Reactivity of serum samples of dogs and horses tested by use of class-specific recombinant-based enzyme-linked immunosorbent assays for detection of granulocytic ehrlichiosis.

OBJECTIVE: To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. SAMPLE POPULATION: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). PROCEDURE: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyelocytic leukemia cells, were conducted on 38 serum samples of horses and 11 serum samples of dogs to verify reactivity to the 44-kd peptide. RESULTS: IgM or IgG antibodies to the HGE agent were detected in 5 to 28% of dog serum samples and 5 to 37% of horse serum samples. Thirty-five of 38 (92%) horse serum samples had corresponding results on both tests (2 positive results for 26 samples and 2 negative results for 9 samples), using an ELISA for IgG antibodies or immunoblotting for total immunoglobulins. All 11 serum samples of dogs had positive results for both methods. CONCLUSION AND CLINICAL RELEVANCE: These ELISA with recombinant 44-kd antigen are suitable for detecting IgM or IgG antibodies to the HGE agent in serum samples of dogs and horses. Positive results for serum samples of horses from Connecticut, New York, Virginia, and Georgia indicate that the HGE agent is widely distributed in tick-infested areas of the eastern United States.

Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses.

OBJECTIVE: To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. ANIMALS: 32 dogs and 43 horses. PROCEDURE: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. RESULTS: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, western immunoblotting, and the ELISA were in complete agreement. Results of the ELISA for 3 canine serum samples known to contain antibodies to Ehrlichia canis and 12 equine serum samples known to contain antibodies to E risticii were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggest that a polyvalent ELISA incorporating a recombinant p44 antigen is suitable for detecting antibodies to E equi in dogs and horses.

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae.

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.

Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States.

OBJECTIVE: To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. DESIGN: Prospective study. ANIMALS: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. PROCEDURE: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: Of the 82 serum samples tested, 37 (45.1%) and 13 (15.9%) had detectable antibodies to B burgdorferi or E equi, respectively. Test results indicated that 12 horses had been exposed to both agents, 11 of these horses had granulocytic ehrlichiosis. The ELISA regularly detected antibodies to the following recombinant protein (p) antigens of B burgdorferi: p29, p37, p39, and p41-G. The use of immunoblot analysis confirmed ELISA results by indicating antibody reactivities to antigens of whole-cell B burgdorferi having molecular masses of predominantly 31, 34, 37, 39, 41, 58, and 93 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Horses living in areas where ticks (Ixodes scapularis) abound are sometimes exposed to multiple pathogens. Analyses for specific recombinant borrelial antibodies using an ELISA can help separate horses with borreliosis from those with granulocytic ehrlichiosis, even when antibodies to both etiologic agents are detected in serum samples. Analysis using immunoblots is sensitive, and along with ELISA or IFA procedures, is suitable for confirming a clinical diagnosis of each disease.

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis.

OBJECTIVE: To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. ANIMALS: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. PROCEDURE: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. RESULTS: Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. CONCLUSION AND CLINICAL RELEVANCE: Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis.

Analysis of inhaled corticosteroid and oral theophylline use among patients with stable COPD from 1987 to 1995.

STUDY OBJECTIVE: To document temporal usage trends for commonly used respiratory medications in patients with COPD. DESIGN: We retrospectively evaluated baseline concomitant medications of 3,720 patients with COPD enrolled in 10 bronchodilator clinical trials from 1987 to 1995. The proportion of patients in each trial using inhaled corticosteroids, inhaled beta-adrenergics, inhaled anticholinergics, oral theophylline, and oral corticosteroids was analyzed using the Cochran-Armitage trend test. PATIENTS: All patients had stable, moderate-to-severe COPD without evidence of asthma or atopy. Reversibility to beta3-agonists was not a requirement. RESULTS: The percentage of patients using inhaled corticosteroids increased significantly over time (p < 0.001) from 13.2% in 1987 to 41.4% in 1995. The percentage of patients receiving oral theophylline decreased significantly (p < 0.001) over this same time interval (63.4 to 29.0%). In addition, the percentage of patients using oral corticosteroids and the percentage using oral beta-adrenergics decreased moderately (p < 0.05) (30.1 to 16.4% and 11.7 to 4.5%, respectively); the percentage of patients using inhaled anticholinergics increased slowly (p < 0.05) (48.2 to 53.8%). The percentage of patients receiving inhaled beta-adrenergics did not significantly (p > 0.05) change. CONCLUSIONS: The observed changes in use of inhaled corticosteroids and theophylline were not likely related to differences in disease severity or other patient characteristics in the evaluated trials, but related to changing prescribing and COPD management practices.

Development and duration of antibody response against Ehrlichia equi in horses.

OBJECTIVE: To characterize antibody response in horses with clinical signs of Ehrlichia equi infection. DESIGN: Prospective study. ANIMALS: 13 horses with confirmed acute E equi infection. PROCEDURE: Sequential serum sampling was performed in Connecticut and New York during 1995 and 1996 to identify horses with naturally acquired equine granulocytic ehrlichiosis (EGE). Horses with clinical signs of EGE (i.e., fever without respiratory involvement) were confirmed as having E equi infection by polymerase chain reaction detection of ehrlichial DNA and by a minimum fourfold increase in total antibody titer by indirect fluorescent antibody staining methods. Infection was corroborated by use of DNA sequencing. RESULTS: 11 of 13 horses did not have detectable antibody in serum samples obtained at onset of disease. Seroconversion was evident in samples obtained 19 to 81 days thereafter. Median time to peak antibody response was 46 days after onset and median titer was 1:320. For 11 of 13 horses, antibody titers were < or = 1:40 by 215 days after onset. CLINICAL IMPLICATIONS: E equi was found in horses in the northeastern United States and caused EGE. Concentration of antibodies to E equi increased within 19 to 81 days of disease onset and were low during early weeks of infection. Therefore, antibody detection may be of limited value for early serologic diagnosis. We suggest that disease may develop from a reinfection, and retrospective serologic studies to determine exposure to E equi may reflect a disproportionate number of negative reactions.

Propagation of granulocytic Ehrlichia spp. from human and equine sources in HL-60 cells induced to differentiate into functional granulocytes.

Ehrlichia spp. from human and equine sources in the northeastern Unites States were detected by PCR, isolated, and propagated in the HL-60 promyelocytic leukemia cell line. Growth of Ehrlichia from both equine and human sources was enhanced by addition of retinoic acid, which causes granulocytic differentiation of the HL-60 cells. DNA sequencing of a portion of the 16S rDNA gene supported the hypothesis that the same pathogen was responsible for both equine and human granulocytic ehrlichiosis.

Ca2+ channel antagonists enhance tension in skinned skeletal and heart muscle fibres.

Striated muscle fibres, both skeletal and cardiac of different species including human, skinned by freeze-drying, were activated in solutions strongly buffered for Ca2+. The single fibres were immersed in solutions with different [Ca2+]. Sarcomere length was set and controlled by laser diffraction. Fibre type was determined by Sr2+ activation. The relation between the negative logarithm of the Ca2+ concentration and the normalized tension, the Ca2+ sensitivity curve, was investigated. The effect on the contractile machinery of three different Ca2+ channel antagonists (verapamil, diltiazem and nifedipine) in a therapeutic concentration (10(-6) M) was investigated. The possible effects on the Ca2+ sensitivity curve were quantified by: (1) the change in maximal tension developed at pCa2+ = 4.4; (2) the change in pCa2+ value at which 50% of the tension induced at pCa2+ = 4.4; (3) the steepness of the Ca2+ sensitivity curve in this point. The three drugs tested, at a therapeutic concentration of 1 microM, all enhanced maximal induced tension by respectively 25, 20 and 7%. The sarcomere length dependency of the effect proved to be dependent upon the drug, but also slightly on fibre type (skeletal or cardiac), or on species. It is concluded that the drug influences the cooperativity of the two different types of binding sites on troponin-C (low- and high-affinity sites). Tension enhancement was due to increased stiffness of the actin-myosin interaction site.

Increased activity of FIM in serum of mice during a Mycobacterium bovis (BCG) infection.

In mice given an intravenous injection of Mycobacterium bovis (BCG), the bacilli proliferated in the spleen, liver and lungs but the peritoneal cavity remained sterile. The numbers of blood monocytes and alveolar macrophages were increased during the first 2 weeks of the infection, whereas the number of peritoneal macrophages remained constant. To study whether factor-increasing monocytopoiesis (FIM) plays a role in the regulation of the monocytosis during the BCG infection, the activity of this factor in the serum of mice at various intervals during the infection was determined. Previous studies have shown that FIM stimulates monocyte production by its effect on the mitotic activity of monoblasts and promonocytes in the bone marrow. The FIM activity of the serum reached a maximum on Day 4 and remained elevated during the first 21 days of the BCG infection. Since FIM is synthesized and secreted by macrophages that have phagocytosed opsonized particles, it is highly probable that FIM occurring in serum originates from macrophages that have ingested BCG. The results of the present study led to the conclusion that FIM plays a role in the monocytosis developing during infection with BCG.

Amyloid in the horse: a report of nine cases.

Out of approximately 16,000 horses referred for clinical examination, nine had amyloidosis. Six of these horses had localised amyloid deposits in the wall of the nasal meatus and ventral turbinates associated with epistaxis. Horse 1 also developed malignant histiolymphocytic lymphosarcomas. The amyloid deposits were potassium permanganate-resistant and tryptophan-positive. Gel filtration of solubilised amyloid fibrils from Horse 1 revealed a major retarded fraction with an apparent molecular weight of 20 kD. This protein had an amino acid composition similar to human AL-amyloid proteins and horse immunoglobulin light chains. On Western blot a strong cross-reaction was observed between horse 1gG2a light chains and the Horse 1 amyloid. Horses 7 to 9 had suppurative verminous aneurysm, tuberculosis and an adrenal cortical adenoma, respectively, and had generalised amyloid deposits in liver and spleen. These amyloid deposits were found to be potassium permanganate-sensitive and positive for tryptophan. Gel filtration of solubilised amyloid fibrils from Horse 8 revealed a major retarded fraction (protein AA) with an apparent molecular weight of 10 kD. Immunoperoxidase-antiperoxidase staining showed the localised deposits to be negative or only weakly positive with antisera against bovine, hamster, dog and human protein AA and to be positive with anti-horse-one amyloid protein. The generalised deposits were found to be positive with the antisera against allogenic protein AA. The results of the potassium permanganate incubation, biochemistry, immunoblotting and immunochemistry, indicate that the localised amyloid of Horse 1 and most likely the amyloid of Horses 2 to 6, is of the AL-type. The generalised amyloid deposits were found to be of the AA type.

Regulation of granulocyte responses in the blood and peritoneal cavity of CBA and B10 mice during an acute inflammation.

The regulatory mechanisms that determine the course of an inflammation induced by an intraperitoneal injection of kaolin were investigated in Listeria-susceptible CBA and Listeria-resistant B10 mice. The magnitude of the granulocyte inflammatory response in the peritoneal cavity was high in B10 mice (area under the curve; AUC0-48 h: 210.9 x 10(6) granulocytes/mouse x h) and lower in CBA mice AUC0-48 h: 136.8 x 10(6) granulocytes/mouse x h), whereas the reverse was seen for the granulocyte response in the peripheral blood (AUC0-48 h: 30.5 and 80.7 x 10(6) granulocytes/mouse x h, respectively). With respect to the presence of humoral factors that affect the number of granulocytes in the circulation, sera of both mouse strains sampled 24 h after the kaolin injection had granulocytosis-inducing effect in CBA recipient mice and did not induce a response in the B10 recipient mice. This divergent sensitivity to serum factors inducing granulocytosis is consistent with the difference in the blood granulocyte response of B10 and CBA mice but does not explain the divergent inflammatory responses in the peritoneal cavity. Computer simulation showed that at least two factors must be taken into consideration to explain the differences in the inflammatory response, i.e., a factor regulating the release of granulocytes from the bone marrow and a factor governing the rate of granulocyte efflux from the site of inflammation.

Macrophages as origin of factor increasing monocytopoiesis.

Earlier investigations had indicated that the factor increasing monocytopoiesis (FIM), present in the serum of mice and rabbits during the onset of an inflammatory response, is released by cells of the inflammatory exudate. The present study was performed to determine which cells produce and secrete this factor and to establish the kinetics of its production and secretion. FIM was assayed in vivo by intravenous injection of samples into untreated mice and monitoring the course of the number of blood monocytes in the recipients. FIM was assayed in vitro by adding samples to cultures of the macrophage cell line PU5 and determining the rate of proliferation of the cells. The results show that only macrophages contain and synthesize FIM. This factor is secreted upon exposure to a phagocytic stimulus, and after the release of preformed FIM, macrophages secrete newly synthesized FIM. Granulocytes and lymphocytes neither contain nor secrete FIM. The characteristics of FIM derived from macrophages are in all aspects similar to those of FIM in serum. Macrophage-derived FIM is a protein with a molecular weight between 10 and 25 X 10(3), its activity is cell-lineage specific and dose dependent, and it stimulates monocyte production in the bone marrow. Macrophage-derived FIM is not identical to either CSF-1 or IL-1, and has no chemotactic activity. Taken together, the present results show that FIM occurring in serum during an inflammatory response originates from macrophages at the site of the inflammation. In this way the macrophages themselves regulate the supply of circulating blood monocytes that can migrate to the site of injury when needed.

Enhancement of amyloid induction by amyloid fibril fragments in hamster.

An extract (fibril amyloid-enhancing factor) prepared by sonication of a suspension of water-purified hamster AA-amyloid fibrils showed an amyloid-enhancing effect upon intraperitoneal injection into hamsters. The fibril amyloid-enhancing factor was identical to the original fibrils according to the results of infrared spectroscopy, gel filtration, gel electrophoresis, and Western blotting; although lyophilized samples of the extract did not show any green birefringence after staining with Congo red. From these results, it was concluded that fibril amyloid-enhancing factor represents small fragments of amyloid fibrils which enhance in vivo formation of amyloid fibrils.

Amyloid enhancing factor in hamster.

An amyloid enhancing factor (AEF) was extracted from spleens and livers of casein-treated hamsters. It shortened the induction time of experimental amyloidosis in recipient hamsters from 14 days to 4 days. Sepharose-4B gel filtration resolved the extract into 4 different fractions, with molecular weights of: higher than IO(7) (Vo-fraction), about 280 000, 59 000 and 12 000 respectively. In all 4 fractions AEF was present, indicating that AEF is probably a low molecular weight substance that easily aggregates, or associates with other compounds present in the spleen extract. AEF was precipitable at 50% ammonium sulphate saturation. On anion exchange chromatography in PBS (pH 7.2) of this precipitate, AEF was found in the fraction eluting at high NaCl concentration. This fraction did not show any relation to hamster protein AA, IgG or albumin with double immunodiffusion. Ultraviolet absorption spectrophotometry indicated nucleotide-like material, suggesting AEF to be related to nucleoproteins.

Neutrophil delivery to wounds of the upper and lower extremities.

Clinical observations and experimental evidence indicate that wounds of the lower extremity are more susceptible to infection than are wounds located elsewhere on the body. The details of this regional relative immunoincompetence are not described. In this initial study of regional inflammatory response, neutrophil delivery to standard wounds of the upper and lower extremities was measured in 15 human volunteer subjects using a quantitative skin-window technique. Neutrophil delivery proved to be relatively deficient in the lower extremity. Neutrophil delivery (mean +/- SEM) was significantly lower to the lower-extremity wounds (5,890 +/- 590 cells per cubic millimeter) than to the upper-extremity wounds (16,600 +/- 1,680 cells per cubic millimeter). This lower neutrophil response may be a part of the lower extremity's increased susceptibility to infection. Further functional study of regionally collected neutrophils may provide more details of differences in regional inflammatory response. The mechanisms underlying these differences remain undescribed.

Differences in the response of inbred mouse strains to the factor increasing monocytopoiesis.

Previous studies have shown that monocyte production during an inflammatory response is controlled by the factor increasing monocytopoiesis (FIM), secreted by macrophages at the site of inflammation. The inflammatory reaction to latex particles and a saline-soluble extract of Listeria monocytogenes (SEL), expressed as the number of monocytes in the circulation and of macrophages at the site of inflammation, was about twice as strong in C57BL/10 mice compared with CBA mice. This raised the question as to the mechanism underlying these differences. One possibility might be that these mouse strains differ with respect to the production of FIM, but this cannot be the case because the maximum levels of FIM activity in the serum of both C57BL/10 and CBA mice given latex or SEL intraperitoneally were almost the same; however, the courses of FIM activity in the two strains after intraperitoneal latex were not exactly synchronous. Another possibility is that the sensitivity of monocyte precursor cells for FIM differs. Evidence for the latter was provided by the finding that the intravenous injection of sera with FIM activity obtained from C57BL/10 and from CBA mice into the C57BL/10 mice evoked monocytosis, whereas CBA mice did not respond to these sera. Earlier studies showed that an increase of monocytes after the injection of serum containing FIM reflects increased monocyte production. Taken together, the results of the present study demonstrate that one of the mechanisms underlying the genetic control of the inflammatory response is, rather than enhanced FIM synthesis, the ability of monocyte precursors in the bone marrow to respond to FIM by increased monocyte production.