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There have been limited studies focused on validation of swine microRNAs (miRNA) with mRNA targets. The objective of this study was to validate a defined set of targets using artificial miRNA mimics transfected into cell lines to confirm specific targets of endogenous miRNAs after administration of Escherichia coli lipopolysaccharide (LPS). Sixteen hours after mimic transfection of 3D4/21 cell lines, the cells were stimulated with 1 μg/ml LPS or phosphate-buffered saline (PBS). The cells were harvested and collected at 0, 1, 3, and 8 h post administration. The selected genes DAD1, IL8, and ESR, which are involved in known pathways of inflammation. and are predicted or validated human targets of either miR-146a, let-7a, or miR-22-3p. These were then evaluated by quantitative real-time-PCR (qRT-PCR) to verify microRNA-mRNA interaction in swine. Using the ROX reference dye, mRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization against the PBS control group. DAD1 and ESR1 were negatively regulated by miR-22-3p and miR-146a-5p, respectively in 3D4/21 cells after LPS stimulation. However, miR-146a-5p may play an indirect positive regulatory role of both DAD1 and IL8 mRNA expression. Furthermore, we found an inverse relationship between LPS stimulation compared with the let-7a-5p overexpression with DAD1. Our inflammation study provides new evidence on the roles and predicted targets of miR-146a, let-7a, and miR-22-3p in swine.

Motor cortex (M1) has been thought to form a continuous somatotopic homunculus extending down the precentral gyrus from foot to face representations1,2, despite evidence for concentric functional zones3 and maps of complex actions4. Here, using precision functional magnetic resonance imaging (fMRI) methods, we find that the classic homunculus is interrupted by regions with distinct connectivity, structure and function, alternating with effector-specific (foot, hand and mouth) areas. These inter-effector regions exhibit decreased cortical thickness and strong functional connectivity to each other, as well as to the cingulo-opercular network (CON), critical for action5 and physiological control6, arousal7, errors8 and pain9. This interdigitation of action control-linked and motor effector regions was verified in the three largest fMRI datasets. Macaque and pediatric (newborn, infant and child) precision fMRI suggested cross-species homologues and developmental precursors of the inter-effector system. A battery of motor and action fMRI tasks documented concentric effector somatotopies, separated by the CON-linked inter-effector regions. The inter-effectors lacked movement specificity and co-activated during action planning (coordination of hands and feet) and axial body movement (such as of the abdomen or eyebrows). These results, together with previous studies demonstrating stimulation-evoked complex actions4 and connectivity to internal organs10 such as the adrenal medulla, suggest that M1 is punctuated by a system for whole-body action planning, the somato-cognitive action network (SCAN). In M1, two parallel systems intertwine, forming an integrate–isolate pattern: effector-specific regions (foot, hand and mouth) for isolating fine motor control and the SCAN for integrating goals, physiology and body movement.

OBJECTIVE To describe an ultrasound-guided technique for central venous catheter placement via the external jugular vein (EJV) in pigs. ANIMALS 96 healthy Landrace–Poland China barrows (approx 16 weeks old with a mean weight of 70 kg). PROCEDURES Pigs were anesthetized. With ultrasound guidance, a needle was inserted into the EJV without a large incision or cutdown procedure. A guidewire was inserted through the needle into the vein. A modified Seldinger technique was used to advance a catheter into the vessel until the tip was in the cranial vena cava near the right atrium. A trocar was used to create a tunnel through the subcutaneous tissues from the catheter insertion site to between the dorsal borders of the scapulae. The free end of the catheter was passed through that tunnel. An extension was attached to the catheter and secured to the skin. Pigs were euthanized and underwent necropsy at completion of the study for which they were catheterized. RESULTS Central venous catheters were successfully placed in all 96 pigs and facilitated collection of serial blood samples with minimal stress. Catheters remained in place for a mean of 6 days (range, 4 to 10 days). Necropsy revealed abscesses along the subcutaneous catheter tract in 9 pigs. Twenty pigs had histologic evidence of phlebitis and fibroplasia in the cranial vena cava. CONCLUSIONS AND CLINICAL RELEVANCE The described technique, in combination with extensive socialization, allowed serial collection of blood samples with minimal stress and restraint and is an alternative to surgical cutdown procedures for catheter placement. (Am J Vet Res 2021;82:760–769)

SUMMARYPrimary motor cortex (M1) has been thought to form a continuous somatotopic homunculus extending down precentral gyrus from foot to face representations1,2. The motor homunculus has remained a textbook pillar of functional neuroanatomy, despite evidence for concentric functional zones3and maps of complex actions4. Using our highest precision functional magnetic resonance imaging (fMRI) data and methods, we discovered that the classic homunculus is interrupted by regions with sharpy distinct connectivity, structure, and function, alternating with effector-specific (foot, hand, mouth) areas. These inter-effector regions exhibit decreased cortical thickness and strong functional connectivity to each other, and to prefrontal, insular, and subcortical regions of the Cingulo-opercular network (CON), critical for executive action5and physiological control6, arousal7, and processing of errors8and pain9. This interdigitation of action control-linked and motor effector regions was independently verified in the three largest fMRI datasets. Macaque and pediatric (newborn, infant, child) precision fMRI revealed potential cross-species analogues and developmental precursors of the inter-effector system. An extensive battery of motor and action fMRI tasks documented concentric somatotopies for each effector, separated by the CON-linked inter-effector regions. The inter-effector regions lacked movement specificity and co-activated during action planning (coordination of hands and feet), and axial body movement (e.g., abdomen, eyebrows). These results, together with prior work demonstrating stimulation-evoked complex actions4and connectivity to internal organs (e.g., adrenal medulla)10, suggest that M1 is punctuated by an integrative system for implementing whole-body action plans. Thus, two parallel systems intertwine in motor cortex to form an integrate-isolate pattern: effector-specific regions (foot, hand, mouth) for isolating fine motor control, and a mind-body interface (MBI) for the integrative whole-organism coordination of goals, physiology, and body movement.

Background Pediatric anxiety disorders are linked to dysfunction in multiple functional brain networks, as well as to alterations in the allocation of spatial attention. We used network-level analyses to characterize resting-state functional connectivity (rs-fc) alterations associated with 1) symptoms of anxiety and 2) alterations in stimulus-driven attention associated with pediatric anxiety disorders. We hypothesized that anxiety was related to altered connectivity of the frontoparietal, default mode, cingulo-opercular, and ventral attention networks and that anxiety-related connectivity alterations that include the ventral attention network would simultaneously be related to deviations in stimulus-driven attention. Methods A sample of children (n = 61; mean = 10.6 years of age), approximately half of whom met criteria for a current anxiety disorder, completed a clinical assay, an attention task, and rs-fc magnetic resonance imaging scans. Network-level analyses examined whole-brain rs-fc patterns associated with clinician-rated anxiety and with involuntary capture of attention. Post hoc analyses controlled for comorbid symptoms. Results Elevated clinician-rated anxiety was associated with altered connectivity within the cingulo-opercular network, as well as between the cingulo-opercular network and the ventral attention, default mode, and visual networks. Connectivity between the ventral attention and cingulo-opercular networks was associated with variation in both anxiety and stimulus-driven attention. Conclusions Pediatric anxiety is related to aberrant connectivity patterns among several networks, most of which include the cingulo-opercular network. These results help clarify the within- and between-network interactions associated with pediatric anxiety and its association with altered attention, suggesting that specific network connections could be targeted to improve specific altered processes associated with anxiety.

The adult human brain is organized into functional brain networks, groups of functionally connected segregated brain regions. A key feature of adult functional networks is long-range selectivity, the property that spatially distant regions from the same network have higher functional connectivity than spatially distant regions from different networks. Although it is critical to establish the status of functional networks and long-range selectivity during the neonatal period as a foundation for typical and atypical brain development, prior work in this area has been mixed. Although some studies report distributed adult-like networks, other studies suggest that neonatal networks are immature and consist primarily of spatially isolated regions. Using a large sample of neonates (n = 262), we demonstrate that neonates have long-range selective functional connections for the default mode, fronto-parietal, and dorsal attention networks. An adult-like pattern of functional brain networks is evident in neonates when network-detection algorithms are tuned to these long-range connections, when using surface-based registration (versus volume-based registration), and as per-subject data quantity increases. These results help clarify factors that have led to prior mixed results, establish that key adult-like functional network features are evident in neonates, and provide a foundation for studies of typical and atypical brain development.

The goal of this study was to compare brain structure between individuals with generalized anxiety disorder (GAD) and healthy controls. Previous studies have generated inconsistent findings, possibly due to small sample sizes, or clinical/analytic heterogeneity. To address these concerns, we combined data from 28 research sites worldwide through the ENIGMA-Anxiety Working Group, using a single, pre-registered mega-analysis. Structural magnetic resonance imaging data from children and adults (5–90 years) were processed using FreeSurfer. The main analysis included the regional and vertex-wise cortical thickness, cortical surface area, and subcortical volume as dependent variables, and GAD, age, age-squared, sex, and their interactions as independent variables. Nuisance variables included IQ, years of education, medication use, comorbidities, and global brain measures. The main analysis (1020 individuals with GAD and 2999 healthy controls) included random slopes per site and random intercepts per scanner. A secondary analysis (1112 individuals with GAD and 3282 healthy controls) included fixed slopes and random intercepts per scanner with the same variables. The main analysis showed no effect of GAD on brain structure, nor interactions involving GAD, age, or sex. The secondary analysis showed increased volume in the right ventral diencephalon in male individuals with GAD compared to male healthy controls, whereas female individuals with GAD did not differ from female healthy controls. This mega-analysis combining worldwide data showed that differences in brain structure related to GAD are small, possibly reflecting heterogeneity or those structural alterations are not a major component of its pathophysiology.

Excessive response to unexpected or "deviant" stimuli during infancy and early childhood represents an early risk marker for anxiety disorders. However, research has yet to delineate the specific brain regions underlying the neonatal response to deviant stimuli near birth and the relation to risk for anxiety disorders. The authors used task-based functional MRI (fMRI) to delineate the neonatal response to deviant stimuli and its relationship to maternal trait anxiety.The authors used fMRI to measure brain activity evoked by deviant auditory stimuli in 45 sleeping neonates (mean age, 27.8 days; 60% female; 64% African American). In 41 of the infants, neural response to deviant stimuli was examined in relation to maternal trait anxiety on the State-Trait Anxiety Inventory, a familial risk factor for offspring anxiety.Neonates manifested a robust and widespread neural response to deviant stimuli that resembles patterns found previously in adults. Higher maternal trait anxiety was related to higher responses within multiple brain regions, including the left and right anterior insula, the ventrolateral prefrontal cortex, and multiple areas within the anterior cingulate cortex. These areas overlap with brain regions previously linked to anxiety disorders and other psychiatric illnesses in adults.The neural architecture sensitive to deviant stimuli robustly functions in newborns. Excessive responsiveness of some circuitry components at birth may signal risk for anxiety and other psychiatric disorders.

Part 1 The Biology of Veterinary Cytokines: Interferons Interleukin-1 Interleukin-2 Biology of Tumour Necrosis Factor Interleukin-6. Part 2 Prophylactic Immunopotentiation: Potentiation of the Immune Response by Cytokines Drug-induced Immunomodulation of Cytokines. Part 3 Role of Cytokines in Animal Health and Disease: Type Interferons and Their Role in Maternal Recognition of Pregnancy and Fecundity in Domestic Ruminant Species Effect of Cytokines on Drug-Metabolizing Enzymes Cytokines, Stress, and Growth Modulation Role of Cytokines in the Pathogenesis and Treatment of Respiratory Disease Role of Cytokines in Intestinal Health and Disease Role of Cytokines in Feline Retrovirus Diseases and Treatment Role of Cytokines in the Prevention and Treatment of Mastitis. Part 4 Future Directions in Veterinary Medicine Mapping Cytokine Genes in Food Animals Future Directions in Animal Cytokine Research and Applictions.

Background Pediatric anxiety disorders involve greater capture of attention by threatening stimuli. However, it is not known if disturbances extend to nonthreatening stimuli, as part of a pervasive disturbance in attention-related brain systems. We hypothesized that pediatric anxiety involves greater capture of attention by salient, nonemotional stimuli, coupled with greater activity in the portion of the inferior frontal gyrus (IFG) specific to the ventral attention network (VAN). Methods A sample of children (n = 129, 75 girls, mean 10.6 years of age), approximately half of whom met criteria for a current anxiety disorder, completed a task measuring involuntary capture of attention by nonemotional (square boxes) and emotional (angry and neutral faces) stimuli. A subset (n = 61) completed a task variant during functional magnetic resonance imaging. A priori analyses examined activity in functional brain areas within the right IFG, supplemented by a whole-brain, exploratory analysis. Results Higher clinician-rated anxiety was associated with greater capture of attention by nonemotional, salient stimuli (F1,125 = 4.94, p = .028) and greater activity in the portion of the IFG specific to the VAN (F1,57 = 10.311, p = .002). Whole-brain analyses confirmed that the effect of anxiety during capture of attention was most pronounced in the VAN portion of the IFG, along with additional areas of the VAN and the default mode network. Conclusions The pathophysiology of pediatric anxiety appears to involve greater capture of attention to salient stimuli, as well as greater activity in attention-related brain networks. These results provide novel behavioral and brain-based targets for treatment of pediatric anxiety disorders.

In veterinary medicine, inflammation in swine is evaluated principally by clinical signs. This method is often unreliable when assessing large animal populations because of inconsistent interpretations of clinical observations. This study examined whether changes in miRNA expression can predict the severity of the inflammatory response in swine after administration of Escherichia coli lipopolysaccharide (LPS). Whole blood from swine challenged with LPS at 0.125 μg/kg to 2.0 μg/kg body weight was collected at 0, 1, 3, and 8 h post LPS-challenge. Mature miRNAs were extracted from plasma and quantitative real-time-PCR (qRT-PCR) was used to evaluate the 84 most abundant swine miRNAs found in plasma. The miRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization with an exogenous control. The results revealed that expression of ssc-let-7e-5p, ssc-mir-22−3p, and ssc-miR-146a-5p were the most significantly changed miRNA over the time course. At 1 h post-LPS, ssc-let-7e-5p decreased as the LPS dosage levels increased from 0.125 to 1.0 μg/kg. Similarly, as the LPS doses increased from 0.125 to 0.5 μg/kg, ssc-miR-22−3p levels significantly decreased at 1 h post-LPS. In the 2.0 μg/kg LPS, ssc-miR-146a-5p levels increased between 0 and 3 h post-LPS; however, expression was downregulated with a 145 % decrease from 3 to 8 h. The three miRNA biomarkers suggest potentially useful surrogate endpoints for the evaluation of inflammatory and endotoxemia responses in swine.

This study was initiated to determine whether a comparative pharmacokinetic (PK) approach could be used to expand the pool of approved anthelmintics for minor ruminant species. Accordingly, the PK profiles of six anthelmintics (levamisole, albendazole, fenbendazole, moxidectin, doramectin, and ivermectin) in sheep, goats, and cattle were determined. The PK values determined for each anthelmintic included Tmax , Tlast , Cmax , AUC, AUC/dose, and Cmax /dose. The results of this study demonstrate that a comparative PK approach does not show commonality in the way these six anthelmintics are individually processed by these three ruminants. While some drugs demonstrated identical PK profiles between sheep and goats, none of these drugs demonstrated PK profiles in sheep and goats comparable to the PK profiles found in cattle. The results from this study suggest drug approval across these three ruminants is not a viable concept. However, the resulting PK profiles for each combination of drug and ruminant species represents a new dataset that can be used to support the US FDA Center for Veterinary Medicine's Minor Use/Minor Species indexing process for drug approvals in minor species such as sheep and goats.

Multiple risk factors that may contribute to the development and severity of pediatric anxiety disorders, one of which is dimensional overcontrol. Overcontrol is a constellation of characteristics including heightened performance monitoring, inflexibility, perfectionism and aversion to making mistakes. In this study, we examined overcontrol in children with anxiety disorders and tested whether the underlying dimension of overcontrol specifically explains altered brain response to errors in pediatric anxiety disorders. Parent-reported scores of child overcontrol were collected in a sample of children (ages 8–12 years) with (n = 35) and without (n = 34) anxiety disorders and the relationship of overcontrol and anxiety symptoms to neural responding to errors during functional magnetic resonance imaging (fMRI) was examined. Results indicated childhood overcontrol was elevated in pediatric anxiety disorders and was significantly associated with anxiety severity, even when controlling for comorbid depression and ADHD. Additionally, overcontrol was associated with reduced neural response to errors versus correct responses in the bilateral dorsal anterior cingulate cortex (dACC) and insula, even when controlling for anxiety symptoms. Overcontrol may serve as an underlying mechanism associated with clinical pediatric anxiety that demonstrates significant associations with aberrant neural error responding. Overcontrol may be an underlying mechanism contributing to pediatric anxiety that could be targeted for early intervention.

Rationale There are no approved animal drugs for management of inflammation in swine due to lack of validated animal models. To assess efficacy, it was essential to develop proteomics approaches to identify suitable biomarkers of inflammation as presented in this study. Methods Serum samples were collected from a group of four pigs prior to (baseline) and 24 and 48 h following lipopolysaccharide (LPS) stimulation to reveal proteomic changes during inflammation. Two other pigs served as untreated controls. Proteins were separated by either one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D) gel electrophoresis (2DE) prior to analysis by nano-flow liquid chromatography (nLC) coupled to tandem mass spectrometry (MS/MS). Results We identified 165 proteins using SDS-PAGE, of which 47 proteins were also detected by 2DE prior to nLC/MS/MS. More than half (72%) of all characterized proteins were modulated as a result of LPS stimulation, many of which are known to be involved with innate and adaptive immunity. Pig serum samples obtained 24 h after LPS initiation of inflammation showed protein modulations of serum albumin, serotransferrin, light and heavy immunoglobulin chains (IGs), and major acute phase proteins including haptoglobin (HPT), serum amyloid A2 (SAA2), C-reactive protein (CRP), β-2-glycoprotein 1 (B-2GP1), alpha-2-HS-glycoprotein (A2HS), α-1-antitrypsin (A1AT), and α-1-acid glycoprotein (A1AG). SAA2 was distinguished from the other SAA isoforms by the unique peptide sequence of SAA2. Conclusions The results provided proteomics analysis of swine serum due to LPS stimulation and indicated the importance of SAA2, which appears to be unique and may be regarded as a potential clinical diagnostic biomarker of inflammation.

Thirty-two Collies were used to determine the impact of ABCB1 genotype and phenotype on the plasma pharmacokinetics of fexofenadine's (Fex) R- and S-enantiomers after bolus Fex administration, as human P-gp exhibits stereoselectivity. Each Collie's ABCB1 genotype and ivermectin (IVM) sensitivity (phenotype) was determined prior to study enrolment. Wild-type (WT) Collies had lower plasma concentrations of the individual enantiomers as compared to heterozygous IVM nonsensitive (HNS), heterozygous IVM-sensitive (HS) and homozygous mutant (MUT) Collies. Based on pairwise statistical comparison, WT Collies had statistically significantly lower (AUC0-last ) and peak (Cmax ) values compared to HS, HNS and MUT Collies. Tmax was not influenced by genotype/phenotype. Inter-individual variability in PK metrics tended to be largest for WT Collies. Although the influence of genotype/phenotype on Fex PK occurred with the individual isomers, impairment of S-Fex absorption, particularly in the MUT dogs, exceeded that associated with R-Fex. Since Fex elimination occurs primarily via biliary excretion via a transporter other than P-glycoprotein, and based upon our understanding of Fex absorption kinetics, we attributed these differences primarily to the absorption portion of the profile. These differences are expressed in a stereo-specific manner. These results demonstrate the potential negative impact on estimates of drug effectiveness and toxicity, especially for P-gp substrates that do not exhibit Central Nervous System toxicities.

In this article, we report on the development of a defined serum-free medium capable of supporting the culture expansion of mesenchymal stromal/stem cells (MSCs) from canine adipose tissue (canine Ad-MSCs). The potential benefits of serum-free media can only be utilized if cells cultured in serum-free media maintain the same functional characteristics as cells cultured in serum-containing media. Therefore, we analyze the characteristics of canine Ad-MSCs cultured in this serum-free medium or in serum-containing medium through evaluation of growth kinetics, clonogenic capacity, senescence, and differentiation capacity. Both, serum-containing medium and our serum-free medium, supported efficient growth and colony formation of canine Ad-MSCs. In addition, canine Ad-MSCs cultured in both media demonstrated similar viability after freeze/thaw, similar cell surface marker expression, and were capable of trilineage differentiation. While canine Ad-MSCs cultured in both media were generally similar, under the conditions of our study, canine Ad-MSCs cultured in serum-free medium demonstrated a shorter lag phase and higher colony-forming capacity, accelerated population doubling, maintained multipotentiality at higher passage numbers, and underwent senescence at higher passage numbers compared to canine Ad-MSCs cultured in conventional serum-containing medium. These results suggest that canine Ad-MSCs cultured in serum-free medium retain the basic characteristics associated with canine Ad-MSCs cultured in serum-containing medium, although some differences in growth kinetics were observed.

This study sought to determine if proteins associated with pain in humans could be measured using a swine in vitro model of inflammation. This would constitute the first step towards using them as surrogate endpoints to help support effectiveness indications for investigational new animal drugs to control pain in swine. Swine whole blood samples were cultured in vitro with E. coli derived-lipopolysaccharide (LPS) or without LPS for 24 h. Supernatants from these cultures were collected to determine the concentration of proteins associated with pain and whether the levels were altered in response to LPS-induced inflammation. Bradykinin protein levels steadily increased over time due to LPS stimulation and returned to 0 h levels after 6 h of culture. Corticotrophin-releasing factor protein levels were not affected by LPS. Substance-P protein trended towards increasing concentrations after LPS stimulation, following a time-concentration profile similar to that observed with bradykinin. These results suggest that 2 biomarkers may be useful as surrogate endpoints for evaluation of pain.

This article summarizes the relevant definitions related to biomarkers; reviews the general processes related to biomarker discovery and ultimate acceptance and use; and finally summarizes and reviews, to the extent possible, examples of the types of biomarkers used in animal species within veterinary clinical practice and human and veterinary drug development. We highlight opportunities for collaboration and coordination of research within the veterinary community and leveraging of resources from human medicine to support biomarker discovery and validation efforts for veterinary medicine.

Background & Aim Introduction Mesenchymal stromal cells (MSCs) are being investigated in clinical trials for treatment of a variety of veterinary conditions based on their anti-inflammatory actions and potential to induce functional tissue repair. However, outcomes of clinical trials have often fallen short of pre-clinical expectations. Heterogeneity of MSC preparations and lack of adequate characterization may be partial contributors to the variability in clinical outcomes. Purpose Identification of subpopulations within heterogenous MSC cultures may improve selection of cells with the desired functionality. Therefore, the functional heterogeneity of different subpopulations of MSCs that vary in size was evaluated, as size is hypothesized to relate to function. Methods, Results & Conclusion Methods A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size. Size sorted MSCs were then characterized and evaluated for the impact of culture conditions on functional heterogeneity. Results We found that larger subpopulations of MSCs with a mean diameter of ∼18 μm proliferate more slowly, senesce at earlier passages, and are inclined to form adipocytes compared to smaller MSC subpopulations (mean diameter of ∼15 μm). Most importantly, size-dependent functions are affected by culture conditions as well as time in culture. Cell surface staining for MSC-specific CD44 & CD90 antigens showed different sized subpopulations of MSCs had comparable expression levels suggesting that this criterion is not relevant to defining subpopulations of MSCs. However, transcriptome analysis showed that differently sized subpopulations of canine Ad-MSCs have distinct gene expression profiles, and culture medium and time in culture further affect gene expression. Conclusion This work provides a strategy for identifying functional subpopulations of canine MSCs based on size that could facilitate the development of quality cellular therapies through a better understanding of functional heterogeneity.

Carrageenan-induced inflammation has long been used as an in vivo model of local inflammation. We developed an in vitro model of inflammation using primary blood cells to characterize gene induction following carrageenan (λ-CGN) stimulation and identify the signal transduction pathway(s) through which λ-CGN worked, using swine whole blood cultures from Yorkshire barrows. Blood samples were divided into stimulated and unstimulated groups. Unstimulated blood was a control for λ-CGN treated cultures to delineate treatment effects from time-in-culture effects. All cultures were collected and separated into two fractions; supernatant for ELISA analyses and white blood cells for mRNA expression. Lambda (λ)-CGN induced MCP-1 at the proteomic and the genomic levels. Lambda-CGN increased IL-8 protein production but had no impact on serum amyloid A protein levels. Alveolar Macrophage-Derived Neutrophil Chemotactic Factor-II (AMCF-II), a swine-specific member of the IL8/GRO family, showed increased gene expression. TNF-α and IL-6 protein levels were not induced by λ-CGN stimulation. Stimulation of HEK-293 cells co-transfected with a single pattern recognition receptor and the secreted embryonic alkaline phosphatase (SEAP) read-out system demonstrated that λ-CGN signals through the TLR-2 and TLR-4 signal transduction pathways. Using silencing RNA to inhibit TLR6 expression in TLR2 transfected HEK-293 cells indicated that λ-CGN works through the TLR2/6 pathway. Silencing TLR6 expression in TLR4 transfected HEK-293 cells showed that λ-CGN stimulation of this cell line worked through a TLR4/6 heterodimer, as lipopolysaccharide (LPS) induced SEAP production through a TLR4 homodimer. These results demonstrate that although carrageenan can stimulate through TLR4 signaling pathways, it initiates an inflammatory response in these cells that differs from a typical endotoxin effect such as LPS stimulation, in terms of the pathways and gene products altered, suggesting that activation of TLR2/6 and TLR4/6 are the predominant pathways through which carrageenan induces inflammatory responses.

Thirty-three Collies (14 male and 19 female) were used in a dose-escalation study to determine the impact of ABCB1 genotype on loperamide pharmacokinetics (PK) and pharmacodynamics (PD). Loperamide was orally administered in four ascending doses (0.01, 0.05, 0.1, or 0.2 mg/kg) over a 4-wk period to fasted Collies. Comparisons were made within each dose to genotype, phenotype, and whether Collies received three (3D) or four (4D) loperamide doses. The 3D and 4D groupings had statistically significant differences in systemic drug exposure (defined by the area under the concentration-versus-time profile estimated from time zero to the last quantifiable drug concentration, AUC0-last). In contrast, statistical differences in AUC0-last only occurred in the comparison between wild-type (WT) Collies versus homozygous mutant (Mut) Collies administered 0.1 mg/kg. Statistical differences in the proportionality relationship were observed when comparing 3D to 4D Collies, and the WT to Mut Collies. Intersubject variability in drug exposure tended to be twice as high between Mut and WT Collies. Associations were observed between systemic drug exposure and ataxia or depression but not between systemic drug exposure and mydriasis or salivation. Thus, Collies expressing the greatest sensitivity to CNS-associated effects of loperamide (Mut) tended to have higher drug exposure compared with those less sensitive to the adverse effects of loperamide. Genotype and phenotype only partially explained differences in loperamide PK and PD, suggesting this relationship may not be straightforward and that other factors need to be considered. Accordingly, the PD and PK of one P-glycoprotein substrate only partially predicted the likelihood of adverse responses to unrelated substrates.

Objective—To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ). Animals—8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT). Procedures—Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates. Results—Compared with control ABCB1-WT mice, ABCB1-1Δ mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1Δ mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1Δ mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ≥ 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1Δ mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network. Conclusions and Clinical Relevance—These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1Δ mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1Δ mutation.

The therapeutic potential of stem cells has fascinated those interested in treating diseases in both human and animal subjects. Although the exact mechanism of action and the definitive effectiveness of stem cell therapies remain unclear, animal owner perceptions and a desire for improved treatment options have fueled the interest of clinicians and stakeholders. Standards do not yet exist to define the critical attributes of mesenchymal stem/stromal cell (MSC)-based products derived from veterinary species such as the dog, cat, and horse. This has led veterinary stakeholders to adopt those guidelines and criteria set forth for human MSC-based products; however, these criteria are not always applicable to MSCs from dogs, cats, and horses (e.g., variability in species-specific cell surface marker expression and antibody cross reactivity). Establishing useful standards and meaningful product quality criteria as well as the understanding of full spectrum of MSC functions and preclinical evidence for safety and therapeutic efficacy for veterinary (companion and recreational animals) MSC-based-products will be critical to furthering product development, and may ultimately facilitate the availability of FDA-approved MSC-based products for use in veterinary medicine.

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato's approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.

Certain dog breeds, especially Collies, are observed to exhibit neurotoxicity to avermectin drugs, which are P-glycoprotein (P-gp) substrates. This neurotoxicity is due to an ABCB1 gene mutation (ABCB1-1Δ) that results in non-functional P-gp expression. A developed Abcb1a knock-in/Abcb1b knock-out mouse model expressing the ABCB1-1Δ canine gene was previously reported and mice exhibited sensitivity upon ivermectin administration. Here, model and wild-type mice were administered P-gp substrates doramectin, moxidectin, and digoxin. While knock-in/knock-out mice exhibited ataxia, lethargy and tremor, wild-type mice remained unaffected. In addition, no neurotoxic clinical signs were observed in either mouse type administered domperidone, a P-gp substrate with no reported neurotoxicity in ABCB1-1Δ Collies. Overall, neurotoxic signs displayed by model mice closely paralleled those observed in ivermectin-sensitive Collies. This model can be used to identify toxic P-gp substrates with altered safety in dog populations and may reduce dog use in safety studies that are part of the drug approval process.

Objectives P-glycoprotein (P-gp) is responsible for the efflux of a broad variety of human and veterinary drugs. Canine P-gp polymorphisms alter drug disposition and toxicity, but their impact on the disposition of enantiomeric drugs is unknown. Using fexofenadine as a model compound, we developed and validated HPLC–fluorescence methods to determine the effect of P-gp on the disposition of fexofenadine and its enantiomers. Methods A chiral CD-Ph column was used for the separation of (R) and (S)-fexofenadine. Determination of racemic fexofenadine was achieved on an XDB-CN column. Fexofenadine and its enantiomers were detected by fluorescence at the excitation wavelength of 220 nm and emission wavelength of 300 nm. These methods were used to measure concentrations of fexofenadine and its enantiomers in Collie plasma after oral administration. Key findings This study demonstrates that P-gp prefers to transport (S)-fexofenadine, and P-gp deficiency causes the increase in both (R)-fexofenadine and (S)-fexofenadine in plasma. Racemic fexofenadine, (R)-fexofenadine and (S)-fexofenadine were increased in ABCB1-1Δ Collies (118.7, 72.0 and 48.3 ng/ml) compared to wild-type Collies (25.0, 16.5 and 7.7 ng/ml) at 1 h postadministration. The results demonstrate that the stereoselectivity of P-gp plays a key role in the disposition of fexofenadine enantiomers. Conclusions The information derived from this drug model will be used to determine whether additional safety or efficacy requirements are necessary for enantiomeric drugs that would be used in dogs or humans.

Objective—To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies. Animals—14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted. Procedures—Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5). Each was observed for clinical signs of toxic effects from 0 to 7 hours following drug administration. Results—After ivermectin administration, considerable differences were observed in drug sensitivity between the 2 types of mice. The genetically engineered mice with the mutated canine ABCB1 gene had signs of severe sensitivity to ivermectin, characterized by progressive lethargy, ataxia, and tremors, whereas the wild-type control mice developed no remarkable effects related to the ivermectin. Conclusions and Clinical Relevance—The ivermectin sensitivity modeled in the transgenic mice closely resembled the lethargy, stupor, disorientation, and loss of coordination observed in ivermectin-sensitive Collies with the ABCB1–1Δ mutation. As such, the model has the potential to facilitate toxicity assessments of certain drugs for dogs that are P-glycoprotein substrates, and it may serve to reduce the use of dogs in avermectin derivative safety studies that are part of the new animal drug approval process.

Non-steroidal anti-inflammatory drugs (NSAID) are a family of chemicals that function to reduce pain, fever, and inflammation, and they are commonly used in people and animals for this purpose. Currently there are no NSAIDs approved for the management of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. A previous in vitro study examining biomarkers of inflammation identified fourteen genes that were significantly altered in response to Escherichia coli lipopolysaccharide (LPS)-induced inflammation. In the present study, five of those fourteen genes were tested in vivo to determine if the same effects observed in vitro were also observed in vivo . Plasma levels of prostaglandin E 2 (PGE 2 ), an essential mediator of fever and inflammation, were also determined. Two groups of swine were stimulated with LPS with the second group also treated with flunixin meglumine. Blood was collected at 0, 1, 3, 6, 8, 24, and 48 h post LPS-stimulation. The RNA was extracted from the blood and quantitative real-time-PCR (qRT-PCR) was utilized to determine the expression patterns of CD1, CD4, serum amyloid A2 (SAA2), Caspase 1, and monocyte chemoattractant protein 1 (MCP-1). The LPS-stimulated animals demonstrated a statistically significant alteration in expression of SAA2 and CD1 at 3 h post-stimulation. Flunixin meglumine treated animals’ demonstrated reduced expression of CD1 in comparison to the LPS-stimulated swine at 24 and 48 h post LPS-stimulation. Flunixin meglumine treated animals exhibited reduced expression of SAA2 at 48 h post-stimulation compared to LPS-stimulated swine. Swine treated with LPS demonstrated statistically significant increases in plasma PGE 2 at 1 h post-stimulation. Swine treated with flunixin meglumine had no increase in plasma PGE 2 levels at any time. These results demonstrate that PGE 2 production, along with two out of five genes (SAA2 and CD1) have the potential to serve as early biomarkers of inflammation as well as indicators of NSAID efficacy.

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.

There are currently no validated animal models or suitable biomarkers with which to ascertain the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in equine, bovine or ovine species during conditions of endotoxemia. This has resulted in approval of only one NSAID, flunixin meglumine, for controlling inflammation due to endotoxemia in bovine and equine animals, while none are approved in ovine animals for this claim. This study aims to investigate biomarkers with which to test efficacy of NSAIDs in these species. To this end, the effects of Escherichia coli lipopolysaccharide (LPS) induced inflammation on gene expression were investigated. Whole blood from each species was cultured and stimulated with LPS, after which RNA was extracted at various times. RNA was analyzed via quantitative RT-PCR (qRT-PCR) to determine differential expression of biomarkers. Results indicated up-regulation of cluster of differentiation 1 (CD1) gene in bovine and serum amyloid A (SAA) gene in ovine cultures. Down-regulation of cluster of differentiation 4 (CD4) gene and Caspase 1 was seen in bovine, and of CD1 in equine cultures. This work demonstrates that LPS stimulation alters expression of these genes in these species. These genes may be useful biomarkers for inflammation which could serve as markers for NSAID efficacy.

In order to study the feasibility of power scaling in resonantly-pumped Er-doped lasers, the fractional heat loads of several laser materials were measured using direct thermocouple calorimetry under low-power illumination with ∼1550 nm light. A sample of 2% Er:YAG exhibited heat loads (relative to the absorbed power) of less than 5.4%, while samples of 1.1% Er-doped fluorophosphate glasses showed similar results, with heat loads of less than 5.3%. A fluorophosphate glass codoped with 2.2% Er and 2.5% Yb showed much higher heat loads of 11–14%, and samples of singly Er-doped and Er/Yb-codoped phosphate glasses both showed even higher heat loads in excess of 17%.

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

λ-Carrageenan (λCGN) is frequently used to model local inflammation, however, the mechanism by which λCGN functions has not been fully examined. Swine whole blood was stimulated with λCGN and biomarkers were evaluated. Monocyte Chemotactic Protein-1(MCP-1) and Interleukin-8 protein levels were increased, while Serum Amyloid A (SAA) and Tumor Necrosis Factor-α (TNFα) protein levels were unchanged. Gene expression of MCP-1, Phosphatidylinositol 3-Kinase, and Alveolar Macrophage Chemotactic–II were increased; gene expression of SAA, Interleukin-6, and TNFα were unchanged; and gene expression of Thrombospondin-2 Precursor were decreased. Assessment of which TLRs (Toll-Like Receptors) were involved in the λCGN activation pathway, HEK-293 cells transfected with one plasmid containing either TLR2 or TLR4, along with a second plasmid to release SEAP (secreted embryonic alkaline phosphatase) upon NFκB activation were stimulated with λCGN. TLR2 and TLR4 both showed increased SEAP release after λCGN stimulation. The lack of TNFα production after λCGN stimulation suggested that it was not working through a classic TLR4 homodimer. To determine if a common mechanism could explain these results, RNA silencing studies were initiated. TLR6 was silenced in SEAP HEK-293 cells containing a plasmid for either TLR2 or TLR4. In both cell lines where TLR6 was silenced, λCGN was unable to induce a SEAP response, while these cells maintained responsiveness to a TLR1/2 ligand and a TLR 4 ligand, respectively. These results suggest that λCGN initiates inflammation via TLR2/6 and TLR4/6 heterodimers.

In this study, six horses each had three subcutaneous tissue chambers. Each horse was pretreated with intravenous saline (control), cimetidine, or aminoguanidine with a 4-week washout period. On day 0, a carrageenan (NaCl inflamed) was instilled in 1tissue chamber. Blood and all tissue chamber fluids were sampled on days -1 to 3 and measured for activity levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6. The Tmax for all cytokines (TNF-a, IL-1, IL-6) was 0.5 and 1 day post-inflammation in tissue chamber fluid. In the tissue chamber fluid, NaCl inflamed (NaCl-I), cimetidine, and aminoguanidine groups showed significant increases in IL-1 production. In the NaCl-I and cimetidine, tissue chamber fluid IL-6 was significantly increased when compared with NaCl noninflamed (NaCl-N) for the periods of study -1 to 1, -1 to 2, and -1 to 3. The cimetidine tissue chamber fluid [TCF] appeared to have a delayed, but greater, IL-6 response than the NaCl-I treatment. Even with cimetidine and aminoguanidine pretreatment, the resulting decrease in NO production demonstrated in our previously reported results in these same animals appeared to be too late to decrease the early cytokine response. However, it may assist in preventing the subsequent NO-induced upregulation of pro-inflammatory cytokine production.

The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Eighteen Holstein dairy cows ranging in body weight from 500-700 kg and with an average milk yield of 37 ± 6 kg/day were used to investigate the depletion of florfenicol (FFL) in milk and plasma of dairy cows. Three groups of six were administered FFL: Group A, intramammary (IMM) infusion of ~2.5 mg FFL/kg BW at three consecutive milking intervals (total amount of ~7.5 mg/kg BW); Group B, one IMM infusion (20 mg/kg BW) into one quarter and Group C, one subcutaneous (SC) treatment (40 mg/kg BW). IMM infusions were into the right front quarter. Cows were milked daily at 06:00 and 18:00 h. The highest concentrations (Cmax ) and time to Cmax (Tmax ) were: 1.6 ± 2.2 μg·FFL/mL milk at 22 h (Group A), 5.5 ± 3.6 μg·FFL/mL milk at 12 h (Group B), and 1.7 ± 0.4 μg·FFL/mL milk at 12 h (Group C). The half-lives (t1/2 ) were ~19, 5.5, and 60 h, for Groups A, B, and C, respectively. FFL was below the limit of detection (LOD) by 60 h in three Group B cows, but above the LOD at 72, 84, and 120 h in three cows. FFL was above the LOD in milk from Group C's cows for 432-588 h. Plasma values followed the same trends as milk. The results demonstrate that IMM-infused FFL is bioavailable and below the LOD within 72-120 h. The concentration of FFL was detectable in both plasma and milk over the course of 2-3 weeks after SC administration. The absence of residue depletion data presents problems in determining safe levels of FFL residues in milk and edible tissues. The data presented here must not be construed as approval for extra-label use in food animals.

Atmosphere is one of the most important noise sources for ground-based cosmic microwave background (CMB) experiments. By increasing optical loading on the detectors, it amplifies their effective noise, while its fluctuations introduce spatial and temporal correlations between detected signals. We present a physically motivated 3d-model of the atmosphere total intensity emission in the millimeter and sub-millimeter wavelengths. We derive a new analytical estimate for the correlation between detectors time-ordered data as a function of the instrument and survey design, as well as several atmospheric parameters such as wind, relative humidity, temperature and turbulence characteristics. Using an original numerical computation, we examine the effect of each physical parameter on the correlations in the time series of a given experiment. We then use a parametric-likelihood approach to validate the modeling and estimate atmosphere parameters from the POLARBEAR-I project first season data set. We derive a new 1.0% upper limit on the linear polarization fraction of atmospheric emission. We also compare our results to previous studies and weather station measurements. The proposed model can be used for realistic simulations of future ground-based CMB observations., Comment: 20 pages, 16 figures

Performance characteristics were evaluated for two lateral-flow test kits, Reveal for Ruminant in Feed (Neogen Corporation) and FeedChek (Strategic Diagnostics Inc.), designed to detect ruminant or terrestrial animal proteins in feeds. The stringent acceptance criteria used were developed by the Center for Veterinary Medicine Office of Research to identify test kits with comparable selectivity and sensitivity to microscopy and PCR assay, the analytical methods used by the U.S. Food and Drug Administration (FDA). Guidelines were developed for evaluating the selectivity, sensitivity, ruggedness, and specificity of these kits. These guidelines further stated that ruggedness and specificity testing would be performed only after a test passed both the selectivity and sensitivity assessments. Acceptance criteria for determining success were developed using a statistical approach requiring 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% bovine meat and bone meal, consistent with the sensitivity of the methods used by the FDA, was required. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of the same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% bovine meat and bone meal. The Reveal test passed the selectivity assessment but failed the sensitivity assessment, detecting only samples fortified at the 2% level and then only 17 to 33% of those samples, when read according to the label directions. The FeedChek test passed the sensitivity assessment but failed the selectivity assessment, with rates for false-positive results ranging from 34 to 38%, depending on the user. The sensitivity of the Reveal test was affected by the concentration of trace minerals present in the feed; concentrations toward the high end of the normal range prevented the detection of true positive feed samples containing bovine meat and bone meal. Better sensitivity assessments were obtained when lamb meal was used either alone or in combination with bovine meat and bone meal. The FeedChek test was not affected by the concentration of trace minerals or by the type of animal meal used. These results indicate that neither of the two tests is adequate for routine regulatory use.

A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.

In this study, we examined the role of different amounts of dietary fishmeal, a component of chicken diet on cell-mediated immune response of chickens. Forty eight laying white Leghorn chickens were randomly divided into 4 groups and fed diets containing 0, 1.5, 3 or 6% fish-meal. Six chickens from each dietary group were euthanized 3 and 9 weeks later and the spleens were removed to isolate cells and assess immune response. Mitogenic response was determined by H-thymidine incorporation and the cell 3 subsets were established by flow cytometry. Three weeks of dietary treatment had no impact on Concanavalin A (Con A) or phytohemagglutinin (PHA) induced proliferation or cell subsets of splenic lymphocytes. However, 9 weeks post dietary treatment PHA-induced proliferation and CD4 cell population were elevated in 3% fishmeal fed hens compared to those fed the control (0% fishmeal) diet. Our results suggest that a moderate increase in dietary fish-meal may enhance the immune response in laying hens. This may prove useful in infections in which cell-mediated immunity plays a role.

Objectives:Actinobacillus pleuropneumoniae causes an often fatal infection of swine due to pleuropneumonia. To determine if inflammatory cytokines are associated with A. pleuropneumoniae-induced pneumonia, infected and noninfected animals were concomitantly administered saline or dexamethasone. Methods: Twenty-four swine were treated with saline, A. pleuropneumoniae, dexamethasone, or A. pleuropneumoniae and dexamethasone (n = 6). The plasma levels of TNF-α, IL-1β, IL-6, IL-8, and IL-10 were examined through time of necropsy (72 h). Gross pathology and histopathology was performed on all animals. Results: Dexamethasone had no effect on A. pleuropneumoniae-induced increases in lung/body weight ratios. Gross pathology of the infected pigs included fibrinous pleuropneumonia with necrosis and hemorrhage in a focal to a multifocal pattern. Histopathology of infected pig lungs revealed necrotizing extensive, fibrinopurulent pneumonia with edema and fibrinopurulent pleuritis. Plasma IL-6 levels were elevated in A. pleuropneumoniae-infected animals beginning 6 h after infection. Dexamethasone treatment did not alter A. pleuropneumoniae-induced plasma IL-6 levels. A. pleuropneumoniae infection did not elicit plasma levels of TNF-α, IL-1β, IL-8, or IL-10. Conclusion: These results suggest that the pneumonia caused by A. pleuropneumoniae infection is not due to the release of systemic inflammatory cytokines.

Objective—To determine the effect of oral administration of low doses of pentobarbital on cytochrome P450 (CYP) isoforms and CYP-mediated reactions in immature Beagles. Animals—42 immature (12-week-old) Beagles. Procedure—Dogs were grouped and treated orally as follows for 8 weeks: low-dose pentobarbital (50 µg/d; 4 males, 4 females), mid-dose pentobarbital (150 µg/d; 4 males, 4 females), high-dose pentobarbital (500 µg/d; 4 males, 4 females), positive-pentobarbital control (10 mg/kg/d; 2 males, 2 females), positivephenobarbital control (10 mg/kg/d; 2 males, 2 females), and negative control (saline [0.9% NaCl] solution; 5 males, 5 females). Serum biochemical and hematologic values were monitored. On necropsy examination, organ weights were determined, and histologic evaluation of tissue sections of liver, kidney, small intestine, testes, epididymis, and ovaries was performed. Hepatic and intestinal drug-metabolizing enzyme activities were measured, and relative amounts of CYP isoforms were determined by western blot analysis. Results—The amount of a hepatic CYP2A-related isoform in dogs from the high-dose pentobarbital treatment group was twice that of dogs from the negative control group. CYP2C was not detectable in small intestinal mucosa of dogs from the negative control group; measurable amounts of CYP2C were found in dogs from the various (low-, mid-, and high-dose) pentobarbital treatment groups and from positive-pentobarbital and positive phenobarbital control groups. Several CYP-mediated reactions increased in a dosedependent manner. The lowest calculated effective dose of pentobarbital ranged from 200 to 450 µg/d. Conclusions and Clinical Relevance—Several CYP isoforms and their associated reactions were induced in dogs by oral administration of low amounts of pentobarbital. (Am J Vet Res 2003;64:1167–1175)

The effect of multiple lipopolysaccharide (LPS) challenges in swine undergoing long-term treatment with porcine somatotropin (PST) was determined. Changes in aspartate serine transaminase (AST) occurred only at 24h following the first LPS challenge dose (P

The impact of Escherichia coli-derived lipopolysaccharide (LPS) on the pharmacokinetic parameters of enrofloxacin in swine was assessed to determine whether this model would substitute for a pleuropneumonia infection model for pharmacokinetic evaluation of drugs. All animals received a single i.v. dose of enrofloxacin (5 mg/kg). Half the animals also received dexamethasone (0.5 mg/kg) to determine the impact of inflammation on any changes in enrofloxacin pharmacokinetics, as most of the effects of LPS are due to elaboration of inflammatory mediators. Administration of LPS alone (2.0 microg/kg) was associated with a decrease in clearance of enrofloxacin. Volume of distribution at steady state was increased in the dexamethasone-treated animals. The terminal elimination half-life of enrofloxacin was significantly increased in the LPS group. Dexamethasone administration, either alone or in combination with LPS challenge, increased the volume of distribution both at steady state and during the elimination phase. Lipopolysaccharide challenge did not affect the volume of distribution. Lipopolysaccharide challenge did not affect urinary excretion of enrofloxacin but did increase the urinary excretion of its principal metabolite, ciprofloxacin. However, the increased excretion did not begin until 24 h after administration of enrofloxacin. Because these pharamcokinetic results are different from those obtained with the pleuropneumonia model using the bacteria Actinobacillus pleuropneumoniae, the results of this study demonstrate that LPS is not a generic substitute for infection for the pharmacokinetic evaluation of drugs.