OspA antibodies inhibit the acquisition of Borrelia burgdorferi by Ixodes ticks

Ixodes ticks are infected by Borrelia burgdorferi when larvae feed on spirochete-infected mice. We studied the acquisition of B. burgdorferi by larval ticks, characterized the production of outer surface protein A (OspA) by spirochetes entering larvae, and examined the effects of OspA antibodies on the establishment of B. burgdorferi infections in ticks. Most larvae were infected by spirochetes 24 to 48 h after placement on mice. OspA antibodies stained the first spirochetes observed in larvae, suggesting that OspA is synthesized early during the colonization of the vector. When OspA antibodies were administered to B. burgdorferi-infected mice and larvae were then placed on the animals, the severity of larval infection and the number of infected ticks (7 of 16) were decreased compared with that of controls (15 of 16). The inhibitory effects of OspA antibodies were observed with passive antibody transfer as well as active host-generated immunity. The lower larval infection rate observed in the presence of OspA antibodies was exacerbated after the larval molt since only 1 of 12 nymphs was infected, and none of the mice that were fed upon by these nymphs became infected with B. burgdorferi. Therefore, an OspA antibody response in mice altered the reservoir competence of the vertebrate host by inhibiting the movement of B. burgdorferi from the host to the vector.     

Novel Borrelia burgdorferi isolates from Ixodes scapularis and Ixodes dentatus ticks feeding on humans

Seven cultures of Borrelia burgdorferi differing from strains B31 and ZS7 were identified from among 99 isolates from Ixodes scapularis ticks and from white-footed mice (Peromyscus leucopus) and 1 isolate from an Ixodes dentatus tick. Five of the six novel isolates from I. scapularis and the isolate from I. dentatus were from ticks feeding on humans. The six isolates from I. scapularis lacked OspA and OspB, four possessed an OspD band, and two reacted with an anti-OspC monoclonal antibody. Restriction fragment length polymorphisms of HindIII-digested DNAs from six OspA-negative isolates did not hybridize with radiolabeled ospA or LA88 DNA, and only isolate 46047 hybridized with the pG gene. Fragments similar to those recorded for the standard B. burgdorferi sensu stricto strains B31 and ZS7 were obtained with the fla and the HSP70 genes. Pulsed-field gel electrophoresis patterns of DNA digested with MluI included the specific B. burgdorferi sensu stricto band at 135 kbp for the five OspA-negative isolates from I. scapularis ticks. The six novel isolates apparently lack the 55-kbp plasmid encoding OspA. The pG-containing plasmid may be missing from all but isolate 46047. The isolate from the I. dentatus tick was similar to previous isolates from I. dentatus ticks feeding on rabbits. None of the isolates could be recovered from inoculated C3H/HeNCrlBR or white-footed mice. All isolates reacted with sera from humans with early or late Lyme disease. Our studies demonstrate that these borreliae occur in ticks feeding on humans, and therefore, at least some humans in the northeastern United States are likely being exposed to borreliae other than the classic B31-type strains that have thus far been isolated from humans.     

Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii

Sequence variability of the outer surface protein (Osp) A among Borrelia burgdorferi sl species suggests that a monovalent OspA vaccine may not protect against the various Borrelia present in Eurasia. Here, we confirmed that a monovalent recombinant OspA (rOspA) vaccine does not protect mice against Ixodes ricinus mediated infection with B. burgdorferi ss, Borrelia garinii and Borrelia afzelii. However, when mice were vaccinated with a cocktail of various rOspA from these three species, they were protected, and all challenge ticks that fed on them were cleared of their spirochetes. These results showed that a multiple OspA antigens vaccine, compatible with human use, was very efficient at protecting mice against B. burgdorferi ss, B. garinii, and B. afzelii.     

Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay

Anxiety levels in a sample of 65 long-term cancer survivors were assessed in a study of the effects of a planned discharge from an oncology clinic. Thirty-one percent of patients scored > or = 8, and 12% > or = 11 on the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS), indicating that anxiety rates in patients in long-standing remission do not greatly differ from patients with active disease. Despite the provision of continued support and guaranteed fast-access return to the clinic if necessary, 28% of patients refused to be discharged. Fear that recurrence would not be detected was the reason most frequently cited. Seventy-five percent of these patients were HADS anxiety cases. A second assessment 4-5 months later of the 41 patients who were discharged showed a slight, but non-significant increase in anxiety rates suggesting that anxiety in cancer survivors may be persistent and not related to clinic attendance.     

Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection

The basic tenet of continuous quality improvement is that there is always room for additional improvement in the clinical care provided to patients. The opportunities for this improvement come from the analysis of information collected during the ongoing monitoring of important elements of care. The provision of clinical psychiatric care is seen as a complex process that is dependent on the effective functioning of all of the health and mental health care organization. The concept of continuous quality improvement is the most recent stage in a long process of defining and redefining the basic goals and tenants of medical and psychiatric quality assurance. The determination of the actual improvement of psychiatric and mental health care due to quality assurance is a substantial and important technical problem. The determination of the value of this improvement in mental health care is an even greater ethical and social problem.     

Amphetamines in hair by enzyme-linked immunosorbent assay

Human hair was collected from the occipital crown region of the head from several subjects; these hair samples were presumptively positive for amphetamines by a previously evaluated immunoassay. Hair was washed briefly with methanol to remove external contamination, then extracted with hot methanol for 2 h to recover the drugs. The extracts were evaporated to dryness, reconstituted in buffer, and analyzed using a new enzyme-linked immunosorbent assay (ELISA) technique adapted for the detection of amphetamines in hair. Gas chromatography-mass spectrometry was used as the reference technique. Cross-reactivity of several related compounds was evaluated by equating the inverse of the ligand concentration at 50% antibody binding to the affinity constant for each compound. The ratio of a compound's affinity constant to that for d-methamphetamine was used to derive percent crossreactivity. These experiments yielded values of 30.8% for d-amphetamine, 7.4% for I-methamphetamine, 4.3% for phentermine, 2.9% for I-amphetamine, and <1% for ephedrine, methylenedioxyamphetamine, and methylenedioxymethamphetamine. Cross-reactivity of unrelated compounds was found to be non-existent. The optimum cutoff concentration was determined by receiver operating characteristic curve analysis to be 300 pg/mg and the observed limit of detection was 60 pg/mg. Intra-assay precision at 300 pg/mg was 3.3% (coefficient of variation, CV), and the interassay CV was 10.5%. The sensitivity and specificity of the method were 83% and 92%, respectively.     

Determination of captopril in human blood by an enzyme-linked immunosorbent assay

An immunoassay for the quantitation of the angiotensin-converting enzyme inhibitor, captopril in human plasma is described. Antisera very specific for captopril were produced by immunization with captopril conjugated to bovine serum albumin or porcine thyroglobulin via the drug's thiol group. The antibodies were used to develop an enzyme-linked immunosorbent assay (ELISA) with a detection limit of 0.3 ng mL-1 and intra- and inter-assay coefficients of variation of 7 and 12%, respectively. Apart from stabilizing captopril by the addition of N-ethyl maleimide, the assay was used to detect the drug in human plasma without further extraction or purification. Our immunoassay provides a very sensitive and rapid (four hours) alternative for the study of captopril pharmacokinetics.     

Geographic distribution of ticks (Acari: Ixodidae) in Michigan, with emphasis on Ixodes scapularis and Borrelia burgdorferi

A 12-yr (1985-1996) passive survey in Michigan based upon tick submissions from citizens yielded 4,755 ticks of 21 species, 16 of which were probably indigenous in the state. Three species of Dermacentor [most common, D. variabilis Say and D. albipictus (Packard)]; 2 species of Amblyomma [most common, A. americanum (L.)]; and 12 species of Ixodes (most common, I. cookei Packard and I. scapularis Say), as well as Haemaphysalis leporispalustris (Packard), Rhipicephalus sanguineus Latreille, and the soft ticks Ornithodoros kelleyi Cooley & Kohls, and Otobius megnini (Duges) were submitted. New state records were I. kingi Bishopp, I. texanus Banks, I. sculptus Neumann, and I. baergi Cooley & Kohls. Examination of gut smears from dissections of 1,037 ticks of 13 species by indirect immunofluorescent assay, using murine monoclonal H9724 as the primary antibody, revealed that 11 of 175 I. scapularis were infected with Borrelia spp. All positive I. scapularis were from Menominee County in the upper peninsula of the state, which also provided 79.8% of all submitted I. scapularis. Surveys for ticks on 5,449 hunter-killed white-tailed deer were conducted from 1988 to 1990, encompassed deer taken from 65 of the state's 83 counties, and showed that although D. albipictus was distributed widely in the northern part of the state, I. scapularis occurred only on deer taken from southern townships of Menominee County. Of 1,218 canine sera tested for antibodies to B. burgdorferi in 1992 and 1993, 25 of 299 (8.0%) from Menominee County were positive but only 1 of 919 sera submitted from 5 counties in the lower peninsula was positive.     

Duration of Borrelia burgdorferi infectivity in white-footed mice for the tick vector Ixodes scapularis under laboratory and field conditions in Ontario

The duration of Borrelia burgdorferi infectivity in white-footed mice (Peromyscus leucopus) experimentally inoculated or infested with infected Ixodes scapularis nymphs was evaluated. Infectivity was assessed by infesting these mice with unfed I. scapularis larvae at 7, 21, 35 and 49 days post-inoculation (DPI) or post-infestation (PI). At 7 DPI, B. burgdorferi was transmitted from 18 of 24 syringe-inoculated mice and all three tick-infected mice to I. scapularis larvae which fed upon them. However, at 21, 35 and 49 DPI, significantly fewer mice were infective. Borrelia burgdorferi was isolated from tissues of 14 of 22 syringe-inoculated mice about 56 DPI, and from all three tick-infected mice. However, the level of agreement between xenodiagnosis and bacterial culture was no greater than would be expected by chance alone. We also determined if B. burgdorferi infectivity of mice varied in relation to periods of tick feeding in the field. White-footed mice were trapped during April, July and August 1993 from two habitats on Long Point peninsula (Ontario, Canada), where B. burgdorferi is endemic. Mice from each habitat were infested with laboratory-reared I. scapularis larvae. Ticks from each mouse were subsequently examined by immunofluorescent assay for B. burgdorferi infection and mice were cultured for B. burgdorferi. None of 3577 I. scapularis larvae fed on 62 mice captured within the cottonwood dune habitat were infected with B. burgdorferi, although it was isolated from six of these mice. Within the maple forest habitat, 0/24, 8/21 (38%) and 1/21 (5%) mice transmitted B. burgdorferi to I. scapularis larvae during April, July and August, respectively. Most mice from the maple forest with B. burgdorferi-positive tissues (14/21) were collected during July, although the level of agreement between xenodiagnosis and tissue culture was poor. Because B. burgdorferi infectivity in mice appears to be of short duration, overwintered I. scapularis larvae and nymphs may have to feed upon infected hosts at the same time of year in order for a cycle of B. burgdorferi infection to be maintained on Long Point. Infected I. scapularis nymphs, rather than persistently infected vertebrate hosts, likely serve as the overwintering "reservoir" for B. burgdorferi on Long Point.     

Killing of Borrelia burgdorferi by antibody elicited by OspA vaccine is inefficient in the absence of complement

A Lyme disease vaccine, based on the Borrelia burgdorferi lipoprotein OspA, has recently undergone phase III trials in humans. The results of one of these trials indicate that vaccine efficacy positively correlates with anti-OspA antibody titer. Spirochete killing within the tick vector midgut, upon which vaccine efficacy appears to depend, may occur chiefly via a mechanism that involves antibody alone, as it has been reported that complement is degraded by tick saliva decomplementing factors. We compared the in vitro killing efficiencies of anti-OspA antibody elicited in rhesus monkeys by the OspA vaccine, in the presence and in the absence of monkey complement. Killing in the absence of complement was between 14 and 3,800 times less efficient than with complement present, depending on the spirochete strain. The relative inefficiency of the complement-independent killing mechanism by anti-OspA antibody may explain why OspA vaccine efficacy is critically dependent on antibody titer.     

Detection of Aeromonas hydrophila serogroup 0:34 in faeces using an enzyme-linked immunosorbent assay

The efficacy of gadopentetate dimeglumine (Gd-DTPA) enhanced magnetic resonance (MR) imaging in the diagnosis and differentiation of soft-tissue, neoplastic and non-neoplastic lesion has not been well established. Thirty patients with soft tissue masses (18 neoplastic and 12 non-neoplastic) were studied, using MR imaging with and without administration of Gd-DTPA. Gd-DTPA proved helpful in characterisation of several entities, including differentiation of solid mass from proteinaceous cyst, demonstration of tumour nodules within haemorrhagic or necrotic masses, and delineation of tumour adjacent to oedema. The use of Gd-DTPA may provide additional information for tissue specificity and, in complicated cases, Gd-DTPA may also provide essential information that cannot be obtained using other methods. We recommend the use of contrast enhanced MR as an adjunct to conventional MR imaging in the initial assessment of musculoskeletal soft tissue masses. However, T2-weighted images show better tissue contrast of the lesions, and are equal to contrast enhanced images in delineation of tumour margins. Non-contrast enhanced images are, therefore, probably adequate for the delineation of lesions for surgical planning when a diagnosis has already been made.     

Prevalence of Borrelia burgdorferi infection in Ixodes ricinus in central Italy

A scalp tumor from a 24-year-old male presenting unusual histological finding is described. The tumor was mostly composed of corpuscles resembling Meissner tactile bodies and contained almost no other components. Immunohistochemical studies revealed the tumor cells to be positive for S-100 protein and neuron-specific enolase, so we considered the tumor to be an unusual nevocellular nevus mostly comprised of nevic corpuscles.     

Analysis of the thrombin inhibitor DuP 714 by an enzyme-linked immunosorbent assay

Resident proteins of the exocytic pathway contain at least two types of information in their primary sequence for determining their subcellular location. The first type of information is found at the carboxyl terminus of soluble proteins of the endoplasmic reticulum (ER) and in the cytoplasmic domain of some ER and Golgi membrane proteins. It acts as a retrieval signal, returning proteins that have left the compartment in which they reside. The second type of information has been found in the membrane-spanning domain of several ER and Golgi proteins and, though the mechanism by which it operates is still unclear, it acts as a retention signal, keeping the protein at a particular location within the organelle. The presence of both a retrieval signal and a retention signal in a trans-Golgi network resident protein suggests that more than one mechanism operates to ensure correct localization of resident proteins along the exocytic pathway.     

Antinuclear antibody screening by an enzyme-linked immunosorbent assay using multiple nuclear antigens

The oxidation of aminopyrine, an N-alkyl aromatic amine, by the hydroperoxidase activity of lipoxygenase was studied. Aminopyrine gave rise to a purple color in the presence of H2O2 and lipoxygenase, the color being proportional to the aminopyrine radical cation. The H2O2/aminopyrine radical cation molar ratio was 0.5. The overall reaction was considered as an enzymic-chemical second order mechanism with substrate regeneration. From the equations, the apparent constant of the radical cation's decomposition (k'app) was evaluated under different experimental conditions. It was found to be inversely proportional to the proton concentration but unaffected by the concentration of aminopyrine. These results suggest a new comprehensive mechanism for N-demethylation, which takes into account the described presence of both nitrogen- and carbon-centered radicals and the marked effect of pH on the stability of the radical cation.     

Epitope characterization of malondialdehyde-acetaldehyde adducts using an enzyme-linked immunosorbent assay

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.     

Detection of Vibrio anguillarum by a sandwich enzyme-linked immunosorbent assay performed with monoclonal antibodies

Monoclonal antibodies (Mabs) against V. anguillarum were produced and characterized by Western blotting analysis, competitive binding assays and cross-reactivity tests. Their ability to detect V. anguillarum in a liquid culture was tested in a sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of these Mabs used as capture or tracer antibodies. One combination was selected as the most suitable for diagnostic applications, showing the highest sensitivity and specificity.     

Detection of rubella virus-specific immunoglobulin G in saliva by an amplification-based enzyme-linked immunosorbent assay using monoclonal antibody to fluorescein isothiocyanate

An immunoglobulin G (IgG)-capture enzyme-linked immunosorbent assay (ELISA) for rubella virus is described. The assay uses a fluorescein isothiocyanate (FITC)-anti-FITC amplification system. The detection limit of the ELISA was approximately 7 IU of rubella virus-specific IgG per ml of serum sample. For saliva samples the performances of the capture ELISA and previously described radioimmunoassay were assessed, and the results of those two assays were compared to the rubella virus-specific IgG result obtained by a commercial ELISA (Behring Enzygnost) with a panel of paired serum and saliva samples. This comparison showed that the capture ELISA with saliva was more sensitive than the radioimmunoassay and that the results correlated better with the serum IgG result than the results of the radioimmunoassay did, with an overall sensitivity of 82% and a rank correlation of 0.68, whereas the sensitivity and rank correlation for the radioimmunoassay were 74% and 0.45, respectively. For subjects of 10 years of age or younger, the ELISA with saliva had a sensitivity of 94% and a specificity of 100% compared to the results of the ELISA (Behring Enzygnost) for rubella virus-specific IgG with corresponding serum samples. The sensitivity was much lower for subjects ages 17 years or older. The assay may have wider epidemiological use with saliva specimens, particularly those from children.     

Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay

Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium. Veillonella, Enterococcus avium and S. typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture. The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids. The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures. The ELISA was capable of detecting 10(4) cells per ml of the bacteria. The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E. avium, S. typhimurium and Veillonella, respectively. The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation.     

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.     

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer beta-sheet

Viruses such as HIV, influenza, picornavirus and others are known stimulators of apoptosis. This individual cellular elimination is a preferential host defense in regenerative tissues. In contrast, if this death occurred in nonregenerating cells, such as neurons of the central nervous system, may result in disease. The target cell for rabies virus is the neuron. Here we studied the outcome of the interaction between rabies virus (CVS-11) and mouse brain cells. Replication of rabies virus in suckling mouse brain cells resulted in brain cell apoptosis, detected by DNA fragmentation and in situ apoptosis within 25 h after infection and before evidence of intracerebral immune activation. Cell death occurred simultaneously with rabies virus replication. There were clinical signs of illness in infected newborn mice within 24 h after the appearance of DNA fragmentation and before infiltration by lymphocytes. This suggested that onset of illness started independently of the immune function. This conclusion was supported by the occurrence of massive apoptosis followed by paralysis in rabies virus-infected immunosuppressed mice. Direct, viral-induced, neuronal apoptosis was the earliest death mechanism detected in these mice. We propose that pathogenesis of this fixed strain of rabies virus in mice begins with the induction of apoptosis by rabies virus replication. Cerebral damage may then be amplified by immunological mechanisms plus an additional unidentified factor. This is followed by increased permeability of the blood brain barrier.