Visceral Marek's disease in white-peafowl (Pavo cristatus) / Doença de Marek visceral em pavão-branco (Pavo cristatus)

Marek's disease (MD) is a lymphoproliferative disorder caused by Gallid herpesvirus 2 (MDV) that infects mainly domestic gallinaceous birds although wild birds may occasionally be affected. The current report describes the anatomopathological and molecular findings of a case of MD in a white-peafowl (Pavo cristatus). The signs included apathy, hyporexia, and diarrhea. Grossly, 0.5 to 1.5cm in diameter, yellow, soft nodules were observed in the skeletal muscle, lung, kidney, air sacs, small intestine, heart, ovary, ventriculus, and proventriculus. Microscopically, numerous atypical round neoplastic cells were noted. The molecular detection of MDV DNA was implemented to amplify part of the meq gene and products were sequenced for the phylogenetic analysis. Template DNA was obtained from tissues of the affected bird and from blood of all the gallinaceous birds of the Zoo. The expected amplicon for the partial amplification of MDV meq gene was obtained and the amplicons were sequenced. Sequences obtained enabled grouping the strain (accession no. KT768121) with MDV serotype 1 strains from the GenBank. Based on the anatomopathological and molecular findings, the diagnosis of MD in a white-peafowl was reached, and to the authors' knowledge, no previous report regarding MD was published in Pavo cristatus.(AU)

Doença de Marek (MD) é uma desordem linfoproliferativa causada pelo Gallid herpesvirus 2 (MDV), que infecta principalmente galináceos domésticos, porém aves silvestres podem ser ocasionalmente afetadas. O presente relato descreve os achados anatomopatológicos e moleculares de um caso de MD em um pavão-branco (Pavo cristatus). Os sinais clínicos incluíram apatia, hiporexia e diarreia. Macroscopicamente, foram observados nódulos macios, de 0,5 a 1,5cm de diâmetro, no músculo esquelético, no pulmão, nos rins, nos sacos aéreos, no intestino delgado, no coração, no ovário, no ventrículo e no proventrículo. Microscopicamente, numerosas células redondas neoplásicas atípicas foram notadas. A detecção molecular do DNA do MDV foi implementada para amplificar parte do gene meq, e os produtos foram sequenciados para análise filogenética. DNA foi obtido de tecidos de aves afetadas e do sangue de todos os galináceos do zoológico. A esperada amplificação de parte do gene meq de MDV amplificado foi ampliada e sequenciada. As sequências obtidas permitiram o agrupamento da cepa (acesso KT768121) com cepas do sorotipo 1 de MDV do GenBank.. O diagnóstico de MD em pavão-branco foi obtido com base nos achados anatomopatológicos e moleculares e, pelo conhecimento dos autores, não há relatos anteriores publicados de MD em Pavo cristatus.(AU)

Determination of human neutrophil antigen-1, -3, -4 and -5 allele frequencies in English Caucasoid blood donors using a multiplex fluorescent DNA-based assay.

BACKGROUND AND OBJECTIVES: A number of DNA-based methods to genotype the alleles coding for HNA have been described, but all require the separate amplification and analysis of each allele. The aim was to develop a DNA-based method for simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles. MATERIALS AND METHODS: An allele-specific primer extension method was used in combination with magnetic beads from Luminex technology. PCR-sequence-specific primers (SSP) was used to resolve the presence of the HNA-1b allele in samples assigned by the Luminex bead assay as HNA-1a/-1b/-1c or HNA-1b/-1c. HNA allele frequencies were determined in a panel of 140 randomly selected English Caucasoid blood donors. RESULTS: HNA allelic types were compared with historical results, and 100% concordance was found. Only eight of the 97 samples used in the validation required additional testing by PCR-SSP. Allele frequencies were determined in the blood donor population as follows: 0·318 for HNA-1a, 0·668 for HNA-1b, 0·014 for HNA-1c, 0·768 for HNA-3a, 0·232 for HNA-3b, 0·882 for HNA-4a, 0·118 for HNA-4b, 0·736 for HNA-5a and 0·264 for HNA-5b. CONCLUSION: A multiplex Luminex bead assay for the simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles is described that enables rapid typing of donors to support HNA alloimmunized patients who require HNA-compatible blood products.

Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay.

Type 1 hereditary haemochromatosis (HH) is a common genetic disorder in Caucasoids resulting from mutations in the HFE gene. Routine diagnostic testing for type 1 HH involves genotyping for two of these described HFE mutations, C282Y and H63D. In some cases typing of a third mutation, S65C is also performed. Several techniques have been reported for HFE genotyping and these include polymerase chain reaction (PCR)-sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), PCR-sequence-specific oligonucleotide probe (SSOP), real-time PCR followed by melting curve analysis and TaqMan assay. The aim of this study was to develop an alternative method to both conventional PCR and real-time PCR/TaqMan assay to detect all three HFE mutations in a single assay using Luminex technology. DNA controls of known genotypes (n = 109) were used to evaluate this approach. These controls were selected to represent the three possible genotypes (wild type, mutant, heterozygous) for each mutation. Subsequently, blind DNA samples (n = 100) were used to validate this method. This new assay was then compared with current techniques (in-house PCR-SSP and TaqMan assay). Comparison of genotypes obtained with the Luminex method with those previously reported by both in-house PCR-SSP and TaqMan assay showed 100% concordance for both DNA controls and blind DNA samples and no discrepancies were observed. Allelic frequency for C282Y, H63D and S65C mutations were 22%, 16% and 2%, respectively. We report here a high-throughput, accurate and robust multiplex luminex bead assay for routine clinical testing of C282Y, H63D and S65C mutations in the HFE gene.

Identification of two novel single nucleotide polymorphisms in the promoter of the human interleukin-18 receptor alpha.

Single-strand conformational polymorphism (SSCP) was used to identify single nucleotide polymorphisms (SNPs) in the promoter region of the human interleukin-18 receptor alpha (IL-18Ralpha). Two SNPs were identified at positions -69 and -638 relative to the transcriptional start site. Two-way comparison of the two SNPs revealed strong linkage disequilibrium (chi2 = 63.45, P < 0.001). Three haplotypes were identified, namely C-69C-638, T-69C-638 and C-69T-638, with frequencies of 0.26, 0.39 and 0.35, respectively.