Sequence-specific Polymerase Chain Reaction Research Articles

Molecular typing of Bordetella pertussis isolates recovered from Belgian children and their household members.

Recently, a moderate increase in the prevalence of pertussis, possibly contracted from adults, has been observed among unvaccinated children. During a 3-year period, we prospectively enrolled 93 index patients with a polymerase chain reaction (PCR) and/or culture result positive for Bordetella pertussis. Among 63 household contacts of 28 index patients, PCR and culture for B. pertussis identified 25 B. pertussis-positive persons. Nineteen of 25 B. pertussis-positive household contacts were asymptomatic. Isolates were available from 10 families of both index patients and household contacts for molecular typing by pulsed-field gel electrophoresis (PFGE) and for genotyping of pertactin and pertussis toxin by sequence-specific PCR and sequencing. PFGE demonstrated homogeneity among the isolates recovered from within each family but heterogeneity among the isolates recovered from different families. B. pertussis isolates recovered from index patients and their household contacts were indistinguishable by molecular typing, demonstrating that identical strains can cause full pertussis disease in children and asymptomatic infection in adults and adolescents.

TAP1, TAP2, and HLA-DR2 alleles are predictors of cervical cancer risk☆

Objective The likelihood of developing cervical cancer has been shown to be increased in persons with certain HLA alleles. We evaluated immune response genes in the HLA region of chromosome 6 to see if individual or interactive associations with cervical cancer risk could be identified. Methods Tissue was obtained from 127 women undergoing surgical treatment for cervical cancer. Blood samples were obtained from 175 control subjects. A combination of polymerase chain reaction (PCR), sequence-specific PCR, and DNA sequencing was used to evaluate polymorphic alleles, including HLA class I B7, TNF α, HLA class II DR2, TAP1, and TAP2 genes. Fisher’s exact test and logistic regression modeling were used for statistical analysis. Results A significantly greater proportion of the patients with cervical cancer were found to have the HLA class II DR2 1501 allele ( P = 0.023) and the TAP2 A/B heterozygous pattern of alleles ( P = 0.0006) than were women without cervical cancer. A proportion of patients with cervical cancer significantly smaller than that of the control women had a polymorphism at the -238 position of the TNF promoter and the TAP1 C/C homozygous pattern of alleles. With logistic modeling, the markers that showed consistent association with the occurrence of cervical cancer were TAP2 A/B, HLA-DR2 1501, and TAP1 C/C. Conclusions We demonstrated a significant association between immune response genes and the risk of cervical cancer. Our data create a compelling argument for a gene or a cluster of genes in the HLA region of chromosome 6 that regulates host immune responses to human papillomavirus infection in a manner that results in inherited susceptibility or resistance to the transforming properties of oncogenic papillomaviruses.

Molecular detection and characterization of Haemobartonella felis in domestic cats in Japan employing sequence-specific polymerase chain reaction (SS-PCR).

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.

MICA4/HLA-DRB1*04/TNF1 haplotype is associated with mixed connective tissue disease in Swedish patients

In order to investigate major histocompatibility complex (MHC) class I chain-related gene A (MICA), tumor necrosis factor (TNFa), −308TNFA, and human leukocyte antigen (HLA-DR/DQ) polymorphisms in mixed connective tissue disease (MCTD), we analyzed 24 patients and 229 healthy controls from Sweden. MICA and TNFa typing was performed by polymerase chain reaction (PCR) and genotyping. HLA-DR and -DQ were genotyped using PCR–sequence specific primers (PCR-SSP) and PCR–sequence-specific oligonucleotide probe (PCR-SSOP), respectively. For analysis of −308TNFA polymorphisms we performed PCR with restriction endonuclease enzymes. We found that the MICA5.1-5.1 genotype was positively associated with MCTD. Shared epitope genes (DRB1*01 and DRB1*04) were also significantly positively associated with MCTD. Polymorphism of −308TNFA was not differently distributed in MCTD patients compared with controls. Furthermore, we demonstrated that frequencies of three estimated haplotypes were increased in MCTD patients compared with controls. Interestingly, the haplotype with MICA allele 4 together with DRB1*04 and TNF1 alleles gives the most specific pattern for MCTD patients compared with controls. Our study demonstrates a clear contribution of HLA loci in susceptibility to MCTD in the Swedish population. Susceptibility to MCTD may be linked to the MICA4/HLA-DRB1*04/TNF1 haplotype and MICA 5.1-5.1 genotype. Mixed connective tissue disease was also associated with shared epitope genes, which in RA has been associated with a more severe disease. Whether these genotypes affect the clinical phenotype of MCTD needs to be determined.

RHCE-D-CE hybrid genes can cause false-negative DNA typing of the Rh e antigen.

DNA typing of the human Rh blood groups generally shows good agreement with serologically defined phenotypes. However, in the present report we describe four individuals who were declared Rh e negative by genotyping although they express the Rh e antigen. Serotyping was performed using mono- and polyclonal Rh antisera. Fluorescent multiplex sequence-specific polymerase chain reactions (PCR-SSPs) identified RHD exons and the polymorphisms usually associated with the Rh E/e or Rh C/c/C(W) antigens. Additional PCR amplification reactions, which were carried out to reveal RHCE-D-CE hybrid genes, analysed exon 5 of the RH genes, the location of the polymorphism (676C-->G) coding for the Rh E and Rh e antigens. Four individuals were identified who expressed Rh e antigens but were negative by PCR-SSP typing for common Rhe-coding sequences. In one family analysed in detail, an RHCE-D5-CE hybrid gene associated with Rh e antigen expression was identified. A concomitant RHcE allele accounted for a seemingly regular typing pattern by conventional RH PCR. The presence of RHCE-D5-CE hybrid alleles may cause false-negative DNA-typing results for the Rh e antigen that are easily overlooked unless appropriate RH hybrid PCR-SSPs are incorporated into conventional DNA-typing protocols. These and previous data strongly caution against an uncritical interpretation of RH DNA-typing results.

Polymorphisms of estrogen receptor α gene in endometrial cancer

It is hypothesized that polymorphisms of estrogen receptor-α (ERα) gene are involved in endometrial cancer. To test this hypothesis, the genotype distributions of six different loci (codon 10 T → C, codon 87 G → C, codon 243 C → T, codon 325 C → G, codon 594 G → A, and intron 1 C → G) of the ERα gene were investigated and their association with endometrial cancer was determined. The DNA from 113 cases of human endometrial cancer was analyzed by sequence-specific polymerase chain reaction. The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The frequency of variant genotype on codon 10 was significantly lower in endometrial cancer patients as compared to controls. Nine of 113 endometrial cancer patients (8.0%) showed genotype 10C/C compared to 27 of 200 healthy controls (13.5%). The relative risk of genotype 10C/C was calculated as 0.44, compared to wild-type. Forty-five of 113 endometrial cancer patients (39.8%) showed genotype T/C on codon 10 compared to 111 of 200 healthy controls (55.5%). The relative risk of genotype 10T/C was calculated as 0.67, compared to wild-type. The polymorphism on codon 87 was not detected both in endometrial cancer patients and in healthy control. Other loci, intron 1, and codons 243, 325, and 594, did not show a correlation with endometrial cancer. The frequency of alleles on codon 10 was also significantly lower in endometrial cancer patients as compared to controls. Sixty-three of 226 alleles (27.9%) of endometrial cancer patients showed allele C compared to 165 of 400 (41.2%) of healthy controls. The relative risk of allele 10C was calculated as 0.67, compared to wild-type. Other loci, intron 1, and codons 243, 325, and 594, did not show a difference between cancer patients and controls. All genotype and allelic distributions were in accordance with the Hardy–Weinberg equilibrium. The present study demonstrates for the first time a protective effect of 10C allele against endometrial cancer. Thus, inherited alterations in ERα may be associated with changes in estrogen metabolism and thereby may possibly explain inter-individual differences in disease incidences of endometrial cancer.

Single nucleotide polymorphisms of estrogen receptor [formula omitted] in human renal cell carcinoma

Estrogens and their receptors (ERs) have been shown to play a role in various cancers. We hypothesize that polymorphisms and genotypic changes of the ERα gene are involved in renal cell carcinoma. To test this hypothesis, DNA samples from 113 cases of human renal cell cancer were analyzed by sequence-specific polymerase chain reaction to determine the genotypic frequency of six different polymorphic loci on ERα gene (codon 10 T→C, codon 87 G→C, codon 243 C→T, codon 325 C→G, codon 594 G→A, and intron 1 C→T). The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The results of this study demonstrate that the distribution of genotypes on codon 10 differs between renal cancer patients and healthy normal controls ( p<0.05). The relative risk of the genotype 10C/C was calculated as 2.51. No differences in genotypes were observed at all other loci. We also analyzed DNA from pairs of cancerous and normal tissues from 96 cases of human renal cell cancer to characterize genotypic changes at these loci. Genotypic changes were detected in nine cancer samples on exon 1 (codons 10 and 87) of ERα, although none were detected at other regions. The present study demonstrates for the first time that codon 10 polymorphism on exon 1 of ERα may be involved in renal cancer risk.

A resistance gene analog useful for targeting disease resistance genes against different pathogens on group 1S chromosomes of barley, wheat and rye.

Comparative sequence analysis of the resistance gene analog (RGA) marker locus aACT/CAA (originally found to be tightly linked to the multiallelic barley Mla cluster) from genomes of barley, wheat and rye revealed a high level of relatedness among one another and showed high similarity to a various number of NBS-LRR disease resistance proteins. Using the sequence-specific polymerase chain reaction (PCR), RGA marker aACT/CAA was mapped on group 1S chromosomes of the Triticeae and was associated with disease resistance loci. In barley and rye, the marker showed linkage to orthologous powdery mildew resistance genes Mla1 and Pm17, respectively, while in wheat linkage with a QTL against fusarium head blight (FHB) disease was determined. The use of RGA clones for R gene mapping and their role in the expression of qualitative and quantitative resistance is discussed.

Polymorphism in the immunoglobulin VH gene V1-69 affects susceptibility to rheumatoid arthritis in subjects lacking the HLA-DRB1 shared epitope.

To investigate the contribution of polymorphism in the immunoglobulin heavy chain variable region V1-69 gene set to genetic susceptibility to rheumatoid arthritis (RA) in Czech and British patients. We used V1-69 gene sequence-specific polymerase chain reaction (PCR) and restriction enzyme digestion to study polymorphism in the V1-69 gene set in germline DNA of 109 Czech and 159 British RA patients and 164 ethnically matched controls. Polymorphism was further studied by nucleotide sequencing of the V1-69 gene locus in germline DNA. We found that all patients and controls had at least one V1-69 gene copy. In the Czech RA cohort, the dimorphic nucleotide in codon 73 of V1-69 (GAA or AAA) was present in the homozygous form 73(A/A) in 31 of 109 (28.4%) RA patients vs 12 of 79 (15.2%) controls [odds ratio (OR)=2.22, P<0.001]. When the RA patients and controls were classified according to HLA shared epitope (SE) status, 73(A/A) was found in 18 of 76 (23.7%) SE(+) patients compared with 13 of 38 (34.2%) SE(-) patients, four of 12 (18.2) SE(+) controls and eight of 57 (14%) SE(-) controls. This suggests that homozygosity for the dimorphic sequence 73(A) contributed to susceptibility to RA in SE(-) Czech individuals (OR=3.2, P<0.001). The most striking observation was that none of the 38 SE(-) Czech patients, compared with 11 of 76 (14.5%) SE(+) RA patients, three of 22 (13.6%) SE(+) and 11 of 57 (19.3%) SE(-) ethnically matched controls, were homozygous for the alternative dimorphic sequence 73(G/G) (OR=9.1, P<0.05). These data, however, were not replicated in a Caucasoid British RA population. The dimorphic sequence at codon 73 (73(A/A)) of the V1-69 gene contributes to genetic susceptibility in SE(-) Czech RA patients.

Real-time sequence-specific primer polymerase chain reaction amplification of HLA class II alleles: a novel approach to analyze microchimerism.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

A new strategy for mannose-binding lectin gene haplotyping.

The mannose-binding lectin 2 (MBL2) gene is polymorphic and codes for a protein with an important role in the innate immune response, whose variants have been associated with a great number of diseases. Point variations have been described in the 5' regulatory region at positions -550 (MBL2*H or *L) and -221 (*X or *Y), in the 5' untranslated sequence at position +4 (*P or *Q), and in the coding sequence of exon 1 at codons 52, 54, and 57 (MBL2*A or D, A or B, and A or C, respectively). These can be in cis or in trans configuration. The different haplotypes influence the immunological phenotype of the individual, which makes MBL2 haplotyping very important. Previously described MBL2-typing methods do not present adequate haplotype resolution or are too complex and costly. We have developed a new MBL2-typing strategy that is economical and renders rapid and reliable results without ambiguities. We typed 202 individuals of European, 32 of African, and 16 of Oriental descent. Only five to six reactions from 10 possible PCR-SSPs (sequence-specific polymerase chain reactions) were sufficient to genotype one individual unambiguously. The reactions were specific for amplification of the variants located upstream of the coding sequence. The results were associated to the results of hybridizations of the amplified products with eight sequence-specific oligonucleotide probes (SSOP). The strategy led to identification of eight alleles: MBL2*HYPA, HYPD, LYPA, LYPB, LYPD, LYQA, LYQC, and LXPA. Their frequencies in each of the groups were similar to those of other populations studied to date, with MBL2*LYPD (g.[-550G>C; -221C>G; 4T>C; 223C>T; 230A>G; 239A>G]) being novel. All samples were found to be in Hardy-Weinberg equilibrium.

RHD gene deletion occurred in the Rhesus box

The Rh blood group antigens derive from 2 genes,RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a deletion of the whole RHD gene occurs with a frequency of approximately 40%. The relative position of the 2 RH genes and the location of the RHD deletion was previously unknown. A model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and nucleotide sequencing. The open reading frames of bothRH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30 000 base pair (bp) that contained the SMP1 gene. The RHD gene was flanked by 2 DNA segments, dubbed Rhesus boxes, with a length of approximately 9000 bp, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1463 bp of identity. PCR with sequence-specific priming (PCR-SSP) and PCR with restriction fragment length polymorphism (PCR-RFLP) were used for specific detection of the RHDdeletion. The molecular structure of the RH gene locus explains the mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of theRHD− genotype is now possible.

A Short Isoform of Col9a1 Supports Alveolar Bone Repair

Bone wound created in intramembranous alveolar bone heals without the formation of cartilage precursor tissue. However, the expression of cartilage collagen mRNAs has been suggested. In this report, we examined the expression and the potential role of type IX collagen in bone restoration and remodeling. The sequence specific polymerase chain reaction demonstrated the exclusive expression of short transcriptional isoform of alpha1(IX) collagen (Col9a1) in alveolar bone wound healing, while the long isoform of Col9a1 transcript was absent. Type IX collagen was immunolocalized in the preliminary matrix organized in granulation tissue before trabecular bone formation in tooth extraction socket. In Col9a1-null mutant mice, there were considerable variations in alveolar bone wound healing with the absence of or abnormally organized trabecular bone. Occasionally, unusual apposition of cortical-bone-like layers in bone marrow space was observed. The Col9a1-null mice indicated no growth retardation, and their facial and long bones maintained the normal size and shape. However, the primary spongiosa region of adult Col9a1 mutant mice showed an abnormal trabecular bone structure associated with abnormal immunostaining with the hypertrophic cartilage specific type X collagen antibody. These data suggest that type IX collagen short transcriptional variant is involved in the restoration and remodeling processes of trabecular bone.

High-resolution sequence typing of HLA-DQA1 and -DQB1 exon 2 DNA with taxonomy-based sequence analysis (TBSA) allele assignment.

High-resolution DNA sequencing of exon 2 of DQA1 and DQB1 genes that uses a taxonomy-based sequence analysis (TBSA) method to assign alleles was developed. The system uses fewer primers for polymerase chain reaction (PCR) amplification and sequencing than other methods and yields accurate DQA1 and DQB1 typing when either homozygous or heterozygous DNA samples are tested. The approach was initially corroborated by the correct typing of 10 blinded samples that had been previously typed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or serology, and subsequently confirmed by sequencing of cloned PCR products. DNA from peripheral blood cell samples of 130 individuals enrolled in a case-control analysis of HLA determinants of abdominal aortic aneurysm were subsequently evaluated. Overall, 8 different DQA1 and 19 DQB1 alleles were identified. All 21 DQA1 heterozygous combinations and 45 of 49 DQB1 heterozygous combinations were successfully resolved with TBSA. The two pairs of heterozygous DQB1 combinations that were not unambiguously typed required sequence specific PCR amplification for correct allele identification. We conclude that the method provides precise analysis for HLA-DQ typing.

Constitutive Expression of Small Subunit Ribosomal RNA Transcripts in Cryptosporidium parvum Oocysts and Intracellular Stages

As in other apicomplexan protozoa, the ribosomal genes of Cryptosporidium parvum are present in small number and dispersed on several chromosomes. Two types of ribosomal small subunit genes have been identified in bovine isolates by sequencing and sequence-specific polymerase chain reaction. Using denaturing gradient gel electrophoresis, ribosomal gene expression was investigated in C. parvum oocysts and intracellular stages grown in tissue culture. In a calf-propageted C. parvum isolate, extra and intracellular stages expressed 2 types of transcripts. This observation contrasts with the differential expression of rRNA, genes in malaria species. Analysis of ribosomal gene expression in oocysts of a different genotype indicates that some C. parvum isolates express only l type of small subunit ribosomal RNA. These observations highlight fundamental differences in ribosomal gene expression among apicomplexan protozoa and within the species C. parvum and support the view that differential expression in malaria is an adaptation to a dixeneous life cycle.

Human Leukocyte Antigen-Associated Susceptibility to Pulmonary Tuberculosis: Molecular Analysis of Class II Alleles by DNA Amplification and Oligonucleotide Hybridization in Mexican Patients

Pulmonary tuberculosis (PTB) develops by a complex combination of environmental factors with genetic susceptibility. In this context, an association between human leukocyte antigens (HLAs) and tuberculosis has been examined in several populations, but results have been controversial. A prospective evaluation of class II HLA genotypes was completed by the polymerase chain reaction (PCR) sequence-specific primer technique and PCR sequence-specific oligonucleotide hybridization in a Mexican population. This study was conducted at the Clinical Service of Tuberculosis and the Department of Immunology, National Institute of Respiratory Diseases, Mexico City, Mexico. Four groups were examined: 95 healthy subjects; 50 nonimmunosuppressed PTB patients; 15 HIV-infected patients (stage IVc in the Centers for Disease Control and Prevention [CDC] classification system for AIDS) with PTB; and 37 HIV-infected patients in the asymptomatic stage (CDC stage II). The frequencies of alleles DQA1*0101 (odds ratio [OR], 6.18; 95% confidence interval [CI], 2.38 to 16.08), DQB1*0501 (OR, 6.16; 95% CI, 2.44 to 17.71), and DRB1*1501 (OR, 7.92; 95% CI, 2.71 to 23.14) were significantly increased in nonimmunosuppressed patients with PTB when compared with healthy subjects. By contrast, frequencies of allele DQB1*0402 and antigens DR4 and DR8 were significantly decreased in patients with PTB. Additionally, a significantly higher frequency of the DRB1*1101 allele was found in HIV-positive subjects (OR, 6.67; 95% CI, 2.13 to 20.83). The genetic influence associated with the HLA system appears to have an important role in the development of PTB, although this susceptibility may not be relevant in patients with severe immunodeficiency diseases such as AIDS.

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal speciesStachybotrys chartarum

The nucleotide sequence of ac936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal speciesStachybotrys chartarumand for other species ofStachybotrysand the related genusMemnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers forS. chartarumin the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo® v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo® 5.0 software. The automated computer searches were unsuccessful in findingS. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that includedS. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for onlyS. chartarumand one other species ofStachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only forS. chartarum. The relative merits of different PCR strategies for the detection ofS. chartarumemploying either one or both of the primers identified in this study are discussed.

RHD genotyping in weak D phenotypes by multiple polymerase chain reactions.

Weak D phenotypes involve a quantitative variation of D. The genomic basis in weak D has been disputed, however. Five sequence-specific polymerase chain reactions (SSP-PCRs) on exons 2, 5, and 7 of the RHD gene were evaluated in 248 white and 98 Japanese blood donors and compared with the results obtained by amplification of intron 4 and serology. All methods and SSP-PCR testing on the 3' non-coding region of the RHD gene were applied to the genotyping of 94 DNA samples derived from individuals expressing weak D phenotypes. Concordant results were obtained with all genotyping and phenotyping methods in testing 201 D-positive and 145 D-negative donors. Four of 94 weak D samples were typed as D-negative by amplification of intron 4 and SSP-PCR on exon 5. Phenotyping with monoclonal antibodies revealed a DVI category in one of these cases and DFR phenotype in three of these cases. One weak D sample, which reacted like normal D-positive cells with all applied monoclonal antibodies, was typed falsely negative by SSP-PCR on exon 5 because of a point mutation at nucleotide 667 (T-->G) that resulted in a Phe223Val amino acid substitution. In this individual, heterozygosity was found at two other amino acid positions (Glu233Gln and Val238Met) by restriction fragment length polymorphism analysis. Genetic diversity in weak D phenotypes is rare. Only 1 of 90 true weak D phenotypes (1.1%) had a genetic variation in testing on seven gene regions of the RHD gene.

Molecular structure and expression of shaker type potassium channels in glial cells of trout CNS.

Two shaker-related potassium channel transcripts (tsha1, tsha2) were identified in glial cells of trout central nervous system (CNS) by polymerase chain reaction (PCR)-cloning and sequencing. While tsha1 was highly similar to the mammalian Kv1.2 subtype of potassium channels, tsha2 did not show a preferential sequence homology with a particular subtype of shaker, but exhibited uniform similarity with mammalian Kv1.1, Kv1.2, and Kv1.3, respectively. Transcripts for the shaw and shal subfamilies of voltage-gated potassium channels were detected in whole brain tissue only but not in freshly dissociated glial cells. Using mRNA extracted from different glial cell types in combination with sequence specific PCR primers, tshal was found restricted to oligodendrocytes and their progenitor cells, while tsha2 was common to astrocytes, as well.

Cecal Spirochetosis Associated with Serpulina pilosicoli in Captive Juvenile Ring-Necked Pheasants

Spirochetes similar to those described in the ceca of broilers with diarrhea and in laying hens with decreased egg production and growth were identified in the ceca of captive-raised juvenile ring-necked pheasants (Phasianus colchicus). The birds were submitted for diagnostic investigation of an illness characterized by a seromucoid ocular discharge, sneezing, swollen infraorbital sinuses, and weight loss. In addition to cecal spirochetosis, the birds had mild enteric coccidiosis, trichomoniasis, and nematodiasis (Heterakis spp.); esophageal capillariasis; and respiratory mycoplasmosis. Weakly beta-hemolytic spirochetes isolated from the ceca of one pheasant were identified as Serpulina pilosicoli with the use of a 16S rRNA sequence-specific polymerase chain reaction amplification assay. Diffuse cecal enterocyte attachment was reproduced in a 1-day-old chick challenged with the pheasant S. pilosicoli isolate. Immunohistochemical staining of sections of ceca from the pheasant and challenged chick with a Serpulina spp. flagellar antigen-specific monoclonal antibody confirmed spirochetal attachment to cecal enterocytes. The etiologic significance of the spirochete infection is unknown because respiratory signs and multiple gastroenteric pathogens dominated the clinicopathologic manifestations and an intestinal disorder was not a clinical complaint.