Identification of FAP locus genes from chromosome 5q21.

Recent studies suggest that one or more genes on chromosome 5q21 are important for the development of colorectal cancers, particularly those associated with familial adenomatous polyposis (FAP). To facilitate the identification of genes from this locus, a portion of the region that is tightly linked to FAP was cloned. Six contiguous stretches of sequence (contigs) containing approximately 5.5 Mb of DNA were isolated. Subclones from these contigs were used to identify and position six genes, all of which were expressed in normal colonic mucosa. Two of these genes (APC and MCC) are likely to contribute to colorectal tumorigenesis. The MCC gene had previously been identified by virtue of its mutation in human colorectal tumors. The APC gene was identified in a contig initiated from the MCC gene and was found to encode an unusually large protein. These two closely spaced genes encode proteins predicted to contain coiled-coil regions. Both genes were also expressed in a wide variety of tissues. Further studies of MCC and APC and their potential interaction should prove useful for understanding colorectal neoplasia.

Direct dideoxy DNA sequencing.

Advances in DNA sequencing techniques have made it possible to routinely sequence long DNA molecules (1,2). Recently double-stranded DNA has been directly sequenced with chain terminators (3), and this eliminates the tedious task of subcloning the fragments into single-stranded M13 vectors. The procedure requires a plasmid DNA template that is denatured to separate the DNA strands and a primer complementary to one of the strands of the plasmid vector adjacent to the cloning site that has been used. Thus by using primers complementary to each strand, the sequence of the insert can be read from both ends. In the presence of the four deoxynucleoside triphosphates, one of which is radioactively labeled, and the Klenow fragment of Escherichia coli DNA polymerase I (or reverse transcriptase, as described in this chapter), primed synthesis occurs across the region of interest. In the presence of competing dideoxynucleoside triphosphates (ddNTPs), specific termination occurs at each of the four different nucleotides, and if this is carried out separately with each of the ddNTPs, then the four sets of reaction products can be analyzed on DNA sequencing gels.

Isolation of high molecular weight DNA suitable for the construction of genomic libraries.

Recent advances in molecular biology have made it possible to construct complete gene libraries for any organism that uses DNA as its carrier of genetic information. A gene library should contain a large number of cloned DNA fragments that in total contain the entire donor genome. The construction of a genomic library first requires the isolation of DNA from the donor organism. To be of maximum use in the construction of genomic libraries, DNA isolated from the donor organism should fulfill the following criteria. First, the DNA must represent all sequences in the genome to be cloned. Second, it must be of high molecular weight. Third, no contaminants must taint the DNA so that its use as a substrate for restriction endonucleases and other enzymes used in genetic engineering is uninhibited.

Effect of sustained release and pulse release anthelmintic intraruminal devices on development of pathophysiological changes and parasite populations in calves infected with Ostertagia ostertagi and Cooperia oncophora.

An experiment was conducted in calves to investigate the effect of sustained release and pulse release anthelmintic intraruminal boli on the development of pathophysiological changes following daily infection with Ostertagia ostertagi and Cooperia oncophora for six weeks. After infection various pathophysiological changes were detected including increases in serum pepsinogen concentration, enteric plasma protein losses and in the catabolic rate of albumin. Such changes developed rapidly in the unprotected calves following patency after 17 days and persisted until the termination of the study. There were indications that the sustained anthelmintic release device was more efficacious than the pulse anthelmintic release device in reducing the worm burdens and early pathophysiological changes associated with infection. It was found at necropsy that the release of anthelmintic by the oxfendazole pulse release bolus had been delayed in several calves.

The influence of dietary protein on live bodyweight, degree of anaemia and erythropoietic responses of Scottish blackface sheep infected experimentally with Trypanosoma congolense.

The present study investigated the influence of dietary protein on the intensity of parasitaemia, degree of anaemia and erythropoietic responses, in sheep experimentally infected with Trypanosoma congolense and given either a high protein diet (116 g digestible crude protein [DCP] per day) or a low protein diet (51.5 g DCP per day). It was observed that infected and control animals on the high protein diet grew at similar rates while infected animals on the low protein diet experienced marked retardation of growth compared with their uninfected controls. Dietary protein had no influence on the degree of anaemia that followed infection. Measurement of blood volumes revealed that low protein infected group had significantly lower mean circulating red cell volume than their controls. Ferrokinetic measurements indicated that plasma iron turnover rates (PITR) and 59Fe incorporation rates were higher in the high protein infected group than in the low protein infected group, although these differences were not significant. These observations indicate that infected animals on a high protein tended to show greater enhancement of erythropoietic activity that infected animals on low protein diet.

Distribution, abundance, and seasonal activities of ticks collected from rodents and vegetation in South Carolina.

Ixodid ticks were collected from live-trapped rodents and by flagging vegetation at sites in the Piedmont, Sandhills, Coastal Plain, and Coastal Zone of South Carolina from May 1994 through December 1995. A total of 1,514 ticks was recovered from 237 live-trapped rodents. Host-attached species included Ixodes minor Neumann (n = 818), Dermacentor variabilis (Say) (n = 346), Amblyomma maculatum Koch (n = 209), Ixodes affinis Neumann (n = 89), and Ixodes scapularis Say (n = 52). Species of questing adult ticks collected from vegetation were Ix. scapularis (n = 1,627), Amblyomma americanum L. (n = 1,052), D. variabilis (n = 649), A. maculatum (n = 134), Ix. affinis (n = 70), and Dermacentor albipictus (Packard) (n = 3). Geographic distribution and seasonal activities of most stages and species are presented. This report includes the first detailed description of the seasonal activities of all active stages of Ix. minor in the United States, and documents that this tick is well established in the southern Coastal Zone of South Carolina.

An epidemiologic and entomologic investigation of a cluster of Rocky Mountain spotted fever cases in Delaware.

BACKGROUND: Rocky Mountain spotted fever (RMSF) continues to be the most common fatal tick-borne illness in the United States. In August of 1996, four children attending a summer camp in Delaware were diagnosed with RMSF. This report summarizes the results of the epidemiologic and entomologic investigation conducted by the Delaware Division of Public Health and the Centers for Disease Control and Prevention regarding this cluster of RMSF cases. Epidemiologic and clinical aspects of RMSF, as well as previously reported clusters of the disease, are also reviewed. METHODS: A questionnaire regarding symptoms and activities was administered via telephone to 163 (73 percent) of the 223 attendees. A suspected case was defined as an illness in a person attending the camp between August 11 and 17 that occurred during the two-week period following the session, characterized by either 1) fever with one or more symptoms (i.e., headache, rash, myalgia, or fatigue) or 2) no fever with two or more symptoms. Cases of RMSF were confirmed by serologic evaluation. RESULTS: Seven of 13 patients with suspected RMSF submitted sera for testing. Four patients had confirmed RMSF; three were males, and the median age was 12.5 years compared with 12 years for all attendees. All confirmed patients reported fever, headache, fatigue, and rash. An increased risk of becoming ill was associated with overnight camping at site A (Odds Ratio (OR) undefined, p = 0.02), visiting or overnight camping at site B (OR undefined, p = 0.003 and 0.002), and leaving the trails when hiking (OR undefined, p = 0.02). CONCLUSIONS: These data suggest that development of RMSF was associated with visiting or camping at specific sites and behavior likely to increase contact with ticks. Camp supervisors were advised to educate campers regarding tick bite prevention measures, reduce underbrush around campsites, and encourage campers to remain on the trails. Health care providers should remain aware of the increased risk for RMSF during the spring, summer, and fall months.

Epidemic typhus meningitis in the southwestern United States.

A patient residing in New Mexico had murine typhus diagnosed. A novel molecular assay was performed at the Centers for Disease Control and Prevention, and Rickettsia prowazekii, the agent of epidemic typhus, was found, rather than R. typhi. To our knowledge, this is the first reported case of epidemic typhus confirmed by means of polymerase chain reaction--based testing of cerebrospinal fluid, and it introduces a novel assay for the molecular diagnosis of both epidemic and murine typhus.

A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping.

We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5'-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.

Survival of Ehrlichia chaffeensis in refrigerated, ADSOL-treated RBCs.

BACKGROUND: The purpose of this study was to investigate the persistence of viable Ehrlichia chaffeensis in ADSOL-treated RBCs stored at 4 to 6 degrees C. STUDY DESIGN AND METHODS: The continuous monocytic cell lines THP-1 and DH82 were infected with E. chaffeensis (St. Vincent isolate). Packed RBC units were inoculated in separate experiments with E. chaffeensis-infected cells as final concentrations of 8.02 x 10(4) (DH82) and 1.43 x 10(4) (THP-1) infected cells per mL. Aliquots were stored at 4 to 6 degrees C for 1 to 42 days. At selected intervals, nucleated cells from the RBC aliquots were obtained by using a ficoll-isopaque separation procedure. Uninfected DH82 cell cultures were inoculated with the harvested nucleated cells or supernatant. The cell cultures were evaluated for infection by weekly examination of Wright's (Diff-Quik) stained cytocentrifuged slides. PCR amplification was also used to test the harvested nucleated cells or supernatant for the presence of E. chaffeensis DNA. RESULTS: In both types of infected cell lines, E. chaffeensis was reisolated in DH82 cells for as long as 11 days from the cellular fraction and for up to 5 days from the supernatant fraction. PCR results were positive throughout the 42-day testing period. CONCLUSION: Cell-associated E. chaffeensis remains viable in ADSOL-treated RBCs stored at 4 to 6 degrees C for at least 11 days. These data suggest that transfusion-acquired infection is possible. Successful reisolation was achieved from the supernatant fraction, which suggests that RBC products treated with a WBC-reduction procedure may still present a risk for transfusion transmission. No correlation between PCR positivity and viability of bacteria was noted.

Family cluster of Rocky Mountain spotted fever.

Soon after a patient from Tennessee died of Rocky Mountain spotted fever (RMSF), several family members developed symptoms suggestive of the disease and were treated presumptively for RMSF. Fifty-four persons visiting the index patient's home were interviewed; serum samples were collected from 35. Three additional cases of RMSF were confirmed, all of which occurred in first-degree relatives. Time spent at the family home and going into the surrounding woods were significantly associated with developing antibodies to Rickettsia rickettsii. Ticks were collected and examined for rickettsiae by polymerase chain reaction analysis. Because hyperendemic foci and family clusters of RMSF can occur, when a case is suspected clinicians should be vigilant for signs and symptoms consistent with R. rickettsii infection in other persons who may have been similarly exposed.

Hidden mortality attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease.

Rocky Mountain spotted fever (RMSF) is the most severe tickborne infection in the United States and is a nationally notifiable disease. Since 1981, the annual case-fatality ratio for RMSF has been determined from laboratory-confirmed cases reported to the Centers for Disease Control and Prevention (CDC). Herein, a description is given of patients with fatal, serologically unconfirmed RMSF for whom a diagnosis of RMSF was established by immunohistochemical (IHC) staining of tissues obtained at autopsy. During 1996-1997, acute-phase serum and tissue samples from patients with fatal disease compatible with RMSF were tested at the CDC. As determined by indirect immunofluorescence assay, no patient serum demonstrated IgG or IgM antibodies reactive with Rickettsia rickettsii at a diagnostic titer (i.e., >/=64); however, IHC staining confirmed diagnosis of RMSF in all patients. Polymerase chain reaction validated the IHC findings for 2 patients for whom appropriate samples were available for testing. These findings suggest that dependence on serologic assays and limited use of IHC staining for confirmation of fatal RMSF results in underestimates of mortality and of case-fatality ratios for this disease.