Correlation between the Circadian Rhythm of Resistance to Extreme Temperatures and Changes in Fatty Acid Composition in Cotton Seedlings.

Fluctuations in fatty acid composition were examined in cotton (Gossypium hirsutum L. cv Deltapine 50) leaves during light-dark cycles of 12:12 h and under continuous light and were correlated to the rhythmic changes in chilling (5[deg]C) resistance (CR) and heat (53[deg]C) resistance (HR). The chilling-resistant and chilling-sensitive phases developed in the dark or the light period, respectively, and this rhythm persisted under continuous light for three cycles. The heat-resistant phase developed in the light period and an additional peak of HR occurred in the middle of the dark period. Under continuous light, only one peak of HR developed, lasting from the middle of the subjective night to the middle of the subjective day. The amounts of palmitic and oleic acids were constant during the light-dark cycle and under continuous light, but those of linoleic and linolenic acids fluctuated, attaining a high level in the middle of the dark period or the subjective night, and a low level in the middle of the light period or the subjective day. A low temperature of 20[deg]C induced CR and affected changes in fatty acid composition similar to those that occurred during the daily CR phase. A high temperature of 40[deg]C induced HR but did not affect changes in fatty acid composition. The results in their entirety show that the CR that develops rhythmically as well as the low-temperature-induced CR coincide with increased levels of polyunsaturated fatty acids. No correlation is found between changes in fatty acid composition and the HR that develops rhythmically or the high-temperature-induced HR.

Tick-induced modulation of the host immune response.

The tick-host-pathogen interface is characterised by complex immunological interactions. Host immune responses to tick infestation and infection with tick-borne pathogens involve cytokines, antibodies, complement and T lymphocyte regulatory and effector pathways. A successful host-parasite relationship is a balance between limiting the parasite by host defenses and the ability of the parasite to modulate, evade or restrict the host response. Hosts acquire immunological based resistance to tick infestation, which reduces engorgement, production of ova and viability. Salivary glands of ixodid ticks produce a complex array of immunogens and pharmacologically active molecules. Tick salivary gland derived material can modulate host cytokine, antibody and cell mediated immune responses. Both immunoregulatory and immune effector pathways of the host are suppressed. Tick feeding impairs the ability to develop a primary immune response to a thymic dependent immunogen. Lymphocytes obtained from tick infested hosts are reduced in their ability to proliferate in vitro to T lymphocyte mitogens, while responses to B lymphocyte polyclonal activators are unaltered. Normal macrophages and lymphocytes were exposed to female tick salivary gland extracts prepared daily during the course of engorgement. All extracts reduced lymphocyte responses to T cell mitogens and enhanced in vitro proliferation in the presence of a B lymphocyte mitogen. Macrophage elaboration of tumour necrosis factor alpha and interleukin-1 are significantly reduced in a differential manner. Production of interleukin-2 and interferon-gamma by T lymphocytes is reduced. Tick modulation of the host immune response could enhance the ability of the arthropod to obtain a blood meal and facilitate pathogen transmission to an immunocompetent host.

Characterization of an immunosuppressant protein from Dermacentor andersoni (Acari: Ixodidae) salivary glands.

A 36-kDa soluble protein was found in the salivary glands of female Dermacentor andersoni (Stiles) ticks that suppressed the in vitro proliferative response of murine splenocytes to concanavalin A (Con A). Incubating the purified protein with splenocytes reduced the incorporation of thymidine into the DNA of proliferating T-lymphocytes by more than 90% compared with cells exposed to Con A and buffer alone. The N-terminal amino acid sequence of the immunosuppressant protein was determined to be NH2-Leu-His-Lys-Ala(Asp)-Lys-Ile-Val-Lys-Leu-Thr -Glu-Glu-Ala -Arg-Lys-Tyr-Val-Gly-Arg-Xxx-Xxx-Thr-Thr-Ala-Leu-Gly-. Although the sequence exhibited a modest degree of similarity with a segment of immunoglobulin-binding protein found in several species of mammals, the mode of action of the immunosuppressant protein is unknown. This protein may play an important role in suppressing the host's acquisition of resistance to ticks.

Influence of repeated infestations with pathogen-free Ixodes scapularis (Acari: Ixodidae) on in vitro lymphocyte proliferation responses of C3H/HeN mice.

In the United States, Ixodes scapularis Say has been implicated as the vector of at least three human pathogens. Tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on in vitro proliferative responses of splenic lymphocytes from C3H/HeN mice. Lymphocytes from repeatedly infested and uninfested mice were exposed to concanavalin A (Con A), Escherichia coli Castellini & Chalmers lipopolysaccharide (LPS), or I. scapularis salivary gland soluble proteins (SGSP), to determine if lymphocyte responses differed between tick-exposed and nonexposed mice. Female C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-d tick-free period between each exposure. After each infestation, tick biology parameters were measured and lymphocyte proliferative responses assessed. Acquired resistance to I. scapularis was not evident in mice subjected to tick feeding. Significant differences in the responses of lymphocytes exposed to I. scapularis SGSP were observed between infested and noninfested mice. In contrast, few differences between infested and noninfested mice were evident for lymphocytes exposed to Con A or LPS. Our results suggest that repeated exposure to I. scapularis nymphs does not affect Con A or LPS-induced proliferation of splenic lymphocytes, but significantly effects lymphocyte responses to tick salivary gland antigens.

Isolation and molecular cloning of a secreted immunosuppressant protein from Dermacentor andersoni salivary gland.

A 36-kDa immunosuppressant protein (Da-p36) was isolated from salivary glands of feeding female ixodid ticks Dermacentor andersoni, using its affinity for UltraLink Biosupport Medium (Pierce, Rockford, Illinois)/protein complexes. Using a nested set of forward degenerate oligonucleotide primers corresponding to Da-p36 N-terminal amino acids, a cDNA encoding the immunosuppressant protein was isolated by 3' rapid amplification of cDNA ends. The resulting 772-base pair cDNA encodes a novel protein with predicted molecular weight of 24.9 kDa. Sequence analysis revealed the presence of 5 potential glycosylation sites and 1 myristylation site. Immunoblot analyses showed native Da-p36 is present in salivary glands and saliva from both male and female D. andersoni but not in salivary glands or saliva from Amblyomma americanum or Ixodes scapularis. Reverse transcription polymerase chain reaction and immunoblot analyses showed that Da-p36 expression is temporally regulated in salivary glands with maximum mRNA levels preceding maximum Da-p36 accumulation that occurred at day 6 of feeding. The levels of Da-p36 mRNA and protein were greatly reduced in salivary glands from near-replete females removed from sheep after 8 days of feeding. These data are consistent with a role of Da-p36 in immunosuppression during feeding.

Prevalence of American trypanosomiasis (Chagas disease) among dogs in Oklahoma.

OBJECTIVE: To determine the prevalence of Trypanosoma cruzi infection among dogs in Oklahoma. DESIGN: Cross-sectional study. ANIMALS: 301 owned or impounded dogs related by ownership or general geographic location to 3 dogs determined to have trypanosomiasis. PROCEDURES: Blood samples were obtained from dogs between November 1996 and September 1997. Infection status was determined by use of a radioimmunoprecipitation assay. Second blood samples were obtained from some of the seropositive dogs for study by hemoculture and polymerase chain reaction (PCR) assay. Sites where infected dogs were found were inspected for triatomine insects, and light traps were used for vector trapping. RESULTS: 11(3.6%) dogs were seropositive for T. cruzi infection. Ten of the 11 were owned rural hunting dogs. Protozoal organisms isolated from the blood of 1 seropositive dog were identified as T. cruzi by PCR testing. Only 1 adult Triatoma sanguisuga was captured in a light trap at a site near infected dogs; this insect was not infected. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that T. cruzi is enzootic in eastern Oklahoma. Measures that would reduce the risk of dogs acquiring T. cruzi infection are unlikely to be acceptable to their owners, and no effective drugs are available for treatment. The presence of T. cruzi-infected dogs poses a threat of transmission to persons at risk of exposure to contaminated blood Veterinarians who practice in the southern United States should be cognizant of this blood borne zoonosis and educate all personnel about appropriate precautions.

Dermacentor andersoni: salivary gland proteins suppressing T-lymphocyte responses to concanavalin A in vitro.

Salivary glands obtained from feeding adult female Dermacentor andersoni (Acari:Ixodidae) were fractionated using differential centrifugation, detergents, centrifugal concentrators incorporating filter membranes with various molecular weight cutoffs, and preparative SDS-PAGE. A lymphocyte proliferation assay was used to evaluate the effects of salivary gland fractions on ConA-induced blastogenesis of normal murine splenocytes. Lipid, soluble, and detergent-soluble fractions were found to significantly suppress ConA-induced proliferation of splenocytes. Fractions containing soluble proteins suppressed splenocyte proliferation by ca. 26%. Suppressant activity in these fractions was due to components with molecular weights greater than 30 kDa. This suppression of splenocyte proliferation occurred with as little as 0.25 microgram protein per well. Salivary gland preparative SDS-PAGE fractions containing one or more soluble polypeptides or proteins with molecular weights in the range 36 to 43 kDa significantly suppressed murine splenocyte responses to ConA in vitro.

Influence of soluble proteins from the salivary glands of ixodid ticks on the in-vitro proliferative responses of lymphocytes from BALB/c and C3H/HeN mice.

In the U.S.A., Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans by the ticks Ixodes scapularis and I. pacificus. Tick modulation of host immunity is an important factor in tick transmission of such pathogens. The proliferative responses of lymphocytes from BALB/c and C3H/HeN mice exposed to the salivary-gland soluble proteins (SGSP) of I. scapularis, I. pacificus or Dermacentor andersoni were therefore compared in vitro. This produced the present report, the first to describe the effects of I. pacificus SGSP on the proliferative responses of a host's lymphocytes in vitro. The effects of four concentrations of SGSP from each tick species were evaluated with unstimulated, and concanavalin-A-stimulated lymphocytes of each mouse strain. The responses of lymphocytes from both mouse strains were significantly effected when exposed to SGSP derived from each tick species. Responses of the unstimulated lymphocytes to SGSP indicated that the proteins from I. pacificus suppressed in-vitro lymphocyte proliferation to a greater degree than those from the other species investigated. For the concanavalin-A stimulated cells, however, suppression of the proliferative responses was greatest for cells exposed to I. scapularis SGSP.

Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks.

Female BALB/c mice were infested four times with pathogen-free Ixodes scapularis nymphs prior to infestation with nymphs infected with Borrelia burgdorferi B31. Each infestation was separated by a 14-day tick-free period. Mean weights of fed ticks and percentage reaching repletion did not indicate development of acquired resistance. Only 16.7% of mice repeatedly infested with pathogen-free ticks prior to infected I. scapularis nymph challenge became positive for B. burgdorferi. One hundred percent of control mice infested only with infected ticks were culture positive for B. burgdorferi.

Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle.

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.