Promoter region of the bovine growth hormone receptor gene: single nucleotide polymorphism discovery in cattle and association with performance in Brangus bulls.

Expression of the GH receptor (GHR) gene and its binding with GH is essential for growth and fat metabolism. A GT microsatellite exists in the promoter of bovine GHR segregating short (11 bp) and long (16 to 20 bp) allele sequences. To detect SNP and complete an association study of genotype to phenotype, we resequenced a 1,195-bp fragment of DNA including the GT microsatellite and exon 1A. Resequencing was completed in 48 familialy unrelated Holstein, Jersey, Brown Swiss, Simmental, Angus, Brahman, and Brangus cattle. Nine SNP were identified. Phylogeny analyses revealed minor distance (i.e., <5%) in DNA sequence among the 5 Bos taurus breeds; however, sequence from Brahman cattle averaged 27.4 +/- 0.07% divergence from the Bos taurus breeds, whereas divergence of Brangus was intermediate. An association study of genotype to phenotype was completed with data from growing Brangus bulls (n = 553 from 96 sires) and data from 4 of the SNP flanking the GT microsatellite. These SNP were found to be in Hardy-Weinberg equilibrium and in phase based on linkage disequilibrium analyses (r(2) = 0.84 and D'= 0.92). An A/G tag SNP was identified (ss86273136) and was located in exon 1A, which began 88 bp downstream from the GT microsatellite. Minor allele frequency of the tag SNP was greater than 10%, and Mendelian segregation was verified in 3 generation pedigrees. The A allele was derived from Brahman, and the G allele was derived from Angus. This tag SNP genotype was a significant effect in analyses of rib fat data collected with ultrasound when bulls were ~365 d of age. Specifically, bulls of the GG genotype had 6.1% more (P = 0.0204) rib fat than bulls of the AA and AG genotypes, respectively. Tag SNP (ss86273136), located in the promoter of GHR, appears to be associated with a measure of corporal fat in Bos taurus x Bos indicus composite cattle.

Associations of DNA polymorphisms in growth hormone and its transcriptional regulators with growth and carcass traits in two populations of Brangus bulls.

Sequence polymorphisms in the growth hormone (GH) gene and its transcriptional regulators, Pit-1 and Prop-1, were evaluated for associations with growth and carcass traits in two populations of Brangus bulls Chihuahuan Desert Rangeland Research Center (CDRRC, N = 248 from 14 sires) and a cooperating breeding program (COOP, N = 186 from 34 sires). Polymorphisms were SNP mutations in intron 4 (C/T) and exon V (C/G) in GH, A/G in exon VI in Pit-1, and A/G in exon III in Prop-1. In the COOP population, bulls of Pit-1 GG genotype had a significantly greater percentage of intramuscular fat than bulls of the AA or AG genotype, and bulls of the Prop-1 AA genotype had significantly greater scrotal circumference than bulls of AG or GG genotypes at ~365 days of age. Also, heterozygous genotypes for the two GH polymorphisms appeared advantageous for traits of muscularity and adiposity in the COOP population. The heterozygous genotype of GH intron 4 SNP was associated with advantages in weight gain, scrotal circumference, and fat thickness in the CDRRC population. The two GH polymorphisms accounted for >/=27.7% of the variation in these traits in the CDRRC population; however, R(2) was <5% in the COOP population. Based on haplotype analyses the two GH SNPs appeared to be in phase; the haplotype analyses also paralleled with the genotype analyses. Polymorphisms in GH and its transcriptional regulators appear to be predictors of growth and carcass traits in Brangus bulls, particularly those with heterozygous GH genotypes.

Associations of DNA polymorphisms in growth hormone and its transcriptional regulators with growth and carcass traits in two populations of Brangus bulls

Sequence polymorphisms in the growth hormone (GH) gene and its transcriptional regulators, Pit-1 and Prop-1, were evaluated for associations with growth and carcass traits in two populations of Brangus bulls Chihuahuan Desert Rangeland Research Center (CDRRC, N = 248 from 14 sires) and a cooperating breeding program (COOP, N = 186 from 34 sires). Polymorphisms were SNP mutations in intron 4 (C/T) and exon V (C/G) in GH, A/G in exon VI in Pit-1, and A/G in exon III in Prop-1. In the COOP population, bulls of Pit-1 GG genotype had a significantly greater percentage of intramuscular fat than bulls of the AA or AG genotype, and bulls of the Prop-1 AA genotype had significantly greater scrotal circumference than bulls of AG or GG genotypes at ~365 days of age. Also, heterozygous genotypes for the two GH polymorphisms appeared advantageous for traits of muscularity and adiposity in the COOP population. The heterozygous genotype of GH intron 4 SNP was associated with advantages in weight gain, scrotal circumference, and fat thickness in the CDRRC population. The two GH polymorphisms accounted for ³27.7% of the variation in these traits in the CDRRC population; however, R2 was <5% in the COOP population. Based on haplotype analyses the two GH SNPs appeared to be in phase; the haplotype analyses also paralleled with the genotype analyses. Polymorphisms in GH and its transcriptional regulators appear to be predictors of growth and carcass traits in Brangus bulls, particularly those with heterozygous GH genotypes

Antenatal perineal massage for reducing perineal trauma.

BACKGROUND: Perineal trauma following vaginal birth can be associated with significant short- and long-term morbidity. Antenatal perineal massage has been proposed as one method of decreasing the incidence of perineal trauma. OBJECTIVES: To assess the effect of antenatal perineal massage on the incidence of perineal trauma at birth and subsequent morbidity. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group Trials Register (30 January 2005), the Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 1, 2005), PubMed (1966 to January 2005), EMBASE (1980 to January 2005) and reference lists of relevant articles. SELECTION CRITERIA: Randomised and quasi-randomised controlled trials evaluating any described method of antenatal perineal massage undertaken for at least the last four weeks of pregnancy. DATA COLLECTION AND ANALYSIS: Both review authors independently applied the selection criteria, extracted data from the included studies and assessed study quality. We contacted study authors for additional information. MAIN RESULTS: Three trials (2434 women) comparing digital perineal massage with control were included. All were of good quality. Antenatal perineal massage was associated with an overall reduction in the incidence of trauma requiring suturing (three trials, 2417 women, relative risk (RR) 0.91 (95% confidence interval (CI) 0.86 to 0.96), number needed to treat (NNT) 16 (10 to 39)). This reduction was statistically significant for women without previous vaginal birth only (three trials, 1925 women, RR 0.90 (95% CI 0.84 to 0.96), NNT 14 (9 to 35)). Women who practised perineal massage were less likely to have an episiotomy (three trials, 2417 women, RR 0.85 (95% CI 0.75 to 0.97), NNT 23 (13 to 111)). Again this reduction was statistically significant for women without previous vaginal birth only (three trials, 1925 women, RR 0.85 (95% CI 0.74 to 0.97), NNT 20 (11 to 110)). No differences were seen in the incidence of 1st or 2nd degree perineal tears or 3rd/4th degree perineal trauma. Only women who have previously birthed vaginally reported a statistically significant reduction in the incidence of pain at three months postpartum (one trial, 376 women, RR 0.68 (95% CI 0.50 to 0.91) NNT 13 (7 to 60)). No significant differences were observed in the incidence of instrumental deliveries, sexual satisfaction, or incontinence of urine, faeces or flatus for any women who practised perineal massage compared with those who did not massage. AUTHORS' CONCLUSIONS: Antenatal perineal massage reduces the likelihood of perineal trauma (mainly episiotomies) and the reporting of ongoing perineal pain and is generally well accepted by women. As such, women should be made aware of the likely benefit of perineal massage and provided with information on how to massage.

Testing plasma pools for markers of viral contamination: the UK experience.

The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.

Medicion de los anticuerpos contra el virus de la inmunodeficiencia humana: estudio internacional en colaboración para evaluar los sueros de referencia de la OMS

Two preparations of human sera, one reactive against human immunodeficiency virus (HIV) and the other unreactive, were evaluated as potential international reference reagents (IRR) in an international collaborative study. Twenty-one laboratories participated and tested these and five other human sera which were found to range from highly reactive to unreactive. The proposed "positive" IRR was found to react strongly in all immunoassays and gave all the expected bands in immunoblot systems using HTLV-III, LAV-I or similar virus strains as antigens. The "unreactive" serum was judged to be negative by ELISA and immunoblots. The end-points determined by ELISAs varied considerably between laboratories, even between those using the same commercial kit. This variation was reduced somewhat when the reactivities of the samples were expressed relative to the proposed IRR

Electron microscopy of human immunodeficiency virus.

Two cell lines of human T lymphocytes (H9 and CEM) chronically infected with isolates of human or simian immunodeficiency viruses were examined by electron microscopy. Scanning electron microscopy of H9 cells showed characteristic morphological changes in the cells after infection with human T cell lymphotropic virus type III (HTLV-IIIB); these included loss of cell microvilli and their replacement by rounded surface protrusions. Virus particles were present over the whole cell surface, but were more numerous between, rather than on, surface protrusions. In contrast, CEM cells infected with three other isolates of the virus showed little change in morphology compared with uninfected cells; they typically had a single large aggregate of virus particles at the posterior end of the cell. Transmission electron microscopy of sections of infected cells showed budding virus in only a small proportion of cells although many had mature virus particles on their surface. The immature particles released by budding had a ring-like morphology in contrast to the mature virus with its characteristic elongated nucleoid. Surface spikes were rarely seen on sectioned virus particles, but were more obvious on the simian immunodeficiency virus than on human isolates. Negative staining of purified virus preparations showed that the peripheral dense material of immature particles had a striated structure. Surface spikes were not seen by negative staining on either immature or mature virus particles. A third type of smaller particle with surface spikes was found in all negatively stained preparations.

Computer matching of oligonucleotide patterns on electrophoretic gels: an application to the epidemiology of cytomegalovirus.

A computer program was written to analyse oligonucleotide patterns displayed by gel electrophoresis following restriction endonuclease digestion of human cytomegaloviral DNA, and was applied to an epidemiological study of the transmission of infection in a hospital special care baby unit, with regard to infant-to-infant and mother-to-infant transmission. The program calculates the molecular weight of oligonucleotides from their mobilities, using a cubic spline curve based on the mobilities of oligonucleotides from the AD169 strain. A matching algorithm then calculates the number of unmatched fragments for each pair of viral isolates. This was used as a similarity measure which successfully distinguished mother and infant isolate pairs from epidemiologically unrelated pairs. The program is not intended to provide fully automatic matching, but could be recommended as a screening device to pick out pairs of strains which are sufficiently similar to suggest a common source of infection, and which may warrant closer comparison. Other applications are discussed, and the possible use of densitometers to automate data entry is considered.

Measurement of antibodies to human immunodeficiency virus: an international collaborative study to evaluate WHO reference sera.

PIP: 21 laboratories participated in an international collaborative study to evaluate potential international reference reagents for measurement of antibodies to human immunodeficiency virus (HIV). Given inherent differences between the principles of currently used assays (based on enzyme-linked or radioimmunosorbance, immunofluorescence, immunoblotting, or immunoprecipitation) and batch-to-batch variations in the preparations of reagents and kits, there is an urgent need for well-characterized reference materials that can be used to define the reliability and sensitivity of the tests, for quality control of batches, and as common references between laboratories. The 7 human sera tested included 1 reactive against HIV and 1 unreactive. The study was designed to identify the coded preparations that reacted with HIV antibodies and to ascertain the minimum amount of the reactive samples that could be detected in the methods routinely used by the participants. Participants carried out single or duplicate assays for individual manufacturer's ELISAs or by their local methods. The proposed positive international reference reagent (coded A) reacted strongly in all immunoassays and gave all the expected bands in immunoblot systems using HIV-related virus strains as antigens. The unreactive serum (coded E) was negative by ELISA and immunoblots. Although the end-points determined by ELISAs varied considerably between laboratories, even when the same commercial kit was used, this variation was reduced somewhat when the reactivities of the samples were expressed relative to preparation A. It is concluded that the proposed international reference reagent A may have value as a qualitative check on the specificity of assays, in calibrating positive controls included in kits and other assays in arbitrary units, in calibrating detection limits in arbitrary units, and for calibrating immunoblots, especially for defining the optimal amounts of antigen and determining the relative mobilities of the major HIV peptides and glycopeptides.^ieng

An improved immunoblotting procedure for the detection of antibodies against HIV.

Immunoblotting ('Western blotting') is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for 'false-positive' results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products.

Cytomegalovirus infection in day nurseries.

One hundred and seventeen children and 41 teachers in day nurseries were screened for cytomegalovirus (CMV) viruria over a period of one year. Thirty two (27%) children and two (5%) teachers were found to be excreting virus on at least one occasion. Restriction endonuclease typing showed that virus strains isolated from the children were dissimilar, with the exception of those from sibling pairs and one unrelated pair. The virus isolate from one teacher matched those from two unrelated children, while the isolate from another teacher could not be distinguished from that from a sibling pair. The CMV serological state of the 41 teachers was not significantly different from 500 matched controls and no seroconversions occurred. It is concluded that although transmission of CMV among children and teachers may occur in day nurseries, the dissimilarity of most of the virus strains indicates that infection predominantly occurs outside. Furthermore, teachers in day nurseries showed no evidence of an increased risk of past CMV infection when compared with matched controls.

Restriction enzyme analysis of cytomegalovirus DNA to study transmission of infection.

Restriction enzyme analysis of cytomegalovirus deoxyribonucleic acid (DNA) has been used to characterise virus isolates and has provided information on patterns of viral transmission. It was shown that virus isolated from a congenitally infected infant was unlikely to have originated from the 13 congenitally infected children with whom the mother, a nurse, had been in contact. Of nine mother and infant pairs, from whom cytomegalovirus was isolated, seven yielded strains that were indistinguishable for mother and child; one pair showed minor differences and one was clearly distinguishable. Virus isolates from seven children attending a day nursery were typed, and only siblings were excreting similar strains of cytomegalovirus. Further examples of the application of this technique to studies of cytomegalovirus in a family environment are given. It is concluded that characterisation of virus strains by restriction analysis of DNA is a valuable epidemiological tool.

A simple technique for endonuclease mapping of cytomegaloviruses.

A simple procedure, suitable for routine epidemiological studies, is described for the differentiation of strains of cytomegaloviruses. Crude DNA from infected cells labelled with 32P was digested with restriction endonucleases and the resultant oligonucleotides were separated by electrophoresis on agarose gels. As few as 10(5) infected cells (i.e. a confluent monolayer of about 1 cm2) provided sufficient DNA for one analysis.