Nordin S. Zeidner

Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi

ABSTRACT 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.

Transmission of the Agent of Human Granulocytic Ehrlichiosis by Ixodes spinipalpis Ticks: Evidence of an Enzootic Cycle of Dual Infection with Borrelia burgdorferi in Northern Colorado

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.

Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors.

ABSTRACT 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.

Epidemiologic and Clinical Aspects of a Rift Valley Fever Outbreak in Humans in Tanzania, 2007

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.

Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed‐tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac‐silenced infected nymphs. SDS‐PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non‐salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood‐feeding in an attempt to block transmission of B. burgdorferi.

Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible mice: an effect mimicked by injection of sialokinins, but not demonstrated in flavivirus resistant mice

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 IFNγ 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 IFNγ 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 IFNγ 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.

Coinoculation of Borrelia spp. with Tick Salivary Gland Lysate Enhances Spirochete Load in Mice and Is Tick Species–Specific

C3H/HeN mice were inoculated with 106 spirochetes, either Borrelia burgdorferi strain N40 or the Portuguese strain of B. lusitaniae, PotiB2. Mice receiving spirochetes coinoculated with salivary gland lysate (SGL) demonstrated significantly higher spirochete loads in target organs as measured by quantitative real-time polymerase chain reaction. This effect was tick dependent, in that Ixodes ricinus SGL specifically enhanced B. lusitaniae load, whereas I. scapularis SGL specifically increased B. burgdorferi N40 load, but did not significantly affect the dissemination of B. lusitaniae. Protein profile analysis indicated at least 5 major protein differences between I. scapularis and I. ricinus SGL, which can possibly account for this specific tick–spirochete interaction.

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFNγ production and promotes an IL-4 response in C3H/HeJ mice

Previously we demonstrated that Borrelia burgdorferi transmission by Ixodes scapularis suppressed IL‐2 and IFNγ production and promoted IL‐4 production in mice. The present studies were conducted to determine whether coinfection with the human granulocytic ehrlichiosis (HE) agent would promote a Th2 cytokine response in mice. Transmission to the spleen of the agent of human granulocytic ehrlichiosis (aoHGE) and B. burgdorferi occurred 4 and 7 days, respectively, after tick infestation. Coinfection synergized to suppress splenic IL‐2 production 7–14 days after tick infestion. Transmission of B. burgdorferi or aoHGE alone significantly decreased splenic IFNγ 4–7 days after tick infestation, while coinfection suppressed IFNγ production 7–14 days after tick infestation. Splenic IL‐4 production was significantly increased 4 days after coinfection, and by day 10, aoHGE plus B. burgdorferi induced greater splenic IL‐4 (57.2 pg/ml, 348% of control values) than either organism transmitted alone (aoHGE, 22.7 pg/ml, B. burgdorferi, 25.1 pg/ml). Coinfection enhanced expansion of splenic T cells, CD4+ lymphocytes and B cells while decreasing CD8+ T cells. These data demonstrate that aoHGE and B. burgdorferi, when cotransmitted, suppress a systemic IL‐2 and IFNγ response, while strongly promoting systemic IL‐4 production in the susceptible host. The antigen(s) responsible for this polarization are unknown and will be the subject of future studies.

An Analysis of Spirochete Load, Strain, and Pathology in a Model of Tick-Transmitted Lyme Borreliosis

Four laboratory-grown, low-passage isolates of Borrelia burgdorferi sensu stricto, B31, JD-1, 910255, and N40, were incorporated into Ixodes scapularis ticks to examine the pathogenesis of these isolates in mice after tick transmission. All isolates induced multifocal, lymphoid nodular cystitis, subacute, multifocal, necrotizing myocarditis, and a localized periostitis and arthritis of the femorotibial joint 6-18 weeks after tick infestation. In terms of the number of mice that demonstrated pathology in bladder, heart, and joint, the highest incidence of lesions occurred 12 weeks after tick bite. Utilizing the Taqman quantitative polymerase chain reaction (q-PCR) fluorogenic detection technology to amplify a conserved region of the flagellin gene, a trend was demonstrated between the number of spirochetes in tissue with duration of pathology. The q-PCR assay developed for this study was sensitive and could reliably measure as few as 1 to 10 spirochetes in the target tissues tested. A higher percentage of B31- and N40-infected mice (92 and 100%, respectively) developed myocarditis than JD-1- or 910255-infected mice (67 and 46%, respectively) 12 weeks after tick bite. The amount of spirochetal DNA that could be amplified for heart at this time point was not statistically different between isolates, indicating a difference in virulence between B31 and N40 relative to JD-1 and 910225. N40-infected mice demonstrated a significantly higher spirochete load (an average of 1.23 spirochetes/mg of tissue, p = 0.045) in femorotibial joints 18 weeks after infection, with 60% of these mice maintaining lesions compared with those infected with B31 (13%), JD-1 (25%), or 910255 (50%), which averaged <0.5 spirochetes/mg of tissue. This mouse model of Lyme borreliosis, including the ability to monitor lesion development and spirochete load, can facilitate the testing of therapeutic regimens for the later stages of tick-transmitted Lyme disease and help investigate aspects of the immunopathogenesis of lesion development.

Laboratory analysis of tularemia in wild-trapped, commercially traded prairie dogs, Texas, 2002.

Oropharyngeal tularemia was identified as the cause of a die-off in captured wild prairie dogs at a commercial exotic animal facility in Texas. From this point source, Francisella tularensis–infected prairie dogs were traced to animals distributed to the Czech Republic and to a Texas pet shop. F. tularensis culture isolates were recovered tissue specimens from 63 prairie dogs, including one each from the secondary distribution sites. Molecular and biochemical subtyping indicated that all isolates were F. tularensis subsp. holarctica (Type B). Microagglutination assays detected antibodies against F. tularensis, with titers as great as 1:4,096 in some live animals. All seropositive animals remained culture positive, suggesting that prairie dogs may act as chronic carriers of F. tularensis. These findings demonstrate the need for additional studies of tularemia in prairie dogs, given the seriousness of the resulting disease, the fact that prairie dogs are sold commercially as pets, and the risk for pet-to-human transmission.