Rumination time around calving: an early signal to detect cows at greater risk of disease.

The main objective of this experiment was to evaluate the use of rumination time (RT) during the peripartum period as a tool for early disease detection. The study was carried out in an experimental freestall barn and involved 23 Italian Friesian cows (9 primiparous and 14 multiparous). The RT was continuously recorded by using an automatic system (Hr-Tag, SCR Engineers Ltd., Netanya, Israel), and data were summarized in 2-h intervals. Blood samples were collected from 30 d before calving to 42 d in milk (DIM) to assess biochemical indicators related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. The liver functionality index, which includes some negative acute-phase proteins and related parameters (albumin, cholesterol, and bilirubin), was used to evaluate the severity of inflammatory conditions occurring around calving. The cows were retrospectively categorized according to RT observed between 3 and 6 DIM into those with the lowest (L) and highest (H) RT. The average RT before calving (-20 to -2d) was 479 min/d (range 264 to 599), reached a minimum value at calving (30% of RT before calving), and was nearly stable after 15 DIM (on average 452 min/d). Milk yield in early lactation (on average 26.8 kg/d) was positively correlated with RT (r = 0.33). After calving, compared with H cows, the L cows had higher values of haptoglobin (0.61 and 0.34 g/L at 10 DIM in L and H, respectively) for a longer time, had a greater increase in total bilirubin (9.5 and 5.7 µmol/L at 5 DIM in L and H), had greater reductions of albumin (31.2 and 33.5 g/L at 10 DIM in L and H) and paraoxonase (54 and 76 U/ml at 10 DIM in L and H), and had a slower increase of total cholesterol (2.7 and 3.2 mmol/L at 20 DIM in L and H). Furthermore, a lower average value of liver functionality index was observed in L (-6.97) compared with H (-1.91) cows. These results suggest that severe inflammation around parturition is associated with a slower increase of RT after calving. Furthermore, more than 90% of the cows in the L group had clinical diseases in early lactation compared with 42% of the H cows. Overall, our results demonstrate the utility of monitoring RT around calving, and in particular during the first week of lactation, as a way to identify in a timely fashion those cows at a greater risk of developing a disease in early lactation.

Rumination time during the summer season and its relationships with metabolic conditions and milk production.

The main objective of this experiment was to monitor the rumination pattern during the summer season in lactating dairy cows and to investigate its relationships with metabolic conditions and physiological markers of heat stress. The study was carried out in an experimental freestall barn located near Piacenza, Italy (45°01'N, 9°40'E; 68 m above sea level), and involved 21 Italian Friesian cows (11 primiparous and 10 multiparous) during the summer season. Rumination time (RT) was recorded by using an automatic system and data were calculated and summarized in 2-h intervals. Microclimatic conditions (temperature and relative humidity) inside the barn were recorded during the trial, and the temperature-humidity index (THI) was calculated. Breathing rates and rectal temperatures were recorded following stable meteorological periods characterized by lower and higher temperatures. At the same times, blood samples were collected to assess biochemical variables related to energy, protein, and mineral metabolism, as well as markers of inflammatory conditions and enzyme activity. Daily milk yield, body weight, nutritional condition, and health status were also recorded. The average RT was 501 min/d, with no significant differences between primiparous and multiparous cows. According to the microclimatic conditions and physiological markers of heat stress, the cows suffered mild to moderate heat stress during the summer. A negative relationship between daily maximum THI and RT was observed (r=-0.32), with a reduction of 2.2 min of RT for every daily maximum THI unit over the threshold of daily maximum THI of 76. Most of the rumination occurred during the night (on average the nighttime RT was 63.2% of daytime and nighttime RT); moreover, the proportion of nighttime RT slightly but significantly increased as THI increased. Rumination time throughout the trial was negatively related to breathing rate and positively related to milk yield. Daily maximum THI was negatively correlated with plasma glucose (r=-0.52) and positively correlated with plasma ß-hydroxybutyric acid (r=0.26). Values of plasma ß-hydroxybutyric acid were positively related to RT through the trial. Our results indicate that hot conditions negatively affect RT and modify its daily pattern. The relationship between RT and the physiological markers used in our trial support the use of RT as a marker of heat stress.

Relationships between rumination time, metabolic conditions, and health status in dairy cows during the transition period.

The main objective of this experiment was to monitor the rumination pattern during the transition period in primiparous (PR) and pluriparous (PL) dairy cows and to investigate its relationships with metabolic conditions, milk yield, and health status. The study was carried out in an experimental free-stall barn and involved 32 Italian Friesian cows (9 PR and 23 PL) during the transition phase. The rumination time (RT) was recorded with an automatic system (HR-Tag), and data were calculated and summarized in 2-h intervals. Blood samples were collected during the transition phase to assess biochemical variables related to energy, protein, and mineral metabolism, as well as markers of inflammatory conditions and some enzyme activity. Daily milk yield, BW, nutritional condition, and health status were also recorded. The average RT before calving (-20 to -6 d) was 463 min/d in PR (range 270 to 620) and 522 min/d in PL (range 411 to 640). In the early lactation [15 to 40 d in milk (DIM)], the average RT was 504 min/d in PR (range 400 to 585) and 562 min/d in PL (range 414 to 685) and was positively correlated with milk yield (r = 0.36; P < 0.001). The RT reached the minimum at calving d (262 min/d in PR and 278 min/d in PL). Before calving the percentage of RT during the nighttime was 60% in PR and 62% in PL, whereas the values decreased after calving (55% in PR and 57% in PL). During the first weeks of lactation, PR showed a shorter RT than PL in the 2-h intervals that included milkings. Cows with reduced RT before calving maintained reduced RT after calving and suffered a greater frequency of disease than cows with greater RT in late pregnancy. Moreover, cows characterized by mild inflammatory conditions and without health disorders or only mild health disorders during the puerperium showed a greater average rumination time (over 520 min/d) during the first 10 d of lactation. Conversely, the decreased RT (450 min/d) during the first few days of lactation was observed in cows with subclinical diseases or health disorders. Cows affected by clinical mastitis during the trial showed a reduction of RT and a change in its variability already some days before the drug treatment. Our results suggest that the automatic measurement of RT is useful to predict calving time and to quickly obtain information on health status of the animals in a period as critical as the transition phase.

Molecular scanning of the ABCA4 gene in Spanish patients with retinitis pigmentosa and Stargardt disease: identification of novel mutations.

PURPOSE: Among the 500 ABCA4 mutations identified so far in the retina-specific ABC transporter (ABCA4) gene, only 20 have been described in patients with autosomal recessive retinitis pigmentosa (arRP). In this study the gene was screened for mutations in a cohort of 25 patients of Spanish origin, to further assess ABCA4 involvement in retinal dystrophies. METHODS: The 50 exons of the ABCA4 gene have been analyzed through denaturing high performance liquid chromatography (DHPLC) and direct sequencing of samples displaying altered elution profiles. RESULTS: Four new and five known mutations were identified in our patients; except for one new deletion detected in a patient with Stargardt disease, all the remaining variations are single nucleotide substitutions resulting in missense mutations. CONCLUSIONS: The results further underline the genetic heterogeneity of retinal disorders, suggesting that more than one gene could differentially contribute to at least some forms of retinal degeneration. Finding a high proportion of novel mutations merits the use of scanning methodologies to analyze the whole coding region of the ABCA4 gene.

Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients.

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.

Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

Seven novel additional small mutations and a new alternative splicing in the human dystrophin gene detected by heteroduplex analysis and restricted RT-PCR heteroduplex analysis of illegitimate transcripts.

Around 35% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients cannot be identified by techniques which identify major DMD rearrangements in the dystrophin gene. In order to characterize the gene defect in these patients, we screened 40 exons of the dystrophin gene by heteroduplex analysis on genomic DNA in 50 affected Italian males. Using conventional heteroduplex analysis and a modified heteroduplex analysis on restricted RT-PCR products of illegitimate transcripts, restricted RT-PCR heteroduplex analysis, we were able to identify 7 novel small mutations and a new alternative splicing involving exon 25 of the dystrophin gene in peripheral blood lymphocytes and skeletal muscle transcripts.

Mutational analysis of muscle and brain specific promoter regions of dystrophin gene in DMD/BMD Italian patients by denaturing gradient gel electrophoresis (DGGE).

In order to characterize the nature of mutations occurring in non-deleted Duchenne (DMD) and Becker muscular dystrophy (BMD) affected males, a total of 40 unrelated Italian patients was studied for the presence of point mutations within the muscle-specific regulatory region of the dystrophin gene. We decided to investigate the dystrophin promoter sequences because nucleotide variations in these regions could impair the expression of the gene and be the underlying molecular defect in some forms of the disease. In four patients suffering from mental retardation, the brain promoter region was also studied. To screen for point mutations, we applied molecular analysis by parallel denaturing gradient gel electrophoresis (DGGE). No sequence alterations were found in either the muscle or the brain promoters, suggesting that mutations in these regions do not represent a common mechanism of mutation in DMD/BMD.

A nonsense mutation (Gln-673-Term) in exon 17 of the human dystrophin gene detected by heteroduplex analysis.

Heteroduplex analysis was used to search for small mutations in a sample of 40 Italian DMD/BMB patients in whom large rearrangements were not found. A novel nonsense mutation in exon 17 of the dystrophin gene, consisting of a C to T transition, is described.

[Polyfructosan and para-aminohippuric acid clearance, comparison of 2 methods for determining glomerular filtration and plasma renal flow]. / Clearance del polifruttosano e dell'acido para-aminoippurico, confronto di due metodiche per la determinazione della filtrazione glomerulare e della portata renale plasmatica.

Glomerular Filtration Rate (GFR) and Renal Plasma Flow (RPF) were measured respectively, Polyfructosan and Para-aminohippuric acid clearances, in 13 patients with different degrees of renal insufficiency. Two different methods were used, the standard method and one without urine collection. The mean GFR (66.4 +/- 27.8 vs. 72.2 +/- 19.0 ml/m, r = 0.74) and RPF (431 +/- 204 vs 490 +/- 188 ml/m, r = 0.76) as well as Filtration Fraction (17.7 +/- 8.3 vs. 16.6 +/- 6.6%) were well correlated. These data show that within the normal range of GRF and RPF the method determining only plasma levels is as reliable as the standard method, giving the advantage to be less troublesome for patients and staff, requiring no bladder catheterization.