In silico identification and mapping of microsatellite markers on sus scrofa chromosome 4.

Marker density of a QTL region on pig chromosome 4 was increased. New microsatellites were identified by in silico mining of BAC-end and genomic shotgun sequences. Among 8,784 BAC-end sequences predicted within the region, 148 microsatellites were identified. In addition, 27,450 CA/TG repeats were identified within the genomic shotgun sequences, of which 157 were most likely located on SSC4q. A selection of 61 new microsatellites was mapped, together with previously mapped markers. The results showed that the human-pig comparative map in combination with BAC-end and genomic sequence resources provides an excellent source for a highly efficient and targeted development of markers.

What is the appropriate screening protocol in Lynch syndrome?

INTRODUCTION: Lynch syndrome families have a substantial risk of developing colorectal cancer (CRC). The recommended surveillance protocol includes colonoscopy every 2 years from age 20-25 years. It is yet unknown whether annual screening of patients aged 40-60 years is more effective than bi-annual screening, whether patients who had an adenoma removed should be re-examined after a year and whether surveillance of second-degree relatives is indicated. The aim of this study was to address these issues. METHODS: All carriers of a mismatch repair gene mutation who participated in the surveillance program were selected from the Dutch Lynch syndrome registry. The results of colonoscopy were prospectively collected. RESULTS: A total of 666 mutation carriers were identified in 110 families. Fourty-one CRCs were detected during endoscopic follow-up, of which 34 (83%) were diagnosed between age 40 and 60 years. In five of 34 patients, CRC was diagnosed within 1 year after colonoscopy, eight cancers were diagnosed between 1 and 2 years and the remaining tumors more than 2 years after colonoscopy. All eight CRCs detected between 1 and 2 years were at local stage. At least one adenoma was diagnosed at 141 examinations. The risk of developing CRC during follow-up in carriers with an adenoma was similar as in carriers without an adenoma at the previous colonoscopy. 280 parent-child couples with at least one Lynch syndrome-related carcinoma were identified in 110 families. In only 19 (6.8%) of these couples, CRC developed earlier in the child than an Lynch syndrome-associated cancer in the parent. CONCLUSION: The current surveillance protocol, i.e., bi-annual colonoscopy in first-degree relatives independent of age and endoscopic findings, appears to be appropriate.

Body weight changes and basal metabolic rates of rural Beninese women during seasons with different energy intakes.

Seasonal influences on the energy balance were studied in rural Beninese women. The average body weight of 130 women showed significant seasonal changes. The lowest average weight, reached in the pre-harvest period in June, was 1.15 +/- 1.27 kg lower than the average weight in the post-harvest period in August. This response was not uniform however: while well-nourished women with a BMI of more than 23 at the beginning of the study showed a marked weight loss in the pre-harvest period, thin women with a BMI of less than 18 did not. Energy intake was studied in a sub-group of 18 women and found to vary between 1550 kcal/day (6485 kJ/day) in May-June to 1900 kcal/day (7950 kJ/day) in March-April. In the pre-harvest period there was a reduction in physical activity. BMR (n = 17) remained at a constant level of about 1330 kcal/day (5556 kJ day) throughout the year.

Continuous intravenous infusion in the unrestrained rat--procedures and results.

1. A method of continuous infusion in the unrestrained rat is described, which provides a scientifically acceptable and easily maintained rodent model for use in toxicological investigations. 2. Sprague Dawley SPF rats had cannulas implanted into the vena cava via the femoral vein, and were continuously infused with physiological saline for a total of 28 or 90 days. 3. The results indicate that there was no change in body weight, food consumption, clinical observations or clinical biochemistry of infused rats when compared to non-infused rats. There were small changes in haematological parameters, however none were toxicologically significant. Urinary volume was increased and urinary specific gravity and osmolality were decreased. At macroscopic and microscopic examination there were findings of scar formation associated with the area of surgery and minimal irritation in the area of the vena cava which accommodated the cannula. 4. These results indicate that implantation of a cannula into the vena cava of a rat and subsequent continuous intravenous infusion of physiological saline produces no toxicological adverse effects over a period of 90 days. Consequently, this model can be recommended for the continuous intravenous administration of test substances to rats.

Continuous intravenous infusion in athymic (nude) rats: an animal model for evaluating the efficacy of anti-cancer agents.

The athymic (nude) rat (rnu/rnu) has been used for a number of years in research into various human tumours involving xenotransplantation. We now report the validation of a continuous intravenous infusion method in nude rats using a tail cuff tether, which enables the study of the efficacy of novel anti-cancer materials in this mutant strain, using intravenous infusion and with no restriction of the animals or of the tumour implantation sites by jackets. Ten animals each had a cannula surgically implanted into the vena cava via the femoral vein and exteriorized via a tail cuff. Animals were housed singly in conventional cages following surgery. Following a recovery period of 5 days all animals were continuously infused with physiological saline at an infusion rate of 0.5 ml/h for a further 37 days. Body weights and food consumption were recorded weekly. Blood samples were taken approximately 14 days post-surgery and analysed for haematology and clinical chemistry parameters. All animals were successfully cannulated, and no unexpected adverse clinical signs were noted during the recovery period and the 37 days of infusion. The results demonstrate that it is possible to surgically cannulate the femoral vein of athymic (nude) rats and infuse them in conventional cages for a period of up to 37 days with minimal adverse effects. The minimal restraint required provides benefits both to the animal and to the conduct of studies such as assessment of tumour growth in the absence of a jacket. Recent work has demonstrated that the same techniques can be successfully applied to the nude mouse.

A novel bile duct cannulation method with tail cuff exteriorization allowing continuous intravenous infusion and enterohepatic recirculation in the unrestrained rat.

A variety of bile duct cannulation methods have been used in the study of biliary excretion in the rat. We now report the validation and use of one such method. In this method, the common bile duct and duodenum were cannulated, the free ends of the cannulae tunnelled through the abdominal wall, passed through a trochar and exteriorized at the ventral aspect of the tail. A purpose-designed stainless steel tail cuff was then attached, to protect the cannulae from the rat. The cannulae were passed through the top of a metabolism cage and attached to a dual swivel that allows the rat freedom of movement within the metabolism cage. Where necessary an additional cannula could be placed in the femoral vein to allow infusion of test material or blood sampling. The results demonstrate that the method is robust and that its use allows a reliable correlation between surgically prepared and intact animals, as physiological parameters are allowed to return to normal prior to inclusion of the animals in the study. The technique allows the animals a great deal of freedom and, as such, is considered to minimize stress associated with the procedure. This fact is reflected in the reliability and reproducibility of the data obtained over the wide range of studies that have been conducted using this method. This method has been in use for over 4 years at Inveresk and this paper describes the authors' experience with the method to date.

[Methotrexate as possible treatment in refractory chronic inflammatory intestinal disease]. / Methotrexaat als mogelijke behandeling bij refractaire chronische inflammatoire darmziekte.

OBJECTIVE: To determine the value of low dose methotrexate therapy in chronic inflammatory bowel disease. SETTING: Catharina Hospital and Diaconessenhuis, Eindhoven, and St. Joseph Hospital, Veldhoven. DESIGN: Descriptive. METHOD: From 1988 until 1993 we treated 15 patients 16 times (one patient was treated twice) with methotrexate 25 mg i.m. once a week during 12 weeks, followed by a tapering oral dose. Our population consisted of 4 men and 11 women with a mean age of 31 year. The diagnosis was Crohn ileitis (1 patient), Crohn colitis (8), ileocolitis (4) and ulcerative colitis (2). The indication for methotrexate was resistance to therapy (9) and steroid dependency (7). Retrospectively the disease activity was determined after 1, 2 and 3 months of therapy. RESULTS: The mean defaecation frequency went down from 7 to 2 times daily after 12 weeks, the ESR from 47 to 17 mm/1st hour, the thrombocytes from 436 x 10(9)/l to 325 x 10(9)/l and the prednisone dose could be lowered from 22 mg to 15 mg after 3 months. In 13/16 treatment cases there was a subjective positive response to methotrexate. Initial response was seen within 4 weeks. No serious side effects were seen. After 10 of the 13 response episodes the disease recurred. CONCLUSION: Methotrexate 25 mg once a week i.m. was associated with a subjective and objective improvement in 12/15 patients, but the risk of recurrence after tapering appears to be large.

[An intrauterine and prenatal fasciola hepatica infection in a dairy herd in South Netherlands]. / Een intra-uteriene en prenatale leverbotinfectie op een rundveebedrijf in Zuid-Nederland.

A intra-uterine infection with Fasciola hepatica in a three years old cow is proved on a milking cow herd in the South of the Netherlands. Two calves showed to have a prenatal infection. Further investigations on the farm revealed a general presence of this parasite.

Validation of the QTL on SSC4 for meat and carcass quality traits in a commercial crossbred pig population.

Porcine chromosome 4 harbours many quantitative trait loci (QTL) affecting meat quality, fatness and carcass composition traits, detected in resource pig populations previously. However, prior to selection in commercial breeds, QTL identified in an intercross between divergent breeds require confirmation, so that they can be segregated. Consequently, the objective of this study was to validate several QTL on porcine chromosome 4 responsible for meat and carcass quality traits. The experimental population consisted of 14 crossbred paternal half-sib families. The region of investigation was the q arm of SSC4 flanked by the markers S0073 and S0813. Regression analysis resulted in the validation of three QTL within the interval: Minolta a* loin, back fat thickness and the weight of trimmed ham. The results were additionally confirmed by factor analysis. Candidate genes were proposed for meat colour, which was the most evident QTL validated in this study.

Variance component analysis of quantitative trait loci for pork carcass composition and meat quality on SSC4 and SSC11.

In a previous study, QTL for carcass composition and meat quality were identified in a commercial finisher cross. The main objective of the current study was to confirm and fine map the QTL on SSC4 and SSC11 by genotyping an increased number of individuals and markers and to analyze the data using a combined linkage and linkage disequilibrium analysis method. A modified version of the method excludes linkage disequilibrium information from the analysis, enabling the comparison of results based on linkage information only or results based on combined linkage and linkage disequilibrium information. Nine additional paternal half-sib families were genotyped for 18 markers, resulting in a total of 1,855 animals genotyped for 15 and 13 markers on SSC4 and SSC11, respectively. The QTL affecting meat color on SSC4 was confirmed, whereas the QTL affecting LM weight could not be confirmed. The combined linkage and linkage disequilibrium analysis resulted in the identification of new significant effects for 14 traits on the 2 chromosomes. Heritabilities of the QTL effects ranged from 1.8 to 13.2%. The analysis contributed to a more accurate positioning of QTL and further characterized their phenotypic effect. However, results showed that even greater marker densities are required to take full advantage of linkage disequilibrium information and to identify haplotypes associated with favorable QTL alleles.

Identification of quantitative trait loci for carcass composition and pork quality traits in a commercial finishing cross.

A QTL study for carcass composition and meat quality traits was conducted on finisher pigs of a cross between a synthetic Piétrain/Large White boar line and a commercial sow cross. The mapping population comprised 715 individuals evaluated for a total of 30 traits related to growth and fatness (4 traits), carcass composition (11 traits), and meat quality (15 traits). Offspring of 8 sires (n = 715) were used for linkage analysis and genotyped for 73 microsatellite markers covering 14 chromosomal regions representing approximately 50% of the pig genome. The regions examined were selected based on previous studies suggesting the presence of QTL affecting carcass composition or meat quality traits. Thirty-two QTL exceeding the 5% chromosome-wise significance level were identified. Among these, 5 QTL affecting 5 different traits were significant at the 1% chromosome-wise level. The greatest significance levels were found for a QTL affecting loin weight on SSC11 and a QTL with an effect on the Japanese color scale score of the loin on SSC4. About one-third of the identified QTL were in agreement with QTL previously reported. Results showed that QTL affecting carcass composition and meat quality traits segregated within commercial lines. Use of these results for marker-assisted selection offers opportunities for improving pork quality by within-line selection.

Genetic parameters for carcass composition and pork quality estimated in a commercial production chain.

Breeding goals in pigs are subject to change and are directed much more toward retail carcass yield and meat quality because of the high economic value of these traits. The objective of this study was to estimate genetic parameters of growth, carcass, and meat quality traits. Carcass components included ham and loin weights as primal cuts, which were further dissected into boneless subprimal cuts. Meat quality traits included pH, drip loss, purge, firmness, and color and marbling of both ham and loin. Phenotypic measurements were collected on a commercial crossbred pig population (n = 1,855). Genetic parameters were estimated using REML procedures applied to a bivariate animal model. Heritability estimates for carcass traits varied from 0.29 to 0.51, with 0.39 and 0.51 for the boneless subprimals of ham and loin, respectively. Heritability estimates for meat quality traits ranged from 0.08 to 0.28, with low estimates for the water holding capacity traits and higher values for the color traits: Minolta b*(0.14), L* (0.15), a* (0.24), and Japanese color scale (0.25). Heritability estimates differed for marbling of ham (0.14) and loin (0.31). Neither backfat nor ADG was correlated with loin depth (r(g) = 0.0), and their mutual genetic correlation was 0.27. Loin primal was moderately correlated with ham primal (r(g) = 0.31) and more strongly correlated with boneless ham (r(g) = 0.58). Backfat was negatively correlated with (sub)primal cut values. Average daily gain was unfavorably correlated with subprimals and with most meat quality characteristics measured. Genetic correlations among the color measurements and water-holding capacity traits were high (average r(g) = 0.70), except for Minolta a* (average r(g) = 0.17). The estimated genetic parameters indicate that meat quality and valuable cut yields can be improved by genetic selection. The estimated genetic parameters make it possible to predict the response to selection on performance, carcass, and meat quality traits and to design an effective breeding strategy fitting pricing systems based on retail carcass and quality characteristics.

Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus.

Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.

Heptatic and rectal pathology in Schistosoma intercalatum infection.

Forty nine needle biopsies and 16 rectal biopsies taken from patients infected with S. intercalatum at the Ndoungué hospital (Department of Mungo, Cameroon) were studied. The histopathological changes were compared with the characteristic liver changes in S. mansoni infections. The pathological findings and clinical data were correlated in order to evaluate the extent of the liver changes in isolated S. intercalatum infections and in S. intercalatum infections assoicated with a concomitant disease. Outside the portal triangle eggs were less frequently found in S. intercalatum infection than in S. mansoni infection. This may be explained by the greater dimensions of S. intercalatum eggs. S. intercalatum granulomata, observed only in the portal triangle, had smaller dimensions than S. mansoni granulomata. S. intercalatum infection did not cause liver-cell destruction. Parenchymal lesions, when present, were always due to other causes (concomitant diseases). The liver cells sometimes showed aspecific changes such as pleomorphism of cells or nuclei or doubling of nuclei as a reaction to the toxic effect of schistosomal metabolites or to egg secretions or the inflammatory process around eggs. These changes were not associated with any impairment of liver function. No profound vascular changes such as are found in S. mansoni hepatosplenic schistosomiasis could be demonstrated; this explains the clinically noted absence of portal hypertension. Pigment formation was extensive in S. intercalatum infection; probably more pigment was present in the liver than in S. mansoni infection. In all patients the Kupffer cells were hypertrophied. The cause of the differences in the liver lesions of S. intercalatum and S. MANSONI INFECTION IN MAN MIGHT BE A LOWER IMMUNE RESPonse to S. intercalatum egg antigens.

Schistosoma intercalatum infection in Syrian hamsters. An experimental and histopathological study.

Syrian hamsters were infected with increasing numbers of circariae of Schistosoma intercalatum and Schistosom mansoni. Various organs were then studied histopathologically. These studies revealed a relatively higher pathogenicity of S. intercalatum in rodents than in human cases reported in a previous communication. Yet the magnitude of infection and its complications were milder in cases of S. intercalatum than in S. mansoni. The absence of liver fibrosis or cirrhosis in S. intercalatum infection is in agreement with our previous experience with human subjects. It is apparent that this parasite produces much more pigment than S. mansoni and the other well known types of Schistosomas. The development of the granulomatous reaction, mainly in liver, rectal mucosa and in lung parenchyma of infected animals seems to be a similar process in all types of Schistosoma infections without major qualitative differences and irrespective of the density of infection. The worm load as roughly indicated by the worm return through perfusion is not always indicative of the severity of infection since a very low worm load in a long term infection can lead to the same results as a short term very dense infection. Some evidence suggesting the possibility of 'reaction' around pulmonary blood vessels, not related to the granulomatous lesion was found. This finding requires further immunological investigation.

Schistosomiasis at Loum, Cameroun; Schistosoma haematobium, S. intercalatum and their natural hybrid.

A survey of 500 schoolchildren in Loum in 1968 revealed an overall infection rate of 54.2% with Schistosoma intercalatum and this was the only species of schistosome encountered. In 1972 a number of children were found to be passing schistosome eggs in their urine and these eggs ranged in shape and size from the forms characteristic for S. haematobium to those of S. intercalatum. Preliminary laboratory studies demonstrated that hybridisation between the two species was occurring. Subsequent field surveys showed that the snail hosts for the two parasites (B. rohlfsi for S. haematobium and B. forskali for S. intercalatum) were both present in the river Mbette and its tributaries in Loum and the distribution of the two snail species coincided closely with the distribution of the schistosomes in the human population. Detailed study of a small group of children passing hybrid eggs in their urine revealed that few of them were passing eggs in their faeces and that those eggs which were found in faeces were not viable. Analysis of schistosome egg-shape by plotting cumulative size-frequency data on probability paper demonstrated that the graph obtained from a natural hybrid series was different from that given by a known mixture of the two separate species. The hybrid series included a number of exceptionally large eggs resembling those of S. bovis but isolation of these eggs and subsequent laboratory passage of the parasites showed that they were part of the series and were not evidence of the presence of a third species. Hybridisation experiments in the laboratory showed that the cross S. haematobium male X S. intercalatum femal is fully viable but that the reverse mating is not successful, thus accounting for the failure of the faecal eggs recovered from children with hybrid infections. Histological results from laboratory passaged hybrids suggest that the Ziehl-positive staining reaction of the egg-shells of S. intercalatum may be a recessive character. The observations reported here indicate that S. haematobium has only recently become established in Loum and that it is, through introgressive hybridisation, replacing the indigenous S. intercalatum. A suggested explanation for the change in the parasite fauna is offered and this depends upon ecological changes resulting from forest clearance and agricultural development providing improved conditions for the spread of B. rohlfsi, the snail host for S. haematobium. It is suggested that, in contrast to recent reports on the spread of S. intercalatum, this species is in fact retreating and being replaced by S. haematobium in areas where forest clearance is taking place. In conclusion it is suggested that introgressive hybridisation of this kind may have been responsible for the evolution of certain characteristic local strains of African schistosomes.

The plain abdominal radiograph in the assessment of constipation.

To investigate the value of the plain abdominal radiograph in the assessment of constipation we prospectively studied 30 patients (20 females, 10 males, mean age 48.6 years, range 21-76 years). These patients underwent the following tests: plain abdominal radiograph in supine position, measurement of stool weight (mean of 5 days) and whole gut transit time (20 radioopaque pellets, fluoroscopy of stools). All patients completed a questionnaire regarding their defaecation characteristics (a.o. average defaecation frequency and faecal consistency). The abdominal films were coded and independently scored for the degree of faecal stasis in ascending, transverse, descending colon and rectosigmoid by four gastroenterologists. To each of these parts of the colon a score of 1 (no faeces) to 4 (loaded with faeces) was assigned. The film scores given by the four observers were significantly correlated (p less than 0.001). Significant correlations were found between the radiograph scores and frequency of defaecation, faecal consistency and stool weight. The strength of these correlations were of the same order of magnitude as those between the other subjective and objective defaecation variables. The scores for the left colon (descending colon and rectosigmoid) showed a better correlation with the other defaecation parameters than the scores for the right colon. It is concluded that in the assessment of constipation a simple plain abdominal radiograph is as reliable as measurement of faecal weight or marker transit and can thus be advocated as the first procedure. When an abdominal radiograph is used for this purpose the stasis in the descending and sigmoid colon provides most information.