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Background

Saliva of hematophagous arthropods contains a diverse mixture of compounds that counteracts host hemostasis. Immunomodulatory and antiinflammatory components are also found in these organisms' saliva. Blood feeding evolved at least ten times within arthropods, providing a scenario of convergent evolution for the solution of the salivary potion. Perhaps because of immune pressure from hosts, the salivary proteins of related organisms have considerable divergence, and new protein families are often found within different genera of the same family or even among subgenera. Fleas radiated with their vertebrate hosts, including within the mammal expansion initiated 65 million years ago. Currently, only one flea species–the rat flea Xenopsylla cheopis–has been investigated by means of salivary transcriptome analysis to reveal salivary constituents, or sialome. We present the analysis of the sialome of cat flea Ctenocephaides felis.

Methodology and Critical Findings

A salivary gland cDNA library from adult fleas was randomly sequenced, assembled, and annotated. Sialomes of cat and rat fleas have in common the enzyme families of phosphatases (inactive), CD-39-type apyrase, adenosine deaminases, and esterases. Antigen-5 members are also common to both sialomes, as are defensins. FS-I/Cys7 and the 8-Cys families of peptides are also shared by both fleas and are unique to these organisms. The Gly-His-rich peptide similar to holotricin was found only in the cat flea, as were the abundantly expressed Cys-less peptide and a novel short peptide family.

Conclusions/Significance

Fleas, in contrast to bloodsucking Nematocera (mosquitoes, sand flies, and black flies), appear to concentrate a good portion of their sialome in small polypeptides, none of which have a known function but could act as inhibitors of hemostasis or inflammation. They are also unique in expansion of a phosphatase family that appears to be deficient of enzyme activity and has an unknown function.

Background

From 1990 to 2006, fifty-five natural villages experienced at least one plague epidemic in Lianghe County, Yunnan Province, China. This study is aimed to document flea abundance and identify predictors in households of villages with endemic commensal rodent plague in Lianghe County.

Methods

Trappings were used to collect fleas and interviews were conducted to gather demography, environmental factors, and other relevant information. Multivariate hurdle negative binomial model was applied to identify predictors for flea abundance.

Results

A total of 344 fleas were collected on 101 small mammals (94 Rattus flavipectus and 7 Suncus murinus). R. flavipectus had higher flea prevalence and abundance than S. murinus, but the flea intensities did not differ significantly. A total of 315 floor fleas were captured in 104 households. Xenopsylla cheopis and Ctenocephalides felis felis were the predominant flea species on the host and the floor flea, respectively. The presence of small mammal faeces and R. flavipectus increased host flea prevalence odds 2.9- and 10-fold, respectively. Keeping a dog in the house increased floor flea prevalence odds 2-fold. Keeping cattle increased floor flea intensity by 153%. Villages with over 80% of houses raising chickens had increased prevalence odds and intensity of floor flea about 2.9- and 11.6-fold, respectively. The prevalence and intensity of floor flea in brick and wood houses were decreased by 60% and 90%, respectively. Flea prevalences of host and floor flea in the households that were adjacent to other houses were increased 7.4- and 2.2-fold, respectively. Houses with a paddy nearby decreased host flea intensity by 53%, while houses with an outside toilet increased host flea intensity by 125%.

Conclusion

Rodent control alone may not be sufficient to control plague risk in these areas. In order to have successful results, plague control programs should pay attention to ecological and hygiene factors that influence flea populations.

Author Summary

Yunnan province is located in southwest China. Plague is still a huge threat to the health of local people in Yunnan where plague epidemics had the most serious impacts than other provinces in China. The risk of plague outbreak is driven by rodent and flea populations. Our research team is conducting a study to identify predictors for abundance of host and floor fleas in households of villages with endemic commensal rodent plague in Yunnan province. The results of this study will contribute to control host and floor flea populations, and therefore to prevent and control plague outbreaks in these areas.

Yersinia pestis, the plague bacillus, infects a variety of mammals throughout the world and is transmitted by fleas. We developed a polymerase chain reaction (PCR) test using primers designed from the Y. pestis plasminogen activator gene to directly detect plague-infected fleas. As few as 10 Y. pestis cells were detected, even in the presence of flea tissue, by PCR and then agarose gel electrophoresis and ethidium bromide staining. The feasibility of the assay was demonstrated by using naturally infected Xenopsylla cheopis fleas. The detection of Y. pestis in fleas by PCR provides a rapid and sensitive way to monitor plaque in wild animal populations, allowing public health officials to better assess the potential risk of transmission to humans.

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The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion comprised of dozens or near one hundred different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy creating unique salivary potions in the form of novel pharmacological use of endogenous substances, and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls

Fleas collected from rats during a three-year period (2000–2003) in 51 areas of all provinces of Cyprus were tested by molecular analysis to characterize the prevalence and identity of fleaborne rickettsiae. Rickettsia typhi, the causative agent of murine typhus, was detected in Xenopsylla cheopis (4%) and in Leptopsylla segnis (6.6%). Rickettsia felis was detected in X. cheopis (1%). This is the first report of R. typhi in X. cheopis and L. segnis from rats, in Cyprus, and the first report of R. felis in X. cheopis in Europe. The role of fleas (mainly X. cheopis) was confirmed in the epidemiologic cycle of murine typhus in Cyprus by interrelation of current results with those of previous studies. The geographic distribution of fleas coincided with the geographic distribution of the pathogen they can harbor, which emphasizes the potential risk of flea-transmitted infections in Cyprus.

The salivary glands of blood sucking arthropods contain a redundant ‘magic potion’ that counteracts their vertebrate host’s hemostasis, inflammation, and immunity. We here describe the salivary transcriptome and proteomics (sialome) of the soft tick Ornithodoros coriaceus. The resulting analysis helps to consolidate the classification of common proteins found in both soft and hard ticks, such as the lipocalins, Kunitz, cystatin, basic tail, hebraein, defensin, TIL domain, metalloprotease, 5′-nucleotidase/apyrase, and phospholipase families, and also to identify protein families uniquely found in the Argasidae, such as the adrenomedullin/CGRP peptides, 7DB, 7 kDa, and the RGD containing single Kunitz proteins. Additionally, we found a protein belonging to the cytotoxin protein family that has so far only been identified in hard ticks. Three other unique families common only to the Ornithodoros genus were discovered. Edman degradation, 2D and 1D PAGE of salivary gland homogenates followed by tryptic digestion and HPLC MS/MS of results confirms the presence of several proteins. These results indicate that each genus of hematophagous arthropods studied to date evolved unique protein families that assist blood feeding, thus characterizing potentially new pharmacologically active components or antimicrobial agents.

After an outbreak of human plague, 95 Xenopsylla cheopis fleas from Algeria were tested for Yersinia pestis with PCR methods. Nine fleas were definitively confirmed to be infected with Y. pestis biovar orientalis. Our results demonstrate the persistence of a zoonotic focus of Y. pestis in Algeria.

Background

The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. Rickettsia typhi, the etiologic agent of murine typhus, is a strictly intracellular gram negative α-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, Xenopsylla cheopis. Thus, R. typhi must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing R. typhi to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37°C to 25°C, mimicking the two different host temperatures in vitro.

Results

Temperature-responsive genes belonged to multiple functional categories including among others, transcription, translation, posttranslational modification/protein turnover/chaperones and intracellular trafficking and secretion. A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database. The microarray results were validated with quantitative real time RT-PCR.

Conclusion

This microarray screen identified various genes that were differentially expressed upon a shift in temperature from 37°C to 25°C. Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.

We investigated a cluster of 5 plague cases; the patients included 4 with severe pharyngitis and submandibular lymphadenitis. These 4 case-patients had eaten raw camel liver. Yersinia pestis was isolated from bone marrow of the camel and from jirds (Meriones libycus) and fleas (Xenopsylla cheopis) captured at the camel corral.

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.

Social animals are susceptible to high infection levels by contact-transmitted parasites due to increased conspecific interaction. Exotic parasites are known to have adverse consequences on native hosts. We examined the relationship between social group size and exotic ectoparasite loads, and adult infection levels with per capita fitness and offspring survival in the plural breeding rodent Octodon degus in central Chile. Degus at our site were almost entirely infected by two exotic ectoparasites: the fleas Leptopsylla segnis and Xenopsylla cheopis. Neither group size nor number of females per group predicted the abundance of either exotic flea species. The per capita number of pups (per capita fitness) that emerged from burrow systems used by known social groups was negatively correlated with abundance of L. segnis but not X. cheopis. On adults, X. cheopis abundance was three times greater than L. segnis but was not significantly correlated with per capita fitness. In females, L. segnis abundance was negatively correlated with peak body mass during pregnancy. Adult ectoparasite load was not correlated with offspring survival. Based on these results, we hypothesize that high infection levels of L. segnis result in decreased reproductive fitness of adult female degus but are not a cost of sociality because parasite loads are not predicted by social group size. Further work is needed to experimentally test this hypothesis and to determine if L. segnis serves as a vector for a deleterious pathogen. Lastly, the lack of native ectoparasites may explain why a previous study at our site determined that behavioral adaptations needed to cope with high ectoparasite burdens (e.g., grooming) are not extensive in degus; they simply have not had the coevolutionary time needed for selection of these behaviors.

Background

Traditionally, efficient flea-borne transmission of Yersinia pestis, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of Xenopsylla cheopis with a wild type strain of Y. pestis and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using Y. pestis-specific antigen or antibody detection assays on mouse tissues.

Results

Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted Y. pestis infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures.

Conclusions

Our results suggest that temperature does not significantly effect the per flea efficiency of Y. pestis transmission by X. cheopis, but that temperature is likely to influence the dynamics of Y. pestis flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether Y. pestis biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.

The virus of Mexican typhus has been shown to multiply abundantly in the following species of fleas: Xenopsylla cheopis, Ceratophyllus fasciatus, Leptopsylla musculi, Ctenocephalus canis, Ctenocephalus felis. In all fleas, Rickettsia prowazeki was demonstrated within the epithelial cells of the stomach and within the cells of the Malpighian tubules. Whereas in infected lice enormous numbers of these organisms are discharged from the disintegrating cells into the intestinal content, only few Rickettsiae are found in the lumen of the fleas' intestines. They are held back by the peritrophic membrane, which covers the mucosa of the entire stomach. Rickettsiae seem to enter the lumen of the gut almost exclusively by the route of the Malpighian tubules. Observations were made which seem to indicate that the fleas recover from the infection and that they are able to regenerate the partly destroyed intestinal mucosa. An explanation is given for the relative harmlessness of fleas as vectors of typhus.

Of 200 individual Xenopsylla cheopis fleas removed from Rattus norvegicus rats trapped in downtown Los Angeles, CA, 190 (95%) were positive for the presence of Bartonella DNA. Ninety-one amplicons were sequenced: Bartonella rochalimae-like DNA was detected in 66 examined fleas, and Bartonella tribocorum-like DNA was identified in 25 fleas. The data obtained from this study demonstrate an extremely high prevalence of Bartonella DNA in rat-associated fleas.

Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sandflies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2β1 or GPVI. Molecules that bind ADP, TXA2, epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbβ3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications.

Abstract

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. Key Words: Cat flea—Ctenocephalides felis—Rickettsia felis.

Early-phase transmission (EPT) is a recently described model of plague transmission that explains the rapid spread of disease from flea to mammal host during an epizootic. Unlike the traditional blockage-dependent model of plague transmission, EPT can occur when a flea takes its first blood meal after initially becoming infected by feeding on a bacteraemic host. Blockage of the flea gut results from biofilm formation in the proventriculus, mediated by the gene products found in the haemin storage (hms) locus of the Yersinia pestis chromosome. Although biofilms are required for blockage-dependent transmission, the role of biofilms in EPT has yet to be determined. An artificial feeding system was used to feed Xenopsylla cheopis and Oropsylla montana rat blood spiked with the parental Y. pestis strain KIM5(pCD1)+, two different biofilm-deficient mutants (ΔhmsT, ΔhmsR), or a biofilm-overproducer mutant (ΔhmsP). Infected fleas were then allowed to feed on naïve Swiss Webster mice for 1–4 days after infection, and the mice were monitored for signs of infection. We also determined the bacterial loads of each flea that fed upon naïve mice. Biofilm-defective mutants transmitted from X. cheopis and O. montana as efficiently as the parent strain, whereas the EPT efficiency of fleas fed the biofilm-overproducing strain was significantly less than that of fleas fed either the parent or a biofilm-deficient strain. Fleas infected with a biofilm-deficient strain harboured lower bacterial loads 4 days post-infection than fleas infected with the parent strain. Thus, defects in biofilm formation did not prevent flea-borne transmission of Y. pestis in our EPT model, although biofilm overproduction inhibited efficient EPT. Our results also indicate, however, that biofilms may play a role in infection persistence in the flea.

Bartonella spp. prevalence in small mammals and their ectoparasites was investigated in Taiwan. Blood samples were obtained from 66 rats, 20 shrews, 276 mites (Laelaps spp.), 74 fleas (Xenopsylla cheopis), 81 lice (Polyplax spp.), and 47 ticks (41 Dermacentor spp. and 6 Ixodes spp.). Bartonellae were isolated or detected in 27 (31.4%) animals. Bartonella DNA was detected in 48 (64.9%) fleas and 11 (64.7%) pooled lice samples, but not in mite and tick samples. Bartonella phoceensis, B. queenslandensis, B. tribocorum, B. elizabethae, and B. rattimassiliensis were isolated or detected in bacteremic mammals. For the first time in Taiwan, B. tribocorum, B. elizabethae, B. queenslandensis, and a B. rochalimae-like strain were detected in fleas, and B. tribocorum, B. phoceensis, and B. rattimassiliensis were detected in lice obtained from small mammals. A broader range of Bartonella species was identified in the ectoparasites than in the small mammals.

Type VI secretion systems (T6SS) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26° C in comparison to 37° C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 hours postinfection for bacterial cells pre-grown at 26° C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37°C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.

Rickettsia typhi and R. felis, etiologic agents of murine typhus and fleaborne spotted fever, respectively, were detected in Oriental rat fleas (Xenopsylla cheopis) collected from rodents and shrews in Java, Indonesia. We describe the first evidence of R. felis in Indonesia and naturally occurring R. felis in Oriental rat fleas.

Background

Little is known about the composition and function of the saliva in black flies such as Simulium guianense, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity.

Results

Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the S. guianense sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in Simulium. Additionally, we found a novel peptide similar to kunitoxin with a structure distantly related to serine protease inhibitors. This study revealed a relative increase of transcripts of the SVEP protein family when compared with Simulium vittatum and S. nigrimanum sialotranscriptomes. We were able to extract coding sequences from 164 proteins associated with blood and sugar feeding, the majority of which were confirmed by proteome analysis.

Conclusions

Our results contribute to understanding the role of Simulium saliva in transmission of Onchocerca volvulus and evolution of salivary proteins in black flies. It also consists of a platform for mining novel anti-hemostatic compounds, vaccine candidates against filariasis, and immuno-epidemiologic markers of vector exposure.

Background

Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae.

Results

As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken.

Conclusions

The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.

Adaptation to vertebrate blood feeding includes development of a salivary ‘magic potion’ that can disarm host hemostasis and inflammatory reactions. Within the lower Diptera, a vertebrate blood-sucking mode evolved in the Psychodidae (sand flies), Culicidae (mosquitoes), Ceratopogonidae (biting midges), Simuliidae (black flies), and in the frog-feeding Corethrellidae. Sialotranscriptome analyses from several species of mosquitoes and sand flies and from one biting midge indicate divergence in the evolution of the blood-sucking salivary potion, manifested in the finding of many unique proteins within each insect family, and even genus. Gene duplication and divergence events are highly prevalent, possibly driven by vertebrate host immune pressure. Within this framework, we describe the sialome (from Greek sialo, saliva) of the black fly Simulium vittatum and discuss the findings within the context of the protein families found in other blood-sucking Diptera. Sequences and results of Blast searches against several protein family databases are given in Supplemental Tables S1 and S2, which can be obtained from http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip.

Rickettsia typhi (prevalence 1.9%) and R. felis (prevalence 24.8%) DNA were detected in rat fleas (Xenopsylla cheopis) collected from mice on Oahu Island, Hawaii. The low prevalence of R. typhi on Oahu suggests that R. felis may be a more common cause of rickettsiosis than R. typhi in Hawaii.

The 5′ nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5′ nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 × 105 copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.