the encountered flea vectors Ctenocephalides orientis and Ctenocephalides felis felis were infected with R. felis. The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsia detected in two Boophilus spp. ticks collected from a cow in Laos may be a new species. Isolation and further characterization will be necessary to specify it as a new species. Bartonella clarridgeiae was detected in 3/90 (3.3%) and 2/360 (0.6%) of examined fleas from Laos and Borneo, respectively. Two fleas collected in Laos and one flea collected in Borneo were co-infected with both R. felis and B. clarridgeiae. Further investigations are needed in order to isolate these agents and to determine their epidemiology and aetiological role in unknown fever in patients from these areas.

Analysis of monitoring results on plague's host animal and its medium insects in Quanzhou city of Fujian province between 2000 and 2009

Objective To examine the composition and distribution of animal reservoir of plague in Quanzhou city, Fujian province, and so as to accumulate basic data for making the plague prevention and control measures. Methods Rodents were captured by cages in six monitoring spots of Quanzhou city between 2000 and 2009. Then rodents and flea species were identified. Rats liver and spleen homogenates were detected of plague F1 antigen by reverse indirect hemagglutination test (RIHA); rats serum were detected of plague F1 antibody by indirect hemagglutination test (IHA). Results A total of 26 264 rodents were captured between 2000 and 2009.Rattus norvegicus and Rattus flavipectus were dominant species, which were accounted for 45.97％ (12 074/26 264)and 32.01％(8407/26 264), respectively. The account of Rattus flavipectus captured between 2005 and 2009 was (26.99 ± 2.46)％ and (37.03 ± 3.79)％ between 2000 and 2004. The difference was statistically significant (t =4.97, P ＜ 0.05). Total rodent densities was (6.86 ± 1.44)％, including(5.36 ± 1.83)％ in mountains and (6.81 ±1.66)％ in coastal areas, respectively, and the difference was not statistically significant(t =1.01, P ＞ 0.05). Total flea index and flea infection rate of rodents were 1.39 ± 0.34 and (35.90 ± 5.34)％, respectively. Xenopsylla Cheopis index was 1.20 and free flea index was 0.009. Flea infection rate was (32.36 ± 0.96)％ between 2005 and 2009, which was lower than (39.44 ± 0.39)％ between 2000 and 2004(t =2.76, P ＜ 0.05). Conclusions The major rodent species found in Quanzhou city is Rattus norvegicus. Xenopsylla Cheopis is the dominant flea species.Spreading of plague among Rattus is not found. Key words: Plague; Host-parasite relations; Fleas; Data collection

Transmission Shifts Underlie Variability in Population Responses to Yersinia pestis Infection

Host populations for the plague bacterium, Yersinia pestis, are highly variable in their response to plague ranging from near deterministic extinction (i.e., epizootic dynamics) to a low probability of extinction despite persistent infection (i.e., enzootic dynamics). Much of the work to understand this variability has focused on specific host characteristics, such as population size and resistance, and their role in determining plague dynamics. Here, however, we advance the idea that the relative importance of alternative transmission routes may vary causing shifts from epizootic to enzootic dynamics. We present a model that incorporates host and flea ecology with multiple transmission hypotheses to study how transmission shifts determine population responses to plague. Our results suggest enzootic persistence relies on infection of an off-host flea reservoir and epizootics rely on transiently maintained flea infection loads through repeated infectious feeds by fleas. In either case, early-phase transmission by fleas (i.e., transmission immediately following an infected blood meal) has been observed in laboratory studies, and we show that it is capable of driving plague dynamics at the population level. Sensitivity analysis of model parameters revealed that host characteristics (e.g., population size and resistance) vary in importance depending on transmission dynamics, suggesting that host ecology may scale differently through different transmission routes enabling prediction of population responses in a more robust way than using either host characteristics or transmission shifts alone.

Acquisition ofRickettsia felisby 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.

Yersinia pestis LOS stimulation of U937 cells shows different miRNA expression compared to Escherichia coli LPS stimulation (37.32)

Abstract Lipopolysaccharide (LPS)-stimulated activation of Toll-like Receptor 4 (TLR4) initiates downstream effects central to the inflammatory response that are important for host-pathogen interaction. In eukaryotes, microRNAs or miRNAs are small regulatory RNAs with multiple mRNA targets. There are ~1000 miRNAs encoded in the human genome, with the potential to regulate ~30% of the transcripts. The lipid A component of E. coli LPS is hexa-acylated, resulting in a highly proinflamatory response in human cells. Yersinia pestis synthesizes different lipid A structures depending on growth temperature, correlating to host conditions. Purified tetra-acylated lipooligosaccharide (LOS) of Y. pestis grown at 37○C, mimicking human infection, does not stimulate the innate immune system, whereas purified hexa-acylated LOS of Y. pestis grown at 25○C, mimicking flea infection, stimulates the innate immune system via TLR4, similar to E. coli LPS. Our hypothesis was that miRNA expression would be altered when human cells are incubated with purified LOS from Y. pestis grown at 25○C vs. 37○C. Initial studies in U937 cells using SABiosciences’ miRNA whole genome PCR arrays showed fewer miRNA expression changes in both LOS treatments than reported for LPS-stimulation. However, one modulation of interest is the 10-fold upregulation of miR-601 with 37○C LOS, compared to a 2-fold upregulation with 25○C. These data hint at a novel LOS-related mechanism, given recent reports of NFκB repression via miR-601.

Rickettsia felisin Fleas, France

To the Editor: Rickettsia felis belongs to the spotted fever group of rickettsia. The pathogenic role of this intracellular Proteobacteria in humans has been reported in patients from the United States (Texas) (1), Mexico (2), Germany (3), Brazil, and France (4). R. felis is widely distributed, is associated with blood-sucking arthropods, and has been isolated from fleas in several countries (5). To obtain new information about the distribution of R. felis in France and potential vectors/ reservoirs of this emerging pathogen, 550 fleas were collected from 82 dogs and 91 cats in 7 widely distributed locations in France (Bordeaux, Toulouse, Cosnes-Cours sur Loire, Dijon, Moulins, Limoges, and Aix-en-Provence). Specimens were collected by combing, recorded, and stored at –20°C. Samples were shipped on dry ice to the entomologic laboratory of the Institute of Comparative Tropical Medicine and Parasitology in Munich, Germany, and species identification was performed by using light microscopy and following the determination key of Hopkins and Rothschild (6). Because infestation levels varied (1–150 fleas/animal), we randomly analyzed 1–8 fleas (mean 3.4) from each host animal. We homogenized fleas individually in 80 μL of phosphate-buffered saline by using 5-mm steel beads in a RETSCH Tissue Lyser Mixer Mill 300 (QIAGEN, Hilden, Germany). A total of 100 μL of ATL buffer and 20 μL of proteinase K (QIAGEN) were added, and the homogenate was incubated at 56°C in a thermomixer (Eppendorf, Hamburg, Germany) until the tissues were lysed. DNA was extracted from each flea by using a QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions (tissue protocol) and stored at –20°C until used in a PCR. PCR amplification of rickettsial DNA was performed by using previously described oligonucleotide primer pairs Rp CS.877p/Rp CS.1258n targeting the citrate synthase (gltA) gene and, for the positive samples, Rr 190.70p/Rr 190.602n targeting the outer membrane protein A (ompA) gene (7). Amplification was conducted in 50-μL volumes that contained 5 μL of DNA, 30 μL of distilled water, 10 μL of 5× Taq buffer (Roche, Mannheim, Germany), 3 μL of 25 mmol/L MgCl2 (Roche), 1 μL of 10 mmol/L deoxyncleotide triphosphates (Roche), 0.25 μL of each primer (100 μM), and 0.5 μL (5 U/mL) of Taq polymerase (Roche). Conditions for the gltA and ompA PCRs were as described by Bertolotti et al. (8). Negative and positive controls were included in all PCRs. All PCR products were separated by electrophoresis on 1.5% agarose gels at 100 V for 60 min and examined under UV light. For both genes, positive samples were purified by using the QIAquick PCR Purification Kit (QIAGEN) and sent for sequencing to the MWG Biotech Company (Martinried, Germany). Sequences were compared with those of previously characterized rickettsia in GenBank by using basic local alignment search tool (BLAST) (www.ncbi.nlm.nih.gov) analysis. Five species of fleas were identified: Ctenocephalides felis (500, 224 from dogs and 276 from cats), C. canis (37 from dogs), Pulex irritans (11 from dogs), Spilopsyllus cuniculi (1 from a cat), and Archaeopsylla erinacei (1 from a cat). Five dogs had mixed populations of fleas; 3 of these had P. irritans and C. felis, and 2 had C. felis and C. canis. One cat had P. irritans and C. felis, and another cat had S. cuniculi and C. felis. A total of 52 (19%) of the 272 fleas from dogs and 44 (16%) of the 278 fleas from cats were positive for both the gltA and ompA genes. Positive samples were obtained from all locations. Prevalence ranged from 6% (Dijon) to 43% (Toulouse) for dogs and from 3% (Moulins) to 37% (Bordeaux) for cats (Table). Of 550 fleas, 96 were positive for both genes (gltA and ompA) and 3 of 5 species of fleas were infected: 10 with C. canis, 85 with C. felis, and 1 with A. erinacei. All sequences matched gltA and ompA genes from R. felis (similarity 99%–100%). Table Prevalence of Rickettsia felis in fleas from dogs and cats, France* Our investigation provides new information about distribution of R. felis and widespread flea infection with R. felis in France. A total of 88% of infected fleas were C. felis, but we found infected C. canis in Bordeaux and Toulouse and infected A. erinacei in Limoges. We report the presence in France of R. felis in C. canis and A. erinacei in France. R. felis in dog fleas in Uruguay and in hedgehog fleas in Algeria has been reported (9,10). Our findings indicate that these 2 flea species may be vectors of human R. felis rickettsiosis in France.

Patterns of parasitism and body size in red squirrels (Tamiasciurus hudsonicus)

Parasites use their hosts for nutrition, shelter, and even dispersal; the latter can result in sex-biased parasite distribution. Host sex-biased parasitism has been well documented in vertebrates, including mammals, and males are often more parasitized than females. Male-biased parasitism is often attributed to sexual size dimorphism, with larger animals being more parasitized. Here, we used a natural population of red squirrels ( Tamiasciurus hudsonicus (Erxleben, 1777)), a species without sexual size dimorphism, to test for sex-biased parasitism in ectoparasites and intestinal helminth parasites. We also tested for size-dependent parasitism to determine the importance of body size on parasitism. We predicted that males would be more parasitized and that larger individuals would be more parasitized. As well, we predicted a male-biased flea distribution on male squirrels. Parasitism fluctuated over the course of 4 months, with flea infection peaking in August and helminth infection peaking in June. We found evidence of male-biased parasitism in helminth and ectoparasite infections. Flea infection was weakly correlated with body size in females but not in males, while no correlation was found between body size and helminth infection. Lastly, fleas had a female-biased population; however, male fleas were more likely to be found on male squirrels, and this could be to maximize dispersal to avoid inbreeding.

Resistance of Yersinia pestis to Complement-Dependent Killing Is Mediated by the Ail Outer Membrane Protein

Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37 degrees C. Ail was expressed at high levels at both 26 and 37 degrees C, but not at 6 degrees C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.

Evaluation of the Role of Constitutive Isocitrate Lyase Activity inYersinia pestisInfection of the Flea Vector and Mammalian Host

Yersinia pestis, unlike the closely related Yersinia pseudotuberculosis, constitutively produces isocitrate lyase (ICL). Here we show that the Y. pestis aceA homologue encodes ICL and is required for growth on acetate but not for flea infection or virulence in mice. Thus, deregulation of the glyoxylate pathway does not underlie the recent adaptation of Y. pestis to arthropod-borne transmission.

Bartonella henselae infection in domestic cat and dog fleas

We studied on the infection of domestic cat and dog fleas with Bartonella henselae by polymerase chain reaction (PCR). A total of 62 fleas (36 Ctenocephalidis felis from cats, 24 C. felis from dogs and 2 Ctenocephalidis canis from dogs), stored in 70% ethanol, were analyzed by PCR for B. henselae specific DNA. Of the 62 fleas, C. felis from cats and dogs were positive for B. henselae specific DNA in 12 of the 36 (33.3%) and in 5 of the 24 (20.8%), respectively, and C. canis from dogs was positive in 2 of the 2 (100%). Our results demonstrated that pet fleas were infected with B. henselae, and suggest that flea transmission of B. henselae between cats or dogs may occur, and direct transmission of B. henselae from pet fleas to human may cause cat scratch disease.

Effect of Yersinia pestis infection on temperature preference and movement of the Oriental rat flea (Xenopsylla cheopis) (Siphonaptera: Pulicidae).

Previous laboratory studies have shown that inoculation of bacterial endotoxin into the hemocoel of some arthropods, or natural infection by a number of pathogens, causes them to seek out a higher ambient temperature. This phenomenon has been called behavioral fever. Yersinia pestis is an endotoxin-producing bacterium that relies on infection of fleas for transmission. Behavioral fever in fleas might enhance the transmission of plague if infected fleas were induced to seek out a warm-bodied host after the death of an infected host. Our study indicates that in thermal gradient Y. pestis infected Oriental rat fleas (Xenopsylla cheopis) do not exhibit behavioral fever and in one experiment sought out a significantly lower temperature.

Epizootiology of epidemic typhus (Rickettsia prowazekii) in flying squirrels.

Vector transmission of Rickettsia prowazekii among wild flying squirrels, Glaucomys volans, was suggested by the occurrence of natural infection of squirrel lice and fleas. Lice, mostly Neohaematopinus sciuropteri Osburn, were found infected in the fall in each of 2 consecutive years; 4 of the 8 pools of this insect tested were infected. Fleas, Orchopeas howardii (Baker), were found infected on two occasions in 1 of the 2 consecutive years. However, only 2 of 14 flea pools were infected. No evidence of infection was found in mites, Haemogamasus reidi Ewing and Androlaelaps fahrenholzi (Berlese). These findings implicate the flying squirrel louse and flea as possible vectors in nature. Serologic tests of flying squirrel sera revealed a maximum incidence of seroconversions in the fall and early winter months, coincident with the maximum increase in abundance of the suspected arthropod vectors. The infection was found to persist form year to year in the same enzootic foci. Infection appeared to spread most rapidly in young, non-immune animals born in the preceding spring and summer after congregating in dense aggregations in the fall. No other animals in the same habitat were found to have been infected. Aspects of the ecology of the ectoparasites associated with the flying squirrels are described, especially seasonal activity and abundance in nests. The potential public health importance of this sylvan disease in flying squirrels and in its ectoparasites, particularly the non-host specific, wide ranging squirrel flea, is noted.

Studies on Pasteurella pestis in fleas. VII. The plague-vector efficiency of Hystrichopsylla linsdalei compared with Xenopsylla cheopis under experimental conditions.

Summary An experimental study is reported on the plague-vector capacity of the wild-rodent flea, Hystrichopsylla linsdalei, compared with the rat flea, Xenopsylla cheopis. The average temperature under which the infected fleas were held (19°C) was lower than that in previous tests with H. linsdalei. Although the H. linsdalei blocked sooner than the X. cheopis, the rate of blocking in the latter species was more rapid and resulted in a greater number of blocked individuals. The plague-vector indices of male and female fleas combined were 0.002 for H. linsdalei and 0.03 for X. cheopis. The lower efficiency of H. linsdalei, under the conditions of the experiment, are partially ascribed to a lower blocking rate and a shorter survival period for the blocked individuals. Thus, the wild rodent flea had fewer opportunities to transmit P. pestis than the rat flea. There was no evidence that infectious feedings by the fleas on different mice with comparable plague septicemia influenced subsequent infection or blocking rates. Rather, differences between the two species suggested the presence of unknown physiological factors which may enter into the receptivity of a flea species to the growth of plague within its intestinal tract. The average death time of mice succumbing to plague transmitted by H. linsdalei was about 5 days, whereas the death time for mice infected by X. cheopis averaged about 4 days. This may have some bearing on the number of bacilli injected by blocked fleas. In confirmation of past work, it was found that culture of feces from, and microscopic observation of, fleas infected with P. pestis are useful procedures, but they do not provide an unequivocal basis for the determination of individual flea infection. The culture of individual fleas is the method of choice.