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.

Influence of lycopene and astaxanthin in feed on metabolic parameters of laying hens, yolk color of eggs and their content of carotenoids and vitamin A when stored under refrigerated conditions.

Orientating investigations were carried out in order to test the influence of oil extracts of lycopene (20, 40 and 60 mg/kg feed) and astaxanthin (10, 20 and 30 mg/kg feed) as feed additives on the metabolic parameters (glucose, creatinine, cholesterol) and enzyme activities (alanine aminotransferase, ALT; aspartate transaminase, AST) of laying hens. Eggs from these hens were stored at refrigerator temperatures of 4°C and 12°C for up to 30 days and analyzed for vitamin A, carotenoid and yolk color. 45 laying hens (Hy-Line W36 cross, 23 weeks of age) were divided in three groups of 15 birds each (control, lycopene fed group, astaxanthin fed group). Blood samples were taken from the hens and laid eggs were collected on days 31, 61, and 91 of the study. The eggs were stored for 30 days in refrigerators. Both lycopene and astaxanthin increased the content of glucose in serum (Р⟨0.05). The content of creatinine and cholesterol, and the activity of ALT, AST and alkaline phosphatase varied dose-dependently. With the exception of cholesterol, metabolite concentrations in the serum of laying hens fed different lycopene and astaxanthin doses did not exceed clinically accepted physiological levels. The carotenoid content and color of the egg yolks from laying hens fed astaxanthin was significantly higher (Р⟨0.05) compared to lycopene fed birds. Refrigerator storage of the eggs did not affect carotenoid content and egg yolk color compared to freshly laid eggs. Both feed additives showed a favorable effect on the metabolism of laying hens and the enrichment of egg yolks with carotenoids, astaxanthin significantly more (Р⟨0.05) than lycopene.

Exploiting metabolic acidosis in solid cancers using a tumor-agnostic pH-activatable nanoprobe for fluorescence-guided surgery.

Cancer cell metabolism leads to a uniquely acidic microenvironment in solid tumors, but exploiting the labile extracellular pH differences between cancer and normal tissues for clinical use has been challenging. Here we describe the clinical translation of ONM-100, a nanoparticle-based fluorescent imaging agent. This is comprised of an ultra-pH sensitive amphiphilic polymer, conjugated with indocyanine green, which rapidly and irreversibly dissociates to fluoresce in the acidic extracellular tumor microenvironment due to the mechanism of nanoscale macromolecular cooperativity. Primary outcomes were safety, pharmacokinetics and imaging feasilibity of ONM-100. Secondary outcomes were to determine a range of safe doses of ONM-100 for intra-operative imaging using commonly used fluorescence camera systems. In this study (Netherlands National Trial Register #7085), we report that ONM-100 was well tolerated, and four solid tumor types could be visualized both in- and ex vivo in thirty subjects. ONM-100 enables detection of tumor-positive resection margins in 9/9 subjects and four additional otherwise missed occult lesions. Consequently, this pH-activatable optical imaging agent may be clinically beneficial in differentiating previously unexploitable narrow physiologic differences.

[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.

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.

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.

Floor space covered by broiler chickens kept at stocking densities according to Council Directive 2007/43/EC.

It is controversially discussed whether the stocking densities set by the EU Directive 2007/43/EC allow a species-appropriate housing of broiler chickens. To calculate the exact area broilers occupy due to their physical size and shape, planimetric measurements using a colour-contrast method were carried out. In total, 1949 photographs of standing and 1482 of squatting chickens, taken from a top view, were analysed. A computer program counted the pixels representing the previously weighed animal in the photograph and calculated the animal area. The average area covered by chickens with 400 g live weight was 116.64±13.12 cm(2) in a standing and 138.61±12.92 cm(2) in a squatting position. These areas increased linearly as a function of live weight to 452.57±58.89 cm(2) (R(2)=0.90 standing) and 513.54±42.70 cm(2) (R(2)=0.82 squatting) at the end of the study (3200 g live weight). Squatting chickens occupied more space compared with a standing position in most of the tested weight classes (P<0.05). Depending on target weights, stocking densities and body positions, broilers occupied 48.5-77.7 per cent of 1 m(2) Thus, from a physical point of view, simultaneous resting is possible at any stocking density provided by the EU Directive and at common target weights.

Aspirin-induced heat stress resistance in chicken myocardial cells can be suppressed by BAPTA-AM in vitro.

Our recent studies have displayed the protective functions of aspirin against heat stress (HS) in chicken myocardial cells, and it may be associated with heat shock proteins (HSPs). In this study, we further investigated the potential role of HSPs in the aspirin-induced heat stress resistance. Four of the most important HSPs including HspB1 (Hsp27), Hsp60, Hsp70, and Hsp90 were induced by aspirin pretreatment and were suppressed by BAPTA-AM. When HSPs were induced by aspirin, much slighter HS injury was detected. But more serious damages were observed when HSPs were suppressed by BAPTA-AM than those cells exposed to HS without BAPTA-AM, even the myocardial cells have been treated with aspirin in prior. Comparing to other HSPs, HspB1 presented the largest increase after aspirin treatments, 86-fold higher than the baseline (the level before HS). These findings suggested that multiple HSPs participated in aspirin's anti-heat stress function but HspB1 may contribute the most. Interestingly, during the experiments, we also found that apoptosis rate as well as the oxidative stress indicators (T-SOD and MDA) was not consistently responding to heat stress injury as expected. By selecting from a series of candidates, myocardial cell damage-related enzymes (CK-MB and LDH), cytopathological tests, and necrosis rate (measured by flow cytometry assays) are believed to be reliable indicators to evaluate heat stress injury in chicken's myocardial cells and they will be used in our further investigations.

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.

In vitro evaluation of aspirin-induced HspB1 against heat stress damage in chicken myocardial cells.

To understand the potential association of heat stress resistance with HspB1 induction by aspirin (ASA) in chicken myocardial cells, variations of HspB1 expression and heat stressed-induced damage of myocardial cells after ASA administration were studied in primary cultured myocardial cells. Cytopathological lesions as well as damage-related enzymes, such as creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), indicated the considerable protective ability of ASA pre-treatment against acute heat stress. Immunostaining assays showed that heat stress caused HspB1 to relocate into the nucleus, while ASA did not. ELISA analysis, revealed that HspB1 expression induced by ASA averaged 45.62-fold higher than that of the control. These results indicated that the acute heat-stressed injuries were accompanied by comparatively lower HspB1 expression caused by heat stress in vitro. ASA pre-treatment induced a level of HspB1 presumed to be sufficient to protect myocardial cells from acute heat stress in the extracorporal model, although more detailed mechanisms will require further investigation.

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.

Acetyl salicylic acid protected against heat stress damage in chicken myocardial cells and may associate with induced Hsp27 expression.

We investigated whether acetyl salicylic acid (ASA) protects chicken myocardial cells from heat stress-mediated damage in vivo and whether the induction of Hsp27 expression is connected with this function. Pathological changes, damage-related enzyme levels, and Hsp27 expression were studied in chickens following heat stress (40 ± 1 °C for 0, 1, 2, 3, 5, 7, 10, 15, or 24 h, respectively) with or without ASA administration (1 mg/kg BW, 2 h prior). Appearance of pathological lesions such as degenerations and karyopyknosis as well as the myocardial damage-related enzyme activation indicated that heat stress causes considerable injury to the myocardial cells in vivo. Myocardial cell injury was most serious in chickens exposed to heat stress without prior ASA administration; meanwhile, ASA pretreatment acted protective function against high temperature-induced injury. Hsp27 expression was induced under all experimental conditions but was one-fold higher in the ASA-pretreated animals (0.3138 ± 0.0340 ng/mL) than in untreated animals (0.1437 ± 0.0476 ng/mL) 1 h after heat stress exposure, and such an increase was sustained over the length of the experiment. Our findings indicate that pretreatment with ASA protects chicken myocardial cells from acute heat stress in vivo with almost no obvious side effects, and this protection may involve an enhancement of Hsp27 expression. However, the detailed mechanisms underlying this effect require further investigation.

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.

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.

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.

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.