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Following confirmation of the first case of the ongoing U.S. HPAI H5N1 epizootic in commercial poultry on February 8, 2022, the virus has continued to devastate the U.S. poultry sector and the pathogen has since managed to cross over to livestock and a few human cases have also been reported. Efficient outbreak management benefits greatly from timely detection and proper identification of the pathways of virus introduction and spread. In this study, we used changes in mortality rates as a proxy for HPAI incidence in a layer, broiler and turkey flock together with diagnostic test results to infer within-flock HPAI transmission dynamics. Mathematical modeling techniques, specifically the Approximate Bayesian Computation algorithm in conjunction with a stochastic within-flock HPAI transmission model were used in the analysis. The time window of HPAI virus introduction into the flock (TOI) and the adequate contact rate (ACR) were estimated. Then, using the estimated TOI together with the day when the first HPAI positive sample was collected from the flock, we calculated the most likely time to first positive sample (MTFPS) which reflects the time to HPAI detection. The estimated joint (i.e., all species combined) median of the MTFPS for different flocks was six days, the joint median most likely ACR was 6.8 newly infected birds per infectious bird per day, the joint median R 0 was 13 and the joint median number of test days per flock was two. These results were also grouped by species and by epidemic phase and discussed accordingly. We conclude that this findings from this and other related studies are beneficial for the different stakeholders in outbreak management. We recommend that combining TOI analysis with complementary approaches such as phylogenetic analyses is critically important for improved understanding of disease transmission pathways. The estimated parameters can also be used to parametrize mathematical models that can guide the design of surveillance protocols, risk analyses of HPAI spread, and emergency preparedness for HPAI outbreaks.

Since August 2014, the University of Minnesota Veterinary Diagnostic Laboratory has received cases of turkey enteritis that are clinically different from previously described cases of poult enteritis syndrome and light turkey syndrome. The birds develop dark green and extremely foul-smelling diarrhea starting at 8–10 wk of age, which may last up to 15–16 wk of age. The affected turkey flocks show poor uniformity, and feed conversion and market weights are reduced. Multiple-age farms are affected more often than the single-age farms. Morbidity varies from flock to flock and in some cases reaches 100%. At necropsy, undigested feed with increased mucus is observed in the intestines along with prominent mucosal congestion and/or hemorrhage. Microscopically, lymphocytic infiltrates expand the villi in duodenum and jejunum to form lymphoid follicles, which are often accompanied by heterophils. Next generation sequencing (Illumina Miseq) on a pool of feces from affected birds identified genetic sequences of viruses belonging to Astroviridae, Reoviridae, Picornaviridae, Picobirnaviridae, and Adenoviridae. On testing pools of fecal samples from apparently healthy (16 pools) and affected birds (30 pools), there was a higher viral load in the feces of affected birds. Picobirnavirus was detected only in the affected birds; 20 of 30 pools (66.7%) were positive. These results indicate that a high viral load of turkey picobirnavirus alone, or in association with novel picornaviruses, may be a cause of this new type of turkey enteritis.

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9–20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase–polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

A reverse-transcriptase–polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) –affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5–35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3–14 PE in freshly collected proventriculi, and on days 1–14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3–10 PE, and by virus isolation on days 1–14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n  =  19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistent with TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P  =  0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.

The clinically approved antioxidant cardioprotective agent dexrazoxane (ICRF-187) was examined for its ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. Hydrolyzed dexrazoxane metabolites prevent site-specific iron-based oxygen radical damage by displacing iron from doxorubicin and chelating free and loosely bound iron. The mitochondrial stain MitoTracker Green FM and doxorubicin were shown by epifluorescence microscopy to accumulate in the myocyte mitochondria. An epifluorescence microscopic image analysis method to measure mitochondrial damage was developed using the mitochondrial membrane potential sensing ratiometric dye JC-1. This method was used to show that dexrazoxane protected against doxorubicin-induced depolarization of the myocyte mitochondrial membrane. Dexrazoxane also attenuated doxorubicin-induced oxidation of intracellular dichlorofluorescin. Annexin V-FITC/propidium iodide staining of myocytes was used to demonstrate that, depending on the concentration, doxorubicin caused both apoptotic and necrotic damage. These results suggest that doxorubicin may be cardiotoxic by damaging the mitochondria and dexrazoxane may be protective by preventing iron-based oxidative damage.

We have examined a variety of sampling strategies for detecting pathogens in turkey flocks undergoing infections with low pathogenicity avian influenza virus (LPAIV). We found that viral RNA was widely distributed in the barn environment of turkey flocks undergoing an active LPAIV infection and was in both water and drinker biofilm samples. Viral RNA was concentrated in drinker biofilm and sediment and was detectable using real-time reverse-transcription polymerase chain reaction (RRT-PCR) and by virus isolation. Drinker biofilm sample results correlated with concurrently collected oropharyngeal (OP) sample results from flocks on a farm with LPAI in which the two sampling strategies were directly compared. To evaluate the utility of biofilm sampling for the detection of highly pathogenic avian influenza virus (HPAIV), biofilm and OP swabs from mortality pools were collected daily from negative turkey flocks on an HPAI-positive premise. The biofilm swabs were positive 1–2 days prior to positives appearing in the OP sample pools. The drinker biofilm sampling strategy overcame the difficulty of finding a subclinical infectious bird in a population by collecting material from a large number of individuals and testing a sample in which a positive signal persists for several days to weeks. The sampling method is convenient for use in turkey barns and has been reliably used in both active and passive surveillance programs for LPAIV and HPAIV using RRT-PCR.

Senior to graduate-level bioinorganic text.

Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31]S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.

Following confirmation of the first case of the ongoing U.S. HPAI H5N1 epizootic in commercial poultry on February 8, 2022, the virus has continued to devastate the U.S. poultry sector and the pathogen has since managed to cross over to livestock and a few human cases have also been reported. Efficient outbreak management benefits greatly from timely detection and proper identification of the pathways of virus introduction and spread. In this study, using changes in mortality rates as a proxy for HPAI incidence in a layer, broiler and turkey flock, mathematical modeling techniques, specifically the Approximate Bayesian Computation algorithm in conjunction with a stochastic within-flock HPAI transmission model, were used to estimate the time window of pathogen introduction into the flock (TOI) and adequate contact rate (ACR) based on the daily mortality and diagnostic test results. The estimated TOI was then used together with the day when the first positive sample was collected to calculate the most likely time to first positive sample (MTFPS) which reflects the time to HPAI detection in the flock. The estimated joint (i.e., all species combined) median of the MTFPS for different flocks was six days, the joint median most likely ACR was 6.8 newly infected birds per infectious bird per day, the joint median R0was 13 and the joint median number of test days per flock was two. These results were also grouped by species and by epidemic phase and discussed accordingly. We conclude that findings from this and related studies are beneficial for the different stakeholders in outbreak management and combining TOI analysis with complementary approaches such as phylogenetic analyses is critically important for improved understanding of disease transmission pathways. The estimated parameters can also inform models used for surveillance design, risk analysis, and emergency preparedness.

Two products, (Co(en) $\\sb2$ (ox))$\\sp+$and (Co(en) $\\sb3$ )$\\sp{3+}$(en = 1,2-diaminoethane; ox $\\sp{2-}$= oxalate(2-)), are detected in the oxidation of cobalt(II) amine complexes by (Co(ox) $\\sb3$ )$\\sp{3-}$ . Kinetic studies reveal two electron transfer pathways: outer-sphere, giving (Co(en) $\\sb3$ )$\\sp{3+}$ , and inner-sphere, giving (Co(en) $\\sb2$ (ox))$\\sp+$ . The enantiomeric excess for the outer-sphere pathway is 9%$\\Delta\\Lambda$ . The inner-sphere pathway involves formation of a bidentate oxalate bridge between the oxidant and reductant, determined by$\\sp{13}$ C-labeled oxalate tracer studies. The stereoselectivity for the (Co(en) $\\sb2$ (ox))$\\sp+$product, 1.5%$\\Delta\\Delta$ , may be explained by the three atom oxalate bridge which inhibits intimate contact between the discriminating non-bridging ligands. Reactions between (Co(ox) $\\sb3$ )$\\sp{3-}$and N-methylated derivatives of the reductant, N,N-dimethylethylenediamine (N,N-Me $\\sb2$ -en) and N,N $\\sp\\prime$ -dimethylethylenediamine (N,N $\\sp\\prime$ -Me $\\sb2$ -en), have been examined. Stereoselectivities for (Co(N,N-Me $\\sb2$ -en) $\\sb2$ )$\\sp{2+}$ , and (Co(N,N $\\sp\\prime$ -Me $\\sb2$ -en) $\\sb2$ )$\\sp{2+}$reductants are 4.3%$\\Delta\\Lambda$and 7.9%$\\Delta\\Delta$ , respectively, reflecting the presence of the additional methyl groups. Kinetics and stereoselectivities in reactions between (Fe $\\sp{\\rm III/II}$ Me $\\sb2$ L(1))$\\sp{+/0}$(Me $\\sb2$ L(1)H $\\sb2$= 3,24-dimethyl-4,7,10,13-tetraazahexadeca-3,13-diene-2,15-dione dioxime) and (Co(edta))$\\sp{2-/-}$or (Co(phen) $\\sb3$ )$\\sp{2+/3+}$(edta $\\sp{4-}$= 1,2-diaminoethane- $N,N,N\\sp\\prime N\\sp\\prime$ -tetraacetate; phen = 1,10-phenanthroline), have been investigated. Trends in data indicate that when electrostatic interactions in the precursor ion-pair are favorable, they govern the magnitude and sense of the observed stereoselectivity. However, when electrostatics are less important, electronic factors may predominate. To provide such interpretation with structural basis, a novel approach to elucidate ion-pair structure directly in solution using proton NMR relaxation techniques has been employed. Investigations of interactions between paramagnetic (Cr(edta))$\\sp-$and (Cr(phen) $\\sb3$ )$\\sp{3+}$ , and diamagnetic (CoMe $\\sb2$ L(1))$\\sp+$isostructural analogs suggest that the chromium(III) reagents approach the cobalt(III) complex differently. Differences in chiral induction in reactions involving bis(oxime-imine) complexes with (Co(edta))$\\sp{2-/-}$and (Co(phen) $\\sb3$ )$\\sp{2+/3+}$are the result of differences in the nature of these interactions.