长角血蜱、嗜群血蜱经期传播莱姆病螺旋体Borrelia garinii的实验研究

目的　探讨长角血蜱、嗜群血蜱经期传播莱病螺旋体的可能性。方法　通过皮下注射KM鼠建立实验感染动物模型 ,以此阳性感染鼠感染试蜱非感染种群 ,观察试蜱的感染能力以及以莱姆病螺旋体的保持能力。结果　通过剌叮阳性KM鼠 ,长角血蜱、嗜群血蜱幼蜱饱血后分别获得 6 1 3%、75 0 %的阳性感染 ,但所感染的螺旋体在感染后 2d即死亡消解 ,不能重新获得分离 ,PCR扩增阳性也只能持续到饱血后 8d ,幼蜱蜕化为若蜱后 ,所有检测结果均为阴性 ;这两种蜱的若蜱也都可通过吸血获得 70 0 %的检测阳性率 ,感染后长角血蜱、嗜群血蜱可分别在在饱血后 5、10d内保持螺旋体的活性 ,PCR检测阳性率可延迟至饱血后 15d。此后直至蜕化成为成蜱 ,所有血、蜱检测结果均呈阴性 ;长角血蜱、嗜群血蜱的不同种群在感染和保持螺旋体能力上表现一致。结论　长角血蜱、嗜群血蜱的幼蜱和若蜱虽可以感染莱姆病螺旋体但不能经期传播到下一发育阶段 ,不具备经期传播能力 ,作为莱姆病主要媒介的可能性不大。     

Hybrid resistance to parental bone marrow-derived cultured mast cells in the skin but not in the peritoneal cavity of (WB X C57BL/6)F1-W/Wv mice

When cultured mast cells of (WB X C57BL/6)F1-+/+(WBB6F1-+/+) and WB-+/+(WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. Although in vitro colony-forming ability is comparable between cultured mast cells of WB mice and those of WBB6F1-+/+ mice, the number of WB mast cells necessary for the appearance of mast cell clusters in the skin of WBB6F1-W/Wv mice was significantly larger than the number of WBB6F1-+/+ mast cells. In spite of the presence of such an apparent hybrid resistance in the skin of WBB6F1-W/Wv mice to mast cells of the WB parent, both WB and WBB6F1-+/+ mast cells grow in the peritoneal cavity of WBB6F1-W/Wv mice with comparable efficiency. This is a demonstration of the tissue-related (nonrecirculating) expression of hybrid resistance against nonmalignant hematopoietic cells.     

Host immune responsiveness to the chigger, Eutrombicula cinnabaris

Chigger infestation is often associated with severe cutaneous reactions. Mice were given four infestations with the pest chigger Eutrombicula cinnabaris, and each exposure was separated by a 14-day mite-free period. Mean duration of engorgement was nine to ten days for a first exposure and four to five days for a fourth exposure. An initial exposure did not elicit macroscopic changes at chigger attachment sites, while all third and fourth exposure animals had marked reactions consisting of erythema, epidermal thickening and serous exudation. Approximately 20% of second exposure animals had macroscopic changes at chigger feeding sites, but these reactions were much less intense than the responses of third and fourth infestation hosts. Third and fourth exposure animals had infiltrates of lymphocytes, eosinophils, basophils and neutrophils at attachment sites, with eosinophil influx the most intense. Cutaneous reactivity to chigger feeding was adoptively transferred with lymphocytes from fourth exposure animals. Passive transfer of serum from fourth infestation donors resulted in heightened reactivity to a challenge infestation. Skin testing, after a fourth infestation, with an extract of whole E. cinnabaris larvae provided evidence for Arthus and delayed type hypersensitivity responses to chigger antigens. Chigger-reactive homocytotropic antibody was not detected by skin testing and active cutaneous anaphylaxis.     

Antigenic profile of Ixodes ricinus: effect of developmental stage, feeding time and the response of different host species

Antigens recognized by host species in response to ectoparasite infestation have been widely reported. Although differences in the immune responses of different host species have been described, only a very few of these studies compare the range of antigens recognized by different host species in response to infestation. We used Western blot analysis to investigate antigenic responses of different host species that were repeatedly infested with Ixodes ricinus ticks. Antigenic profiles of larval and nymphal whole tick homogenates were compared with the respective salivary gland extract (SGE) samples using sera from rabbits repeatedly infested with either adults, nymphs or larvae. SGE samples were also analysed using sera from hamsters infested with adults, nymphs or larvae. Sera from BALB/C mice, Apodemus flavicollis (yellow-necked mouse) or Clethrionomys glareolus (bank vole) repeatedly infested with larvae were used to compare the antigenic profiles of SGE and larval homogenate samples. We also investigated different sources of tick antigens, using rabbit sera, by comparing midgut extracts from female adult ticks and SGE from unfed ticks and from ticks throughout the 6-day feeding period with whole tick homogenates of female and male adults, nymphs and larvae. The pattern of antigenic tick-molecules recognized by infested host species varies with the period of feeding, developmental stage and the particular host species parasitized.     

Humoral immune responses of Hereford cattle vaccinated with midgut antigens of the cattle tick, Boophilus microplus

Vaccination of cattle with midgut membrane (GM) antigen derived from the cattle tick, Boophilus microplus, infected with the adjuvant Quil A, resulted in significant increases in total immunoglobulins, mainly in the IgG1 and IgG2 fractions of the serum. Analysis of the anti-GM antibody levels of vaccinated cattle showed that the levels of IgG, IgG1 and complement-fixing antibodies were significantly correlated to protection against infestation with cattle ticks. Anti-GM antibodies of the IgG2 and IgM isotype were not correlated to protection against infestation with cattle ticks. Anti-GM antibodies fixed complement (C') in the presence of GM, larval membrane antigen and live, midgut cells, but not in the presence of live, larval cells. Anti-GM antibodies were able to fix C' equally well in the presence of GM antigen and live, midgut cells. None of the antigens tested activated the alternate pathway of complement under the conditions tested. Levels of anti-GM IgG1 antibodies were used to develop a regression model for predicting levels of protection against infestation with cattle ticks in vaccinated cattle.     

Factors influencing the distribution of larval blacklegged ticks on rodent hosts

Because of differences among hosts in reservoir competence for tick-borne diseases, the distribution of larval blacklegged ticks on hosts might determine tick infection prevalence and disease risk to humans. We conducted a three-part study to determine the factors responsible for greater burdens of larval blacklegged ticks on white-footed mice than on eastern chipmunks. A microhabitat study indicated that questing ticks have higher encounter rates with mice than with chipmunks. Laboratory experiments demonstrated that ticks oriented more strongly toward mice. However, larval ticks fed more successfully from chipmunks. Our results strongly suggest that mice are both more likely to use larval tick-infested microhabitats and to attract questing larvae than are chipmunks, leading to a dramatically higher initial infestation rate, which is then reduced by greater grooming activity by mice. The high mortality rate of larvae that were experimentally introduced onto mice suggests that grooming is a significant cause of mortality to larval blacklegged ticks.     

Reconstitution of mucosal mast cells in W/Wv mice by adoptive transfer of bone marrow-derived cultured mast cells and its effects on the protective capacity to Strongyloides ratti-infection

Bone marrow-derived cultured mast cells (BMMC) were transferred intravenously into W/WV mice to examine if they could reconstitute defective mucosal mast cell response or defective protective capacity against infection with Strongyloides ratti. When mast cell growth factor-producing activity of W/WV mice were examined, mesenteric lymph node cells obtained at 7 to 14 days after infection could produce this factor in vitro by stimulation with S. ratti-adult worm antigen. A single injection of BMMC (1 X 10(7] on day 7 post-infection (p.i.) neither caused an increase in number of intestinal mucosal mast cells not altered the kinetics of faecal larval output (LPG). On the other hand, serial injections of BMMC (5 X 10(6] from day 5 to 10 p.i. (total 3 X 10(7) cells) resulted in the significant increase in number of intestinal mucosal mast cells. However, this treatment too could not alter the kinetics of LPG. Therefore, adoptive transfer of BMMC could cause the increase in number of histologically detectable-mucosal mast cells, but these cells are, by themselves, not sufficient to cause the expulsion of S. ratti adult worms from the intestine.     

Resistance to ixodid tick infestation induced by administration of tick-tissue culture cells

Primary tissue culture cells of developing larvae of Amblyomma americanum were administered to guinea-pigs never previously exposed to ixodid ticks. Guinea-pigs were given 1 X 10(6) primary culture cells on Days 0, 7 and 21 by subcutaneous injection and challenged with male and female A. americanum on Day 35. A significant degree of induced host tick resistance was expressed by reduced engorgement weight of females, reduced oviposition by those females which did obtain a blood meal, and by death of ticks at the attachment site. Resistance induced by A. americanum primary culture cells stimulated a significant degree of resistance to infestation with Dermacentor andersoni adults.     

Mast cells, eosinophils and antibody-mediated cellular cytotoxicity are not critical in resistance to Trichuris muris

The murine intestinal nematode Trichuris muris provides an invaluable model of human infection with T. trichiura. Hence, analysis of the immunological responses in the mouse may elucidate the mechanisms of immunity to trichuriasis in man. The work described here investigates the roles of eosinophils, mast cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in the elimination of T. muris from the host gut. Following ablation of IL-5, and hence eosinophilia, mice usually resistant to T. muris infection remained so. Further, blocking the stem cell factor receptor, c-kit, to facilitate complete ablation of mast cells over the period of parasite expulsion in resistant mice had no effect on the development of protective immunity. Therefore it can be deduced that eosinophils and mast cells are not critical in resistance. In addition to these studies, the role of antibody-mediated cellular cytotoxic mechanisms was investigated via the analysis of an infection time course in Fc gamma R-/- mice. These animals, on a resistant background, were fully immune and expelled the parasites before development of the adult stage. Thus this model provides evidence against a major role for ADCC in resistance to infection with T. muris. The studies described here have eliminated some of the major effector mechanisms traditionally associated with helminth infection, and work continues to elucidate the critical immune responses associated with resistance.     

Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae

The growth rates of Manduca sexta (tobacco hornworm) larvae feeding on tomato plants constitutively expressing a prosystemin antisense gene were approximately 3 times higher than growth rates of larvae feeding on nontransformed control plants. The levels of proteinase inhibitor I and inhibitor II proteins in leaves of tomato plants expressing the antisense prosystemin gene remained at undetectable levels until the sixth day of larval feeding and then increased throughout the plants to 100-125 microg/g of leaf tissue after 14 days. In control plants, levels of proteinase inhibitor I and II proteins increased rapidly from the second day of larval feeding and by the eighth day contained levels of 225 microg/g of leaf tissue and 275 microg/g of leaf tissue, respectively, and then increased slowly thereafter. Prosystemin mRNA levels in antisense and control plants after 6 days and 12 days of larval feeding correlated with levels of inhibitor I and II protein levels. These experiments demonstrate that resistance of plants toward an insect pest can be modulated by genetically engineering a gene encoding a component of the inducible systemic signaling system regulating a plant defensive response.     

Antibody to interleukin 5 prevents blood and tissue eosinophilia but not liver trapping in murine larval toxocariasis

Mice previously sensitized by infective-stage larvae of the canine nematode, Toxocara canis, trap large numbers of challenge larvae within the liver; trapped larvae are found within eosinophilic granulomas. To investigate the role of eosinophils in this phenomenon we examined larval trapping in mice depleted of blood and tissue eosinophils by treatment with a monoclonal antibody (MoAb) (TRFK-5) produced against recombinant murine interleukin 5 (rmIL-5). Control mice received either an isotype-matched control MoAb or PBS. On day 0 test mice were given a sensitization dose of 125 infective T. canis eggs. Test and challenge control mice received 500 infective eggs on day 28. All mice were killed on day 42 and larval numbers within the liver were determined. Liver samples were also collected for histopathological and morphometric examination. When compared to test mice treated with PBS or the isotype control, the level of circulating eosinophils in anti-IL-5-treated test mice was reduced by 94–96% on days 14 and 27,99% on day 35, and 100% on day 42; the level of tissue eosinophils within liver granulomas on day 42 was reduced by 92–95%. The total area of inflammation within the liver was similar among all test groups. However, the highly eosinophilic infiltrates, present in control sections, were replaced in anti-IL-5-treated mice by lymphocytes, macrophages, and foreign-body giant cells. No difference was found in larval trapping between antibody-treated groups. These findings suggest that the eosinophil is not necessary for liver trapping in murine larval toxocariasis.     

Transmission route efficacy and kinetics of Anaplasma phagocytophilum infection in white-footed mouse, Peromyscus leucopus

Anaplasma phagocytophilum was used to infect Peromyscus leucopus mice by three routes of inoculation: infected tick infestation and intraperitoneal (IP) and subcutaneous (SQ) injection of infected tissue culture cells. A set of 12 mice were infected (four tick, four IP, and four SQ), and blood was drawn at 1, 3, 6, 9, 12, 15, 21, 28, 35, and 60 days post-infection and analyzed by use of a quantitative PCR assay to assess the level of infection. An additional set of 108 mice were infected (36 tick, 36 IP, 36 SQ) and euthanized at 1, 3, 6, 9, 12, 15, 21, 28, and 35 days post-infection (four mice/time point), and blood, spleen, bone marrow, and bladder tissue samples were analyzed. Tick infection generally produced the highest average levels of infection and peaked at 9 days post-infestation in blood, spleen, and bone marrow and at 6 days after infestation in the bladder. IP injection resulted in levels of infection that peaked on day 6 (spleen) or 12 (bladder, bone marrow, and blood). A. phagocytophilum injected SQ showed low levels of infection, and the day of peak infection varied. The average level of infection in the blood drawstressed mice was consistently higher and peaked earlier than infection in the non-stressed, euthanized mice. Xenodiagnosis was used to assay a third set of 12 mice (four tick, four IP, and four SQ) on days 7 and 14 post-infection and ticks fed on tick-infected mice showed the highest rate of PCR-positive test results at both time points (day 7, 22.2%; day 14, 17.3%). These data indicate that P. leucopus mice can be infected by tick infestation, IP injection, or SQ injection but that the kinetics and level of infection are quite variable among individual mice, may be influenced by the route of inoculation, and may be further altered by common laboratory procedures such as repeated collection of blood samples.     

Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W/Wv mice

The normal skin and other tissues of adult mast cell-deficient WBB6F1- W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. As a result, genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice are widely used for studies of mast cell differentiation and function. We found that mast cells developed at sites of idiopathic chronic dermatitis in WBB6F1-W/Wv mice and that the number of mast cells present in the skin of WBB6F1-W/Wv mice was proportional to the severity of the dermatitis (in ear skin, there were 33 +/- 4 mast cells/mm2 of dermis at sites of severe dermatitis v 9 +/- 3 at sites of mild dermatitis, 0.8 +/- 0.3 in skin without dermatitis, and 100 +/- 7 in the normal skin of congenic WBB6F1-+/+ mice; in back skin, the corresponding values were 2.0 +/- 0.6, 1.1 +/- 0.9, 0.025 +/- 0.025, and 26.2 +/- 3.2). The development of mast cells was a local, not systemic, consequence of the dermatitis. Thus, WBB6F1-W/Wv mice with severe dermatitis lacked mast cells in skin not showing signs of dermatitis and also in the peritoneal cavity, stomach, cecum, and tongue. Idiopathic chronic dermatitis was not associated with the local development of mast cells in WCB6F1-Sl/Sld mice, a mutant whose mast cell deficiency is due to a mechanism distinct from that of WBB6F1-W/Wv mice. These findings may have implications for understanding the nature of the mast cell deficiency in WBB6F1-W/Wv and WCB6F1-Sl/Sld mice and for the use of these mutants to analyze mast cell differentiation and function.     

Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice

OBJECTIVE: Previously, we established a mouse model of scleroderma induced by repeated subcutaneous bleomycin injections. In this model, increased numbers of mast cells were observed in the lesional skin of dermal sclerosis, and degranulation of mast cells was prominent prior to the increase of mast cell numbers. Mast cells have been suggested to play an important role in tissue fibrosis. In this study, we investigated whether dermal sclerosis is also induced by bleomycin administration in genetically mast cell deficient WBB6F1-W/W(V) mice. METHODS: Bleomycin was subcutaneously injected every day in WBB6F1-W/W(V) and their normal littermate WBB6F1-+/+ mice for 4 weeks, and mice were analyzed for histological sclerosis, mast cell number, plasma histamine level, and hydroxyproline content. RESULTS: Four weeks' injections of bleomycin effected histological dermal sclerosis in both mast cell deficient and control strains; however, at 1 week, dermal sclerosis was induced only in WBB6F1-+/+ mice. Mast cells gradually increased in number around or on the edge of sclerotic lesions in WBB6F1-+/+ mice, as the dermal sclerosis developed. Hydroxyproline content of the skin of WBB6F1-+/+ mice was higher than that of WBB6F1-W/Wv mice at 1 week, but was not statistically significant. After 2 weeks' treatment with bleomycin, the hydroxyproline content of the skin was similar in both strains. The number of infiltrating macrophages and CD4+ T cells also gradually increased in both strains; however, the difference did not reach significance during the course of bleomycin treatment. CONCLUSION: These results show that mast cell is not necessary for inducing dermal sclerosis by bleomycin, and other types of inflammatory cells such as infiltrating macrophages or T lymphocytes may play a role in triggering induction of dermal sclerosis via fibrogenic cytokines. However, mast cell releasing mediators or cytokines may play a role in accelerating formation of dermal sclerosis, in particular, at an early phase of the sclerotic process, and not merely as a result of sclerosis.     

Early stages of Ascaris suum induce airway inflammation and hyperreactivity in a mouse model

The inflammatory and functional changes that occur in murine lung after infection with 2500 infective Ascaris suum eggs were studied in this work. A sequential influx of neutrophils, mononuclear cells and eosinophils occurred into airways concomitantly with migration of larvae from liver to the lungs. Histological analysis of the lung showed a severe intra-alveolar haemorrhage at the peak of larval migration (day 8) and the most intense inflammatory cell infiltrate on day 14. Ascaris L3 were found in alveolar spaces and inside bronchioles on day 8. The number of eosinophils was elevated in the blood on days 8 and 14. The peak of eosinophil influx into the lung was at day 14, as indicated by the high levels of eosinophil peroxidase activity, followed by their migration into the airways. The antibody response against egg and larval antigens consisted mainly of IgG1 and IgM, and also of IgE and anaphylactic IgG1, that cross-reacted with adult worm antigens. Total IgE levels were substantially elevated during the infection. Measurement of lung mechanical parameters showed airway hyperreactivity in infected mice. In conclusion, the murine model of A. suum infection mimics the Th2-induced parameters observed in pigs and humans and can be used to analyse the immunoregulatory properties of this helminth.     

Agents of human anaplasmosis and Lyme disease at Camp Ripley, Minnesota

The transmission dynamics of Anaplasma phagocytophilum (Ap) and Borrelia burgdorferi (Bb) among Ixodes scapularis (Is) and mammalian hosts was investigated at Camp Ripley, an area representative of central Minnesota. Prevalence of white-footed mouse infection with Ap and Bb were 20% and 42%, respectively, with a coinfection level of 14%. Peak levels of infection with both agents occurred in May. The average levels of seropositivity to Ap and Bb were 29.3% and 48%, respectively. Of the mice infected with Ap, 47.5% were able to eliminate the pathogen as compared with 19.4% of mice infected with Bb. Ap was detected in 88.4% of 43 eastern chipmunks examined and isolated from 44.7% of the animals. Bb was present in 72.7% of 11 chipmunks examined, and 100% of the animals were also infected with Ap. The seasonality of tick activity differs from that reported for the New York area. Is infestation of mice began in May with peak nymphal infestation also occurring in May (7.4 per infested mouse) and overlapping with peak larval infestation in June (77.1 per infested mouse). Infestation ranged from 100% in May to 34.5% in October. Is comprised 98.4% of the ticks infesting the mice. The temporal pattern of the developmental stages of Is infesting chipmunks was the same as for mice, except that the tick burdens were greater. The nymphal stage peaked in May (81.3 per animal), and the larval stage peaked in June (164.7 per animal). Infestation was 100% in May-August, and >99% of the ticks were Is. Antibodies to Ap were present in >80% of the white-tailed deer examined, but they were infected with the Ap-1 variant rather than the Ap strain infecting mice and humans. Antibodies to Bb were detected in >80% of the deer, but Bb DNA was only detected in 1.5% of blood specimens.     

Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes.

A pure population of mast cells was obtained after 14 days of culturing mouse bone marrow cells in the presence of medium derived from concanavalin A-stimulated mouse spleen cells. The cells were characterized as mast cells by their morphologic appearance and histologic staining, by their histamine content (450 ng per 10(6) cells) and by the demonstration of IgE receptors on their surface (150,000--440,000 receptor sites per cell). The histamine content and the number of IgE receptors remained constant for at least 7 wk of culture. These mast cells could be passively sensitized to mice hybridoma IgE. They then released 43% of their histamine content upon incubation with anti-mouse hybridoma IgE.     

Prevalence of Human-Active and Variant 1 Strains of the Tick-Borne Pathogen Anaplasma phagocytophilum in Hosts and Forests of Eastern North America

Anaplasmosis is an emerging infectious disease caused by infection with the bacterium Anaplasma phagocytophilum. In the eastern United States, A. phagocytophilum is transmitted to hosts through the bite of the blacklegged tick, Ixodes scapularis. We determined the realized reservoir competence of 14 species of common vertebrate hosts for ticks by establishing the probability that each species transmits two important strains of A. phagocytophilum (A. phagocytophilum human-active, which causes human cases, and A. phagocytophilum variant 1, which does not) to feeding larval ticks. We also sampled questing nymphal ticks from ∼150 sites in a single county over 2 years and sampled over 6 years at one location. White-footed mice (Peromyscus leucopus) and Eastern chipmunks (Tamias striatus) were the most competent reservoirs for infection with the A. phagocytophilum human-active strain. Across the county, prevalence in ticks for both strains together was 8.3%; ticks were more than two times as likely to be infected with A. phagocytophilum human-active as A. phagocytophilum variant 1.     

Development of human mast cells in vitro.

Nucleated cells of human umbilical cord blood were cocultured with mouse skin-derived 3T3 fibroblasts. After 7-8 weeks in culture, when the number of the other hematopoietic cells declined, metachromatic granule-containing mononuclear cells appeared in the cultures, and the number of the cells increased up to 12 weeks. After 11-14 weeks in culture, the metachromatic mononuclear cells comprised a substantial portion of the cultured cells. These cells contained 1.8-2 micrograms of histamine per 10(6) cells and bore receptors for IgE. All of the cells contained tryptase in their granules. Electron microscopic analysis showed that these cells were mature human mast cells, clearly different from the basophilic granulocytes or eosinophils that arise in a variety of circumstances in cord blood cell cultures. Most of the cultured mast cells expressed some granules with regular crystalline arrays and contained both tryptase and chymase, and thus resembled human skin mast cells.