Linkage of in situ ruminal degradation of crude protein with ruminal degradation of amino acids and phytate from different soybean meals in dairy cows.

The objectives of this study were to determine the range in ruminal degradability of crude protein (CP) and intestinal digestibility of rumen undegradable protein in commercial soybean meal (SBM) and to investigate the range in in situ ruminal AA and phytate (InsP6) degradation and their relationship to CP degradation. An in situ study was conducted using 3 lactating Jersey cows with permanent rumen cannulas. Seventeen SBM variants from Europe, Brazil, Argentina, North America, and India were tested for ruminal CP and AA degradation, and in vitro intestinal digestibility of rumen undegradable protein. Nine variants were used to investigate the ruminal degradation of InsP6. The estimated rapidly degradable fraction (a) of CP showed an average value of 4.5% (range: 0.0%-9.0%), the slowly degradable fraction (b) averaged 95% (91%-100%), and the potential degradation was complete for all 17 SBM variants. The degradation of fraction b started after a mean lag phase of 1.7 h (1.1-2.0 h) at an average rate (c) of 10% per hour, but with a high range from 4.5% to 14% per hour. Differences in the degradation parameters induced a considerable range in CP effective degradation at a rumen passage rate of 6% per hour (CPED6) from 38% to 67%; hence, the concentration of rumen undegradable protein varied widely from 33% to 62%. The range in AA degradation between the SBM variants was high, with Ser showing the widest range, from 28% to 96%, and similar for the other AA. The regression equations showed close relationships between CP and AA degradation after 16 h of in situ incubation. However, the slopes of the linear regressions were significantly different between AA, suggesting that degradation among individual AA differs upon a change in CP degradation. The concentrations of InsP6 and myo-inositol pentakisphosphate in bag residues in the in situ study decreased constantly with longer ruminal incubation times. The ruminal degradation parameters of InsP6 ranged from 11% to 37% for fraction a, 63% to 89% for fraction b, and from 7.7% to 21% per hour for degradation rate c, with average values of 21%, 79%, and 16% per hour, respectively. The calculated InsP6 effective degradation at a rumen passage rate of 6% per hour (InsP6ED6) varied from 61% to 84% among the SBM variants. Significant correlations were detected between InsP6ED6 and CPED6 and between InsP6ED6 and chemical protein fractions A, B1, B2, B3, and C. Linear regression equations were developed to predict ruminal InsP6 degradation using CPED6 and chemical protein fractions B3 and C chosen by a stepwise selection procedure. We concluded that a high range in CP, AA, and InsP6 degradation exists among commercial SBM, suggesting that general degradability values may not be precise enough for diet formulation for dairy cows. Degradation of CP in SBM may be used to predict rumen degradation of AA and InsP6 using linear regression equations. Degradation of CP and InsP6 could also be predicted from the chemical protein fractions.

Two different prenatal imaging cerebral patterns of tubulinopathy.

To illustrate the prenatal cerebral imaging features associated with tubulinopathy, we report on five affected fetuses from unrelated families, with a de-novo heterozygous variant in a tubulin gene (TUBA1A, TUBB2B or TUBB3). We identified two distinct prenatal imaging patterns related to tubulinopathy: a severe form, characterized by enlarged germinal matrices, microlissencephaly and a kinked brainstem; and a mild form which has not been reported previously in the prenatal literature. The latter form is associated with non-specific features, including an asymmetric brainstem, corpus callosal dysgenesis, a lack of Sylvian fissure operculization and distortion of the anterior part of the interhemispheric fissure with subsequent impacted medial borders of the frontal lobes, the combination of which, in the absence of additional extracerebral anomalies, is highly suggestive of tubulinopathy. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Inhibition of heat stress-related apoptosis of chicken myocardial cells through inducing Hsp90 expression by aspirin administration in vivo.

1. This experiment investigated the anti-apoptosis effects and the mechanism of aspirin action in the heat shock response of chicken myocardial cells in vivo, via changes in the heat stress (HS) protein Hsp90 and the rate of apoptosis. Broiler chickens were administered aspirin (1 mg/kg body weight) 2 h before exposure to HS, and then exposed to 40 ± 1°C for 0, 1, 2, 3, 5, 7, 10, 15 and 24 h. 2. The induction and consumption of the HS factor heat shock factor (HSF)-1, and reductions of HSF-2 and HSF-3 induced by HS led to a delay in Hsp90 expression. HSF-1, 2 and 3 regulation of hsp90 expression in turn inhibited the synthesis and activation of protein kinase ß (Akt), which resulted in a significant increase in caspase-3 at 2 and 10 h, caspase-9 from 1 to 7 h (except at 5 h), and the heat-stressed apoptosis of the myocardial cells. 3. Administration of aspirin changed the expression patterns of HSF-1, 2 and 3 such that the expression of Hsp90 protein was significantly upregulated (by 2.3-4.1 times compared with that of the non-treated cells). The resultant increase in Akt expression and activation, compared with the HS group, inhibited caspase-3 and caspase-9 activities and reduced the myocardial cells apoptosis rate (by 2.14-2.56 times). 4. Aspirin administration could inhibit heat-stressed apoptosis of myocardial cells in vivo and may be closely associated with its promotion of HS response of chicken hearts, especially Hsp90 expression.

[Catheter-based atrial appendage closure-current data and future developments]. / Der katheterbasierte Vorhofohrverschluss ­ aktuelle Daten und künftige Entwicklungen.

In Germany more than 1.6 million patients suffer from atrial fibrillation (AF). Within the next decades this number will substantially increase due to current demographic trends with the increasing average age of the population. When untreated, patients with atrial fibrillation have a five times higher risk for stroke as compared with a control cohort. A potent stroke prevention therapy reducing the risk of stroke by approximately 70-80% is primarily treatment with new oral anticoagulants (NOACs). The risk scores for stroke (CHA2DS2-VASc) and major bleeding (HAS-BLED) in patients with atrial fibrillation share common variables, so that patients with the highest stroke risk often carry a very high bleeding risk. A significant number of patients (ca. 20-30%) are, however, not eligible for long-term anticoagulation, e.g. because of a high bleeding risk. For this population there is an urgent need for alternative stroke prevention strategies, such as catheter-based percutaneous left atrial appendage closure. Current data about the efficiency and safety of this treatment as well as a discussion of ongoing recruitment for randomized studies are discussed in this review.

Determination of body width in brown and white layer pullets by image analyses.

1. Specific legal requirements for keeping pullets are not available in the European Union. However, two of the most important rearing factors for pullets are sufficient perching and feeder space. Both factors represent horizontal space dimensions which derive from the body width of the birds. 2. The body width of two strains of layer pullets (brown (BL) and white (WL) layer pullets) based on the measurement of distances in digital images was conducted on front-view digital photographs of BL and WL pullets taken at 8, 12 and 19 weeks of life. 3. Depending on live weight, age and body position, BL pullets measured an average body width between 10.70 ± 1.10 and 13.96 ± 1.11 cm. The width of WL pullets ranged from 10.30 ± 0.86 to 13.00 ± 1.14 cm. 4. Compared with WL, BL pullets occupied more horizontal space during rearing. Age influenced the body width of BL and WL pullets at the end of rearing. The tested body positions of the pullets did not affect the measured body width. 5. The biometric data obtained in this study are a useful basis for developing legal requirements for pullets, especially for defining minimum perch width and feeder space allowances.

Probing Stereoselectivity in Ring-Opening Metathesis Polymerization Mediated by Cyclometalated Ruthenium-Based Catalysts: A Combined Experimental and Computational Study.

The microstructures of polymers produced by ring-opening metathesis polymerization (ROMP) with cyclometalated Ru-carbene metathesis catalysts were investigated. A strong bias for a cis,syndiotactic microstructure with minimal head-to-tail bias was observed. In instances where trans errors were introduced, it was determined that these regions were also syndiotactic. Furthermore, hypothetical reaction intermediates and transition structures were analyzed computationally. Combined experimental and computational data support a reaction mechanism in which cis,syndio-selectivity is a result of stereogenic metal control, while microstructural errors are predominantly due to alkylidene isomerization via rotation about the Ru═C double bond.

Spatial requirements of poultry assessed by using a colour-contrast method (KobaPlan).

Sufficient floor space is a fundamental precondition for poultry to perform normal behavioural patterns. To calculate and determine stocking densities, it is essential to know the absolute minimum surface area required by any given animal (body space). Additional space is required for characteristic behaviours (behavioural space) and for adequate inter-individual distances, group sizes and room to perform social interactions have to be taken into account. To calculate body space, planimetric measurements were carried out by the colour contrast method "KobaPlan" in various poultry species in standing and sitting positions and at a number of different ages. They included laying hens (Lohmann brown (LB), Lohmann selected Leghorn (LSL)), broiler breeders (Ross, both genders), broiler chickens (Ross 308, both genders), turkeys (BUT 6, males), Peking ducks (Cherry Valley, both genders) and Muscovy ducks (Canedins R51, males). Depending on live weight, age, plumage condition and body position, LB hens occupied an average area between 401 cm(2) and 542 cm(2), LSL hens between 353 cm(2) and 445 cm(2), broiler breeder females between 440 cm(2) and 537 cm(2), broiler breeder males 623 cm(2) up to 945 cm(2), broiler chickens up to 434 cm(2), male fattening turkeys up to 1808 cm(2), Muscovy drakes up to 873 cm(2) and Peking ducks up to 627 cm(2). The values can be regarded as necessary minimum spatial requirements for the measured poultry species and genotype. The current method offers the potential to record the area occupied by animals exhibiting species-specific behavioural patterns.

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress.

To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

Association of heat shock protein 70 expression with rat myocardial cell damage during heat stress in vitro and in vivo.

To investigate the mechanism of sudden death as a result of stress-induced damage to heart tissue and myocardial cells and to investigate the cardioprotective role of Hsp70 during heat stress, the distribution and expression of Hsp70 was evaluated in the heart cells of heat-stressed rats in vivo and heat-stressed H9c2 cells in vitro. After exposure to heat stress at 42°C for different durations, we observed a significant induction of CK, CK-MB, and LDH as well as pathologic lesions characterized by acute degeneration, suggesting that cell damage occurs from the onset of heat stress. Immunocytochemistry showed that Hsp70 was distributed mainly in the cytoplasm of myocardial cells in vivo and in vitro. Hsp70-positive signals in the cytoplasm were more prominent in intact areas than in degenerated areas after 60 min of heat stress. Hsp70 protein levels in myocardial cells in vitro decreased from the beginning to the end of heat stress. Hsp70 protein levels in rat heart tissues in vivo decreased gradually with prolonged heat stress, with a slight increase at the beginning of heat stress. These results indicate that Hsp70 plays a role in the response of cardiac cells to heat stress and that decreased Hsp70 levels are associated with damage to rat myocardial cells in vitro and in vivo. Significant differences were found in hsp70 mRNA, which began to increase after 20 min of heat stress in vitro and after 40 min in vivo. This indicates that hysteresis is involved in mRNA expression after heat stress in vivo.

Expression of heat shock protein 90 alpha (Hsp90α) in primary neonatal rat myocardial cells exposed to various periods of heat stress in vitro.

The objective of this study was to investigate the mechanism of heat shock protein 90 alpha (Hsp90α) protection against heart damage resulting from heat stress by detecting Hsp90α mRNA, Hsp90α protein, protein localization, and cell damage in primary myocardial cells of neonatal rats in response to heat stress in vitro. The cells were heat-stressed at 42°C in an incubator with 95% air and 5% CO2 for different periods. Levels of Hsp90α, protein localization, enzymes, and cytopathological lesions were detected using Western blot, immunocytochemistry enzymatic assays, and cytopathological techniques. Aspartate aminotransferase, lactate dehydrogenase, and creatine kinase enzyme levels were elevated during heat stress, and acute cellular lesions that were characterized by vacuolar degeneration and necrosis were observed. Hsp90α levels decreased between 10 and 60 min of heat stress and increased after 360 and 480 min, while Hsp90α mRNA decreased after 360 min. These results indicate that heat stress might induce irreversible damage in certain myocardial cells. The elevated Hsp90α level at the end of heat stress and its positive signal in the cytoplasm of myocardial cells after heat stress could be associated with its protective role. Additionally, the consumption of Hsp90α exceeded its production in the first period of treatment.

Evaluation of Hsp47 expression in heat-stressed rat myocardial cells in vitro and in vivo.

The aim of the present study was to identify the correlation between expression of heat shock protein 47 (Hsp47) and stress injury in heat-stressed myocardial cells and to compare variations in Hsp47 expression in rat myocardial cells exposed to different heat stress for varying periods in vitro and in vivo. Exposure to heat stress at 42°C resulted in similar induction patterns of the heart damage-related enzyme aspartate aminotransferase in the supernatants of H9c2 cells and in the serum of rats. Histological analysis revealed that both H9c2 cells and heart tissues displayed cellular degeneration in response to different periods of heat stress. Hsp47 was constitutively expressed in the cytoplasm of H9c2 cells at all time points during heat stress, which was consistent with observations in heart fibers in vivo. Immunoblotting analysis revealed no significant difference between the expression of Hsp47 in H9c2 cells and heart tissue. However, the expression of hsp47 mRNA in response to heat stress was significantly increased in H9c2 cells at 60 min (P < 0.01) and 100 min (P < 0.01), which was comparable to that at 100 min (P < 0.01) in the rat heart. Thus, Hsp47 was elevated significantly after hyperthermia at the mRNA level but not at the protein level both in vitro and in vivo. The results suggest that Hsp47 turnover may increase during heat stress or that Hsp47 consumption exceeds its production.

Association of Hsp60 expression with damage to rat myocardial cells exposed to heat stress in vivo and in vitro.

To investigate the protective role of Hsp60 against stress damage and its role in the sudden death of stressed animals, changes in the levels of Hsp60 protein and hsp60 mRNA of myocardial cells in vivo and in vitro were studied. In addition, the relationship between Hsp60 expression and heat-induced damage was also studied. Rats were exposed to a temperature of 42° ± 1°C for 0, 20, 40, 60, 80, or 100 min. More than 50% of the rats died suddenly within 100 min. With increasing heat stress duration, hsp60 mRNA levels significantly increased in both in vivo and in vitro rat myocardial cells; however, a similar trend was not observed for Hsp60 protein levels. Although the changes observed in Hsp60 expression in myocardial cells in vitro were inconsistent with those of rat heart tissues in vivo, Hsp60 expression levels were consistent with the histopathological damage observed in myocardial cells both in vivo and in vitro. Differences in Hsp60 expression may reflect the degree of injury sustained by myocardial cells in vivo and in vitro. As a mitochondrial protein, Hsp60 represents a potential biomarker of heat stress, and may protect against heat stress induced myocardial cellular damage both in vivo and in vitro.

First Report of Citrus leprosis virus Nuclear Type in Sweet Orange in Colombia.

Colombia is ranked 18th in the world in citrus production and contributed 0.9% of the total world share. Among four important citrus-producing regions of Colombia, the Orinoco region (3 to 6°N, 68 to 74°W) consists of two citrus-producing states, Meta and Casanare. Citrus leprosis is the most important viral disease of citrus in Colombia (1,3). Three types of Citrus leprosis virus (CiLV) infect citrus, producing leprosis-like lesion symptoms. Two of the three CiLV species, Citrus leprosis virus cytoplasmic type (CiLV-C) and cytoplasmic type 2 (CiLV-C2), produce particles only in the cytoplasm (3). The other species, Citrus leprosis virus nuclear type (CiLV-N), produces particles in both the cytoplasm and nucleus (4). CiLV-C is more prevalent and destructive while CiLV-N has been reported only in Brazil, Panama, and Mexico (4). Interestingly, both CiLV-C and -C2 were reported from the same regions of Meta and Casanare States in Colombia in 2004 and 2012 (1,3). CiLV-C lesions are usually rounded (initially 2 to 3 mm in diameter and extending up to 30 mm), have dark-brown or greenish central chlorotic spots, and are surrounded by yellow halos. CiLV-N lesions have been described as smaller in size and form three well-defined regions including a necrotic center with an intermediate orange color halo and an outer chlorotic halo (2). In 2013, 'Valencia' sweet orange (Citrus sinensis L.) leaves with suspected CiLV-N symptoms were collected from 8 plants in Casanare State and shipped under permit to the USDA-APHIS-PPQ-CPHST, Beltsville, MD. Total RNA from symptomatic and healthy sweet orange leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR primers specific to CiLV-C, CiLV-C2 (3), and CiLV-N nucleocapsid (N) (CiLV-N-NPF: 5'-ATGGCTAACCCAAGTGAGATCGATTA-3'; CiLV-N-NPR: 5'-AGTTGCCTTGAGATCATCACATTGGT-3') and putative matrix protein (M) genes (CiLV-N-MF: 5'-ATGTCTAAACAGATTAATATGTGCACTGTG-3'; CiLV-N-MR: 5'-CTAACCACTGGGTCCCGC-3') were utilized to identify the CiLV associated with the leprosis-affected leaf samples from Casanare. RT-PCR with CiLV-C primers failed to produce any amplicon, but CiLV-N primers successfully amplified the partial N gene (681 bp) and entire M gene (552 nt) amplicons from multiple leaves of all leprosis samples. In addition, a 795-bp amplicon specific to CiLV-C2 also was amplified from the CiLV-N suspected samples. Similar results were obtained when the vector, flat spider mite (Brevipalpus spp.) total RNA was used as template for RT-PCR. For further confirmation, each amplicon was cloned and sequenced. Sequencing of the N and M gene amplicons of CiLV-N (accession nos. KJ195893 and KJ195894) and coat protein gene of CiLV-C2 showed 97 to 99% nucleotide sequence identity with the CiLV-N M2345 isolate sequence (KF209275) from Mexico (4) and CiLV-C2 L147V1 isolate sequence (JX000024) from Colombia (3), respectively. Phylogenetic analyses of these N and M protein gene sequences confirmed a mixed infection of the same plant with two viruses, one from an unassigned new genus Dichorhavirus (CiLV-N) and another from genus Cilevirus (CiLV-C2). This is the first report of CiLV-N in Colombia, and also the first report of an occurrence of CiLV-N in mixed infection with CiLV-C2. All three known species of CiLV occur in the Orinoco region of Colombia. References: (1) M. G. León et al. Plant Dis. 90: 682, 2006. (2) J. P. R. Marques et al. Anais da Academia Brasileira de Ciências 82:501, 2010. (3) A. Roy et al. Phytopathology 103:488, 2013. (4) A. Roy et al. Genome Announc. 1(4): e00519-13, 2013.

Occurrence of livestock-associated methicillin-resistant Staphylococcus aureus in Turkey and broiler barns and contamination of air and soil surfaces in their vicinity.

The emission of microorganisms, especially resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), from poultry farms is of public interest, and its occurrence and relevance are controversially discussed. So far, there are limited data on this issue. In this study, we investigated the occurrence of livestock-associated (LA)-MRSA inside and outside previously tested MRSA-positive poultry barns in Germany. In total, five turkey and two broiler fattening farms were investigated four and three times, respectively. In a longitudinal study during one fattening period, samples were collected from animals, the animals' environment inside the barn, including the air, and the barns' surroundings, such as ambient air and boot swabs of ground surfaces at different distances from the barn. Moreover, a cross-sectional study was carried out once inside the barns on five turkey and four broiler farms during the last third of the fatting period. In the cross-sectional study, LA-MRSA was detected in the air of most barns (7 of 9, 77.8%), as well as in many samples originating from animals, with detections levels of 50 to 54% in broiler and 62 to 77% in turkey farms. In the longitudinal study, LA-MRSA was found in the ambient air outside two turkey barns and on the ground surface on the downwind side of many (44.4%) turkey and broiler farms. The same spa types of isolates were observed inside and outside the barns. Transmission of MRSA within poultry farms, as well as emission via the airborne route, seems to be possible.

Temporal variations of Hsp60 and HSF-1 in primary rat myocardial cells in vitro under heat stress.

The mechanisms involved in sudden animal death due to acute heart failure during heat stress are not well understood. We examined the relationship between heat stress-induced variations of protective Hsp60 and expression of its regulatory factor, HSF-1, in heat-stressed primary myocardial cells of neonatal rats in vitro through cardiac enzyme detection, immunoblotting, immunocytochemistry, and qPCR. Increases in cardiac damage-related enzyme levels demonstrated injury to myocardial cells after heat exposure at 42°C. Hsp60 expression levels fluctuated during heat stress; they decreased significantly after 20 min, then increased at 120 min and decreased again at 360 min after initiation of heat stress. The highest levels of Hsp60 were observed at 240 min, while the lowest were at 60 min. Damage to myocardial cells was characterized by increases in cardiac enzyme levels and low levels of Hsp60 due to functional disorder of myocardial cells at early stages of heat stress. However, the significant induction of hsp60 mRNA levels from the beginning up to 240 min of heat stress was not consistent with the classic regulatory mechanisms that link transcription and translation, suggesting that Hsp60 expression is delayed due to loss of Hsp60 during the early stages of heat stress. hsf-1 mRNA levels were significantly increased from 10 min of heat stress; however, HSF-1 protein levels did not simultaneously increase, indicating that HSF-1 is not the sole regulator of Hsp60 expression.

The role of Hsp90α in heat-induced apoptosis and cell damage in primary myocardial cell cultures of neonatal rats.

To understand the mechanism underlying the sudden animal death caused by acute heart failure during heat stress, the relationships among the heat-induced pathological changes and apoptosis and the variations in the levels of protective Hsp90α and its mRNA in the heat-stressed primary myocardial cells of neonatal rats in vitro were studied by cytopathological observation, immunoblotting, RT-PCR, and analysis of the related enzymes. After a period of adaptive cell culture, the myocardial cells were immediately exposed to heat stress at 42°C for 10, 20, 40, 60, 120, 240, 360, and 480 min. Levels of creatine kinase increased from the beginning of heat stress, and the cells exposed to heat stress showed acute cellular lesions characterized by vacuolar degeneration and necrosis after 40 min of heat stress, suggesting that the myocardial cells in vitro were obviously stressed and damaged by higher temperature. The levels of cleaved caspase-3 and cytochrome C, which were related to apoptosis, increased significantly after 40 min of heat stress while the Hsp90α protein level significantly decreased. In contrast, after 6 h of exposure to heat stress, the levels of cleaved caspase-3 and cytochrome C decreased while those of Hsp90α significantly increased, suggesting that early depletion of Hsp90α coincides with a high rate of necrosis and apoptosis in heat-stressed myocardial cells, while the Hsp90α level in surviving cells increases again with significantly less apoptosis after 6 h of heat stress. These findings also indicate that apoptosis of myocardial cells occurs through the activation of the cytochrome C and caspase-3 pathway. The cell repair capacity of Hsp90α is overstrained in the early phase of heat treatment and needs some hours to stabilize. As a result, in the primary myocardial cells in vitro, Hsp90α shows protective activity against damage at the end period of the heat exposure.

Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the ß-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Survival of Campylobacter jejuni in naturally and artificially contaminated laying hen feces.

Infected laying hens regularly excrete large amounts of Campylobacter jejuni with their feces, which represent a reservoir of infection within the flock and for animals in the region. However, the knowledge about survival times of C. jejuni in these feces is still scarce. Therefore, orienting laboratory experiments were carried out under controlled conditions to estimate the survival times of C. jejuni both in artificially and naturally contaminated laying hen feces. In 6 different laying hen flocks (3 Campylobacter-free and 3 Campylobacter-positive flocks), fresh excreta were randomly collected and pooled in 20-g samples per flock. In the laboratory, each of the 3 pooled samples from the Campylobacter-free barns were homogenized and mixed with 10 mL of a freshly prepared C. jejuni suspension (3 × 10(8) cfu/mL). The other 3 samples were homogenized only. The 6 samples were stored at 20 ± 1°C and 40 to 60% RH in 2 different incubators. Specimens of 2 g were taken from all 6 samples 1 h after storage and daily at the same time during the next 10 consecutive days and investigated on culturable C. jejuni. The survival times of culturable C. jejuni ranged from 72 to 96 h in artificially inoculated feces and varied from 120 to 144 h in naturally colonized flocks. The flaA typing by RFLP confirmed that the isolates from the artificially contaminated feces were identical with the added strain. A total of 5 different flaA types were identified from the naturally contaminated feces, and survival of these isolates was dependent on flaA type. The demonstrated survival times indicate that contaminated fresh feces are an important reservoir of C. jejuni, representing a permanent source of infection over at least 6 d after excretion. It shows the considerable potential of fresh feces in transmitting the agent within and between flocks during that period. This 6-d span should be considered when poultry manure is applied to land as organic fertilizer.

Hsp110 expression changes in rat primary myocardial cells exposed to heat stress in vitro.

We investigated and described the kinetics of heat shock protein (Hsp) 110 expression and distribution in rat primary myocardial cells exposed to heat stress in vitro. After incubation at 37°C for 72 h, myocardial cells were heat stressed at 42°C for 0, 10, 20, 40, 60, 120, 240, 360, and 480 min. Significant increases in aspartate transaminase, lactate dehydrogenase, and creatine kinase enzymatic activities in the myocardial cell culture media were observed during heat stress, suggesting that the integrity of the myocardial cells was altered. Immunocytochemical analysis revealed that the expressed Hsp110 was constitutively localized in the cytoplasm and in the nuclei in small amounts characterized by a granular pattern. Nuclear Hsp110 levels increased significantly after 240 min of heat stress compared with levels in the control. The overall levels of Hsp110 expression increased significantly after 20 min. After 240 min, Hsp110 levels were approximately 1.2-fold higher than those in the control. Increasing levels of hsp110 messenger RNA detected using real-time quantitative polymerase chain reaction were observed after 20 min of heat stress, and the levels peaked with a 10-fold increase after 240 min of heat stress. These results indicate that the expression of Hsp110 in primary myocardial cells in vitro is sensitive to hyperthermic stress and that Hsp110 is involved in the potential acquisition of thermotolerance after heat stress. Therefore, Hsp110 might play a fundamental role in opposing and alleviating heat-induced damage caused by hyperthermic stress in primary myocardial cells.