Corneal Opacity in Domestic Ducks Experimentally Infected With H5N1 Highly Pathogenic Avian Influenza Virus.

Domestic ducks can be a key factor in the regional spread of H5N1 highly pathogenic avian influenza (HPAI) virus in Asia. The authors performed experimental infections to examine the relationship between corneal opacity and H5N1 HPAI virus infection in domestic ducks (Anas platyrhyncha var domestica). A total of 99 domestic ducks, including 3 control birds, were used in the study. In experiment 1, when domestic ducks were inoculated intranasally with 2 H5N1 HPAI viruses, corneal opacity appeared more frequently than neurologic signs and mortality. Corneal ulceration and exophthalmos were rare findings. Histopathologic examinations of the eyes of domestic ducks in experiment 2 revealed that corneal opacity was due to the loss of corneal endothelial cells and subsequent keratitis with edema. Influenza viral antigen was detected in corneal endothelial cells and some other ocular cells by immunohistochemistry. Results suggest that corneal opacity is a characteristic and frequent finding in domestic ducks infected with the H5N1 HPAI virus. Confirming this ocular change may improve the detection rate of infected domestic ducks in the field.

Dynamics of AV coupling during human atrial fibrillation: role of atrial rate.

The causal relationship between atrial and ventricular activities during human atrial fibrillation (AF) is poorly understood. This study analyzed the effects of an increase in atrial rate on the link between atrial and ventricular activities during AF. Atrial and ventricular time series were determined in 14 patients during the spontaneous acceleration of the atrial rhythm at AF onset. The dynamic relationship between atrial and ventricular activities was quantified in terms of atrioventricular (AV) coupling by AV synchrogram analysis. The technique identified n:m coupling patterns (n atrial beats in m ventricular cycles), quantifying their percentage, maximal length, and conduction ratio (= m/n). Simulations with a difference-equation AV model were performed to correlate the observed dynamics to specific atrial/nodal properties. The atrial rate increase significantly affected AV coupling and ventricular response during AF. The shortening of atrial intervals from 185 ± 32 to 165 ± 24 ms (P < 0.001) determined transitions toward AV patterns with progressively decreasing m/n ratios (from conduction ratio = 0.34 ± 0.09 to 0.29 ± 0.08, P < 0.01), lower occurrence (from percentage of coupled beats = 27.1 ± 8.0 to 21.8 ± 6.9%, P < 0.05), and higher instability (from maximal length = 3.9 ± 1.5 to 2.8 ± 0.7 s, P < 0.01). Advanced levels of AV block and coupling instability at higher atrial rates were associated with increased ventricular interval variability (from 123 ± 52 to 133 ± 55 ms, P < 0.05). AV pattern transitions and coupling instability in patients were predicted, assuming the filtering of high-rate irregular atrial beats by the slow recovery of nodal excitability. These results support the role of atrial rate in determining AV coupling and ventricular response and may have implications for rate control in AF.

Applicability and performance of heart failure prognostic scores in dilated cardiomyopathy: the real-world experience of an Italian referral center for cardiomyopathies.

BACKGROUND: The performance of heart failure (HF) risk models is validated in the general population with HF but in specific aetiological settings, and specifically in dilated cardiomyopathy (DCM), has scarcely been explored. We tested eight of the main prognostic scores used in HF in a large real-world population of patients with DCM. METHODS: We included 784 consecutive DCM patients enrolled, both inpatients and outpatients, enrolled between January 2000 and December 2017. The risk of 1 and/or 3-year all-cause mortality/heart transplantation/durable left ventricular assist device (LVAD) implantation (D/HTx/LVAD) was estimated in our cohort according to the following risk scores SHFM, 3-CHF, CHARM, MAGGIC, GISSI-HF, MECKI, Barcelona Bio-HF, Krakow score and their accuracy calculated through the receiver operator characteristic (ROC) curve analysis. RESULTS: During a median follow-up of 5.8 years (Interquartile Range 3.2-7.6 years), 191 patients (20%) died or underwent HTx/LVAD (158 deaths, 30 heart transplantations, and 3 LVAD implantations). The high missing rate allowed to calculated only four prognostic models (MAGGIC, CHARM, 3-CHF and SHFM). All the scores overestimated the rate of D/HTx/LVAD. The prognostic accuracy was suboptimal for MAGGIC (AUC 0.754) and CHARM (AUC 0.720) scores and only modest for 3-CHF (AUC 0.677) and SHFM (AUC 0.667). CONCLUSIONS: Main prognostic scores for the risk stratification of HF are only partially applicable to real-world patients with DCM. MAGGIC and CHARM scores showed the best accuracy, despite the overestimation of risk. Our findings corroborate the need of specific risk scores for the prognostic stratification of DCM. CLINICAL PERSPECTIVE: What is new? The present study is the largest analysis in literature which investigate how the main existing heart failure prognostic risk scores performed in a real-world of dilated cardiomyopathy population, both in- and outpatients. What are the clinical implications? DCM is a stand-alone model of heart failure, where the performance of multiple heart failure prognostic scores for the risk stratification is quite limited. The need for contemporary, dedicated prognostic scores in this disease is increasingly evident.

Delta-ADC (apparent diffusion coefficient) analysis in patients with idiopathic normal pressure hydrocephalus.

We have developed the delta-apparent diffusion coefficient (ADC), a new parameter of the water dynamics of brain tissue using MRI. Delta-ADC is the changes in regional ADC values of the brain during the cardiac cycle. The study included 6 idiopathic normal pressure hydrocephalus (iNPH) patients (iNPH group) and 12 healthy volunteers (control group). ECG-triggered single-shot diffusion echo planar imaging (b = 0 and 1,000 s/mm(2)) was used on a 1.5-T MRI. The delta-ADC image was calculated from the maximum minus the minimum ADC value of all cardiac phase images (20 phases) on a pixel-by-pixel basis. Delta-ADC values in the white matter of the frontal, temporal, and occipital lobe were obtained. Delta-ADC values in the iNPH group were significantly higher than those in the control group in all regions. ADC values in the iNPH were also significantly higher than those in the control group, but the differences in the ADC between the groups in each region were much lower than those for the delta-ADC. Although the changes in the delta-ADC and ADC values were similar, there was no significant correlation between the delta-ADC and the ADC. These results suggest that the ADC and the delta-ADC may reflect different kinds of water dynamics. The ADC depends on the water content in brain tissue. On the other hand, delta-ADC depends on not only the water content, but also on the degree of the fluctuation of the water molecules. Delta-ADC analysis makes it possible to obtain non-invasively new and more detailed information on the regional brain condition in iNPH.

Modeling Framework for the Generation of Synthetic RR Series during Atrial Arrhythmias.

We introduced a modeling framework for the generation of realistic ventricular interval (RR) series to be used in the validation of atrial arrhythmia detection algorithms. The framework included three previously proposed models, which reproduced the specific variability properties of RR series in normal sinus rhythm, atrial flutter (AFL) and atrial fibrillation (AF). Transitions between the three rhythms were governed by a three-state continuous-time Markov chain model, which could be tuned to obtain arrhythmic episodes of the requested length. As a representative application, the modeling framework was used to generate a database of RR series for the validation of a previously proposed AF detection algorithm, which was based on RR pattern similarity. The validation showed the deterioration of detector performance in presence of simulated AFL episodes. Thanks to the detailed reproduction of the specific features of the two most common atrial arrhythmias, our modeling framework may constitute a novel tool for the assessment and comparison of detection algorithm performance.

Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens.

Typically highly pathogenic avian influenza (HPAI) viruses spread very rapidly among chickens within sheds. However, the spread was slower than expected for the initial 10 days of the index farm in Japan during 2004. This slow spread, as well as the lack of gross lesions, clinical signs, or high mortality, hindered the field veterinarian from reporting a suspected HPAI outbreak to the veterinary office. To understand the field conditions for the slow virus spread, we examined contact and airborne transmission of the H5N1 virus to chickens in a negative-pressure isolator using various numbers of infected chickens and separate compartments. We found that the contact transmission did occur inefficiently when one or two chickens were infected, whereas the transmission was efficient when four chickens were infected. Airborne transmission of the HPAI virus was also dependent on the number of infected chickens and was less efficient than contact transmission. These data together with field observations suggested that number of infected chickens, chicken house types, and amount of environmental contamination might affect the virus transmission efficiency to chickens.

Non-invasive measurement of intracranial compliance using cine MRI in normal pressure hydrocephalus.

The aim of this study is to clarify biophysics of normal pressure hydrocephalus (NPH) based on non-invasive intracranial compliance measurement using magnetic resonance imaging (MRI). Patients with NPH after subarachnoid hemorrhage (NPH group, n = 5), brain atrophy or asymptomatic ventricular dilation (VD group, n = 5), and healthy volunteers (control group, n = 12) were included in this study. Net blood flow (bilateral internal carotid and vertebral arteries, and jugular veins) and cerebrospinal fluid (CSF) flow in subarachnoid space at the C2 level of cervical vertebra were measured using phase-contrast cine MRI. CSF pressure gradient and intracranial volume changes during a cardiac cycle were calculated based on Alperin's method. Compliance index (Ci = delta V/delta P) was obtained from the maximum pressure gradient and volume changes. Pressure volume response (PVR) was measured in the NPH group during a shunt operation. Ci in the NPH group was the lowest among the three studies groups. No difference was found between the control and VD groups. There was a linear correlation between Ci and PVR. In conclusion, intracranial compliance can be determined by cine MRI non-invasively. It is well known that NPH has relatively low intracranial compliance, this non-invasive method can be used for the diagnosis of NPH.

Control of an outbreak of highly pathogenic avian influenza, caused by the virus sub-type H5N1, in Japan in 2004.

An outbreak of highly pathogenic avian influenza (HPAI), caused by the virus sub-type H5N1, occurred at four premises in three prefectures in Japan during January and March 2004. As a result, 274,654 poultry died or were slaughtered. This was the first outbreak of HPAI in Japan since 1925. (The earlier outbreak was caused by H7N7.) The disease was successfully eradicated within three-and-a-half months, following an eradication campaign that included depopulating the affected premises, implementing movement controls and intensive surveillance. Control measures were conducted in accordance with the National Manual of HPAI Control. However, during the eradication campaign, some key issues arose, such as delays in notification by the affected farmers. As a result of these experiences, the relevant laws and HPAI Control Manual have been appropriately revised.

Regulation of aquaporin-4 expression in astrocytes.

Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation.

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Hypothermia modulates induction of hsp70 and c-jun mRNA in the rat brain after subarachnoid hemorrhage.

We investigated expression of hsp70 and c-jun mRNA with in situ hybridization for evaluating hypothermia effect on the brain exposed to subarachnoid hemorrhage (SAH). SAH was induced in Wistar rats with endovascular perforation. Animals were divided arbitrarily into normothermic and hypothermic groups, and they were sacrificed at 3 h or 12 h after SAH. The SAH induced hsp70 and c-jun mRNAs in the cerebral cortex, hippocampus, thalamus, hypothalamus, and caudoputamen. Mild hypothermia depressed hsp70 mRNA expression in the cortex, thalamus, and hippocampus. The c-jun mRNA expression was reduced by hypothermia in the cortex, thalamus, and CA1 of the hippocampus. Based on these findings, we speculate that hypothermia protects the brain exposed to SAH by reducing this stress response. Although it is yet difficult to employ hypothermia in the clinical settings, this study suggests its utility to those patients sustaining severe subarachnoid hemorrhage.

Acute and transient increase of lipocalin-type prostaglandin D synthase (beta-trace) level in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

We measured the concentration of lipocalin-type prostaglandin D synthase (PGDS) in cerebrospinal fluid (CSF) and serum in patients 1, 3, 5, 7, 9, 11, 14 and 17 days after subarachnoid hemorrhage (SAH) due to ruptured cerebral aneurysms. The PGDS level in lumbar CSF increased about two-fold at day 3 (20.85 +/- 2.71 microg/ml, mean +/- SE) and at day 5 (25.24 +/- 3.76), as compared with the level at day 1 (11.25 +/- 1.07). The CSF level gradually decreased and returned to the day 1 level at day 17. The serum PGDS level was much lower than the CSF level (0.39 +/- 0.06 at day 1) and almost unchanged until day 17. The neuron-specific enolase level in CSF, as an index of brain damage, was maximum at day 1 (29.83 +/- 7.32 ng/ml) and decreased at day 3 and at day 5 (18.28 +/- 2.65 and 11.95 +/- 1.82, respectively). These results suggest that the transient and delayed increase in the PGDS level in CSF is due to its induction of PGDS in the arachnoid membrane after SAH.

Endovascular stent placement for multiple aneurysms of the extracranial internal carotid artery: technical case report.

A patient with multiple extracranial internal carotid artery aneurysms that caused cerebral infarction was treated by endovascular placement of a metallic stent. Complete disappearance of the aneurysms was angiographically confirmed within 15 weeks. Endovascular observation 6 months after the stent placement revealed that the endoluminal surface was totally covered by normal endothelium. The patient returned to his regular work routine and had no complication or neurological deterioration during the 17-month follow-up period.

Acoustic noise analysis in echo planar imaging: multicenter trial and comparison with other pulse sequences.

The purpose of this study was to evaluate acoustic noise in echo planar imaging (EPI) at various magnetic resonance imaging (MRI) centers and to compare EPI acoustic noise with that in other fast pulse sequences. We measured A-weighted root-mean-square sound pressure levels and peak impulse sound pressure levels for EPI, under the same conditions, in eleven clinical super-conducting MRI systems. We also compared sound pressure levels for the EPI and six different pulse sequences and analyzed the acoustic noise spectra. Sound pressure levels during the use of the EPI differed greatly among institutions. Moreover, sound pressure levels of the EPI were not significantly different from those of other fast pulse sequences and were within permissible noise exposure levels. In comparison to other fast sequences, the EPI had significantly greater acoustic noise in the high-octave band frequency.

Quantification of chicken anaemia virus by competitive polymerase chain reaction.

A quantitative method for chicken anaemia virus (CAV) was developed using competitive polymerase chain reaction (PCR). Competitive template was constructed by deletion of 33 nucleotides from a wildtype DNA clone of CAV. Quantification of CAV DNA molecules by the competitive PCR was rapid and highly reproducible when compared with conventional infectivity titration methods. The ratios of the viral DNA molecules and infectivity titres in MDCC-MSB1 cells varied between 1.3 and 3.55 log(10) among several isolates, suggesting the existence of different infection efficiencies to MDCC-MSB1 cells by isolates. The competitive PCR will be useful for studying CAV infection in vivo and/or in vitro.

Induction of hydropericardium in one-day-old specific-pathogen-free chicks by adenoviruses from inclusion body hepatitis.

The pathogenicities of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) strains of adenovirus for specific-pathogen-free (SPF) chicks were compared. One-day-old SPF chicks inoculated intramuscularly with the DPI-2 (serotype 2), S-PL1 (serotype 2), TR630 (serotype 8), and Saga97 (serotype 8) strains from IBH and with the LVP-1 strain (serotype 4) from HPS exhibited the mortality, liver enlargement, and hydropericardium characteristic of gross change found in HPS. The chicks inoculated with the IBH and HPS strains exhibited similar histologic and immunohistochemical changes. Neither mortality nor pathologic changes occurred in 3-wk-old SPF chicks inoculated with IBH strains, although HPS strain induced HPS lesions in them. This study indicates that IBH strains of adenovirus can also reproduce HPS lesions and mortality in 1-day-old SPF chicks and that IBH and HPS strains may have similar pathogenicities except for their different virulence for older chickens.

Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains.

In order to study replication efficiency of infectious bursal disease virus (IBDV) in lymphoid tissues, both the virus titers and the virus antigen titers in four lymphoid tissues were compared among chickens inoculated with Ehime/91 (about 50% mortality), J1 (no mortality), or K (attenuated) IBDV strains during 1-7 days postinoculation (PI). IBDV antigens in tissue homogenates were detected by an enzyme-linked immunosorbent assay. In the bursa of Fabricius, higher virus titers were maintained for 1-3 days PI in chickens inoculated with Ehime/91 or J1 strains, whereas the virus titers increased gradually and reached to the peak on 3 days PI in chickens inoculated with the K strain. There were no clear differences in both the virus and the virus antigen titers in bursae and thymus between chickens inoculated with Ehime/91 and J1 stains. However, the virus and/or the virus antigen titers in spleen and bone marrow of chickens inoculated with Ehime/91 strain were higher than those of the J1-inoculated chickens. Immunohistochemical analyses indicated that larger numbers of IBDV antigen-positive cells were detected in spleen and bone marrow of the Ehime/91 group than in those of the J1 group. There was almost no detectable virus and virus antigens in thymus, spleen, and bone marrow of the K-inoculated chickens throughout the experiment. These results suggest that efficient replication of IBDV not only in the bursa but also in the spleen and the bone marrow may be required for clinical infectious bursal disease.

A highly sensitive, broad-spectrum infectivity assay for infectious bursal disease virus.

In order to develop an infectivity assay for infectious bursal disease virus (IBDV), an enzyme-linked immunosorbent assay (ELISA) for detecting IBDV antigens was developed using a rabbit antiserum to IBDV. The ELISA detected both serotypes 1 and 2 of IBDV, purified virus proteins at a concentration of about 0.1 ng/well and about 10(4) to 10(5) infectious units/well, and was about 10(3) times more sensitive than an agar gel precipitin test. By using the ELISA for detecting IBDV antigens in the cultured fluids, infectivity titration could be determined within 5 days of cultivation in seven of the 10 virus-cell combinations when chicken embryo fibroblasts (CEFs), BGM-70 cells, and LSCC-BK3 cells were used. IBDV antigens were not detected in three combinations remaining after 7 days of cultivation. When 18 IBDV strains, including highly virulent, classically virulent, and attenuated strains, were tested for growth in four types of cells using the ELISA, eight strains in CEFs, seven strains in chicken kidney cells, five strains in BGM-70 cells, and 17 strains in LSCC-BK3 cells were detected. These results indicated that LSCC-BK3 cells had the broadest spectrum for IBDV. When 26 field bursal homogenates were tested for infectious IBDV using LSCC-BK3 cells, all 19 field IBDV isolates that were detected by immunostaining of the cells were also detected by the ELISA. These results indicate that this infectivity assay for IBDV using LSCC-BK3 cells and the ELISA has a high sensitivity and a broad spectrum for IBDV and is especially useful for large-scale applications.

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.

No evidence for adaptation of current egg drop syndrome 1976 viruses to chickens.

In order to determine whether the current field strains of egg drop syndrome (EDS) 1976 viruses adapt to chickens, we compared the growth efficiency of three Japanese field strains (PA-1/79, AWI/98, Gifu/01) in chicken and duck embryo liver cells. The growth efficiency in chicken or duck embryo liver cells was almost similar in these strains. The fiber protein may carry the type-specific antigen and the hemagglutination activity, and hexon protein may contain the subgroup-specific antigenic determinants. Therefore, the fiber head and hexon loop 1 DNA domain sequences of the six Japanese field strains UPA-1/79, ME/80, 44/81, Kyoto/91, AWI/98, Gifu/01) were compared, but these DNA domains were identical among the six field strains. Our data suggested that the EDS virus was maintained without discernible changes for the last two decades in the field.