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Transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice.

Vesicular stomatitis is an economically important arboviral disease of livestock. Viremia is absent in infected mammalian hosts, and the mechanism by which insects become infected with the causative agents, vesicular stomatitis viruses, remains unknown. Because infected and noninfected insects potentially feed on the same host in nature, infected and noninfected black flies were allowed to feed on the same host. Viremia was not detected in the host after infection by a black fly bite, but because noninfected black flies acquired the virus while co-feeding on the same host with infected black flies, it is concluded that a viremic host is not necessary for an insect to be infected with the virus. Thus co-feeding is a mechanism of infection for an insect-transmitted virus.

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Toxicant-disease-environment interactions associated with suppression of immune system, growth, and reproduction.

The effects of marginal malnourishment , infections, and environmental chemicals on growth and reproductive success in Swiss-Webster white mice and wild deer mice were studied with fractional factorial designs. Interaction effects were discovered. For example, malnourished mice were more sensitive to virus exposure and environmental chemicals (a plant growth regulator or polychlorinated biphenyls). Since several commercial plant growth regulators also appear to suppress the immune system, these results cast doubt on the adequacy of current toxicity testing procedures in which factors are studied individually and not in combination.

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Natural Distribution of the Ixodes dammini spirochete.

Spirochetes believed to be the cause of Lyme disease were isolated from white-footed mice and white-tailed deer, the preferred natural hosts of Ixodes dammini, the tick vector. Evidence suggests that deer act as a reservoir of the disease and provide an overwintering mechanism for both spirochetes and adult ticks. Some tick larvae may acquire the spirochete by transovarial passage and the nymphal stage may transmit the disease to humans.

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Effect of prior exposure to noninfected ticks on susceptibility of mice to Lyme disease spirochetes.

To determine whether prior exposure to Nearctic Ixodes vector ticks protects native reservoir mice from tick-borne infection by Lyme disease spirochetes, we compared their infectivities for white-footed mice and laboratory mice that had been repeatedly infested by noninfected deer ticks. Nymphal ticks readily engorged on tick-exposed laboratory mice, but their feeding success on white-footed mice progressively declined. Tick-borne spirochetes readily infected previously tick-infested mice. Thus, prior infestation by Nearctic ticks does not protect sympatric reservoir mice or Palearctic laboratory mice from infection by sympatric tick-borne spirochetes.

Animals↗

Early course of infection in susceptible and resistant strains of mice, using [3H]uridine-labeled Trypanosoma brucei subsp. brucei.

A radioisotopic technique utilizing [3H]uridine was developed to determine the tissue distribution of intravenously inoculated Trypanosoma brucei spp. brucei in susceptible (Swiss-Webster) and resistant (deer) strains of mice. The reliability of the technique was tested by using unincorporated [3H]uridine and heat-killed (labeled) trypanosomes in parallel experiments. The inoculations with viable (labeled) organisms showed that during the initial 24 h T. brucei subsp. brucei was cleared from the bloodstream of deer mice to a significantly greater extent than in Swiss-Webster mice. In addition, significantly more radioactivity was found in the liver, spleen, and kidneys of the deer mice. Autoradiographs of trypanosomes in selected tissues supported the distribution observed with labeled organisms. All of the above experiments involved the highly virulent, monomorphic bloodstream form of T. brucei subsp. brucei. Similar experiments with less virulent tissue culture-adapted trypanosomes showed that these organisms were readily cleared from the bloodstream, even in highly susceptible Swiss-Webster mice. The results suggest that avoidance of phagocytosis may be an important virulence factor of T. brucei subsp. brucei and contributes to the variation observed in species and strain susceptible to trypanosomiasis.

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Borrelia burgdorferi erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria.

Borrelia burgdorferi, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the erp genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35 degrees C than in those maintained at 23 degrees C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.

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Isolation of Treponema hyodysenteriae from wild rodents.

Rodents from swine-producing farms were examined for the presence of Treponema hyodysenteriae. Wild mice (n = 257) and rats (n = 41) were trapped on eight farms. Ceca were removed aseptically, and the contents and mucosal scrapings were cultured on selective medium (blood agar containing 400 micrograms of spectinomycin per ml). T. hyodysenteriae was detected in the cecal scrapings of four mice from three different farms where swine dysentery had occurred. Gross lesions were detected in the ceca in two of the four mice. In addition, Treponema innocens was detected in the cecal scrapings of 12 mice and 13 rats. Three of the four T. hyodysenteriae isolates were pathogenic when inoculated intragastrically into swine. The results of this investigation suggest that wild rodents may be carriers of T. hyodysenteriae.

Animal Population Groups↗

Rickettsiae and Borrelia burgdorferi in ixodid ticks.

Nymphs and adults of hard-bodied ticks were collected in Connecticut and tested by direct and indirect immunofluorescence staining methods for rickettsiae and Borrelia burgdorferi. Of the 609 Ixodes dammini ticks examined, 59 (9.7%) harbored rickettsialike microorganisms in hemocytes (blood cells). These bacteria reacted with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of canine ehrlichiosis. Prevalence of infection ranged from 6.8 to 12.7% for males and females, respectively. Although the specific identities of the hemocytic rickettsialike organisms are unknown, they share antigens with ehrlichiae. Electron microscopy revealed rickettsiae in ovarian tissues of I. dammini that also had infected hemocytes. Rickettsialike organisms were also observed in the hemocytes of 5 (6.9%) of 73 Dermacentor variabilis ticks. In analyses for B. burgdorferi, 146 (23.7%) of 617 I. dammini ticks harbored these spirochetes in midguts. Hemocytic rickettsialike microorganisms coexisted with B. burgdorferi in 36 (6.7%) of the 537 nymphs and adults of I. dammini examined. I. dammini, with its broad host range, has the potential to acquire multiple microorganisms.

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Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans.

Current biological and serological techniques for demonstrating infections by Borrelia burgdorferi can be inconclusive. In order to monitor Lyme borreliosis, we developed a rapid and sensitive assay for B. burgdorferi antigens in infected hosts. Polyclonal rabbit antisera were raised against membrane vesicles and an 83-kDa vesicle-associated protein band that was purified from in vitro B. burgdorferi cultures. Immunoglobulin G (IgG) antibodies were recovered from these sera and tested for a species-specific reaction with several geographically diverse Borrelia isolates by immunoblot analysis. Parlodion-coated electron microscope grids were activated with anti-vesicle F(ab')2 fragments and then incubated with confirmed or experimental sources of spirochetal antigens. Such sources included cultured spirochetes; spirochete culture supernatants; samples of urine, blood, or serum from mice, dogs, and humans; triturates of Ixodes ticks; and bladder, spleen, liver, kidney, heart, or brain tissues from infected or control mice. Captured antigens were assayed by immune electron microscopy by using anti-83-kDa IgG antibodies and protein A-colloidal gold conjugates. The results indicated that B. burgdorferi appears to shed surface antigens which are readily detectable in urine, blood, and several organs from infected hosts. Such antigens were detectable in mouse urine at dilutions exceeding 10(-6). Intact spirochetes were frequently observed on grids incubated with blood, spleen, or bladder preparations, and B. burgdorferi was reisolated from the urinary bladders of all experimentally infected mice. These results indicated that B. burgdorferi antigens arise in a variety of host materials. Such antigens can be captured and identified with specific polyclonal antibodies, providing a sensitive assay for monitoring and studying Lyme borreliosis.

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Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California.

Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.

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Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis.

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.

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