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.

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.

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.

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.

Carbon-carbon bond formation in regio- and stereoselective palladium-catalyzed cyclization of allene-substituted conjugated dienes.

Regio- and stereoselective palladium-catalyzed reactions of allene-substituted 1,3-dienes 1 in acetic acid at room temperature lead to cyclization with formation of a carbon-carbon bond between the middle carbon of the allene and the terminal carbon of the 1,3-diene. Two different types of reactions, both that constitute 1,4-carboacetoxylations of the 1,3-diene, have been developed. In one of the reactions, Pd(II) catalyzes the oxidation of 1 to bicyclic compounds 2, and in the other, Pd(0) catalyzes the transformation of 1 to bicyclic compounds 3. The products 2 are useful for further synthetic transformations and undergo Diels-Alder reactions with dienophiles to give polycyclic ring systems.

Numerical dating of the Eckfeld maar fossil site, Eifel, Germany: a calibration mark for the Eocene time scale.

Sediments of the Eckfeld maar (Eifel, Germany) bear a well-preserved Eocene fauna and flora. Biostratigraphically, Eckfeld corresponds to the Middle Eocene mammal reference level MP (Mammals Paleogene) 13 of the ELMA (European Land Mammal Age) Geiseltalian. In the maar crater, basalt fragments were drilled, representing explosion crater eruption products. By 40Ar/39Ar dating of the basalt, for the first time a direct numerical calibration mark for an Eocene European mammal locality has been established. The Eckfeld basalt inverse isochron date of 44.3 +/- 0.4 Ma suggests an age for the Geiseltalian/Robiacian boundary at 44 Ma and, together with the 1995 time scale of Berggren et al., a time span ranging from 49 to 44 Ma for the Geiseltalian and from 44 to 37 Ma for the Robiacian, respectively. Additional 40Ar/39Ar dating on a genetically related basalt occurrence close to the maar confirms a period of volcanism of ca. 0.6 m.y. in the Eckfeld area, matching the oldest Eocene volcanic activity of the Hocheifel volcanic field.

Analysis of non-covalent protein complexes up to 290 kDa using electrospray ionization and ion trap mass spectrometry.

Non-covalently-bound subunit complexes of proteins have been measured by an ion trap mass spectrometer equipped with an orthogonal electrospray ionization source. For the analysis of the generated molecular ions with high mass/charge ratios, the mass/charge range of the ion trap was extended by increasing its radio frequency (rf) voltage to 15 kV (V(0-p)) and by resonant ion ejection. Ions of the non-covalent dimer of bovine serum albumin (BSA), as well as of subunit complexes of alcohol dehydrogenase (ADH) from bakers' yeast and from horse liver, have been detected at mass/charge values between 3000-9000 Th. The maximum observed molecular weight was that of a non-covalently-bound subunit-octamer of bakers' yeast ADH (two non-covalently-bound subunit-tetramers) at ca. 290 kDa.

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.

Domain 5 binds near a highly conserved dinucleotide in the joiner linking domains 2 and 3 of a group II intron.

Photocrosslinking has identified the joiner between domains 2 and 3 [J(23)] as folding near domain 5 (D5), a highly conserved helical substructure of group II introns required for both splicing reactions. D5 RNAs labeled with the photocrosslinker 4-thiouridine (4sU) reacted with highly conserved nucleotides G588 and A589 in J(23) of various intron acceptor transcripts. These conjugates retained some ribozyme function with the lower helix of D5 crosslinked to J(23), so they represent active complexes. One partner of the gamma x gamma' tertiary interaction (A587 x U887) is also in J(23); even though gamma x gamma' is involved in step 2 of the splicing reaction, D5 has not previously been found to approach gamma x gamma'. Similar crosslinking patterns between D5 and J(23) were detected both before and after step 1 of the reaction, indicating that the lower helix of D5 is positioned similarly in both conformations of the active center. Our results suggest that the purine-rich J(23) strand is antiparallel to the D5 strand containing U32 and U33. Possibly, the interaction with J(23) helps position D5 correctly in the ribozyme active site; alternatively, J(23) itself might participate in the catalytic center.

The occipital torus and developmental age of Sangiran-3.

Since its discovery in 1938 Sangiran-3 has been considered a juvenile Pithecanthropus (Homo) erectus, and therefore, excluded from studies of adult H. erectus. Although morphological features align Sangiran-3 with H. erectus, its age designation rests on an unconvincing reconstruction of the occipital torus and lack of sutural fusion. Evaluation of the occipital shows the original reconstruction is faulty and that the current midline occipital torus is actually the right lateral torus. The new reconstruction of Sangiran-3 results in midline toral morphology and development that is comparable with that in Sangiran-2. Compared with juvenile and adult H. erectus and Homo sapiens Sangiran-3 has three fully developed layers of vault bone with localized hypertrophy of the outer table into a sagittal keel, bregmatic eminence, and occipital torus. Sangiran-3's absolute vault thickness is also within the range of adult H. erectus. In addition, the coronal suture is fully interdigitated and sagittal sutural complexity is consistent with adult H. erectus. Sangiran-3's parietal sagittal contours are indistinguishable from adult H. erectus, whereas sagittal vault contours of juvenile H. erectus are usually more rounded than adults. These features indicate that Sangiran-3 is best considered a young adult H. erectus and should be included in metric and non-metric analyses of this taxon.

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.

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.

Catalytically critical nucleotide in domain 5 of a group II intron.

Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems.

Thermal activation of a group II intron ribozyme reveals multiple conformational states.

Conformational changes often accompany biological catalysis. Group II introns promote a variety of reactions in vitro that show an unusually sharp temperature dependence. This suggests that the chemical steps are accompanied by the conversion of a folded-but-inactive form to a differently folded active state. We report here the kinetic analysis of 5'-splice-junction hydrolysis (SJH) by E1:12345, a transcript containing the 5'-exon plus the first five of six intron secondary structure domains. The pseudo-first-order SJH reaction shows (1) activation by added KCl to 1.5 M; (2) cooperative activation by added MgCl2, nHill = 4.1-4.3, and [MgCl2]vmax/2 approximately 0.040 M; and (3) a rather high apparent activation energy, Ea approximately 50 kcal mol-l. In contrast, the 5'-terminal phosphodiester bond of a domain 5 transcript (GGD5) was hydrolyzed with Ea approximately 30 kcal mol-1 under SJH conditions; the 5'-GG leader dinucleotide presumably lacks secondary structure constraints. The effect of adding the chaotropic salt tetraethylammonium chloride (TEA) was also investigated. TEA reduced the melting temperatures of GGD5 and E1:12345. TEA also shifted the profile of rate versus temperature for SJH by E1:12345 toward lower temperatures without affecting the maximum rate. TEA had little effect on the rate of hydrolysis of the 5'-phosphodiester bond of GGD5. The high apparent activation enthalpy and entropy for SJH along with the effect of TEA on these parameters imply that conversion of an inactive form of E1:12345 to an active conformation accompanies enhanced occupation of the transition state as the temperature is raised to that for maximum SJH. Analytical modeling indicates that either a two-state model (open and disordered, with open being active) or a three-state model (compact, open, and disordered) could account for the temperature dependence of kSJH. However, the three-state model is clearly preferable, since it does not require that the activation parameters for phosphodiester bond hydrolysis exhibit exceptional values or that the rates for the chemical steps of SJH respond directly to TEA addition.

Domain 5 interacts with domain 6 and influences the second transesterification reaction of group II intron self-splicing.

The role of domain 5 (d5) from the self-splicing group II intron 5 gamma of the COXI gene of yeast mitochondrial DNA in branching and 3' splice site utilization has been studied using a substrate transcript lacking d5 (delta d5 RNA). This RNA is completely unreactive in vitro, but releases 5' exon by hydrolysis under various reaction conditions when d5 RNA is added in trans. Under an extreme reaction condition, some accurate branching and splicing occur. Much more efficient use of a 3' splice site is obtained when delta d5 RNA is complemented by a transcript containing the wild-type domains 5 and 6 plus the 3' exon. While most delta d5 RNA molecules in that protocol still react by hydrolysis at the 5' splice site, the branching that occurs uses only the d6 tethered to d5 that is provided in trans. The use of this d6 and the 3' splice site also linked to d5, along with the observed indifference to the other d6 and 3' splice site resident in the delta d5 RNA, indicates that d5 plays a key role in positioning d6 for the first reaction step as well as in 3' splice site use. Two models for the manner by which d5 interacts with d6 are discussed.

Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5.

The 5' splice junction (5'SJ) of Group II intron transcripts is subject to a specific hydrolysis reaction (SJH). This reaction occurs either within a single transcript containing intron sequences through domain 5 (D5) or by cooperation of two separate transcripts, one bearing the 5'SJ and another contributing D5 (1). In this report we describe the latter reaction in terms of its kinetic parameters. A minimal D5 RNA of 36 nts (GGD5) was sufficient to promote SJH of a second transcript containing the 5' exon plus intron domains 1, 2, and 3 (E1:123). Equimolar production of two RNAs, the 5' exon (E1) and an intron fragment containing domains 1, 2, and 3 (123) was observed. The kinetic coefficients were evaluated by an excess GGD5 approach. The apparent Km was complex, varying with GGD5 concentration. This behavior indicates heterogeneity in E1:123 with respect to GGD5 binding. The binding heterogeneity may result from formation of E1:123 dimers or from nicks in some molecules of each E1:123 preparation. The heterogeneity was always evident, but to a variable degree, regardless of the procedure by which E1:123 was isolated. The system may be described in terms of parameters analogous to kcat and Km. At infinite dilution of GGD5, the characterizing values were: k2 degrees (the analog of kcat) = 0.0055 min-1 and Km degrees = 0.22 microM. In the limit of GGD5 saturation, the values were: k2 infinity = 0.012 min-1 and Km infinity = 4.5 microM. A natural variant D5, representing the sequence from intron 1 of the yeast cytochrome-b gene, was also functional in SJH. This GGD5b1 was governed by similar Km degrees and Km infinity values, but was only one-third as active over the entire D5 concentration range. A different D5 isomer was entirely ineffective for SJH.

Accurate preparation of the patella during total knee arthroplasty.

The development of a resection guide that facilitates accurate preparation of the patella during total knee arthroplasty is reported. Use of the guide prevents tilting of the prosthesis, as well as over or underresection of the patella.

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.