Dexamethasone-induced cytokine changes associated with diminished disease severity in horses infected with Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the zoonotic cause of granulocytic anaplasmosis. We hypothesized that immune response, specifically gamma interferon (IFN-γ), plays a role in disease severity. To test this, horses were infected and IFNG expression was pharmacologically downregulated using corticosteroids. Eight horses were infected with A. phagocytophilum; 4 received dexamethasone on days 4 to 8 of infection. Clinical signs, hematologic parameters, and transcription of cytokine/chemokine genes were compared among treated and untreated horses. Infection was quantitated by msp2 real-time PCR and microscopy. As anticipated, there was significantly greater leukopenia, thrombocytopenia, and anemia in infected versus uninfected horses. The A. phagocytophilum load was higher for dexamethasone-treated horses. Dexamethasone reduced IFNG transcription by day 12 and IL-8 and IL-18 by days 7 to 9 and increased IL-4 on day 7. The ratio of IL-10 to IFNG was increased by dexamethasone on day 9. There were no hematologic differences between the infected horses. Dexamethasone suppression of proinflammatory response resulted in delayed infection-induced limb edema and decreased icterus, anorexia, and reluctance to move between days 6 and 9 and lower fever on day 7. These results underscore the utility of the equine model of granulocytic anaplasmosis and suggest that Th1 proinflammatory response plays a role in worsening disease severity and that disease severity can be decreased by modulating proinflammatory response. A role for Th1 response and macrophage activation in hematologic derangements elicited by A. phagocytophilum is not supported by these data and remains unproven.

Co-infection of Ornithodoros coriaceus with the relapsing fever spirochete, Borrelia coriaceae, and the agent of epizootic bovine abortion.

The soft tick, Ornithodoros coriaceus (Koch) (Acari: Argasidae), is a common mammalian parasite of livestock in many arid regions of the western U.S.A. The tick is a known vector of the undescribed bacterial pathogen that causes epizootic bovine abortion (EBA), which results in late-term abortions in beef cattle and subsequent economic loss, which can be considerable, to producers. A second reported bacterial pathogen, Borrelia coriaceae, a member of the relapsing fever complex, has also been identified in this tick and was at one time hypothesized to be the aetiological agent of EBA. In order to test whether bacterial infections in ticks overlapped geographically and to determine the prevalence of co-infection in O. coriaceus populations, we used molecular methods to detect bacterial DNA from ticks collected from a wide variety of habitats in California, Nevada and Oregon. Of the 15 sites at which ticks tested positive for the agent of EBA (aoEBA), eight also contained ticks positive for Borrelia spp. by polymerase chain reaction assay. Additionally, two ticks were co-infected; both of these were collected from the same location. Univariate risk analysis indicated the presence of juniper-dominated habitat at the collection site and geographic location to be significantly associated with infection of the tick vector by either pathogen.

Lyme neuroborreliosis in 2 horses.

Lyme neuroborreliosis--characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis--was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto-specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

Humoral immune response associated with lyme borreliosis in nonhuman primates: analysis by immunoblotting and enzyme-linked immunosorbent assay with sonicates or recombinant proteins.

The immune response to Borrelia burgdorferi, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of B. burgdorferi. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.

Central and peripheral nervous system infection, immunity, and inflammation in the NHP model of Lyme borreliosis.

The relationship between chronic infection, antispirochetal immunity, and inflammation is unknown in Lyme neuroborreliosis. In the nonhuman primate model of Lyme neuroborreliosis, we measured spirochetal density in the nervous system and other tissues by polymerase chain reaction and correlated these values to anti-Borrelia burgdorferi antibody in the serum and cerebrospinal fluid, and to inflammation in tissues. Despite substantial presence of Borrelia burgdorferi, the causative agent of Lyme borreliosis, in the central nervous system, only minor inflammation was present there, though skeletal and cardiac muscle, which contained similar levels of spirochete, were highly inflamed. Anti-Borrelia burgdoferi antibody was present in the cerebrospinal fluid but was not selectively concentrated. All infected animals developed anti-Borrelia burgdorferi antibody in the serum, but increased amplitude of antibody was not predictive of higher levels of infection. These data demonstrate that Lyme neuroborreliosis is a persistent infection, that spirochetal presence is a necessary but not sufficient condition for inflammation, and that antibody measured in serum may not predict the severity of infection.

Infection of mice with the agent of human granulocytic ehrlichiosis after different routes of inoculation.

Population kinetics of the agent of human granulocytic ehrlichiosis (aoHGE) were examined after needle and tickborne inoculation of C3H mice. Blood, skin, lung, spleen, liver, kidney, brain, lymph node, and bone marrow samples were analyzed by using real-time polymerase chain reaction (PCR) at various intervals after inoculation, using a p44 gene target. The highest number of copies of the p44 gene target occurred in blood and bone marrow samples, emphasizing aoHGE leukocytotropism. Numbers of copies of the p44 gene target in other tissues reflected vascular perfusion rather than replication. Needle-inoculated infected mice had earlier dissemination, but kinetics of infection in both groups were parallel, with declining rates of infection by day 20 and recovery in some mice on days 20-60 after inoculation. On the basis of an aoHGE lysate ELISA, mice seroconverted by day 10 after inoculation. Therefore, real-time PCR is useful for quantitative studies with the aoHGE in experimental infections, and results showed that needle inoculation can be used to study the aoHGE infection because of its similarity to tickborne inoculation.

Evaluation of diagnostic methods for Helicobacter bilis infection in laboratory mice.

Disease-susceptible (C3H) and -resistant (B6) immunocompetent and immunodeficient (C3H-scid and B6-rag1) mice were examined up to 10 weeks after inoculation with Helicobacter bilis (a prototype species of proven virulence). Infection was monitored weekly by use of fecal culture, polymerase chain reaction (PCR) nucleic acid amplification, membrane extract enzyme-linked immunosorbent assay (ELISA), and histologic examination. All mice became infected by three to five weeks after inoculation, on the basis of results of culture and PCR analysis of feces. The PCR analysis was more sensitive than culture at determining infection status, particularly during early infection. None of the mice had evidence of disease by week 10. Immunoglobulin G seroconversion was detectable in C3H mice by week eight and in B6 mice by week nine. Results indicated that culture and PCR analysis are more sensitive than is membrane extract ELISA serologic testing for detecting early infection in individual mice, regardless of genotype or immune status. Results underscore the need for improved seroassays for this important group of murine pathogens.

Acquisition dynamics of Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis at the host-vector interface.

The population dynamics of two cotransmitted tick-borne pathogens, Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis (aoHGE), were assessed at the skin-vector interface at intervals after tick attachment on infected mice. Quantitative real-time polymerase chain reaction targeting the B. burgdorferi flagellin gene revealed consistent decreases in spirochete numbers in skin at the sites of tick attachment compared with non-tick attachment sites. This phenomenon was found during early (2 weeks) and late (8 weeks) infection and at 24, 48, and 72 h after tick attachment. A nonspecific inflammatory stimulus, implantation of suture material, did not have this effect. In contrast to B. burgdorferi, copy numbers of an aoHGE p44 target gene target were significantly increased at the sites of tick attachment, compared with non-tick sites. The non-specific stimulus of suture material had the same effect on aoHGE recruitment as tick attachment in aoHGE infected mice. These results reinforce the concept that B. burgdorferi interfaces with its vector by virtue of its non-systemic dermatotropism, and not via systemic hematogenous acquisition. In contrast, results indicate that the aoHGE relies upon hematogenous acquisition. Thus, these two cotransmitted tick-borne pathogens utilize distinctly different means of vector acquisition.

[Characteristics of the early posttransplantation period in kidney transplantation]. / Karakteristike ranog posttransplantacionog perioda u bubreznoj transplantaciji.

INTRODUCTION: Renal transplantation has become the procedure of choice and the most cost-effective strategy for the management of patients with end stage renal disease. Posttransplant period is very important because possible complications, which have to be detected and treated according to approve statements. The aim this paper with is to show all characteristics of early postransplant period in patients operated at the Clinical Center of Tuzla. METHODS AND RESULTS: Eighteen patients with end stage renal diseases has been analyzed with average age 32 + 8.6 years. Glomerulonephritis as primary kidney diseases has been found in 16 patients, lupus nephritis and reflux nephropathy in one patient. We paid attention on: creatinine level, urine output and balance, level of cyclosporin, body weight, ultrasound fallow-up, number episodes of acute rejection and number of additional dialysis. Clinical and labs sign of acute rejection have been found in 6 patients. Two of them recidive pulse dose of corticosteroides and four ATG. Additional haemodialysis has been performed in 5 patients. One patient died because of the rupture aneurism of aorta. Other 17 patients have been discharged after average hospitalisation of 20.87 8.18 days. CONCLUSION: We can say it's very important to recognise the sings of acute rejection and to start with therapy. In patients with cardiovascular risk, postoperative period has to be guided careful.

Lyme arthritis resolution with antiserum to a 37-kilodalton Borrelia burgdorferi protein.

A 37-kDa protein from Borrelia burgdorferi (the agent of Lyme disease) was identified as a target for immune-mediated resolution of Lyme arthritis. Studies in a mouse model have shown that arthritis resolution can be mediated by antibodies (against unknown target antigens) within immune sera from actively infected mice. Immune sera from infected mice were therefore used to screen a B. burgdorferi genomic expression library. A gene was identified whose native product is a putative lipoprotein of approximately 37 kDa, referred to here as arthritis-related protein (Arp). Active and passive immunization of mice with recombinant Arp or Arp antiserum, respectively, did not protect mice from challenge inoculation. However, when Arp antiserum was administered to severe combined immunodeficient (SCID) mice with established infections and with ongoing arthritis and carditis, treatment selectively induced arthritis resolution without affecting the status of carditis or influencing the status of infection, including spirochetemia. The selective arthritis-resolving effect of Arp antiserum mimics the activity of immune serum from immunocompetent mice when such serum is transferred into SCID mice with established infections. The arp gene could not be amplified from unrelated B. burgdorferi isolates but hybridized with those isolates only under very-low-stringency conditions. Arp antiserum reacted against proteins of similar size in a wide range of B. burgdorferi isolates.

Stability of Borrelia burgdorferi outer surface protein C under immune selection pressure.

Outer surface protein (Osp) C immune pressure during persistent infection with Borrelia burgdorferi was examined in relation to genetic variation of ospC. Mice were infected with clonal B. burgdorferi sensu stricto (s.s.) N40 or B. afzelii PKo and then were hyperimmunized with homologous recombinant OspC or with decorin-binding protein A (DbpA) (controls). After 6 months, B. burgdorferi isolates were subjected to restriction enzyme analysis of the amplified ospC genes and were found to have no differences among 9 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC hyperimmune mice or among 10 B. burgdorferi s.s. N40 and 10 B. afzelii PKo isolates from DbpA hyperimmune mice, compared with input inocula. Comparison of gene sequences among 4 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC-immunized mice revealed no ospC variation from input inocula. Variation in ospC among B. burgdorferi isolates and species during chronic infection is not likely to be an important mechanism for immune evasion.

Acquisition and transmission of the agent of human granulocytic ehrlichiosis by Ixodes scapularis ticks.

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with Ixodes scapularis ticks. The median infectious dose for C3H mice was 10(4) to 10(5) organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.

Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice.

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.

PTSD symptoms in Bosnian refugees 1 year after resettlement in the United States.

OBJECTIVE: The authors' goal was to describe the characteristics of posttraumatic stress disorder (PTSD) symptoms on resettlement in the United States and at 1-year follow-up among Bosnian refugees as well as possible factors affecting the PTSD symptom profile among these refugees. METHOD: They used standardized instruments to assess 34 Bosnian refugees for PTSD at resettlement in the United States and 1 year later. RESULTS: Fifteen of the refugees were diagnosed with PTSD at 1-year follow-up, compared with 25 at initial assessment. The average PTSD severity score at follow-up was 12.5, compared with 20.6 at initial assessment. At 1-year follow-up, 25 of the refugees experienced a decrease in severity of PTSD symptoms, one remained the same, and eight experienced an increase in severity. Older refugees were significantly more likely to have PTSD than younger refugees, and older refugees had more severe symptoms. CONCLUSIONS: The level of PTSD diagnosis and symptoms in Bosnian refugees remained substantial 1 year after their resettlement in the United States, although there were notable overall decreases. Older refugees appeared to be at greater risk.

Granulocytic ehrlichiosis in tick-immune guinea pigs.

We investigated whether Ixodes scapularis-mediated host immunity interrupts transmission of the agent of human granulocytic ehrlichiosis (aoHGE) to guinea pigs. Ticks infected with aoHGE readily transmitted aoHGE to tick-immune guinea pigs, despite incomplete tick engorgement and host attachment. Although tick immunity can prevent Lyme borreliosis, protection is not afforded against granulocytic ehrlichiosis.

Granulocytic ehrlichiosis in the laboratory mouse.

C3H mice that were inoculated with ehrlichiae isolated from a patient with human granulocytic ehrlichiosis (HGE) developed anemia and leukopenia, but by day 24, they returned to normal values. Granulocytic morulae were present in peripheral blood and spleen smears on days 5 and 10, and there was a reduction in morulae on day 17. Ehrlichiae were present in HL-60 cell cultures of blood and spleen from all mice at all intervals. Pathogenicity, but not infectivity, waned with mouse passage but could be resurrected by SCID mouse passage. Various methods were tested for their relative sensitivity in detecting infection: blood smears, HL-60 cell cultures, polymerase chain reaction (PCR) amplification of a 16S recombinant DNA target, and a mouse infectivity assay. All assays detected the HGE agent in blood during early infection, but PCR and the mouse infectivity assay were most sensitive during late infection. Xenodiagnosis demonstrated that mice remain persistently infected through 55 days.

Individual change after genocide in Bosnian survivors of "ethnic cleansing": assessing personality dysfunction.

The authors used the SCID-DES (disorders of extreme stress) instrument to assess for personality change in Bosnian survivors of "ethnic cleansing." Twenty four refugees underwent systematic, trauma-focused, research assessments, including the SCID-DES interview. Overall, this group of Bosnian survivors had been severely traumatized as a result of the Serbian nationalists' genocide. However, no subject met diagnostic criteria for DES. The SCID-DES yields far lower rates of trauma-related personality change in Bosnian survivors of genocide than in adult survivors of prolonged early life traumas. Therefore, the DES construct may have better application to prolonged, interpersonal, early life traumas than to the prolonged, communal traumas of genocide.

Immune evasion by tickborne and host-adapted Borrelia burgdorferi.

Immune sera from mice infected with the Lyme disease spirochete, Borrelia burgdorferi, have strong biologic activity against spirochetes cultured in vitro. Recent studies with rodents and ticks infected with B. burgdorferi indicate that spirochetes undergo major changes in protein expression as they adapt to the diverse environments encountered by a vectorborne pathogen. The purpose of this study was to explore the susceptibility of three different adaptive forms of B. burgdorferi (in vitro cultured, host-derived, and tickborne) to immune sera. Passive transfer of immune sera protected mice when they were challenged with spirochetes cultured in vitro. Immune sera did not protect mice from tickborne spirochetes or spirochetes derived from infected mice. These results indicate that spirochetes that have adapted within either the feeding tick or host are relatively invulnerable to the protective effects of immune sera, unlike spirochetes grown in vitro, which are highly susceptible.