High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.

Culicoides variipennis (Diptera:Ceratopogonidae) host selection in Colorado.

Drop traps containing either steer or ewe bait were used to investigate host selection and crepuscular period host seeking activity of Culicoides spp. at 2 farms in Colorado. Of the 1,103 Culicoides spp. collected, 755 were Culicoides variipennis (Coquillett). Of these, 86% were collected from steers and 14% from ewes. The engorgement rate of C. variipennis was 31% on steers and 6% on ewes. Culicoides crepuscularis Malloch was the 2nd most abundant species. Of the 253 collected, 39% were from the steers, and 61% were from ewes: The engorgement rate of the C. crepuscularis collected was 71% on ewes and 34% on steers. Thus, C. variipennis preferentially selected and fed upon cattle, whereas C. crepuscularis preferentially selected and fed upon sheep.

Characterization of two unusual clinically significant Francisella strains.

We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F. tularensis biovar novicida, and F. philomiragia. Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar. Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase. They were additionally shown to belong to the species F. tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene. Also, RNA probe hybridization confirmed their belonging to the species F. tularensis. However, the new strains, which are not identical, are distinguished from other F. tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests. Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative. Commercial F. tularensis antiserum agglutinated stock F. tularensis strains but not Fx1, Fx2, F. tularensis biovar novicida, or F. philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate. Fx1 and Fx2 produced beta-lactamase. Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used.

Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat.

We wished to develop a cost-effective, rapid strategy to detect and identify Bartonella species in the clinical laboratory and to determine the prevalence of Bartonella infection in the Houston veteran population. Bartonella colonies were identified by colony morphology, Gram stain, RapID ANA, repetitive extragenic palindromic-PCR (REP-PCR) and whole-cell fatty acid (CFA) analysis, and these methods were compared for their usefulness. A new test order for "Rochalimaea culture" (the genus Bartonella was previously known as the genus Rochalimaea) was instituted, and in addition, all blood specimens submitted for fungal culture (obtained in an isolator tube) were processed for Bartonella culture. Over a 16-month period we isolated Bartonella henselae from only 0.4% (2 of 533) of total cultures but from 1% (2 of 204) of human immunodeficiency virus-positive patients. After sufficient growth, identification of the Bartonella isolates to the species level could be obtained in 2 days. The REP-PCR allowed discrimination of all known species, whereas CFA analysis distinguished all except B. henselae and Bartonella quintana. The RapID ANA results failed to differentiate between B. henselae and B. quintana, and results for other species differed by only one or two tests. Blood obtained from a kitten which had been introduced into the household of one patient 2 months before the onset of fever yielded a Bartonella strain which was shown to be different from the strain from the patient and distinct from other Bartonella species by a combination of REP-PCR, CFA, and growth characteristics. Subsequent analysis of the citrate synthase gene sequence showed only an 86% similarity with any of the other known Bartonella species, suggesting that this isolate represents a distinct, previously uncharacterized species of Bartonella.

Polymerase chain reaction approaches to Culicoides (Diptera: Ceratopogonidae) identification.

Molecular genetic approaches such as polymerase chain amplification (PCR) of target genomic sequences are finding wide application in systematic and taxonomic studies of arthropods. PCR-based techniques that preclude the need for target DNA sequence information of the species of interest facilitate molecular taxonomic studies. Two such techniques, tDNA-PCR (DNA encoding tRNAs is the analyte) and RAPD-PCR (randomly amplified polymorphic DNA) were investigated for their ability to differentiate certain North American Culicoides spp. larvae and adults.

[Acute tonsillitis: clinical symptoms; bacteriologic culture and rapid test as deciding criteria for the use of antibiotics]. / Akute Tonsillitis: Klinische Symptomatik; bakteriologische Kultur und Schnelltest als Entscheidungskriterien für Antibiotikaeinsatz.

In order to prevent late sequelae from an untreated streptococcal pharyngitis all patients with streptococcal tonsillitis/pharyngitis have to be treated with antibiotics, preferably penicillin. A correct diagnosis by clinical criteria is only achievable in 72% of patients with streptococcal pharyngitis. Additional criteria such as diagnosis by culture are, therefore, mandatory. Time is a major disadvantage of traditional culture methods. Slide agglutination tests show an acceptable sensitivity of 92% and a specificity of 93%. Due to the possibility of false negative test results, with consequent withholding of adequate antimicrobial chemotherapy, these test results should be used only as a valuable guide. Decision to administer an antibiotic should still be based on clinical criteria.