Automated marker enzyme analysis of density gradient fractions using the Cobas-Bio centrifugal analyzer.

An automated method for analytical subcellular fractionation which utilizes routinely available reagents and equipment is described. Tissues are fractionated in Percoll density gradients and marker enzyme analysis of gradient fractions is performed with the Cobas-Bio centrifugal analyzer. Manual assays for cytochrome oxidase and protein were adapted for use with the instrument along with commercially available 5'-nucleotidase, lactate dehydrogenase, and acid phosphatase assay kits. Crude liver homogenate fractions containing different levels of enzyme activity were used to examine the linearity of the enzyme assays. The assays were shown to be linear at high and low levels of activity. Within-run precision studies using high and low activity liver homogenate pools were also performed, and a coefficient of variation of less than or equal to 5.5% was obtained for all assays. This method was used to analyze subcellular fractions from a Percoll density gradient separation of mouse liver homogenate material. The method allowed for a complete marker analysis, consisting of 96 separate kinetic assays, within 2 hours. The versatility of this method and the ease with which it can be performed should expand the use of analytical subcellular fractionation in the diagnostic laboratory.

Increase of leptospirosis in dengue-negative patients after a hurricane in Puerto Rico in 1996 [correction of 1966].

Leptospirosis has rarely been reported in Puerto Rico, although in the period from 1948 to 1952, 208 cases of leptospirosis and an island-wide seroprevalence of antibody to Leptospira of 14% were documented. In Puerto Rico in October 1996, following rainfall and a period of flooding generated by Hurricane Hortense, serum specimens of 4 patients with suspected dengue fever that were negative for dengue tested positive for Leptospira-specific IgM antibodies in a dipstick assay. Subsequently, we used an island-wide dengue laboratory-based surveillance system to determine the increase in leptospirosis after hurricane-generated floods. All anti-dengue IgM-negative patients (n = 142) with disease onset from August 8 to October 6, 1996 from prehurricane and posthurricane groups were investigated for leptospirosis. Laboratory-confirmed leptospirosis cases were defined as microscopic agglutination test titers > or = 1 :400 to 1 or more serovars, or positive immunohistochemistry in autopsy tissues. Four (6%) of 72 prehurricane and 17 (24%) of 70 posthurricane patients had laboratory-confirmed cases of leptospirosis (relative risk [RR] = 4.4, 95% confidence interval [CI] = 1.6-12.4). The mean age of case-patients was 34 years (range = 13-64). Eighteen (86%) of 21 confirmed case-patients were males, including one patient who died (31 years old). Patients were located in 18 (38%) of 48 municipalities that submitted serum samples. Clinical features significantly associated with leptospirosis were eye pain (RR = 1.5, 95% CI = 1.3-1.9), joint pain (RR = 1.4, 95% CI = 1.1-1.6), diarrhea (RR = 1.7, 95% CI = 1.2-2.5), and jaundice (RR = 3.3, 95% CI = 1.5-7.2). This study demonstrates the utility of a dengue laboratory-based surveillance system for the detection of an increase of leptospirosis, which most likely would have gone unrecognized. Leptospirosis is treatable with antibacterial agents; knowledge of this diagnosis may significantly reduce morbidity and mortality.

Asymptomatic infection and risk factors for leptospirosis in Nicaragua.

As part of an investigation of a 1995 outbreak of leptospirosis in Nicaragua, a cross-sectional serologic survey was conducted in the town of El Sauce. Of 566 persons, 85 (15%) were positive for IgM anti-Leptospira antibodies, indicating recent leptospirosis infection. Asymptomatic leptospirosis infection was common, with only 25 (29.4%) of the 85 seropositive inhabitants reporting a febrile illness in the 2 months before the survey. Multivariable analysis revealed that having an indoor water source remained independently protective against leptospirosis. Gathering wood was independently associated with infection. These findings suggest that asymptomatic infection with Leptospira is common in endemic areas of Leptospira transmission. Improvement in water sanitation and behavioral modifications to reduce environmental exposure may reduce the risk of leptospirosis in endemic regions.

Identification of Leptospira inadai by continuous monitoring of fluorescence during rapid cycle PCR.

Seven new Leptospira isolates from rats, a buffalo, and contaminated media showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the microscopic agglutination test (MAT). Because of these inconclusive results, the 16S rDNA sequences of these isolates were determined and found to resemble that of the type strain of Leptospira inadai (L. inadai), serovar lyme strain 10, which is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences from databases revealed a L. inadai-specific signature sequence, against which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid thermal cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous monitoring of fluorescence emission were accomplished by the LightCycler; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity, as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. inadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these experiments showed that the PCR protocol was robust because target DNA of different conditions, which were extracted by either 1 of the 4 methods used, could be detected.

Detection and characterization of feline Bartonella henselae in the Czech Republic.

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats (n=6) and one from a shelter cat without any ectoparasites (n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene (gltA) and DNA-DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S-23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S-23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping (PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.

What's hot in animal biosafety?

In recent years, the emergence or re-emergence of critical issues in infectious disease and public health has presented new challenges and opportunities for laboratory animal care professionals. The re-emergence of bioterrorism as a threat activity of individuals or small groups has caused a heightened awareness of biosecurity and improved biosafety. The need for animal work involving high-risk or high-consequence pathogens and for arthropod-borne diseases has stimulated renewed interest in animal biosafety matters, particularly for work in containment. Application of these principles to animals retained in outdoor environments has been a consequence of disease eradication programs. The anticipated global eradication of wild poliovirus has prompted the promulgation of new biosafety guidelines for future laboratory and animal work. Increased concern regarding the use of biologically derived toxins and hazardous chemicals has stimulated a new categorization of facility containment based on risk assessment. Recognition that prion disease agents and other high-consequence pathogens require safe handling and thorough destruction during terminal decontamination treatment has led to the development of new biosafety guidelines and technologies. The implementation of these guidelines and technologies will promote state-of-the-art research while minimizing risk to laboratory animals, researchers, and the environment.

Fatal mycotic dermatitis in captive brown tree snakes (Boiga irregularis).

Cutaneous fungal infections occurred in four captive brown tree snakes (Boiga irregularis). The ventral scales were most commonly affected, and lesions began as areas of erythema and edema with vesicle formation, followed by development of caseous brown plaques. Lesions usually started where ventral scales overlapped and spread rapidly. All snakes died within 14 days after clinical signs were first noted. The deaths of three of the snakes were directly attributable to the cutaneous disease; the other snake died from renal failure and visceral gout, most likely induced by gentamicin therapy. Histologically, lesions consisted of epidermal hyperplasia and hyperkeratosis, with foci of epidermal necrosis, intraepidermal vesicle formation, and subacute inflammation of the underlying dermis. These lesions were associated with bacteria and numerous septate, branched fungal hyphae within the epidermis and overlying serocelluar crusts. Hyphae that penetrated through the superficial surface of the epidermis often formed terminal arthroconidia. The same species of fungus was isolated in pure culture from the skin of three snakes, but fungal cultures were not performed on samples from the fourth snake. The fungus has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii based on its formation of solitary dermatophytelike aleurioconidia and alternate and fission arthroconidia. The source of the fungus in this outbreak was not determined; however, the warm, moist conditions under which the snakes were housed likely predisposed them to opportunistic cutaneous fungal infections.

Bactericidal activity of antibiotics against Legionella micdadei (Pittsburgh pneumonia agent).

The bactericidal activity of five antibiotics for Legionella micdadei was determined by the construction of time-kill curves. Erythromycin, rifampin, penicillin G, cephalothin, and gentamicin were bactericidal for L. micdadei at readily achievable concentrations. The minimal bactericidal concentrations, defined as those producing 99.9% killing within 24 h, were: erythromycin, 4.6; rifampin, 0.13; penicillin G, 0.25; cephalothin, 2.5; and gentamicin, 0.25 micrograms/ml. The ratios of the minimal bactericidal to minimal inhibitory concentrations for these antibiotics ranged from 1 to 8. Thus, the poor in vivo activity of beta-lactam and aminoglycoside antibiotics against L. micdadei cannot be ascribed to a lack of killing by these agents.

Cellular fatty acid composition of Lautropia mirabilis.

Ten strains of Lautropia mirabilis (ATCC 51599(T) and nine phenotypically similar clinical isolates) were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by gas-liquid chromatography. CFA profiles were generated by using a commericial software package (MIDI, Newark, Del.). All strains tested had an identical CFA profile characterized by major amounts of 16:1omega7c (41%) and 16:0 (44%); smaller amounts (1 to 4%) of 3-OH-10:0, 12:0, 14:0, 15:0, and 18:1 omega7c; trace amounts (<1%) of 10:0, 18:2 and 18:0; and no cyclopropane acids. This profile was similar to the CFA profiles of Acidovorax delafieldii, Comamonas terrigena, and strains of an unclassified Centers for Disease Control group designated weak oxidizer group 1. CFA analysis, when supplemented by phenotypic characterization, is useful for the identification of L. mirabilis isolates.

Destruction of human microvascular endothelial cell capillary-like microtubules by Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius.

When grown in the presence of Matrigel, monolayers of an immortalized human microvascular cell line (HMEC-1) form capillary-like microtubule networks. Previous work, using HMEC-1 monolayers, demonstrated a significant difference in in vitro cytotoxicity between Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (HAE) strains and non-BPF-associated HAE strains. The present study demonstrates that BPF-related cytotoxic differences can also be observed in HMEC-1 microtubule networks. At a multiplicity of infection (MOI) of 2 x 10(-2) bacteria/tissue culture cell, BPF-associated strain F3031 disrupted the microtubule network, producing random clumps of rounded cells at 48 h of incubation. Infection with non-BPF-associated strain F1947 at the same MOI produced no observable microtubule disruption. The ability of HMEC-1 microtubule model to differentiate virulent and avirulent HAE in vitro will further aid in the study of BPF pathogenesis. In addition, the fact that the HMEC-1 cells can be induced to form microtubules make it an excellent model system for the general study of many of the agents of vascular purpura.

Intracellular multiplication and toxic destruction of cultured macrophages by Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a gram-negative rod that causes opportunistic infections resulting in bacteremia, septicemia, meningitis, and death in immunocompromised, splenectomized, and alcoholic individuals. Infections caused by a related species, Capnocytophaga cynodegmi, remain localized at the site of the wound where the organism is introduced. Both organisms are part of the normal canine oral flora and are introduced through puncture wounds via dog bites. We found that both C. canimorsus and C. cynodegmi attach, are phagocytized, and multiply intracellularly in J774 mouse macrophage cells. After 48 h of infection by C. canimorsus, large sections of the macrophage cell layer were observed to detach and lyse, while the monolayer infected with C. cynodegmi demonstrated no cytotoxic effects. Tissue culture supernatants from the C. canimorsus-infected J774 cells filtered through a 0.22-micron-pore membrane produced a similar effect on fresh monolayers, while filtrates from C. cynodegmi and uninfected controls produced no effect. No endotoxin release was observed in these supernatants. We conclude that the cytotoxic phenotype of C. canimorsus is the likely result of a toxin produced by this organism.

Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF.

Fulminating bacteremia and pneumonia due to Bacillus cereus.

We present two cases of rapidly progressing, fatal pneumonia caused by Bacillus cereus. These cases are interesting in that B. cereus, even from blood or sputum specimens, may often be considered a contaminant and receive inadequate attention. Also of interest was the fact that the two patients resided in the same area of the state, were welders by trade, and became ill within a few days of each other, yet there was no epidemiologic link between them.

Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies.

DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).

Characterization of Neisseria elongata subsp. glycolytica isolates obtained from human wound specimens and blood cultures.

Four slightly yellow-pigmented, alpha-hemolytic, gram-negative coccobacilli, three from wound specimens and one from multiple blood cultures of a patient with endocarditis, were identified as Neisseria elongata subsp. glycolytica on the basis of their overall biochemical and genetic similarities to this subspecies. These strains resembled N. elongata in their guanine-plus-cytosine contents (55.6 to 57.1 mol%) and in their overall cellular fatty acid profiles, which are characterized by large amounts of 16:0, 16:1 omega 7c, and 18:1 omega 7c fatty acids. Their identities were confirmed by species-level DNA relatedness (hydroxyapatite method) to the type strains of all three N. elongata subspecies. The biochemical profiles and cultural characteristics of these strains resembled those of the type strain of N. elongata subsp. glycolytica except for the production of a weak yellow growth pigment and alpha-hemolysis on sheep blood agar. They differed from N elongata subsp. elongata by the production of catalase, by the production of alpha-hemolysis on sheep blood agar, and by acid production from D-glucose. They differed from N. elongata subsp. nitroreducens by the production of catalase and an inability to reduce nitrate. These studies suggest a pathogenic potential for N. elongata subsp. glycolytica, usually considered to be a transient colonizer in humans.

Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3.

Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysis of cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains of Brevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens. Twenty-two strains were shown to be B. casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus. The remaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacterium. They were not further identified, but they were biochemically distinguishable from B. casei. Eleven of the clinical strains of B. casei were isolated from blood, and two each were isolated from cerebrospinal fluid and from pleural fluid. At least five isolates were from multiple blood or cerebrospinal fluid cultures. To our knowledge, these strains are the first described clinical isolates identified as B. casei, which was previously considered to be a nonpathogenic species.