Studies of human polyomaviruses, with HPyV7, BKPyV, and JCPyV present in urine of allogeneic hematopoietic stem cell transplanted patients with or without hemorrhagic cystitis.

BACKGROUND: BK polyomavirus (BKPyV) can cause hemorrhagic cystitis (HC) in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients and polyomavirus-associated nephritis in renal transplant patients, while JC polyomavirus (JCPyV) can generate progressive multifocal leukoencephalopathy in immunocompromised individuals. Since 2007, additional human polyomaviruses (HPyVs) have been identified. In this study, we examined the urines of allo-HSCT patients for possible presence of polyomaviruses BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and HPyV10 (MWPyV). METHODS: A total of 185 urinary samples obtained 2002-2007 from 105 allo-HSCT patients, 32/105 with HC, were tested for the above-listed HPyVs by a bead-based multiplex assay. Of these, 142 urine samples had previously been tested for BKPyV and JCPyV by nested polymerase chain reaction (PCR). RESULTS: Aside from BKPyV and JCPyV, which dominated, HPyV7 was detected in 5 BKPyV-positive urinary samples from 1 patient. The multiplex assay was more sensitive and specific than the nested PCR. BKPyV and/or JCPyV were found in all but 1 of the previously BKPyV- or JCPyV-positive samples, although 6 previously BKPyV-positive cases were now JCPyV-positive or the reverse. Furthermore, 18/79 previously negative samples were found to be BKPyV and/or JCPyV positive, and a total of 21 double infections were found. Lastly, in 1/29 HC patients, only JCPyV was detected. CONCLUSION: HPyV7 was found for the first time in urine of an allo-HSCT patient, and BKPyV and JCPyV were more commonly found in urine samples using the bead-based assay compared to testing by nested PCR. Finally, only JCPyV was detected in the urine of 1 HC patient.

Lack of detection of Porcine circovirus 3 (PCV-3) in formalin-fixed, paraffin- embedded tissues from porcine abortions in Switzerland.

INTRODUCTION: The novel Porcine circovirus 3 (PCV-3) has been associated in the past years to different porcine diseases, including reproductive failure. The potential occurrence of PCV-3 in abortions from Swiss pig herds has not been investigated so far. Thus, we conducted a retrospective study on pig aborted cases submitted to our laboratory in the University of Bern during the last 10 years with the main aim of investigating the possible presence of PCV-3 in foetal and/or placental tissue. Twelve out of the 53 studied cases showed mild histopathological changes as previously described in PCV-3 positive cases. However, in none of the cases, PCV-3 genetic material could be detected in the examined formalin-fixed, paraffin-embedded tissues. In only one third of the cases, a cause for the abortion was found, which is similar to other studies. Our survey suggests that PCV-3 was not involved in the porcine abortion cases submitted over the last decade at our institution in Switzerland.

INTRODUCTION: Le nouveau Circovirus porcin 3 (PCV-3) a été associé ces dernières années à différentes maladies porcines, y compris des troubles de la reproduction. La présence potentielle du PCV-3 dans les avortements de porcs en Suisse n'a pas été étudiée jusqu'à présent. Nous avons donc mené une étude rétrospective sur les cas d'avortements de porcs soumis à notre laboratoire de l'Université de Berne au cours des 10 dernières années, dans le but principal d'étudier la présence éventuelle du PCV-3 dans les tissus fœtaux et/ou placentaires. Douze des 53 cas étudiés présentaient des changements histopathologiques légers, tels que décrits précédemment dans les cas positifs au PCV-3. Cependant, dans aucun des cas, le matériel génétique du PCV-3 n'a pu être détecté dans les tissus examinés fixés au formol et inclus en paraffine. Dans un tiers des cas seulement, une cause d'avortement a été trouvée, ce qui est similaire à d'autres études. Notre étude suggère que le PCV-3 n'a pas été impliqué dans les cas d'avortements porcins soumis au cours de la dernière décennie dans notre institution en Suisse.

Genetic relationships among mastitis and alternative somatic cell count traits in the first 3 lactations of Swedish Holsteins.

The objectives of this study were to estimate heritabilities of, and genetic correlations among, clinical mastitis (CM), subclinical mastitis (SCM), and alternative somatic cell count (SCC) traits in the first 3 lactations of Swedish Holstein cows, and to estimate genetic correlations for the alternative traits across lactations. Data from cows having their first calving between 2002 and 2009 were used. The alternative SCC traits were based on information on CM and monthly test-day (TD) records of SCC traits of 178,613, 116,079, and 64,474 lactations in first, second, or third parity, respectively. Sires had an average of 230, 165, or 124 daughters in the data (parities 1, 2, or 3, respectively). Subclinical mastitis was defined as the number of periods with an SCC >150,000 cell/mL and without a treatment for CM. Average TD SCC between 5 and 150 d was used as a reference trait. The alternative SCC traits analyzed were 1) presence of at least 1 TD SCC between 41,000 and 80,000 cell/mL (TD41-80), 2) at least 1 TD SCC >500,000 cells/mL, 3) standard deviation of log SCC over the lactation, 4) number of infection peaks, and 5) average days diseased per peak. The same variables in different parities were treated as distinct traits. The statistical model considered the effects of herd-year, year, month, age at calving, animal, and residual. Heritability estimates were 0.07 to 0.08 for CM, 0.12 to 0.17 for SCM, and 0.14 for SCC150. For the alternative traits, heritability estimates were 0.12 to 0.17 for standard deviation of log SCC, TD SCC >500,000 cells/mL, and average days diseased per peak, and 0.06 to 0.10 for TD41-80 and number of infection peaks. Genetic correlations between CM with SCM were 0.62 to 0.74, and correlations for these traits with the alternative SCC traits were positive and very high (0.67 to 0.82 for CM, and 0.94 to 0.99 for SCM). Trait TD41-80 was the only alternative trait that showed negative, favorable, genetic correlations with CM (-0.22 to -0.50) and SCM (-0.48 to -0.85) because it is associated with healthy cows. Genetic correlations among the alternative traits in all 3 parities were high (0.93 to 0.99, 0.92 to 0.98, and 0.78 to 0.99, respectively). The only exception was TD41-80, which showed moderate to strong negative correlations with the rest of the traits. Genetic correlations of the same trait across parities were in general positive and very high (0.83 to 0.99). In conclusion, these alternative SCC traits could be used in practical breeding programs aiming to improve udder health in dairy cattle.

Characterization of an outbreak of equine coronavirus infection in adult horses in Switzerland.

INTRODUCTION: Outbreaks of equine coronavirus (ECoV) infections have been described in different parts of the world including Europe. The aim of this report was to describe clinical signs, diagnostic work-up and outcome of the first documented outbreak of ECoV in Switzerland in order to raise the awareness for the disease and its various clinical presentations. The outbreak occurred on a farm with 26 horses. Of these, seven horses developed clinical disease ranging from mild signs such as fever and anorexia to severe signs of acute colitis. One horse died due to severe endotoxemia and circulatory shock secondary to severe acute necrotizing enteritis and colitis. Out of the 26 horses, five horses tested positive for ECoV, including two ponies without any clinical signs of infection. The low number of positive cases should nevertheless be interpreted with caution as testing was only performed on one occasion, over a month after the onset of clinical signs in the first suspected case. This report highlights the importance of diagnostic testing and early implementation of biosecurity measures on a farm with an ECoV outbreak. It should furthermore raise the awareness for unspecific and mild clinical signs such as fever and anorexia in affected animals that are potentially able to spread the disease.

INTRODUCTION: Des foyers d'infection à coronavirus équin (ECoV) ont été décrits dans différentes parties du monde, y compris en Europe. L'objectif de ce rapport est de décrire les signes cliniques, le diagnostic et les conséquences du premier foyer d'ECoV documenté en Suisse, afin de sensibiliser le public à cette maladie et à ses différents aspects cliniques. L'épidémie s'est produite dans une écurie comptant 26 chevaux. Parmi ceux-ci, sept chevaux ont développé une forme clinique allant de signes légers tels que la fièvre et l'anorexie à des signes sévères de colite aiguë. Un cheval est mort en raison d'une endotoxémie sévère et d>un choc circulatoire secondaire à une entérite nécrosante aiguë sévère et à une colite. Sur les 26 chevaux, cinq ont été testés positifs à l>ECoV, dont deux poneys sans aucun signe clinique d'infection. Le faible nombre de cas positifs doit néanmoins être interprété avec prudence car les tests n'ont été effectués qu'à une seule occasion, plus d'un mois après l'apparition des signes cliniques chez le premier cas suspect. Ce rapport souligne l'importance des tests de diagnostic et de la mise en œuvre rapide de mesures de biosécurité dans une exploitation où un foyer d'ECoV est détecté. Il devrait en outre sensibiliser à la présence de signes cliniques peu spécifiques et bénins tels que la fièvre et l'anorexie chez les animaux atteints qui sont potentiellement capables de propager la maladie.

Phenotypic and genetic characterization of novel somatic cell count traits from weekly or monthly observations.

The objectives of this study were (1) to explore traits that better capture weekly or monthly changes in somatic cell counts (SCC) than does the commonly used lactation-average SCC, (2) to estimate their heritabilities and relationships to clinical mastitis (CM), and (3) to determine if these traits are feasible for use in monthly testing schemes. Clinical mastitis and weekly test-day (TD) records of SCC and milk production traits from 1,006 lactations of Swedish Red and Holstein cows collected from 1989 to 2004 were used (data set W). A data subset was also created to mimic monthly recording (data set M, 980 lactations). Twenty SCC traits were defined, taking into account SCC general levels and variation along the lactation curve, time and level of infection, and time of recovery. To reduce dimensionality, cluster and stepwise logistic regression procedures were applied. In data set W, 3 traits, "standard deviation of SCC over the lactation," a discrete (0/1) indicator of "at least one TD with SCC >500,000 cells/mL", and "number of days sick in the widest SCC peak" (DWidest) were the variables kept both with cluster procedures and a stepwise logistic regression with the logit of CM as dependent variable. In data set M, DWidest was replaced by "number of SCC peaks" and "average number of days sick per peak" (ADSick). Lactation-average SCC (in the first 150 d or between 150 and 305 d) did not enter into the logistic regression. Heritability estimates obtained for these new traits under a Bayesian setting and a Gibbs sampling approach were 10 to 16% (except for ADSick: 5%). Heritabilities were at least as high in the monthly data set as in the weekly data set. Thus, these SCC traits seem promising for use in breeding programs based on monthly milk recording.

Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.

Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Regulation of apolipoprotein A-I synthesis in lymph-drained rats.

The effect of lymph diversion on plasma apolipoprotein A-I levels was studied. In lymph fistula rats apolipoprotein A-I levels in plasma stayed constant in spite of a loss of an equivalent of one-half plasma pool of apolipoprotein A-I per day through the lymph fistula. This indicates that synthesis of apolipoprotein A-I increases or that catabolism of apolipoprotein A-I decreases in a compensatory manner as intestinal apolipoprotein A-I is diverted. By using incorporation of [3H]leucine into newly synthesized apolipoprotein A-I it was shown that 2.6-times as much [3H]leucine was incorporated into apolipoprotein A-I in thoracic duct drained animals compared to controls. In experiments in which 125I-labeled HDL was injected intravenously into rats, it was shown that catabolism of HDL and apolipoprotein A-I was not decreased in lymph-drained rats. These data thus suggest that an increased synthesis of apolipoprotein A-I occurs when the intestinal contribution of apolipoprotein A-I diminishes. This is probably due to an increase in liver protein synthesis.

Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase.

The binding of NADH to uridine diphosphate glucose dehydrogenase has been examined by equilibrium dialysis. There is an absolute requirement for the presence of UDP-glucose for the binding of NADH. Other analogs such as UDPxylose, UDPgalactose and UDPglucuronic acid cannot replace UDPglucose as an effector of NADH binding. UDPxylose competes with UDPglucose for the UDP-sugar-binding site, and in so doing releases the bound NADH. The binding of NADH to UDPglucose dehydrogenase in the presence of UDPglucose reaches a saturation limit of 3 mol NADH bound per enzyme hexamer, and displays positive cooperativity, Hill number = 1.34. The effects of UDP-sugars on the fluorescence of UDPglucose dehydrogenase derivatized at the catalytic sites with a fluorophore have also been studied. Two classes of UDPxylose-binding site have been detected. One class has high affinity (Kdiss = 3 microM, determined by equilibrium dialysis) but does not affect fluorophore fluorescence, and the other has lower affinity (Kdiss = 120 microM) and leads to red-shifted fluorescence quenching, presumably by effecting exposure of the fluorophore to solvent. The high-affinity sites are identified as the UDP-sugar subsites of the underivatized catalytic sites, and the low-affinity sites as UDP-sugar subsites of the fluorophore-labeled catalytic sites.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.

Methanol Emission from Leaves (Enzymatic Detection of Gas-Phase Methanol and Relation of Methanol Fluxes to Stomatal Conductance and Leaf Development).

We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall [1993] Atmos Environ 27A: 1709-1713). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from several species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methanol exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 [mu]g g-1 fresh weight in young leaves to 10.0 [mu]g g-1 fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.

Low serum apolipoprotein A-I in acute myocardial infarction survivors with normal HDL cholesterol.

High density lipoprotein (HDL) cholesterol, apolipoprotein A-I (apo A-I), gamma-glutamyl-transferase, testosterone and oestradiol were determined in plasma in non-diabetic males who had survived an acute myocardial infarction (AMI) before the age of 60. They also had serum levels of cholesterol below 7.0 mmol/l 1 year after the AMI. On the basis of diastolic blood pressure they were subdivided into 2 groups with diastolic blood pressures below 90 mm Hg (n = 39), and with or above 95 mm Hg (n = 21) and then compared with an age-matched male non-diabetic reference group (n = 32). There were no significant differences in the levels of HDL cholesterol, gamma-glutamyl-transferase, and sex hormones between the AMI groups and the reference group. Reduced plasma levels of apo A-I were found in the AMI groups.

Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy.

Low density lipoprotein (LDL) induced accumulation of cholesterol esters was analyzed by the digital imaging fluorescence microscopy (DIFM) in murine tumor macrophages. To analyze cholesterol ester accumulation, P388D1 macrophages were incubated with increasing quantities of unmodified or acetylated human LDL, washed, and live stained with a lipophylic fluorescent dye Nile Red. The increase in fluorescence intensity was quantitatively determined by the interactive laser cytometer (ACAS 470) and compared with the accumulation of cellular cholesterol esters determined by the gas liquid chromatography. Correlation between the two methods was highly significant (r greater than 0.9, P less than 0.001). A good agreement between the two methods was also found in terms of sensitivity and reproducibility. With the use of 589 nm narrowband interference filter in the light path of emitted light the intensity of fluorescence correlated well with cellular cholesterol ester content even in the presence of relatively high concentrations of triglycerides. Therefore, digital imaging fluorescence microscopy appears to be a reliable method for quantification of cholesterol ester accumulation at the single cell level offering new possibilities of studying interactions between cells and cholesterol ester rich lipoproteins.

The effects of smoking, hypertension and cardiovascular disease on the estimation of unscheduled DNA synthesis and covalent binding to DNA induced by N-acetoxy-2-acetylaminofluorene in resting human mononuclear leukocytes.

The levels of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis (UDS) and of NA-AAF binding to DNA have been determined in resting mononuclear leukocytes from individuals with various smoking habits, heart infarct patients and subjects diagnosed for hypertension. Age-matched and blood-pressure-controlled smokers (n = 99) had significantly elevated levels of NA-AAF-induced UDS and NA-AAF binding to DNA when compared to nonsmokers (n = 75) similarly corrected for age and blood pressure. Heart infarct patients without any history of risk factors, as well as diagnosed hypertensives with normalized blood pressure, were not significantly different from matched controls when assessed by the NA-AAF method. Our results support the theory that increased mutagen sensitivity is associated with smoking and high blood pressure but not with cardiovascular disease itself via some mechanism of genetic selection.