Dermatoglyphics of a high altitude Peruvian population and interpopulation comparisons.

Studies of genetic structures of Andean human populations have not been numerous, even though these studies could be used to answer questions concerning migration routes of the indigenous peoples who populated America. Such studies could provide basic genetic information and clarify uncertainties surrounding genetic relatedness of South American indigenous peoples. This present work describes, quantifies, and analyzes the digital and palmar dermatoglyphics of 120 people in the community of San Pedro de Casta, Perú. The results were then compared using distance analysis to all other Peruvian population values studied to date and other South American populations. The dermatoglyphic indicators studied were the distribution of digital pattern frequencies, the total ridge counts (TRC), the pattern intensity index (PII), the atd angle, and the a-b ridge counts. The results did not show statistically significant differences for digital patterns between hands, neither within a sex nor between sexes. The means and standard deviations of PII and TRC were 12.32 +/- 3.97 and 112.18 +/- 45.09, respectively. The means and standard deviations for the other two indicators were the following: atd angle, 94.85 degrees +/- 12.33; and a-b ridge counts, 81.57 +/- 9.06. The distance analyses results suggest the existence of two different genetic lines among high altitude populations, as well as the need for further research.

Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils.

Supernatants prepared from disrupted Coxiella burnetii possess acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M(r) of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 degrees C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 degrees C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-linked glycan residues since its treatment with N-glycosidase F (PNGase F) decreased its M(r) to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phosphocompounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP > 5'-AMP. The ACP also dephosphorylated (32)P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C. burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells.

Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment.

Bacterial cell attachment to the surfaces of self-assembled monolayers formed by the adsorption of omega-substituted alkanethiols on transparent gold films has been studied under defined bacterial culture and flow conditions. Phase contrast microscopy was used to quantify the attachment of two organisms, one of medical (Staphylococcus epidermidis) and one of marine (Deleya marina) importance. Self-assembled monolayers terminated with hexa(ethylene glycol), methyl, carboxylic acid and fluorocarbon groups were investigated. Over the range of experimental conditions, self-assembled monolayers formed from HS(CH2)11(OCH2CH2)6OH were found to be uniformly resistant to bacterial attachment, with a 99.7% reduction of attachment for both organisms when compared to the most fouled surface for each organism. On other surfaces, S. epidermidis and D. marina were shown to exhibit very different attachment responses to the wettability of the substratum. While the attachment of S. epidermidis correlated positively with surface hydrophilicity, D. marina showed a preference for hydrophobic surfaces. This study suggests that surfaces incorporating high densities of oligo(ethylene glycol) are good candidates for surfaces that interact minimally with bacteria.

A cationic antimicrobial peptide enhances the infectivity of Coxiella burnetii.

Purified Coxiella burnetii (Nine Mile, phase I) ricketssiae were exposed to a synthetic peptide (CAP37(20-44)) based on the amino acid sequence of CAP37--a 37 K human neutrophil granule-associated cationic antimicrobial protein--and their capacity to infect L929 mouse fibroblast cells was assessed during a 10-day post-exposure period. Because the parasite thrives within the acidic phagolysosome we anticipated that CAP37(20-44) would have no adverse effect on the organism. This was borne out by the experiments; however, to our surprise, treated C. burnetii had a much greater capacity to infect L cells than the non-treated counterpart. We speculate that the peptide exhibits opsonin-like properties, enhancing attachment of the rickettsia to the host cell surface and subsequent entry.

Survival of the Q fever agent Coxiella burnetii in the phagolysosome.

The Q fever agent, Coxiella burnetii, thrives in the acidic environment of the phagolysosome of the host cell. How this obligate intracellular agent manages to survive within this hostile milieu is unknown; however, several of its enzymes may eliminate or prevent the formation of toxic oxygen metabolites by the host cell. Also implicated as virulence factors are its surface lipopolysaccharide and plasmids.

Acid phosphatase activity in Coxiella burnetii: a possible virulence factor.

High-speed supernatant fluids derived from sonicated Coxiella burnetii contained considerable acid phosphatase activity when assayed by using 4-methylumbelliferylphosphate; they also contained a factor that blocked superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe. The pH optimum of the enzyme was approximately 5.0. The level of phosphatase activity detected in several isolates of C. burnetii implicated in acute (Nine Mile) and chronic (S Q217, PRS Q177, K Q154) Q fever was 25 to 60 times greater than that reported in other microorganisms, including Leishmania and Legionella spp. The enzyme was found in rickettsiae grown in different hosts (L929 cells and embryonated eggs) and, in the case of L929 cells, for both short periods (less than a month) and the long term (years). Cytochemical techniques coupled with electron microscopy localized the phosphatase activity to the periplasmic gap in the parasite. Ion-exchange chromatography revealed a major species of the enzyme and showed that the enzyme of the parasite was distinct from that of the host cell (L929 fibroblasts); its apparent molecular weight was 74,000. Phosphatase inhibitors (i.e., molybdate heteropolyanions) had differential effects on the phosphatases of the parasite and host cell. C. burnetii supernatant fluid inhibited superoxide anion production by formyl-Met-Leu-Phe-stimulated human neutrophils; molybdate inhibitors reversed the inhibition. Treatment of C. burnetii-infected L929 cells with one of the molybdate compounds (complex B') significantly reduced the level of infection and did not affect the viability or growth of the host cell. These data suggest that the acid phosphatase of the parasite may be a major virulence determinant, allowing the agent to avoid being killed during uptake by phagocytes and subsequently in the phagolysosome.

Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.

Entry of Coxiella burnetii into host cells.

The attachment to and entry into L mouse fibroblast cells of viable phase I and phase II Nine Mile Coxiella burnetii was investigated. The use of 32P-labelled rickettsiae showed that phase II C. burnetii attached more readily to L cells than phase I organisms; this probably accounts for the more rapid establishment of infection of host cells by the phase II agents. Two lines of evidence indicated that C. burnetii plays a passive role in both attachment and entry into host cells: (1) inactivation of rickettsiae by either heat or glutaraldehyde did not affect either process, and (2) metabolic inhibitors of L cell phagocytic function--NaF and cytochalasin B and D--abolished rickettsiae uptake. These results indicate that it is an endocytotic event. While the presence of purified phase I lipopolysaccharide (LPS) did not interfere with attachment of rickettsiae to the surface of host cells, it markedly impaired entry of C. burnetii in both phases. This suggests that LPS is not an adhesin and that it is toxic to the host cell. Treatment of L cells with either pronase, subtilisin or subtilopeptidase A significantly reduced the number of C. burnetii that adhered to the host cell surface; this result suggests that proteins are either proximate to or components of the C. burnetii attachment site.

DNA probes for detecting Coxiella burnetii strains.

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Coxiella burnetii isolates in cell culture and the expression of parasite-specific antigens on the host membrane surface.

Coxiella burnetii isolates may be classified into several groups based on plasmid character. These groups may also be correlated with disease syndrome--chronic or short-term acute. L929 mouse fibroblast cells were exposed, independently, to two members of the three major C. burnetii groups, and their growth/morphological characteristics analysed by light and electron microscopy, including High Voltage Electron Microscopy. The fates of the isolates were followed. Two acute isolates [Nine Mile (RSA 493) and Henzerling (RSA 331), QpH1-type plasmids] and two chronic isolates (S Q217 and L Q216, plasmidless) readily infected L929 cells in static culture. Priscilla (Q177) and F (Q228) isolates (QpRS-type plasmid, and implicated in causing chronic Q fever) took longer to infect cells, and, unlike the members of the other two groups, gradually disappeared when shifted to suspension culture. Cells infected with Q177 and Q228 exhibited a higher degree of vacuolation than cells infected with the other isolates. Parasite-specific antigens on the surfaces of the host cells were analysed by immunofluorescence/flow cytometry. The acute and plasmidless isolates caused the display of significantly more C. burnetii-specific antigen on the host cell membrane than the two QpRS plasmid-containing isolates. This host cell model system clearly reveals biological differences among the C. burnetii groups.

Mechanisms that may account for differential antibiotic susceptibilities among Coxiella burnetii isolates.

The Nine Mile, S Q217, and Priscilla isolates, representative of the three major genetic groups of Coxiella burnetii, are known to differ in their susceptibilities to antibiotics. Mechanisms potentially responsible for these differences were investigated. Accumulation of antibiotics by infected L929 cells and purified isolates was measured. In addition, C. burnetii plasmid-transformed Escherichia coli HB101 cells were used to study the possibility that different C. burnetii plasmids are responsible for disparate antibiotic susceptibilities of the isolates. L929 cells recently or persistently infected with the Priscilla isolate exhibited a significantly reduced accumulation of [3H]tetracycline as compared with that in L929 cells infected with either the Nine Mile or S Q217 isolates; accumulation of this drug was greater in cells recently infected each isolate. In contrast, L929 cells recently or persistently infected with the different isolates accumulated [3H]norfloxacin to an equivalent extent. [3H]tetracycline accumulation was approximately the same among the purified isolates. However, as measured by both scintillation and spectrofluorometry, norfloxacin accumulation was significantly diminished in the purified Priscilla isolate. pH had no apparent effect upon isolate permeabilities. The presence of C. burnetii QpH1 or QpRS plasmids did not alter the antibiotic susceptibility of E. coli. Collectively, these results indicate that differential susceptibilities to tetracyclines or fluoroquinolones in C. burnetii isolates may be the result of distinct mechanisms involving altered host-cell (tetracyclines) or isolate-specific (fluoroquinolones) permeabilities.

Pathogenesis of rickettsial infections emphasis on Q fever.

The underlying mechanisms at the organismic, cellular and molecular levels that account for rickettsial pathogenesis are beginning to be revealed. In the case of Coxiella burnetii infection, relatively recent genetic and biochemical data, as well as drug susceptibility studies, indicate a correlation between isolate type and clinical disease--chronic or short-term acute. The use of cultured cells as model host systems has revealed that, indeed, different isolates from the major classified strains of C. burnetii cause different host cell responses. Use of this and other models (guinea pigs, mice) have revealed other characteristics and properties of the rickettsiae and the infected hosts and host cells that may account, in part, for acute disease and persistent infection culminating in chronic disease. The virulence factors involved apparently include the agent's surface lipopolysaccharide; other unidentified factors have not been excluded. Molecular cloning will play a major role in elucidating the roles of these factors and in identifying other virulence determinants.

Unexpected antibiotic susceptibility of a chronic isolate of Coxiella burnetii.

Evidence is mounting in support of the idea that different isolates of Coxiella burnetii, the etiologic agent of Q fever, are responsible for the distinct disease syndromes observed clinically. Recent studies have shown distinct antibiotic susceptibilities of different isolates of C. burnetii implicated in distinct clinical Q fever syndromes. With this in mind, we performed antibiotic susceptibility testing of the "S" isolate, a chronic-type isolate retrieved from a human patient with chronic disease. Antibiotics with differing efficacies upon the Nine Mile and Priscilla isolates were tested for their abilities to control acute and persistent "S" isolate infection of L-929 cells in vitro. The efficacies of doxycycline, rifampin, and three fluoroquinolone drugs--ciprofloxacin, ofloxacin, and norfloxacin--were tested. Compared to the chronic Q fever-implicated Priscilla isolate, which has been shown to exhibit a significant resistance to these antibiotics, the "S" isolate was much more susceptible. In persistently infected cells (greater than 300 d), the "S" isolate proved to be significantly more resistant to doxycycline, slightly more resistant to ciprofloxacin, slightly more susceptible to rifampin, and equally sensitive to ofloxacin and norfloxacin compared to the acute Q fever-implicated Nine Mile isolate. As with both the Nine Mile and Priscilla isolates, the "S" isolate was more susceptible to doxycycline, rifampin, and ofloxacin in recently infected cells (22 d) compared to cells having been persistently infected. With respect to the resistant nature of the chronic Q fever-implicated Priscilla isolate, as well as the lack of success in treating the "S" isolate in vivo, these results were unexpected. Such data supports an evolving hypothesis that the distinct C. burnetii isolates which may be responsible for the clearly different Q fever syndromes exhibit a spectrum of antibiotic susceptibility ranging from very susceptible (acute-implicate), Nine Mile isolate), to moderately susceptible (chronic-implicated "S" isolate), to moderately resistant (chronic-implicated Priscilla isolate).

DNA probes for the identification of Coxiella burnetti strains.

Isolation of Coxiella Burnetii in the standard laboratory setting is hazardous; therefore most diagnoses are based on retrospective detection of a rising antibody titer to C. burnetti. As a result, this disease is usually undiagnosed or misdiagnosed. Methods for the rapid detection of C. burnetti have now been developed that utilize specific hybridization of labeled DNA probes to nucleic acid in clinical samples. One method detects the presence of C. burnetii 16S ribosomal RNA (rRNA); another uses plasmid sequences. We have developed a probe that detects C. burnetii and one that differentiates between Coxiella strains capable of causing chronic disease and those that cause the acute form. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. A third method differentiates between chronic (endocarditis-causing) strains and those that cause acute Q fever. This method uses the polymerase chain reaction (PCR), in which the target regions of DNA are amplified by iterative cycles of Taq I DNA polymerase chain extension to produce up to a 10(6) amplification of the target sequences. When Southern blotting is used in conjunction with PCR, the test detects as few as 2-9 C. burnetti cells.

Strategy for detection and differentiation of Coxiella burnetii strains using the polymerase chain reaction.

A method for the rapid detection of Coxiella burnetii and differentiation between strains that cause endocarditis and those that cause acute Q fever is based on the observation that the different strains contain unique plasmid sequences. This method employs the polymerase chain reaction (PCR) and requires knowledge of specific DNA sequences in the region (target) of DNA to be amplified. To detect and differentiate between C. burnetii isolates, two sets of primers are required. The first set was derived from a fragment of plasmid QpH1 which has been detected in all C. burnetii isolates. A second PCR reaction was conducted using primers specific for DNA sequences that are shared only by QpRS plasmid-containing strains of C. burnetii. The first reaction detects the presence of C. burnetii. The second PCR is necessary to determine whether the isolate contains DNA sequences associated with strains causing chronic disease. These procedures detect as few as one to ten organisms.

Phylogenetic diversity of the Rickettsiae.

Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the alpha subdivision of the purple bacteria. E. risticii also belongs to the alpha subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the gamma subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.

Antibiotic susceptibilities of two Coxiella burnetii isolates implicated in distinct clinical syndromes.

Antibiotic susceptibility testing of two isolates of the Q-fever agent, Coxiella burnetii, was performed with recently and persistently infected L929 fibroblast cells. The two genetically distinct isolates, Nine Mile and Priscilla, are implicated in two different clinical disease syndromes, acute and chronic Q fever, respectively. We compared the efficacies of rifampin, doxycycline, and five 4-quinolone compounds (ciprofloxacin, difloxacin, ofloxacin, norfloxacin, and pefloxacin) in reducing persistent C. burnetii infection of L929 fibroblasts. In persistently infected cells, the Priscilla isolate was less susceptible to all antibiotics tested when compared with the Nine Mile isolate. The most effective antibiotics against the Priscilla isolate were ofloxacin, pefloxacin, and ciprofloxacin (50% inhibitory concentrations of 0.5, 2.2, and 2.5 micrograms/ml, respectively). In persistently infected cells, the Nine Mile isolate was highly susceptible to all antibiotics tested except doxycycline. In contrast, the Priscilla and Nine Mile isolates in recently infected cells were somewhat susceptible to doxycycline; the Priscilla isolate was significantly more susceptible to ofloxacin and rifampin in recently infected host cells than in persistently infected cells. Persistently infected L929 cells were also treated with antibiotic combinations. Although ciprofloxacin and doxycycline had no synergistic effect on the Priscilla isolate, ciprofloxacin and rifampin acted synergistically. Collectively, these in vitro results are in accord with the fact that chronic Q fever in humans is generally not successfully managed with antibiotics. They also indicate that early diagnosis may be essential and that combination antibiotic therapy that includes quinolones may be effective in treating chronic Q fever.

Susceptibility of Coxiella burnetii to pefloxacin and ofloxacin in ovo and in persistently infected L929 cells.

The relative lack of efficacy of the antibiotic treatment of chronic Q fever endocarditis justifies the further evaluation of the susceptibility of Coxiella burnetii to the modern quinolone antibiotics. We evaluated the efficacies of pefloxacin and ofloxacin in controlling the Nine Mile isolate of C. burnetii by using an embryonated egg assay and persistently infected L929 cells in culture. Pefloxacin was effective in controlling the intracellular parasite at a concentration of 50 micrograms per egg and 1 microgram/ml in cultures of infected cells. Ofloxacin was effective at a concentration of 25 micrograms per egg and 0.5 microgram/ml in infected-cell cultures. In light of the fact that the concentrations of antibiotics used fall within physiological ranges used in humans, ofloxacin and pefloxacin may be useful in the clinical management of chronic Q fever, for which, to date, results have been poor.

In vitro susceptibility of Coxiella burnetii to antibiotics, including several quinolones.

Antibiotic susceptibility testing of the rickettsial Q fever agent Coxiella burnetii was performed by using persistently infected L929 fibroblast cells. The efficacies of a variety of antibiotics with different metabolic targets were tested and compared. The most effective antibiotics in bringing about the elimination of the parasite from infected cells included several quinolone compounds and rifampin. Of the quinolone compounds tested, difloxacin (A-56619) was the most effective, followed by ciprofloxacin and oxolinic acid. These three quinolones were apparently rickettsiacidal. After 48 h of exposure to microgram amounts of the compounds (ranging from 2 micrograms of difloxacin per ml to 5 micrograms of the other two antibiotics per ml), the number of intracellular parasites markedly declined; after 10 days of treatment, very few intracellular rickettsiae were detected. Rifampin (1 microgram/ml) was also very effective in eliminating the parasites. Some of the 13 other antibiotics tested that were somewhat effective included chloramphenicol, doxycycline, and trimethoprim. The persistently infected L929 cells were found to provide a convenient system for the relatively rapid determination of the susceptibility of C. burnetii to antibiotics.