RESUMEN
In the United States, few intramammary antimicrobials exist that are approved for treatment of bovine mastitis; thus, ensuring judicious use of these products is a priority. The objectives of this study were to determine phenotypic susceptibility and presence of selected antimicrobial resistance genes from staphylococci, streptococci, and streptococcal-like organisms recovered from cases of clinical mastitis occurring in cows on large Wisconsin farms. Staphylococcus aureus (n=35 from 19 herds), coagulase-negative staphylococci (n=51 from 30 herds), Streptococcus spp. (n=78 from 36 herds), and streptococcal-like organisms (n=31 from 19 herds) were used in this study. All Staphylococcus spp. were susceptible to ceftiofur, cephalothin, and the combination of penicillin and novobiocin. Of all staphylococci, only a single Staphylococcus epidermidis exhibited phenotypic resistance to oxacillin. Phenotypic susceptibility to erythromycin was observed in only 8.6 and 15.7% of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Approximately 20% of staphylococci and 13 to 22% of streptococci and streptococcal-like organisms exhibited phenotypic resistance to pirlimycin. All Streptococcus spp. exhibited phenotypic susceptibility to ceftiofur, cephalothin, and oxacillin. The proportion of isolates exhibiting phenotypic susceptibility to pirlimycin and sulfadimethoxine differed among Streptococcus dysgalactiae and Streptococcus uberis. All streptococcal-like organisms exhibited phenotypic susceptibility to ceftiofur, cephalothin, oxacillin, penicillin, and the combination of penicillin and novobiocin. Of all organisms tested, 36.9% did not carry any of the resistance genes (ermC, blaZ, tetK, or tetM), 35.4% carried 1 gene, and 27.7% carried multiple genes (usually blaZ in combination with a tet gene). Eighteen (51.4%) Staph. aureus and 12 (48.0%) Staphylococcus chromogenes carried multiple resistance genes. Six (12.2%) Strep. dysgalactiae and no Strep. uberis carried multiple resistance genes. Results indicate that most gram-positive mastitis organisms were susceptible to most antimicrobials used for intramammary administration, but some resistance to drugs used for systemic treatment of mastitis was noted. The presence of selected resistance genes was not proportional to the occurrence of phenotypic resistance.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/genética , Mastitis Bovina/microbiología , Pruebas de Sensibilidad Microbiana/veterinaria , Animales , Antibacterianos/uso terapéutico , Antiinfecciosos/uso terapéutico , Bovinos , Industria Lechera , Femenino , Mastitis Bovina/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/veterinaria , Staphylococcus/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/veterinaria , Streptococcus/efectos de los fármacos , WisconsinRESUMEN
This study compared the fatty-acid profiles of Brucella canis blood culture isolates obtained from infected dogs in the UK, Germany, Japan, South Africa, Peru, Mexico, Colombia, and Argentina, and from a human clinical case in Argentina, to a bank of isolates obtained from canine outbreaks in the USA. Analysis of a total of 42 B. canis isolates and one reference strain found a marked variation within the species. Fatty-acid analysis showed that only the isolates from Argentina, Colombia, and Mexico, which included the human B. canis isolate, contained a specific fatty acid, 19:0 cyclopropane (lactobacillic acid), w8c (cis-11,12-methylene octadecanoic acid), and that this fatty acid, when present, made up a large percentage of overall fatty-acid content. Prior to this study, the cellular fatty-acid 19:0 cyclopropane had been identified in all of the species of Brucella considered to be pathogenic to humans (B. abortus, B. melitensis, B. suis) except for B. canis. Discovering that this fatty acid not only occurs in B. canis, but also that it is only present in some strains of the species provides a new focus for investigations aimed at identifying the cause of reported geographical variability in human B. canis infection, and at finding predictors of biological behaviour and human pathogenicity within this Brucella species.
Asunto(s)
Brucella canis/química , Brucella/clasificación , Brucelosis/microbiología , Ácidos Grasos , Animales , Brucella/química , Brucelosis/veterinaria , Cromatografía de Gases , Perros , Mapeo Geográfico , Alemania , Humanos , Japón , México , Sudáfrica , América del Sur , Especificidad de la Especie , Reino Unido , Estados UnidosRESUMEN
Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of S. suis type 2 and used to screen an S. suis gene library in an effort to identify protective antigen(s) and antigens of diagnostic importance. A clone that produced a 45-kDa S. suis-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 45-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size as the recombinant protein in S. suis strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally infected with a virulent strain of S. suis type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect S. suis infection. Activity staining showed that the S. suis GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha-ketoglutarate as the substrate. Highly virulent strains of S. suis type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the S. suis GDH protein is exposed at the surface of intact cells.
Asunto(s)
Glutamato Deshidrogenasa/genética , Infecciones Estreptocócicas/diagnóstico , Streptococcus suis/enzimología , Animales , Anticuerpos Antibacterianos/análisis , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Regulación Bacteriana de la Expresión Génica , Glutamato Deshidrogenasa/inmunología , Glutamato Deshidrogenasa/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Infecciones Estreptocócicas/veterinaria , Streptococcus suis/inmunología , Streptococcus suis/ultraestructura , PorcinosRESUMEN
Canine otitis externa and cutaneous pyoderma are common problems that are often associated with Staphylococcus intermedius, Pseudomonas aeruginosa, and Malassezia pachydermatis. In vitro activity of two topical products against these organisms isolated from canine referral patients were evaluated. Organisms were grown and diluted to a concentration equivalent to 10(7) colony-forming units (CFU) per mL and exposed to either a 0 or 1/5 dilution of Hexadene Flush with Spherulites (Virbac Animal Health Inc, Fort Worth, TX) or a 1/5 or 1/25 dilution of ResiCHLOR Lotion with Spherulites (Virbac Animal Health Inc, Fort Worth, TX) at time intervals from 1 to 30 minutes. Results showed that all three organisms were killed within 1 minute of contact time at 0 and 1/5 dilution of the flush. The lotion diluted to 1/5 also killed all three organisms. At 1/25 dilution, this lotion killed S. intermedius and P. aeruginosa within 1 minute of contact time, whereas M. pachydermatis was killed after 1 minute. The findings suggest that the two topical products exhibit efficacy against these common skin pathogens in vitro and can be useful in their clinical management.
Asunto(s)
Antiinfecciosos/farmacología , Clorhexidina/farmacología , Enfermedades de los Perros/microbiología , Otitis Externa/veterinaria , Piodermia/veterinaria , Animales , Antiinfecciosos/administración & dosificación , Química Farmacéutica , Clorhexidina/administración & dosificación , Perros , Malassezia/efectos de los fármacos , Malassezia/aislamiento & purificación , Otitis Externa/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Piodermia/microbiología , Streptococcus intermedius/efectos de los fármacos , Streptococcus intermedius/aislamiento & purificaciónRESUMEN
A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.
Asunto(s)
Genes Bacterianos , Proteínas Hemolisinas/genética , Infecciones Estreptocócicas/microbiología , Streptococcus suis/genética , Animales , Secuencia de Bases , Southern Blotting , Western Blotting , Sondas de ADN , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Compuestos Orgánicos , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Análisis de Secuencia de ADN , Streptococcus suis/clasificación , Streptococcus suis/metabolismo , Streptococcus suis/patogenicidad , VirulenciaRESUMEN
Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.
Asunto(s)
Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/metabolismo , Animales , Anticuerpos Monoclonales/análisis , Antígenos Virales/análisis , Antígenos Virales/inmunología , Bovinos , Línea Celular , Regulación Viral de la Expresión Génica , Vectores Genéticos/metabolismo , Herpesvirus Bovino 1/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Spodoptera/citología , Spodoptera/genética , Spodoptera/virología , Fracciones Subcelulares/química , Fracciones Subcelulares/virología , Proteínas Virales/genéticaRESUMEN
Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal. Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter. F. necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples. Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli. EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison. Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI. The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples. None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates. The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F. necrophorum isolates of liver abscesses originate from the rumen.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Infecciones por Fusobacterium/veterinaria , Fusobacterium/genética , Absceso Hepático/veterinaria , Rumen/microbiología , Alimentación Animal/efectos adversos , Animales , Técnicas de Tipificación Bacteriana , Bovinos , Enfermedades de los Bovinos/etiología , Fusobacterium/clasificación , Fusobacterium/aislamiento & purificación , Infecciones por Fusobacterium/etiología , Infecciones por Fusobacterium/microbiología , Genes Bacterianos , Absceso Hepático/etiología , Absceso Hepático/microbiología , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genéticaRESUMEN
The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP). The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies. The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples. The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens. BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen. Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera. The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization. In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/biosíntesis , Diarrea Mucosa Bovina Viral/diagnóstico , Pestivirus/genética , Proteínas Virales/biosíntesis , Animales , Anticuerpos Monoclonales , Diarrea Mucosa Bovina Viral/inmunología , Bovinos , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Técnica del Anticuerpo Fluorescente Indirecta , Genes Virales , Pruebas de Neutralización , Radioinmunoensayo , Proteínas Recombinantes/biosíntesis , Spodoptera , TransfecciónRESUMEN
Steptococcus suis is a Gram-positive, facultatively anaerobic coccus that has been implicated as the cause of a wide range of clinical disease syndromes in swine and other domestic animals. In swine, the disease has spread worldwide but is more prevalent in countries with intensive swine management practices. The disease syndromes caused by S. suis in swine include arthritis, meningitis, pneumonia, septicaemia, endocarditis, polyserositis, abortions and abscesses. S. suis has also been implicated in disease in humans, especially among abattoir workers and swine and pork handlers. In humans, S. suis type 2 can cause meningitis, which may result in permanent hearing loss, septicaemia, endocarditis and death. The pathogenic mechanism of S. suis is not well defined. Several virulence factors have been identified, but their roles in pathogenesis and disease have not been well elucidated. Much work is in progress on characterization of virulence factors and mechanisms, with emphasis on the control of the disease. Because of the non-availability of suitable immunoprophylaxis, control of S. suis infection has depended mainly on the use of antimicrobials.
Asunto(s)
Infecciones Estreptocócicas/veterinaria , Streptococcus suis , Enfermedades de los Porcinos , Aborto Veterinario/microbiología , Animales , Antibacterianos/uso terapéutico , Profilaxis Antibiótica/veterinaria , Femenino , Embarazo , Infecciones Estreptocócicas/fisiopatología , Infecciones Estreptocócicas/prevención & control , Streptococcus suis/patogenicidad , Porcinos , Síndrome , VirulenciaRESUMEN
Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.
Asunto(s)
Técnicas de Tipificación Bacteriana , Enfermedades de los Bovinos/microbiología , Fusobacterium necrophorum/clasificación , Fusobacterium necrophorum/genética , Absceso Hepático/veterinaria , Rumen/microbiología , Animales , Bovinos , Sondas de ADN , Enzimas de Restricción del ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Fusobacterium necrophorum/patogenicidad , Absceso Hepático/microbiología , Ratones , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Especificidad de la Especie , Virulencia/genéticaRESUMEN
Whole-cell chromosomal digests of 54 isolates of Streptococcus suis encompassing all known serotypes from a geographically varied collection were examined by PstI restriction fragment length polymorphisms and then hybridized with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli MRE600. The hybridization patterns showed genetic heterogeneity within and between S. suis serotypes. Most isolates (87%) representing 28 serotypes contained a common band at approximately 1.8 kb. However, 13% of the isolates representing seven serotypes lacked the 1.8-kb band, indicating that the species as currently defined is diverse. Nonetheless, the 1.8-kb band may be a useful genotypic marker for identification of most S. suis isolates. We tested the ability of this technique to discriminate between virulent and avirulent S. suis type 2 isolates. A virulent strain of S. suis type 2 could be distinguished from avirulent strains by the presence of specific bands. No correlation was obvious between band pattern and hemolysin production.
Asunto(s)
Polimorfismo de Longitud del Fragmento de Restricción , ARN Bacteriano/genética , ARN Ribosómico/genética , Streptococcus suis/genética , Animales , ADN Bacteriano/genética , ADN Complementario , Marcadores Genéticos , Variación Genética , Humanos , Serotipificación , Streptococcus suis/clasificación , Streptococcus suis/aislamiento & purificación , Porcinos/microbiologíaRESUMEN
A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.
Asunto(s)
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico , Técnicas Bacteriológicas , Secuencia de Bases , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Estudios de Evaluación como Asunto , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Tuberculosis Pulmonar/microbiologíaRESUMEN
A Listeria monocytogenes-specific, acridinium-ester-labelled DNA probe was evaluated in a chemiluminescent homogeneous protection assay (HPA) for the rapid confirmation of suspect L. monocytogenes colonies from blood agar plates. The HPA uses an acridinium-ester-labelled chemiluminescent DNA probe in a free-solution hybridization format. After the DNA probe hybridized with the target ribosomal RNA, the acridinium label on the unhybridized probe was inactivated by a chemical differential hydrolysis step. Formation of a hybrid between probe and target was detected in a luminometer after the addition of a detection reagent. The assay can be completed in 30 to 45 min and allows for simultaneous processing of several (50-100) samples. The probe showed 100% sensitivity and 100% specificity for L. monocytogenes when evaluated in the HPA against L. monocytogenes, other Listeria species and other Gram-positive bacteria. The lower detection limit of the HPA was between 10(4) and 10(5) cells. In an evaluation with 296 bacterial colonies isolated from food, the HPA colony confirmation showed 100% agreement with conventional biochemical characterization. HPA will be useful for the rapid confirmation of L. monocytogenes isolated from food and clinical specimens.