Persing, David H.

ABSTRACTWe examined the role of the cytokines gamma interferon (IFN-γ) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1Babesia. Mice genetically deficient in IFN-γ-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-γ and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-γ-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenicBabesiaWA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-γ, and induction of macrophage-derived effector molecules like NO.

Clinical microbiology is in the midst of a new era. Methodology that is based on nucleic acid detection has slowly appeared in the diagnostic laboratory, and is revolutionizing our ability to assist physicians in the diagnosis and management of patients suffering from infectious diseases. Much like the introduction of immunoassays built around hybridoma technology in the 1980s, considerable doubt and promise exist hand in hand in the 1990s. Conventional testing that is technically straight forward, informative, and timely will always be a part of clinical microbiology; however, considerable room for improvement exists with organisms/diseases for which laboratory methods are limited. Nucleic acid methodology will have its greatest and long-awaited impact in this arena.

ABSTRACTInfection of severe combined immunodeficient mice withBabesiasp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. Thescidmutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course ofBabesiainfection in these strains. In contrast, thescidmutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.

ABSTRACT Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti . To develop more sensitive and specific assays for B. microti , we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.

ABSTRACT In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group ( n = 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group ( n = 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results ( P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

ABSTRACTMurine Lyme borreliosis, caused by infection with the spirocheteBorrelia burgdorferi, results in acute arthritis and carditis that regress as a result ofB. burgdorferi-specific immune responses.B. burgdorferi-specific antibodies can attenuate arthritis in mice deficient in both B cells and T cells but have no effect on carditis. Because macrophages comprise the principal immune cell in carditis, T-cell responses that augment cell-mediated immunity may be important for carditis regression. To investigate this hypothesis, we examined the course of Lyme carditis in mice selectively deficient in B cells or αβ T cells. Our results show that carditis regresses in B-cell-deficient B10.Akmice but not in αβ T-cell-deficient mice, independently of the mouse strain background. Despite prominent macrophage infiltrates, hearts fromB. burgdorferi-infected αβ T-cell-deficient mice had less mRNA for tumor necrosis factor alpha as measured by reverse transcription-PCR compared to infected control mice. Anti-inflammatory cytokine mRNA levels were equivalent. Adoptive transfer of gamma interferon-secreting CD4+T cells into infected αβ T-cell-deficient mice promoted carditis resolution. These results show that αβ T cells can promote resolution of murine Lyme carditis and are the first demonstration of a beneficial role for CD4+T helper 1 cells in this disease.