A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.Lyme disease and human anaplasmosis (formerly human granulocytic ehrlichiosis) have emerged as two of the most common vector-borne bacterial illnesses in the United States, where Borrelia burgdorferi and Anaplasma phagocytophilum have been identified as their respective etiologic agents (3)(4)(5)20). Both B. burgdorferi and A. phagocytophilum share common tick vectors and rodent reservoirs, with Ixodes scapularis characterized as the principal vector species and the white-footed mouse, Peromyscus leucopus, as a major reservoir species (3,14,19,22,25). Lyme disease and human anaplasmosis are found in overlapping regions of the United States, with most cases occurring in the northeast and upper midwest regions, areas that have large populations of both rodents and I. scapularis ticks.The screening of tick and rodent samples for B. burgdorferi and A. phagocytophilum has been used for studies designed to assess the prevalence of these agents in nature and the risk they pose to the human population. Numerous methods for the detection of B. burgdorferi and A. phagocytophilum have been described and include antibody and antigen detection assays (immunofluorescence assay, enzyme-linked immunosorbent assay, and Western blotting), culture isolation, and PCR-based assays. PCR assays have been shown to be both sensitive and specific for the detection of B. burgdorferi and A. phagocytophilum in clinical and field-collected samples (5,11,17,18,23,26). However, PCR assays can be time-consuming and laborintensive, particularly when testing for multi...

Abstract. In January 2007, an outbreak of Rift Valley fever (RVF) was detected among humans in northern Tanzania districts. By the end of the outbreak in June, 2007, 511 suspect RVF cases had been recorded from 10 of the 21 regions of Tanzania, with laboratory confirmation of 186 cases and another 123 probable cases. All confirmed RVF cases were located in the north-central and southern regions of the country, with an eventual fatality rate of 28.2% ( N = 144). All suspected cases had fever; 89% had encephalopathy, 10% hemorrhage, and 3% retinopathy. A total of 169 (55%) of the 309 confirmed or probable cases were also positive for malaria as detected by peripheral blood smear. In a cohort of 20 RVF cases with known outcome that were also positive for human immunodeficiency virus, 15 (75%) died. Contact with sick animals and animal products, including blood, meat, and milk, were identified as major risk factors of acquiring RVF.

Quantitative real-time PCR was used to assay spirochetes in feeding ticks. Spirochetes in tick midguts increased sixfold, from 998 per tick before attachment to 5,884 at 48 h of attachment. Spirochetes in tick salivary glands increased >17-fold, from 1.2 per salivary gland pair before feeding to 20.8 at 72 h postattachment. The period of the most rapid increase in the number of spirochetes in the salivary glands occurred from 48 to 60 h postattachment; this time period coincides with the maximal increase in transmission risk during nymphal tick feeding.Nymphal Ixodes scapularis ticks are the principal vectors of Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) in North America. These spirochetes appear to be well adapted to their hosts (1) and tick vectors (12). Questing nymphal I. scapularis ticks contain spirochetes restricted mainly to the tick midgut. When an infected tick successfully finds a host and starts to take a blood meal, dramatic changes occur in the spirochete population: spirochetes multiply rapidly in the midgut of infected ticks, thus increasing the overall spirochete density (3). In parallel with increased density, a shift occurs in the dominant outer surface protein expression of the spirochetes, from OspA to OspC (4,17). Other proteins, such as the vlsE protein, become more heterogeneous upon tick attachment (12). Spirochetes then migrate through the hemolymph of the feeding nymphal tick to the salivary glands, where they are subsequently transmitted to the skin of the vertebrate host (2,12,21).A basic understanding of spirochete dynamics within feeding ticks is central to an appreciation of why nymphal I. scapularis ticks infected with B. burgdorferi sensu stricto removed during the first 2 days of attachment do not transmit infection to tick bite victims whereas those feeding for longer periods efficiently transmit infectious spirochetes (6,10,12). In addition, the efficacy of the newly licensed recombinant OspA human vaccine in use in the northeastern United States is apparently based on the ability of anti-OspA antibodies to enter the tick midgut and kill spirochetes before they can migrate to the salivary glands to be transmitted to the victims of tick bite (4, 5). This vaccine does not appear to act against the spirochetes within the vertebrate host (4,20). Thus, studies on the dynamics of spirochete populations in feeding ticks will improve our understanding of how ticks transmit spirochetes and how to prevent such transmission. A better appreciation of the interactions between ticks, hosts, and spirochetes may also lead to a better understanding of the epidemiology and ecology of these important human pathogens (11).Spirochete populations in ticks have been quantified principally through the use of microscopic tools. Rough estimates of the number of spirochetes viewed in tick smears through the use of standard epifluorescent, electron, or confocal microscopy have been reported (2, 12, 21). Recently, extremely sensitive and accurate quantitative fluorogenic-detection PCR ...

Vector-borne viruses are naturally transmitted when a vector salivates during feeding on a vertebrate host. Most laboratory studies of infection disregard the role that the vector plays in the pathogenesis of the virus. In this study, intradermal inoculations of Aedes aegypti salivary gland extract (SGE) and Sindbis virus (SINV) were used to investigate the effect of mosquito feeding on the vertebrate immune response to infection with an arthropod-borne virus. Murine cytokine expression in the skin was quantified by means of real-time RT-PCR. In response to co-inoculation of SINV with SGE, interferon (IFN)-beta expression at 24 and 72 h post inoculation was significantly reduced by 2.2- and 2.3-fold, respectively, when compared to injection of virus alone. IFN-gamma expression in response to SINV infection was significantly decreased by 1.6-fold at 24 h post inoculation when SGE was co-inoculated. In contrast, interleukin (IL)-4 expression was significantly up regulated when SGE was co-inoculated at 24 h post inoculation becoming a 3.3-fold increase by 72 h post inoculation. Compared to expression with SINV alone, IL-10 expression showed a 7.6-fold increase by 72 h post inoculation in mice receiving SGE concurrently with virus. This study suggests that the response to virus is significantly different when an infection is initiated in the presence of mosquito salivary factors, and we identify a possible mechanism for potentiation of viral infections initiated by the natural mosquito vector or in the presence of mosquito saliva.

Culex pipiens and Aedes aegypti mosquitoes were fed on C3H/HeJ mice and systemic cytokine production was quantified from stimulated lymphocytes harvested four to ten days after feeding. Mosquito feeding on C3H/HeJ mice significantly down regulated IFN gamma production seven to ten days post feeding by Cx. pipiens and seven days after Ae aegypti feeding. Th2 cytokines, IL-4 and IL-10, were significantly up regulated 4-7 days after Cx. pipiens and Ae. aegypti feeding. The immunosuppressive effect of Cx. pipiens feeding on systemic cytokine production was not evident in congenic flavivirus resistant (C3H/RV) mice, as systemic IFN gamma and IL-2 were significantly up regulated at days 7 and 10, correlating with a significant decrease in IL-4 10 days after feeding by Cx. pipiens mosquitoes. Inoculation of 5-1000 ng of sialokinin-I into C3H/HeJ mice mimicked the effect of Ae. aegypti feeding by down regulating Th1 cytokines and significantly up regulating Th2 cytokines four days post inoculation. Injections of sialokinin-II resulted in only moderate effects on IFN gamma and IL-4 production seven and ten days after injection. Thus natural feeding by two arbovirus vectors had a profound T cell modulatory effect in vivo in virus susceptible animals which was not demonstrated in the flavivirus resistant host. Moreover, sialokinin-I and sialokinin-II mimicked the effect of mosquito feeding by modulating the host T cell response. These results may lend new insight into specific aspects of the role of the mosquito vector in potentiating virus transmission in the mammalian host.

A 3-yr community-based study was conducted on residential properties on Mason's Island, Mystic, CT, to determine the efficacy of a rodent-targeted acaricide (fipronil) to control immature Ixodes scapularis (Say) on Peromyscus leucopus. Results indicated that modified commercial bait boxes were effective as an acaricide delivery method for reducing nymphal and larval tick infestations on white-footed mice by 68 and 84%, respectively. Passive application of fipronil significantly reduced the infection rate of Borrelia burgdorferi among white-footed mice by 53%. Moreover, the abundance of questing I. scapularis adults on treated properties was reduced by 77% and fewer were infected with spirochetes (31%) compared with untreated sites (47%) after 3 yr of treatment. Likewise, the abundance of host-seeking nymphs was significantly reduced on treated properties by >50%. Finally, infection rates in flagged nymphal ticks for both B. burgdorferi and Anaplasma phagocytophilum were reduced by 67 and 64%, respectively, after only 2 yr of treatment. Results from this 3-yr trial indicate that the use of fipronil passively applied to reservoir animals by bait boxes is an environmentally acceptable means to control ticks, interrupt the natural disease transmission cycle, and reduce the risk of Lyme disease for residents of treated properties.

Previous work described an enzootic cycle of Borrelia burgdorferi sensu lato (hereafter referred to as B. burgdorferi) maintained by the rodent Neotoma mexicana and the tick Ixodes spinipalpis in northern Colorado. We investigated the incidence of coinfection among rodents with the agent of human granulocytic ehrlichiosis (aoHGE). aoHGE was detected in 23.5% of 119 rodent spleens examined. Biopsy results indicated that 78 (65.5%) of the 119 rodents were positive for B. burgdorferi, whereas 22 (78.5%) of the 28 animals that harbored aoHGE were also infected with B. burgdorferi. In 14 of 25 I. spinipalpis tick pools, aoHGE was detected by amplifying both the 16s rRNA and p44 gene of aoHGE. The ability of I. spinipalpis to transmit aoHGE was examined in C3H/HeJ mice. aoHGE was detected in their blood 5 days after I. spinipalpis infestation. This study confirms that both B. burgdorferi and aoHGE can be transmitted by I. spinipalpis ticks and that there is a high incidence of coinfection in rodents, predominantly Peromyscus maniculatus and N. mexicana, that inhabit the foothills of northern Colorado.

Down-regulation of mammalian cytokine production has been demonstrated during tick feeding. To examine the hypothesis that reconstitution of cytokines during tick feeding could facilitate immune containment of Borrelia burgdorferi, the following experiments were done. C3H/HeJ mice were given cytokines for 10 days after Ixodes scapularis attachment. At day 21, ear biopsies were analyzed for B. burgdorferi. Polymerase chain reaction analysis indicated a protection rate of 95% in mice receiving tumor necrosis factor (TNF)-alpha. Mice that received interleukin (IL)-2 or interferon (IFN)-gamma had infection rates of 30%-45% compared with 83% for untreated controls. No correlation was noted between neutralizing antibody, reactivity by Western blot, and subsequent protection. Culture of B. burgdorferi in cytokine-conditioned media indicated that TNF-alpha, IFN-gamma, and IL-2 were not cytotoxic for B. burgdorferi. These data suggest that cytokine-induced protection from B. burgdorferi infection was immune-mediated and that cellular immunity may be associated with protection from I. scapularis-induced infection.