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This study describes the identification of Borrelia parkeri spirochetes in Colorado. Two isolates of B. parkeri (6230 and 6232) were recovered from Ornithodoros parkeri Cooley ticks collected at an inactive prairie dog town in Moffat County. Both isolates were partially characterized by sequencing and subsequent parsimony and neighbor-joining analyses of appropriate regions of the 16S ribosomal RNA, flagellin and P66 genes. Analyses of the 16S gene sequences from the Colorado isolates indicated that they were more closely related to B. parkeri and B. tucatae than to B. hermsii or the other species of Borrelia investigated in this study. Additional analyses of amino acid sequences for flagellin and P66, however, clearly demonstrated that isolates 6230 and 6232 were most closely related to B. parkeri. The possible significance of B. parkeri as an agent of human disease is discussed.

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Exposure to cats infected with Yersinia pestis is a recently recognized risk for human plague in the US. Twenty-three cases of cat-associated human plague (5 of which were fatal) occurred in 8 western states from 1977 through 1998, which represent 7.7% of the total 297 cases reported in that period. Bites, scratches, or other contact with infectious materials while handling infected cats resulted in 17 cases of bubonic plague, 1 case of primary septicemic plague, and 5 cases of primary pneumonic plague. The 5 fatal cases were associated with misdiagnosis or delays in seeking treatment, which resulted in overwhelming infection and various manifestations of the systemic inflammatory response syndrome. Unlike infections acquired by flea bites, the occurrence of cat-associated human plague did not increase significantly during summer months. Plague epizootics in rodents also were observed less frequently at exposure sites for cases of cat-associated human plague than at exposure sites for other cases. The risk of cat-associated human plague is likely to increase as residential development continues in areas where plague foci exist in the western US. Enhanced awareness is needed for prompt diagnosis and treatment.

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Sylvatic plague, or plague of wild rodents is caused by Yersinia pestis and entered California (USA) from Asia about 1899. Extensive sampling during the 1930's and 1940's documented the spread of plague to approximately its current distribution in North America. Records from the Centers for Disease Control and Prevention document plague in Kansas (USA) between 1945 and 1950, but since then there has been no documentation of plague in the state. Following a die-off of a black-tailed prairie dog (Cynomys ludovicianus) colony on the Cimarron National Grassland, in the southwestern corner of Kansas (3710'N, 10145'W), we sampled fleas from burrows in June 1997, and tested them for Yersinia pestis. Twelve of 13 pools of Oropsyla hirsuta and one of two Pulex sp. were positive. A similar sample of fleas, from another colony where black-tailed prairie dogs were active at the time, yielded no positive fleas.

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The 1993 U.S. hantavirus pulmonary syndrome (HPS) outbreak was attributed to environmental conditions and increased rodent populations caused by unusual weather in 1991- 92. In a case-control study to test this hypothesis, we estimated precipitation at 28 HPS and 170 control sites during the springs of 1992 and 1993 and compared it with precipitation during the previous 6 years by using rainfall patterns at 196 weather stations. We also used elevation data and Landsat Thematic Mapper satellite imagery collected the year before the outbreak to estimate HPS risk by logistic regression analysis. Rainfall at case sites was not higher during 1992-93 than in previous years. However, elevation, as well as satellite data, showed association between environmental conditions and HPS risk the following year. Repeated analysis using satellite imagery from 1995 showed substantial decrease in medium- to high-risk areas. Only one case of HPS was identified in 1996.

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The hypothesis that regions of low blood velocity in a membrane oxygenator, as predicted by computational fluid dynamics (CFD), would correspond with regions of clinical thrombotic deposition was investigated. Twenty heparin-coated oxygenators were sectioned following use in adult extracorporeal membrane oxygenation. The activated clotting time (ACT) was maintained at approximately 180 s via heparin infusion throughout the support period. Cross-sections were systematically photographed, and slides made to allow image projection upon a digitizing pad. Thrombotic deposition was traced to allow creation of a device cross-section image with an overlaid color scale representing thrombotic deposition frequency. A two-dimensional CFD model was developed to predict blood velocities throughout the oxygenator cross-section. Direct spatial comparisons were made between maps of CFD modeled blood speed and thrombotic deposition. Theoretical oxygenator design modification was performed within the CFD model to investigate flow paths which might minimize regions of low blood velocity. CFD results demonstrated that low velocity regions qualitatively matched regions with a high incidence of thrombotic deposition. Thrombotic deposition was also correlated to longer perfusion periods. This technique of coupling clinical data and CFD offers the potential to relate flow characteristics to thrombotic deposition and represents a potentially powerful new methodology for the optimization of oxygenator flow-related biocompatibility.

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We used a quantitative competitive polymerase chain reaction assay to quantify Yersinia pestis loads in fleas and bacteremia levels in mice that were used as sources of infectious blood meals for feeding the fleas. Xenopsylla cheopis, the Oriental rat flea, achieved higher infection rates, developed greater bacterial loads, and became infectious more rapidly than Oropsylla montana, a ground squirrel flea. Both flea species required about 10(6) Y. pestis cells per flea to be able to transmit to mice. Most fleas that achieved these levels, however, were incapable of transmitting. Our results suggest that at the time of flea feeding, host blood must contain > or = 10(6) bacteria/ml to result in detectable Y. pestis infections in these fleas, and > or = 10(7) bacteria/mL to cause infection levels sufficient for both species to eventually become capable of transmitting Y. pestis to uninfected mice. Yersinia pestis colonies primarily developed in the midguts of O. montana, whereas infections in X. cheopis often developed simultaneously in the proventriculus and the midgut. These findings were visually confirmed by infecting fleas with a strain of Y. pestis that had been transformed with the green fluorescent protein gene.

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We used a quantitative competitive polymerase chain reaction (PCR) (QC-PCR) to determine bacterial loads in 669 fleas collected in areas of confirmed and suspected plague epizootics. Fleas were collected out of rodent burrows (67.9%) and off of captured animals (24.1%) and rodent carcasses (8.1%). An initial PCR screening assay indicated that 12.1% (81/669) of all fleas were positive for Yersinia pestis. Fleas collected from burrows had significantly lower (chi2 = 264.9, P < 0.0001) infection rates (6.8%) but significantly higher (Student t-test, P < 0.0001) bacterial loads (mean = 10(5.6) Y. pestis per flea) than fleas collected off of rodent carcasses (infection rate = 92.6%; mean bacterial load = 10(4.8) Y. pestis per flea). None of the fleas collected off of captured animals were positive for Y. pestis by PCR, although seven of the 176 captured animals were serologically positive for Y. pestis.

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Plague occurs episodically in many parts of the world, and some outbreaks appear to be related to increased abundance of rodents and other mammals that serve as hosts for vector fleas. Climate dynamics may influence the abundance of both fleas and mammals, thereby having an indirect effect on human plague incidence. An understanding of the relationship between climate and plague could be useful in predicting periods of increased risk of plague transmission. In this study, we used correlation analyses of 215 human cases of plague in relation to precipitation records from 1948 to 1996 in areas of New Mexico with history of human plague cases (38 cities, towns, and villages). We conducted analyses using 3 spatial scales: global (El Niño-Southern Oscillation Indices [SOI]); regional (pooled state-wide precipitation averages); and local (precipitation data from weather stations near plague case sites). We found that human plague cases in New Mexico occurred more frequently following winter-spring periods (October to May) with above-average precipitation (mean plague years = 113% of normal rain/ snowfall), resulting in 60% more cases of plague in humans following wet versus dry winter-spring periods. However, we obtained significant results at local level only; regional state-wide precipitation averages and SOI values exhibited no significant correlations to incidence of human plague cases. These results are consistent with our hypothesis of a trophic cascade in which increased winter-spring precipitation enhances small mammal food resource productivity (plants and insects), leading to an increase in the abundance of plague hosts. In addition, moister climate conditions may act to promote flea survival and reproduction, also enhancing plague transmission. Finally, the result that the number of human plague cases in New Mexico was positively associated with higher than normal winter-spring precipitation at a local scale can be used by physicians and public health personnel to identify and predict periods of increased risk of plague transmission to humans.

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The "gold standard" for identifying Yersinia pestis-infected fleas has been inoculation of mice with pooled flea material. Inoculated mice are monitored for 21 days, and those that die are further analyzed for Y. pestis infection by fluorescent-antibody assay and/or culture. PCR may provide a more rapid and sensitive alternative for identifying Y. pestis in fleas. To compare these assays, samples were prepared from 381 field-collected fleas. Each flea was analyzed individually by both PCR and mouse inoculation. Sixty of the 381 flea samples were positive for Y. pestis by PCR; 48 of these PCR-positive samples caused death in mice (80.0% agreement). None of the 321 PCR-negative samples caused death. Among the 12 mice that survived inoculation with PCR-positive samples, 10 were later demonstrated by serology or culture to have been infected with Y. pestis. This suggests that death of inoculated mice is less reliable than PCR as an indicator of the presence of Y. pestis in flea samples. Mouse inoculation assays produce results that are comparable to PCR only when surviving as well as dead mice are analyzed for infection. The rapidity and sensitivity (10 to 100 CFU of Y. pestis) of PCR suggest that it could serve as a useful alternative to mouse inoculation for routine plague surveillance and outbreak investigations.

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In July 1995, an outbreak of acute febrile illness affected 11 (48%) of 23 family members from Nebraska and Kansas who had vacationed at a Colorado cabin in June. Similar symptoms were identified among five (17%) of 30 additional persons from Nebraska, Kansas, Florida, and Texas who had vacationed at the same cabin. Symptoms suggested tick-borne relapsing fever (TBRF). Although no spirochetes were detected in available blood smears from five case-patients, Borrelia hermsii was cultured from the blood of one case-patient and two chipmunks trapped near the cabin. Case-patients were more likely than non-ill cabin visitors to have slept on the floor (odds ratio [OR] = 28.0, 95% confidence interval [CI] = 3.0-258) or in the top bunk bed (OR = 5.2, 95% CI = 1.1-25.1). Tick-borne relapsing fever should considered in the differential diagnosis of fever in patients who have stayed overnight in mountain cabins in the western United States.

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The prevalence of infectivity within a vector population is a critical factor in arthropod-borne disease epidemiology but it is difficult to estimate. In the case of bubonic plague, infective flea vectors contain large numbers of Yersinia pestis within a bacterial mass that blocks the flea's foregut, and only such blocked fleas are important for biologic transmission. A bacterial quantitation method could therefore be used to assess the prevalence of plague-infective (blocked) fleas in a population. We developed a standard, curve-based, competitive polymerase chain reaction (PCR) procedure to quantitate Y. pestis in individual fleas. The quantitative PCR (Q-PCR) method equaled a colony count reference method in accuracy and precision when evaluated using mock samples and laboratory-infected fleas. The Q-PCR was more reliable than colony count, however, for field-collected fleas and for blocked fleas collected after their death. In a sample of fleas collected from a prairie dog colony in the aftermath of a plague epizootic, 48% were infected but less than 2% contained numbers of Y. pestis indicative of blockage. The method provides a means to monitor plague epizootics and associated risks of flea-borne transmission to humans, and is applicable to the study of other vector-borne diseases.

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Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful.

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The efficacy of a liquid permethrin-treated bait tube controlling fleas and ticks on Mexican wood rats, Neotoma mexicana Baird, was evaluated during a 1-yr study in north-central Colorado. Results indicated that the bait tubes were effective for reducing flea and tick infestations on wood rats. The effects of treatment persisted throughout the study, despite the fact that bait tubes were replenished with bait and permethrin only during the first 4 mo (4 replenishments). Our results suggest that these bait tubes provide an effective, economical, and environmentally acceptable means of controlling vectors of flea or tick-borne diseases, although slight modifications of the basic bait tube design might be required to maintain the effectiveness of the tube under different ecological conditions.

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During an outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States, trained environmental assessment teams conducted surveys at 17 case-patient homes and matched controls from June through August 1993. Variables related to rodent abundance were quantified and standardized rodent trapping was conducted around and within households. The majority of households were located in pinon-juniper vegetation zones, and there were no significant differences in the type of house in which cases and controls lived. The only environmental factor that distinguished case households from controls was significantly higher small rodent densities (median trap success for case sites = 17.3%, 12.7% for near controls, and 8.3% for far controls). Frequency of hantaviral infection in deer mice (Peromyscus maniculatus) did not vary significantly among households of cases and controls, with a range of 27.5-32.5% antibody-positive. Indices of rodent fecal contamination were slightly higher in case houses. The data indicate that higher rodent densities were associated with households in which HPS cases occurred. Strategies that control rodent numbers and decrease rodent access to dwellings may reduce risk of human infection.

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An intensive enzootic cycle of Borrelia burgdorferi was seen in populations of the Mexican wood rat, Neotoma mexicana, and Ixodes spinipalpis ticks in northern Colorado. Cultures of rodent ear tissue and ticks yielded 63 spirochetal isolates: 38 N. mexicana, 2 Peromyscus difficilis, and 23 I. spinipalpis. All 63 isolates were identified as B. burgdorferi sensu lato by polymerase chain reaction; a representative subset was characterized as B. burgdorferi by SDS-PAGE and immunoblotting. A tick-derived spirochete isolate was infectious to laboratory mice and I. scapularis, the principal vector of Lyme disease in endemic areas of the United States. The risk of human contact with infected I. spinipalpis appears to be minimal from this epidemiologically silent focus in northern Colorado, since this tick is restricted to wood rat nests in this semiarid environment.

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An outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States was etiologically linked to a newly recognized hantavirus. Knowledge that hantaviruses are maintained in rodent reservoirs stimulated a field and laboratory investigation of 1696 small mammals of 31 species. The most commonly captured rodent, the deer mouse (Peromyscus maniculatus), had the highest antibody prevalence (30%) to four hantavirus antigens. Antibody also was detected in 10 other species of rodent and in 1 species of rabbit. Reverse transcriptase-polymerase chain reaction (RT-PCR) products of hantavirus from rodent tissues were indistinguishable from those from human HPS patients. More than 96% of the seropositive P. maniculatus were positive by RT-PCR, suggesting chronic infection. Antibody prevalences were similar among P. maniculatus trapped from Arizona (33%), New Mexico (29%), and Colorado (29%). The numeric dominance of P. maniculatus, the high prevalence of antibody, and the RT-PCR findings implicate this species as the primary rodent reservoir for a new hantavirus in the southwestern United States.

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We used the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery and others to rapidly identify rickettsiae in naturally infected ticks. Unlike previously described methods, our PCR assays type rickettsiae directly from tick tissues without first isolating the organisms. We collected 226 adult Dermacentor andersoni ticks in the Bitterroot Mountains of western Montana and analyzed them for possible rickettsial infection by hemolymph test using the Gimenez stain. Thirteen (5.8%) of these ticks were positive by hemolymph test and selected for further analysis using the above PCR/RFLP typing system. The PCR assays performed using the first primer set (RpCS) resulted in amplification of fragments of the predicted size from nine of the 13 hemolymph test-positive tick samples. Only four of these nine tick samples were also positive in similar PCR assays performed with a second primer set (Rr190) that is presumed to be spotted fever group specific. The RFLP analyses of material amplified from these four ticks indicated they were infected with Rickettsia rickettsii (one sample) and R. rhipicephali (three samples). The PCR/RFLP analyses of the five PCR-positive tick samples that were positive only in assays performed with the RpCS primer set indicated that these ticks were infected with R. bellii. The remaining four of 13 hemolymph test-positive tick samples gave negative PCR results with both the RpCS and Rr190 primer sets. Infected hemocytes from these PCR-negative ticks contained organisms of distinctive bacillary morphology that appeared similar to those described previously as long forms, and it is possible that these organisms belong to a genus other than Rickettsia. We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results. Multiple isolates of R. montana (nine isolates), R. bellii (five isolates), R. rickettsii (Hlp-like) (four isolates), and R. canada (two isolates) were tested and no significant variations in PCR/RFLP patterns were observed between members of the same serotypes. However, among the five isolates of R. rhipicephali tested, two slightly different RFLP patterns were noted. Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsiae directly from D. andersoni or D. variabilis tick tissues.

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The relatively unrelated spotted fever group rickettsia Rickettsia rhipicephali conferred on guinea pigs protective immunity against challenge with virulent R. rickettsii. Immunity was conferred at all doses of R. rhipicephali used in the study. Because of the serologic unrelatedness of these two rickettsiae, determined by the use of microimmunofluorescence and other serological assays, further studies were performed to define the nature of the immune response elicited by R. rhipicephali and the characteristics of the rickettsial antigens that evoke cross-reactive antibody responses. Animals immune to R. rhipicephali tested at the time of challenge showed a complete cross-reactive lymphocyte proliferative response to rickettsial antigens prepared from each species. In fact, spleen cells from R. rhipicephali-immune animals responded better to R. rickettsii antigens than to homologous immunizing antigens. Serum samples were obtained from R. rhipicephali-infected animals at various times after infection and tested by the use of Western immunoblot assay for antibodies that were cross-reactive with antigens of R. rickettsii. By 10 days after infection with R. rhipicephali, antibodies to antigens of both species were noted, and by 37 days after infection, sera from immune animals showed strong reactivity to antigens of R. rhipicephali with apparent molecular masses of 107 and 151 kDa. The cross-reactive antibody response to antigens of R. rickettsii was relatively strong and involved predominantly the rOmpB protein and the rickettsial lipopolysaccharide. These findings establish the presence of T-cell-dependent epitopes associated with antigens of R. rhipicephali, which confer protective immunity against challenge with R. rickettsii. Results of Western immunoblot assays support the contention that the R. rickettsii rOmpB surface antigen contains important protective epitopes.

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Eight species of small mammals were evaluated as potential hosts for American dog ticks, Dermacentor variabilis (Say), in an upland, tallgrass prairie study site in central Oklahoma. Only hispid cotton rats, Sigmodon hispidus, and deer mice, Peromyscus maniculatus, were found to be important hosts for immature D. variabilis. Although D. varibilis larvae and nymphs frequently infested both cotton rats and deer mice, cotton rats were the most important host species for both immature stages in the study area. Cotton rats constituted 63.2% of the total 530 small mammals captured and were hosts to 85.2% of all larvae and 88.7% of all nymphs. Deer mice accounted for 19.8% of all small mammals captured and were hosts for 14.5% of the larvae and 10.8% of the nymphs recovered. The remaining small mammal species were hosts for less than 1% of the immature ticks collected. Larval infestations peaked during summer, whereas summer and spring peaks were noted for the nymphal infestations. The relative importance of cotton rats and deer mice as hosts for immature ticks could be largely, but not completely, explained by cotton rats being more than three times as abundant as deer mice. Attachment site data indicated that differences in grooming behavior also might be partially responsible for the larger infestations observed on cotton rats. Other possible ecological and behavioral explanations of the heavy infestations observed on cotton rats are discussed.

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