Molecular and clinical evidence of Ehrlichia chaffeensis infection in Cameroonian patients with undifferentiated febrile illness.

In the U.S.A., human monocytotropic ehrlichiosis (HME) caused by Ehrlichia chaffeensis is an emerging tick-transmitted zoonosis. In Cameroon, where E. canis, E. chaffeensis and E. ewingii have recently been detected in dogs and/or ticks (Rhipicephalus sanguineus), the potential exists for human infections. Patients from the coastal region of Cameroon who had acute fevers of unknown aetiology were therefore checked for ehrlichial infection, using a real-time PCR that amplifies part of a genus-specific gene (dsb) that codes for a disulphide-bond formation protein. Ehrlichial blood was detected in the peripheral blood from 12 (10%) of the 118 patients investigated by PCR. When the 12 amplicons from the positive cases were sequenced, they were found to be identical to each other and to the corresponding dsb sequence of an Arkansas strain of E. chaffeensis. The 12 patients who were PCR-positive for E. chaffeensis suffered from fever (100%), headache (67%), myalgia (42%), arthralgia (58%), pulmonary involvement (17%) and/or a diffuse rash (17%).

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii.

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.

Diffuse intimal fibromuscular dysplasia with multiorgan failure.

A woman presented with a rapid onset of hypertension, angina pectoris, peripheral vascular disease, renal involvement, and a large liver cyst. Surgical removal of the liver cyst precipitated renal and liver failure and a terminal arrhythmia. At autopsy, there was intimal fibromuscular dysplasia involving the arteries to the heart, liver, kidneys, and intestines and evidence of recent infarction of the intestines, kidney, and liver. This case illustrates that intimal fibromuscular dysplasia (FMD) can be a diffuse and rapidly progressive disease. Some treatments currently being evaluated for preventing restenosis following angioplasty may find use in treating this uncommon disease.

Supraventricular tachycardia treated with continuous infusions of propranolol.

Because oral therapy is often contraindicated in hospitalized patients we assessed the safety and efficacy of continuous intravenous propranolol infusions in nine patients with refractory supraventricular tachycardia. Standard pharmacokinetic formulas predicted a loading dose (52.2 +/- 38.3 micrograms/kg), steady-state plasma concentration, and the initial maintenance dose (16.1 +/- 16.2 micrograms/kg/hr; range 6.1 to 56.0 micrograms/kg/hr) to control heart rate. Subsequent maintenance doses (3.9 to 74.9 micrograms/kg/hr) were determined by clinical response. Heart rate decreased from 146 +/- 22 to 98 +/- 16 beats/min (p less than 0.0001). This decrease persisted throughout the infusion. Measured propranolol levels (28 +/- 21 ng/ml) did not differ significantly from the predicted levels (23 +/- 17 ng/ml). The duration of the infusion averaged 97 +/- 77 hours. A side effect, transient wheezing, occurred in only one patient. This resolved when the infusion rate was decreased. We conclude that continuous propranolol infusions appear safe and effective in treating these patients with supraventricular tachycardia.

Characterization of the complete transcriptionally active ehrlichia chaffeensis 28 kDa outer membrane protein multigene family.

The 28kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family. The purpose of this study was to determine all the p28 gene sequences and their transcriptional activities. There were 21 members of the p28 multigene family located in a 23kb DNA fragment in the genome of E. chaffeensis. The p28 genes each contained 816-903 nucleotides with intergenic spaces of 10-605 nucleotides. All the genes were complete and were predicted to have a signal sequence. The molecular masses of the mature proteins were predicted to be 28-32kDa. The amino acid sequence identity of the P28 proteins was 20-83%. Ten p28 genes were investigated for transcriptional activity by using RT-PCR amplification of mRNA. Six of 10 tested p28 genes were actively transcribed in cell-culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were divergent among isolates of E. chaffeensis also. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that P28s may be involved in immune avoidance.

A conserved, transcriptionally active p28 multigene locus of Ehrlichia canis.

Antigenic diversity of Ehrlichia chaffeensis and Ehrlichia canis may involve independent or differential expression of the P28 outer membrane proteins genes, enabling persistent infections of the natural hosts. In this study, we analyzed the transcriptional activity of a five gene locus in E. canis encoding homologous, but non-identical, p28 genes. The p28 multigene locus contained three previously identified complete gene sequences and one partial gene sequence. A new p28 gene was identified and sequenced, and the complete sequence of a second partial p28 gene was determined. The new p28 gene joined two previously separate loci, forming the single p28 multigene locus. The amino acid homology of the E. canis P28 proteins ranged from 51 to 74%. The nucleic acid sequence of regions compared within the locus spanning four p28 genes from two geographically distinct E. canis isolates was completely conserved. Analysis of the five p28 genes demonstrated that all were transcriptionally active in in-vitro cultures of E. canis incubated at the vertebrate host (37 degrees C) and ambient tick temperatures (27 degrees C). Polycistronic copies of multiple p28 genes were not detected by RT-PCR, but monocistronic p28 mRNA transcripts were detected by Northern blotting from E. canis infected DH82 cells, indicating that the genes are transcribed as monocistronic messages.

Esophageal gastric tube airway vs endotracheal tube in prehospital cardiopulmonary arrest.

We evaluated the efficacy of the esophageal airway (EA) by prospectively randomizing 175 prehospital cardiopulmonary arrest patients to receive either an esophageal gastric tube airway (EGTA) or an endotracheal tube (ET). If attempts with the initial airway failed, the alternate airway was attempted. The cost of training paramedics in EA use was considerably less than the ET ($80 vs $1,000). Survival to the emergency room, to hospitalization and to discharge in ET and EGTA groups were 64.4 percent, 25.6 percent, 11.1 percent, and 54.1 percent, 27.1 percent, 12.9 percent, respectively--differences not statistically significant. The incidence of neurologic residual (ET 50 percent, EGTA 36.4 percent) and congestive heart failure (ET 40 percent, EGTA 45.5 percent) in surviving ET and EGTA patients did not differ (NS). An additional 125 consecutive patients with only the opportunity to receive an EA were also evaluated and did not differ in mortality, neurologic residual, or congestive heart failure from ET patients. We conclude that the EA is a satisfactory alternative to the ET for short-term prehospital use in cardiopulmonary arrest patients.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica.

Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34-37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40-43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher (P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly (P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.

Primary and anamnestic responses of bovine bronchoalveolar and peripheral blood lymphocyte subsets to aerosolized Pasteurella haemolytica A1.

Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.

Memory and CD8+ are the predominant bovine bronchoalveolar lymphocyte phenotypes.

Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8+ while peripheral blood T cells were predominately CD4+. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25+), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of gammadelta T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.

PCR detection of acute Ehrlichia canis infection in dogs.

A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.

Detection of antigenemia by enzyme-linked immunosorbent assay in horses with experimental Ehrlichia risticii infection.

Four horses were inoculated with Ehrlichia risticii contained in either infected murine P388 D1 cells or heparinized blood from an infected horse. All 4 horses produced serum antibody, plasma antigen, and clinical signs of the disease. An enzyme-linked immunosorbent assay was used to detect antibody in the serum and was also used in conjunction with an anti-E. risticii monoclonal antibody to detect antigenemia. These laboratory and clinical findings were correlated to determine the efficiency of the antigen detection method for discerning E. risticii infection.

Detection of humoral antigen and antibody by enzyme-linked immunosorbent assay in horses with experimentally induced Ehrlichia equi infection.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antigen in plasma and antibody in serum of 3 horses inoculated with Ehrlichia equi. Clinical signs, including rectal temperature, were correlated with the antigen and antibody detection. ELISA was very efficient in detection of serum antibody. Antigen detection using monoclonal antibodies to E. equi and ELISA should be considered as a diagnostic method.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica after intrapulmonary inoculation.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of an indwelling bronchial catheter model of bovine pneumonic pasteurellosis for evaluation of therapeutic efficacy of various compounds.

A model of bovine pneumonic pasteurellosis, using an indwelling bronchial catheter for inoculation and subsequent lavage of a single main stem bronchus of the lung, was evaluated in a preliminary efficacy trial of an experimental therapeutic compound. Inoculation of 10(7) Pasteurella haemolytica organisms into the bronchus consistently induced a focal pneumonic lesion with typical morphology of pneumonic pasteurellosis in the left or right caudal lung lobe. The experimental treatment caused significant (P less than 0.05) reduction in lung lesion volume, compared with that of a saline-treated control. It also caused significant (P less than 0.05) reduction in lavage fluid bacterial counts at 48 hours after inoculation, compared with counts in the controls. The inflammatory cell count and the percentage of neutrophils increased markedly in lavage fluids 8 hours after inoculation, but differences were not detected between treatments. Significant differences between treatments were not found in clinical signs, rectal temperature, or histologic changes. This model appears to be a sensitive indicator of treatment efficacy and has the advantage over previous models of pneumonic pasteurellosis of allowing sequential monitoring of the primary lesion site.

Variable first-pass elimination of propranolol following single and multiple oral doses in hypertensive patients.

The disposition of orally administered propranolol has been studied in twelve patients with mild to moderate hypertension. Each patient received single doses of 40, 80, and 160 mg. Serial blood samples were obtained and quantitated using a sensitive gas chromatographic analytical technique. Ten of the twelve patients received 40 mg doses of propranolol every 6 hours for 5 doses. Blood samples were obtained after administration of the first, second, third, and fifth doses. Substantial intersubject variability in the areas under the bloodconcentration-time profiles (AUC) was observed. Evidence for a nonlinear first-pass effect was not obtained in all patients. The patients displaying a nonlinear relationship between dose and AUC for single propranolol doses consistently showed a similar relationship during multiple dosing. Blood levels obtained following the evening dose (08h00 to 14h00) appeared to be lower than expected based on multiple-dosing pharmacokinetic principles. These findings suggest that monitoring propranolol blood levels is the most viable way to ascertain therapeutic concentrations of this drug.

Female security officers: a discussion of the possible advantages for the female in the hospital security setting.

Female security officers may be an unrecognized resource for specific performance attributes which are valuable to the security task in hospitals. This article discusses some of the advantages the female security officer may contribute to desirable performance outcomes on the job in hospital security.

Genetic diversity of Ehrlichia canis in Brazil.

Canine monocytic ehrlichiosis is a highly prevalent disease in Brazil, where the genetic diversity of Ehrlichia canis remains undefined. In this study, we used the TRP36 gene to examine the genetic diversity of E. canis strains from naturally infected dogs residing in five distinct geographic regions in Brazil. E. canis DNA was detected in 82/126 (65%) dogs by dsb-specific PCR and E. canis was isolated in cell culture from 13 dogs. Sequences obtained from dsb genes amplified from the isolates were identical to the US E. canis strain. An extended molecular characterization based on the TRP36 gene identified two major genogroups based on differences among eight isolates. Isolates with tandem repeat amino acid sequence (TEDSVSAPA) identical to the previously reported TRP36 sequence were found in the midwest, northeast and southeast regions of Brazil, and classified into the US genogroup. A novel Brazilian genotype with a different tandem repeat sequence (ASVVPEAE) was also identified in midwest, northern and southern regions. Similarity in the N-terminal sequence of a US genogroup member with the Brazilian genogroup suggested that genomic recombination between the two genogroups may have occurred. Other subtypes within the Brazilian genogroup were also identified using C-terminal amino acid divergence. We identified two distinct major Brazilian genogroups and several subtypes based on analysis of TRP36, and such information will be useful for further genotyping and possible associations with disease severity, understanding of the genetic and antigenic variability of E. canis, and for developing strain-specific vaccines and diagnostic methods based on TRP36.

Optical fiber imaging for high speed plasma motion diagnostics: applied to low voltage circuit breakers.

An integrated portable measurement system is described for the study of high speed and high temperature unsteady plasma flows such as those found in the vicinity of high current switching arcs. An array of optical fibers allows the formation of low spatial resolution images, with a maximum capture rate of 1 x 10(6) images per second (1 MHz), with 8 bit intensity resolution. Novel software techniques are reported to allow imaging of the arc; and to measure arc trajectories. Results are presented on high current (2 kA) discharge events in a model test fixture and on the application to a commercial low voltage circuit breaker.